free for authors, free for readers, free from publisher, free free neuropathology 1:2 (2020) opinion piece the “neuroepithelial tumor”: exchanging our trash can for an industrial size dumpster? arie perry division of neuropathology, department of pathology, university of california, san francisco, usa corresponding author: arie perry, department of pathology, university of california, 505 parnassus avenue, san francisco, ca 94117, usa arie.perry@ucsf.edu submitted: 30 november 2019 accepted: 07 december 2019 published: 01 january 2020    https://doi.org/10.17879/fnp-2020-2611   if “a rose by any other name would smell as sweet”, then does garbage by any other name smell as foul? before i argue the affirmative, i must first fully disclose that like every other neuropathologist, i’ve previously used the term “neuroepithelial tumor” in my own reports and manuscripts. nevertheless, i’ve been increasingly concerned that its usage is now pursuing an alarming crescendo with an inversely decreasing specificity. in the 2015 world health organization (who) consensus meeting in heidelberg, a decision was made to abandon the term, “primitive neuroectodermal tumor” or pnet from the subsequent 2016 scheme1 and this was generally hailed as a major breakthrough, with the promise of enhancing our diagnostic accuracy for central nervous system (cns) tumor classification. nonetheless, it was recognized that even with improved definitions, one still encounters occasional “pnet-like” cases that do not conform to currently known entities. as such, it was decided to introduce the term, cns embryonal tumor, nos for such cases. of course, everyone recognized that this was essentially trading in one trash can for another, but with the notion that the new trash can was smaller and with the hope that as additional entities are elucidated over time, eventually this category would disappear altogether. also, given that in 2013, the soft tissue and bone blue book similarly ditched “peripheral pnet” in favor of ewing sarcoma2, this new approach essentially eliminated the diagnosis of “pnet” altogether. unfortunately, since the who 2016 publication1, i feel that our trash can is yet again expanding, given that the term “neuroepithelial tumor” (net) is gaining momentum, both within the literature and in clinical practice. for instance, whereas we previously had only two known net entities, dysembryoplastic neuroepithelial tumor (dnet) (don’t even get me started on “dysembryoplastic”) and cribriform neuroepithelial tumor (crinet), we’ve since added: 1) high-grade neuroepithelial tumor (hgnet) with mn1 alteration (hgnet-mn1), 2) hgnet with bcor exon 15 internal tandem duplication (hgnet-bcor), 3) neuroepithelial tumor with h3 g34 mutation (net-h3-g34), and 4) polymorphous low-grade neuroepithelial tumor of the young (plnty)3-9. additionally, a fifth hgnet or hgnet, not elsewhere classified (hgnet-nec) is now being used in some clinical reports for malignant cns neoplasms that don’t fit neatly into a well-delineated tumor type, although some of these descriptive diagnoses are eventually replaced by a more specific one with further molecular testing. the oxford dictionary definition of neuroepithelium is: “1. a type of epithelium containing sensory nerve endings and found in certain sense organs (e.g. the retina, the inner ear, the nasal membranes, and the taste buds)” or more pertinent to net, “2. (in embryology) ectoderm that develops into nerve tissue.” https://www.lexico.com/en/definition/neuroepithelium). other definitions similarly focus on brain development. for instance, according to wikipedia, “neuroepithelial cells, or neuroectodermal cells, form the wall of the closed neural tube in early embryonic development. the neuroepithelial cells span the thickness of the tube's wall, connecting with the pial surface and with the ventricular or lumenal surface. they are joined at the lumen of the tube by junctional complexes, where they form a pseudostratified layer of epithelium called neuroepithelium. neuroepithelial cells are the stem cells of the central nervous system, known as neural stem cells, and generate the intermediate progenitor cells known as radial glial cells, that differentiate into neurons and glia in the process of neurogenesis.” (https://en.wikipedia.org/wiki/neuroepithelial_cell). this explains the intended use of neuroepithelial tumor in the original 1988 description of dnet10, wherein the authors emphasized their view that dnet is likely related to a developmental disorder or malformation, given the frequent histologic findings resembling focal cortical dysplasia in adjacent cortex. in other circumstances, net is utilized in a broader fashion to state a belief that a neoplasm is derived from cns precursor cells. unfortunately, net is now often utilized in an even less specific manner, essentially meaning: “i think this is probably a cns tumor because it’s located there, but i wouldn’t swear to it under oath in a court of law”. as long as the entire oncology team knows that this diagnosis represents our mea culpa of ignorance, then there’s no harm in using this term as a placeholder until we know more. however, busy people (including oncologists) often generalize and may assume that given the similar terminology, hgnet, nec is equivalent to hgnet-mn1 (replacing mostly what was previously diagnosed as astroblastoma, mainly behaving as who grade ii) or to hgnet-bcor and net-h3-g34 (both behaving predominantly as who grade iv tumors). in other words, one could falsely assume that all hgnets are biologically related and should therefore be treated in a similar fashion clinically. another major source of confusion comes from very different uses of “net” by various experts. as already mentioned, in the past, it was an abbreviation for neuroectodermal tumor within both central and peripheral forms of pnet. in neuropathology, it is now being used for neuroepithelial tumor as already discussed, but outside the cns, net is currently utilized far more commonly as an abbreviation for neuroendocrine tumor11. this newly sanctioned who term represents the lower grade or well differentiated subtype of “neuroendocrine neoplasm”. in other words, this is the more favorable tumor type, but nevertheless one that occasionally behaves more aggressively; in turn, net needs to be distinguished from neuroendocrine carcinoma, which is the overtly malignant and high-grade form of disease. within neuropathology, the most common manifestation of this newly proposed nomenclature is the pituitary neuroendocrine tumor or pitnet, in place of pituitary adenoma12,13. nonetheless, with so many different versions now entering the medical lexicon, no-one should be surprised if one net subtype is confused for another. in conclusion, by discarding pnet (i.e., who grade iv small blue cell tumor with neuronal features) in favor of net or hgnet, have we essentially exchanged our trash can for an industrial size dumpster? i occasionally wake up in a sweat from dreaming of a dystopic future wherein the who scheme is simply composed of a long list of entities all entitled “neuroepithelial tumor with ___ molecular alteration”. wouldn’t it be preferable to go as far as we can with what we know? in other words, if a tumor shows compelling astrocytic features, why not invoke astrocytoma or astrocytic neoplasm in the name? if the tumor has glioneuronal features, why not say so? if indeed, neuroepithelial tumor is the best we can do, then at least, let’s make a concerted effort to replace the name once we know more. of course, this is just one man’s opinion and an opinion is only worth the price one pays for it! references 1.       louis dn, ohgaki h, wiestler od, et al. who classification of tumours of the central nervous system (revised 4th edition). lyon, france: iarc; 2016. 2.       fletcher cdm, bridge ja, hogendoorn pcw, mertens f, eds. who classification of tumours of soft tissue and bone. 4th ed. lyon, france: iarc; 2013. 3.       sturm d, orr ba, toprak uh, et al. new brain tumor entities emerge from molecular classification of cns-pnets. cell. 2016;164(5):1060-1072. 4.       korshunov a, capper d, reuss d, et al. histologically distinct neuroepithelial tumors with histone 3 g34 mutation are molecularly similar and comprise a single nosologic entity. acta neuropathol. 2016;131(1):137-146. 5.       paret c, theruvath j, russo a, et al. activation of the basal cell carcinoma pathway in a patient with cns hgnet-bcor diagnosis: consequences for personalized targeted therapy. oncotarget. 2016;7(50):83378-83391. 6.       yoshida y, nobusawa s, nakata s, et al. cns high-grade neuroepithelial tumor with bcor internal tandem duplication: a comparison with its counterparts in the kidney and soft tissue. brain pathol. 2018;28(5):710-720. 7.       huse jt, snuderl m, jones dt, et al. polymorphous low-grade neuroepithelial tumor of the young (plnty): an epileptogenic neoplasm with oligodendroglioma-like components, aberrant cd34 expression, and genetic alterations involving the map kinase pathway. acta neuropathol. 2017;133(3):417-429. 8.       ferris sp, velazquez vega j, aboian m, et al. high-grade neuroepithelial tumor with bcor exon 15 internal tandem duplication a comprehensive clinical, radiographic, pathologic, and genomic analysis. brain pathol (in press). 9.       andreiuolo f, lisner t, zlocha j, et al. h3f3a-g34r mutant high grade neuroepithelial neoplasms with glial and dysplastic ganglion cell components. acta neuropathol commun. 2019;7(1):78. 10.    daumas-duport c, scheithauer bw, chodkiewicz jp, laws er, jr., vedrenne c. dysembryoplastic neuroepithelial tumor: a surgically curable tumor of young patients with intractable partial seizures. report of thirty-nine cases. neurosurgery. 1988;23(5):545-556. 11.    rindi g, klimstra ds, abedi-ardekani b, et al. a common classification framework for neuroendocrine neoplasms: an international agency for research on cancer (iarc) and world health organization (who) expert consensus proposal. mod pathol. 2018;31(12):1770-1786. 12.    asa sl, casar-borota o, chanson p, et al. from pituitary adenoma to pituitary neuroendocrine tumor (pitnet): an international pituitary pathology club proposal. endocr relat cancer. 2017;24(4):c5-c8. 13.    villa c, vasiljevic a, jaffrain-rea ml, et al. a standardised diagnostic approach to pituitary neuroendocrine tumours (pitnets): a european pituitary pathology group (eppg) proposal. virchows arch. 2019;475(6):687-692.   62nd meeting of the french society of neuropathology meeting abstracts feel free to add comments by clicking these icons on the sidebar free neuropathology 1:34 (2020) meeting abstracts 62nd meeting of the french society of neuropathology meeting abstracts december 4th, 2020 the french society of neuropathology was created in 1989, succeeding the french club of neuropathology set up in 1965. the society organizes two scientific meetings per year.   submitted: 11 december 2020 accepted: 14 december 2020 published: 15 december 2020 https://doi.org/10.17879/freeneuropathology-2020-3134 keywords: french society of neuropathology, sfnp, meeting abstracts, 62nd meeting dec. 2020 short communications:     plu i, evrard b, duchesne m, regnault b, pérot p, chrétien d, eloit m, seilhean d (2020) etiological diagnosis of a plasma cell encephalitis by next generation sequencing. free neuropathol 1, 34: 2 etiological diagnosis of a plasma cell encephalitis by next generation sequencing isabelle plu1, bruno evrard2, mathilde duchesne3, béatrice regnault4, philippe pérot4, delphine chrétien4, marc eloit4, danielle seilhean1 1 department of neuropathology, pitie salpetriere hospital, ap-hp sorbonne university, paris, france 2 intensive care department, university hospital limoges, france 3 department of pathology, university hospital limoges, france 4 pathogen discovery unit, department of virology, pasteur institute, paris a 59-year-old man, working as a forester, developed a fever and temporal headache associated with cranial nerves deficits. etiological investigations including multiplex pcr and the search for autoantibodies remained negative. his condition quickly worsened leading to coma and death after 2 months.   neuropathology revealed lymphoplasmacytic infiltrates in the leptomeninges, cranial nerve roots and around the vessels in cerebral gray and white matter. perivascular cuffs were associated with astrocytic gliosis and microglial activation. immunochemistry against known viruses was negative. the new generation dna sequencing (ngs) without a priori has led to the identification of a type 1 lyssavirus of the european bat (eblv1), the reservoir of which is an insectivorous bat. only two cases of transmission to human have been reported to date. this case of zoonosis illustrates the interspecies transmission of an unusual virus leading to a potential emerging disease. ngs coupled with neuropathology provide a valuable tool for identifying new causes of encephalitis.     teyssou e, muratet f, del mar amador m, gyorgy b, guegan j, marie y, meininger v, salachas f, millecamps s, seilhean d (2020) a novel mutation in sod1 causing unusual neuropathological findings. free neuropathol 1, 34: 3 a novel mutation in sod1 causing unusual neuropathological findings elisa teyssou1*, françois muratet1*, maria del mar amador1,2, beata gyorgy1, justine guegan1, yannick marie1, vincent meininger2,3, françois salachas1,2, stéphanie millecamps1, danielle seilhean1,4 1 inserm u1127, cnrs umr7225, sorbonne university-umrs1127, brain institute icm, paris, france 2 department of neurology, reference center for als ile de france, pitie salpetriere hospital, ap-hp sorbonne university, paris, france 3 peupliers hospital, ramsay générale de santé, paris, france 4 department of neuropathology, pitie salpetriere hospital, ap-hp sorbonne university, paris, france * equal contribution a 63-year-old man with no personal or family history developed amyotrophic lateral sclerosis (als) at age 63, fatal in 17 months. in the anterior horns of the spinal cord, neuronal loss was moderate. cystatin c immunohistochemistry (ihc) showed numerous bunina bodies. ihc of ubiquitin, p62 or tdp43, did not show skein-like inclusions. swollen neurons in clusters, were labelled with antiubiquitin, sod-1 and phosphorylated neurofilaments. they were different from inclusions usually observed in sod-1 mutations.   whole exome sequencing analysis identified a novel sod1 mutation c.164c>t, p.thr55ileu, confirmed by sanger sequencing. no other rare variant was identified in any other als-related genes. association of neuropathology and whole exome sequencing can provide a useful tool for the identification of unknown forms of the disease, better understanding of the physiopathology and lead to new therapeutic targets.     siegert e, dittmayer c, schneider u, preuße c, goebel hh, stenzel w (2020) myositis in scleroderma – capillary pathology is fundamental. free neuropathol 1, 34: 4 myositis in scleroderma – capillary pathology is fundamental elise siegert, carsten dittmayer, udo schneider, corinna preuße, hans-hilmar goebel, werner stenzel department of neuropathology, charité universitätsmedizin, corporate member of freie universität berlin, humboldt-universität zu berlin, and berlin institute of health (bih), berlin, germany systemic sclerosis is a chronic disease of connective tissues characterized by fibosis, vasculopathy and autoimmunity. affected patients show signs of the skin, internal organs and sometimes overlap myositis. the vasculopathy is considered obliterative but the pathogenic basis is not known to date.   we are presenting light-microscopic and ultrastructural as well as clinical data of 18 patients suffering form scleroderma and myositis. we have applied a new electron microscopical technique which we call large scale electron microscopy allowing a ‘pan and zoom’ approach similar to ‘google earth’ viewing.   this analysis allows to study >1000 capillaries of patients and controls, highlighting reduplications of basement.   we show that this type of ultrastructural changes is membranes, endothelial activation and proliferation of pericytes specific for a subtype of scleromyositis and discus possible pathogenic mechanisms.     boluda s, wallon d, rovelet-lecrux a, campion d, nicolas g, duyckaerts c (2020) neuropathological variability of four tauopathy cases with mapt microduplication. free neuropathol 1, 34: 5 neuropathological variability of four tauopathy cases with mapt microduplication susana boluda1,2, david wallon3,4,5, anne rovelet-lecrux3,4,6, dominique campion3,4,6, gaël nicolas3,4,6, charles duyckaerts1,2 1 department of neuropathology, pitie salpetriere hospital, ap-hp sorbonne university, paris, france 2 inserm u1127, cnrs umr7225, sorbonne university-umrs1127, brain institute icm, paris, france 3 inserm u1245, university of normandy, unirouen, rouen, france 4 cnr-maj, normandy center for genomic and personalized medicine, rouen, france 5 department of neurology, university hospital, rouen, france 6 department of genetics, university hospital, rouen, france we report the neuropathology of four mapt duplication carriers, a rare chromosomal rearrangement involving the 17q21.31 chromosomal region that causes an early onset dementia (eod) clinically mimicking alzheimer disease or an atypical extrapyramidal syndrome, as recently described. they were three males and one female with ages ranging between 37 and 57 years. they were all tauopathies with a variability in the morphology and distribution of the aggregates. the cases either mimicked pick disease with neuronal globular aggregates similar to pick bodies and pathology involving predominantly the cortical and limbic regions and subcortical nuclei or they resembled progressive supranuclear palsy with tufted astrocytes with major involvement of the brain stem and subcortical nuclei. the tau isoform expression also varied from either only 3r, only 4r or a mixed 3r/4r expression.   in conclusion, mapt duplication may lead to a tauopathy spectrum with a range of 3r and 4r expression.     seilhean d, mokhtari k, plu i, boluda-casas s, mathon b, cao a, hervé d, mégarbane b, bielle f, levavasseur e, malet i, marot s, el hachimi h, marty s, prigent a, duyckaerts c, potier mc, haïk s, delatour b, marcelin ag (2020) multiple cerebral angiopathy in sars-cov-2 infection. free neuropathol 1, 34: 6 multiple cerebral angiopathy in sars-cov-2 infection danielle seilhean1,2, karima mokhtari1, isabelle plu1, susana boluda-casas1,2, bertrand mathon3, albert cao4, dominique hervé5, bruno mégarbane6, franck bielle1,2, etienne levavasseur2, isabelle malet7, stéphane marot7, hamid el hachimi2, serge marty2, annick prigent2, charles duyckaerts1,2, marie-claude potier2, stéphane haïk1,2, benoît delatour2, anne-geneviève marcelin7 1 department of neuropathology, pitie salpetriere hospital, ap-hp sorbonne university, paris, france 2 inserm u1127, cnrs umr7225, sorbonne university-umrs1127, brain institute icm, paris, france 3 department of neurosurgery, pitie salpetriere hospital, ap-hp sorbonne university, paris, france 4 department of neurology, neuro-icu, pitie salpetriere hospital, ap-hp sorbonne university, paris, france 5 department of neurology, lariboisière hospital, ap-hp nordparis university, paris, france 6 department of intensive care, lariboisière hospital, ap-hp nordparis university, paris, france 7 department of virology, inserm umrs 1136, pitie salpetriere hospital, ap-hp sorbonne university, paris, france sars-cov-2 is the cause of a pandemic characterized by its severity in elderly subjects or those presenting metabolic or vascular risk factors. brain damage, although relatively rare, is often fatal. from four cases (one autopsy and three brain biopsies) we analyzed the involvement of small cerebral arteries associated with white matter lesions, without signs of vasculitis or encephalitis. viral rna has not been detected in the brain. sars-cov2 spike protein (s) has been detected in the golgi apparatus of endothelial cells, colocalized with a host protease. our observations suggest the possibility of hematogenous neuroinvasion. the interaction of a small amount of protein s with endogenous proteases is thought to be able to disrupt the permeability of brain endothelial cells causing vascular damage. this result could provide therapeutic avenues to prevent or cure severe brain forms in patients at risk.     bourhis a, peyre m, bielle f (2020) a rare tumor of the peripheral nerve mimicking a schwannoma. free neuropathol 1, 34: 7 a rare tumor of the peripheral nerve mimicking a schwannoma amélie bourhis1, matthieu peyre2,3, franck bielle2,4 1 department of pathology, university hospital, brest, france 2 inserm u1127, cnrs umr7225, sorbonne university-umrs1127, brain institute icm, paris, france 3 department of neurosurgery, pitie salpetriere hospital, ap-hp sorbonne university, paris, france 4 department of neuropathology, pitie salpetriere hospital, ap-hp sorbonne university, paris, france a 38 year-old man presented with pain of the left forearm for five years. he had a history of old hiv infection and several past opportunistic infections. the pain was paroxysmal, favored by pronation and was increasing for two years. mri evidenced a tumor nodule on the path of the posterior interosseous nerve mimicking a schwannoma. intracapsular tumor resection stopped the pain. microscopic examination showed a tumor proliferation of fascicled eosinophilic fusiform cells. immunostaining showed the expression of the muscular markers smooth muscle actin and desmin, and the absence of expression of sox10. in situ hybridization for eber rna of ebv was positive in tumor cells. we diagnosed an ebv-associated leiomyoma of the immunocompromised, which was located in the peripheral nerve, a localization not previously reported. we discuss the tumorigenesis of this rare neoplasm.   conferences:     eloit m (2020) virus spillover from animal reservoirs and vectors to human detection using agnostic tools. free neuropathol 1, 34: 8 virus spillover from animal reservoirs and vectors to human detection using agnostic tools marc eloit head of pathogen discovery laboratory, pasteur institute, paris, france oie collaborating centre for detection and identification in humans of emerging animal pathogens, pasteur institute, paris, france professor of virology, veterinary school of maisons alfort, france viruses from wildlife including arboviruses vectored by mosquitoes have caused dramatic outbreaks over the last 25 years. on the one hand, new sequencing capacities have deeply affected the means by which new viruses are discovered, leading to the identification of ever more viruses in animal reservoirs and arthropods. on the other hand, it is worth noting that the continually growing knowledge regarding these viruses does not per se serve to identify potential human or animal threats. our laboratory aims to fill this gap by identifying, in selected ecosystems, whether as yet unknown, unexpected or neglected mosquito or wildlife viruses are responsible for frequent but sub-clinical or mild infection in humans highly exposed to wild life/arthropods, and thus represent good candidates for global spreading. it combines high throughput screening of viruses in animal/arthropods linked to antibody screening in healthy exposed human populations and virus search in patients presenting with severe diseases of unknown etiology. i will show our methodology and some results.     ludes b (2020) migration of siberian populations of the past: contribution of a multi-genetic markers approach. free neuropathol 1, 34: 9 migration of siberian populations of the past: contribution of a multi-genetic markers approach bertrand ludes university of paris-cnrs, fre 2029 head of the forensic institute of paris (institut médico-légal de paris) molecular markers of nuclear dna (autosomes and y chromosome) and mitochondrial dna from human samples help clarifying the migrations of the first peoples of the eurasian steppes. we analyzed samples dating from the middle of the 2nd millennium bc to 4th century ad and originating from the krasnoyarsk region (south siberia). we confirmed that, during the bronze and iron ages, southern siberia was a region dominated by european peoples, suggesting an eastward migration of the kurgan peoples across the russo-kazak steppe. the results further showed that at that time, the inhabitants of southern siberia must have had fair eyes, skin and hair.     copyright: © 2020 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. free for authors, free for readers, free from publisher, free free neuropathology 1:1 (2020) editorial free for authors, free for readers, free from publisher, free formatting and free opinion: this is free neuropathology werner paulus institute of neuropathology, university hospital muenster, muenster, germany corresponding author: werner paulus, institute of neuropathology, pottkamp 2, 48149 muenster, germany werner.paulus@uni-muenster.de submitted: 05 december 2019 published: 01 january 2020    https://doi.org/10.17879/fnp-2020-2610   as reader, author, referee and editor i have often discussed and reflected upon the nature of the "perfect" journal what are the qualities that make up a great scientific journal? the following ingredients came to my mind: •    excellent science •    interesting and relevant content •    high visibility •    a high impact factor •    short turnaround times (from submission to first decision, from accept to online publication) •    fair reviews •    no unnecessary major revisions just to satisfy referees •    a responsive and supportive editorial office •    insightful editors •    no subscription rates for readers (open access) •    low (ideally no) article processing fees for authors •    a convenient and easy electronic submission system •    high technical standard (figures, copyediting, layout) •    a clear and informative website •    electronic tools for enabling interaction between authors and readers i am not aware of a journal that fulfills all of these requirements. since the great majority of established journals are published by commercial publishers, some of these features are out of control of the editor, such as quality of copyediting, layout design, time until online publication, charges for authors and/or readers, promotion, and appearance and content of the website. however, there is no need for these latter features to fall into the responsibility of the publisher. should scientists be willing to perform these activities themselves, they are in principle well capable to found, shape and run a wonderful journal that fulfills the expectations of the scientific community. it was precisely this insight that sparked the development of free neuropathology. why is the new journal called free neuropathology? the word "free" bears various meanings. first, it means no cost, like in "free beer". accordingly, the journal is free of cost for everyone. when discussing the name, a few colleagues expressed concern that "free" may have a connotation of "poor quality", because something without price might be considered as having no value. i am not convinced of this argument, because some of our highest ethical values cannot be bought for money (and thus are for free), and because some  universities with the word "free" in their name are among the most distinguished academic institutions, such as frije universiteit amsterdam and freie universität berlin. second, "free" means having the freedom to do something your own way. in fact, at free neuropathology we are able to decide on every aspect of the journal without having to obey non-scientific paper-shufflers, and we put much emphasis on freedom from unnecessary formalities and bureaucracy that increasingly abound in the publishing business. third, we encourage authors to submit not only original papers, letters and reviews, but also opinion pieces, critiques and annotations, because we feel that frank views, open-minded discussion and critical analysis of prevailing approaches and trendy hypotheses are integral parts of science. in the end, the name of the journal reflects a mix of freebie, freedom and liberty. free for authors and free for readers most scientists, scientific organizations and politicians believe that publishing is expensive and that someone has to pay for the publication process, either the reader through subscription models or the author through open-access models. i believe that there is a third option if the activities of the publisher are rendered by scientists working in the field of the journal, i.e. by authors, referees and editors who have put the most time, energy and enthusiasm into the journal anyway. it is true that some technical infrastructure and manpower is necessary to keep a journal running and that this is not without cost, but compared to the time that scientists spend as voluntary referees and editors (let alone the work of authors), the additional time and expenses for taking over the publishers´ classical activities are moderate and they can easily be covered by scientific institutions. furthermore, while in past years handling of manuscripts, layout and printing of journals required considerable time, staff, technical skills and money, digital publishing has made these activities easier, increasingly automated and cheaper. at free neuropathology we take advantage of the open journal systems platform for the management of peer-reviewed academic journals. many thanks to the developers of this open-source software and to my university library! green open access means self-archiving of the accepted version of the manuscript in the authors´ format. in the gold open access model, articles are made immediately and freely available, while authors must pay article processing fees. diamond open access (also referred to as platinum open access), as will be employed at free neuropathology, means that the whole process, including submission, handling by the editorial team, peer review, copyediting, layout and retrieving full text content, is completely free. these tasks are taken over by colleagues who provide high quality editorial, peer reviewing and publishing services. free neuropathology´s diamond open access model is based on the enthusiasm of volunteers who love to be engaged in the scientific publication process, thereby serving science and society. we also believe that volunteers will enjoy career benefits and recognition from institutions for doing the work. our young members of the layout/copyediting board feel that this is a great opportunity to learn about publishing and a great way to expand personal networks. free from publishers virtually everyone in science criticizes the high profit margins and pricing policies of commercial publishers. it has been estimated that the major fifty-seven academic publishers generate a combined revenue of 60 billion € per year, with profits in the range of 20 to 50%. increases in subscription fees by 60% per year for individual journals are not unusual – the more prestigious the journal, the more impertinent the increase. while from an economical point of view this is understandable because shareholders and private equity must be satisfied; however, the excessive cash outflow endangers science. this scenario is even more absurd, because scientists working voluntarily as authors, referees and members of editorial boards do the bulk of the work, thereby serving as useful dupes for publishing houses and maximizing the profit of people who have zero interest in science per se. the authors, who are largely funded by governments, even offer their work to publishers for free, who then sell it back to government-funded institutions at astronomical prices. publishers are inventive in obfuscating their business models. the cash flow is often organized in a way so that the individual scientist does not see the problem, because subscription fees or article processing charges are covered by universities or funding agencies. needless to say that, in the end, all scientists have to pay the bill because the money transferred to publishers needs to be detracted from personnel, infrastructure and funding of scientific institutions. furthermore, publishers have invented a variety of new services that nobody needs, in order to justify their prices. finally, publishers sell not only their few top journals but also bunches of hundreds of low-impact or irrelevant journals to large scientific organizations. nobody is in need of these journals, but all scientists have to pay for them. established publishers tend to condemn so-called predatory publishers because they rip off scientists, but do they mean themselves? let me relate to you a parable (it´s a bit lengthy and needs pondering, so if you have little time feel free to skip to the next paragraph): an artist has created a painting after spending years of work, money, creativity, care and enthusiasm. because this is the way it has always been, the artist does not try to sell the opus, but prefers to donate it to a company called jumpartize, which is owned by private equity that operates amusement parks and museums. the businesspeople of jumpartize do not understand art nor do they appreciate it, but they are very good at making the most money out of it. artists must pay art processing charges (apc) for the art they donate, and they find this ok because jumpartize builds and maintains museums, frames and dusts the paintings, employs staff (custodians, cleaners, clerks), and counts how often each piece of art is mentioned in newspaper articles and social media, resulting in the artificialfactor®. alternatively, artists can commit to lifelong work for one week per year in one of jumpartize´s amusements parks as clowns or as animate figures in haunted houses (open joy program). many artists love to become famous and they try to endow their artwork to the most prestigious museums boasting the highest artificialfactor®. jumpartize asks several art historians to evaluate the offered paintings and to write up art critiques within two weeks for free, which they happily accept because they consider the invitation to be an honor. admission fees for the museum are high at about 120 € and they rise by 30 % every year, but the public accepts this because art is considered to be high value and because prices for dusters have increased recently. the artists themselves must also pay admission fees to see their own works, but they are allowed to hang a low-quality poster of their paintings in their private rooms. some museums of jumpartize offer free entrance for everyone if artists are willing to defray the costs of running the museum and serve as building workers for jumpartize´s new 20-story headquarters building. politicians and several national academies of fine arts are very proud of having successfully negotiated with jumpartize that artists are allowed to terminate work in haunted houses by the age of 80 (plan artistique or in short plan a). meanwhile jumpartize, including all fun parks, museums and artwork has been sold to another private equity investor for double the original price. if you find this scenario absurd or crazy, what does it mean for the behavior of scientists? scientists, librarians and politicians complain about costs of journals but at the same time they continue to support commercial publishers. science politicians have been very proud of negotiations or declarations such as plan s or deal, whereby article processing charges are limited or somewhat reduced, but in the end they have surrendered. financially supporting journals by national or international funding organizations (such as gates open research) is also not helpful, because funding is usually restricted to a limited period of time, so that permanent structures which are mandatory for scientific journals are endangered. at first glance, journals that are owned by scientific societies and published by commercial publishers may be in a more comfortable position, because societies can replace the publisher in case of disagreement or disservice. however, the problem is that scientific societies usually develop dollar signs in their eyes as soon as the publisher offers sharing part of the profit. this corrupts science. i am deeply convinced that the purpose of scientific journals is publishing science, ideally the best science, but definitely not making money, for whomsoever. there are other good reasons for proceeding without publishers. we, the scientists and developers of a journal, will be in a position to decide on website, layout, copyediting and promotion, and we no longer depend on publishers´ decisions which are led by financial considerations. for example, publishers tend to decline suggestions of modifying individual journal websites because, for economical or branding reasons, they want the websites of their hundreds or thousands of journals to appear identical. publishers promote journals at commercial exhibitions at scientific meetings (often largely unnoticed by scientists), while scientists know their field, their colleagues and the appropriate communication channels better than publishers do. scientists therefore could perform more efficiently and more cost-effectively in undertaking these classical publishers´ activities. it is true that publishers have much more experience because at least the handful of oligopolistic publishers have published thousands of journals before. but we will learn and we will learn fast. and yes, we may be somewhat naïve, we will make mistakes and some technical issues will not work perfectly right from the start, but in my experience scientists work more efficiently and creatively than publishers´ staff, and in the end our approach will be successful. publishers like to talk about ethics. they request from authors disclosures of potential conflicts of interest such as shareholding of family members in relevant companies, they ask for reference numbers of ethical committees, they request statements of author contributions, and they analyze papers using software for detecting plagiarism and manipulation of figures. while these activities may be considered to be measures for increasing quality and transparency (as well as subscription rates and article processing fees), publishers play the role of science police, thereby undermining the general credibility of scientists and considering them as potential cheaters and criminals. to be clear, i find that ethical standards and guidelines are important, but they should be developed and released by scientific societies and not by individual publishers who just try to increase sales and the prestige of their products. publishers stress the highest ethical standards, but in the end they are interested in nothing more than profit and they harness scientists in their dull business. commercial publishers have had a detrimental effect on science. publishing scientific work together with commercial publishers should be discontinued for reasons of economy, quality and ethics. free formatting many scientists are concerned and annoyed by the increasing number of formal requirements when submitting papers, such as rigid regulations on references, tables, figures, organization of manuscript, font type/size, abbreviations and nomenclature. we believe that most of these regulations are unnecessary and they detract from the gist of the purpose, i.e. prompt publication of good science. at free neuropathology authors can format their paper as they like, as long as formatting is 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critique, ideas or stimulating suggestions: send me a note. molecular clarification of brainstem astroblastoma with ewsr1-bend2 fusion in a 38-year-old man feel free to add comments by clicking these icons on the sidebar free neuropathology 2:16 (2021) case report molecular clarification of brainstem astroblastoma with ewsr1-bend2 fusion in a 38-year-old man matthew a. smith-cohn,1,2 zied abdullaev,3 kenneth d. aldape,3 martha quezado,3 marc k. rosenblum,4 chad m. vanderbilt,4 fausto j. rodriguez,5 john laterra,**2 charles g. eberhart**5 1 neuro-oncology branch, national cancer institute, national institutes of health, bethesda, md, usa 2 department of neurology, the johns hopkins university school of medicine, baltimore, md, usa 3 laboratory of pathology, center for cancer research, national cancer institute, bethesda, md, usa 4 department of pathology, memorial sloan-kettering cancer center, new york, ny, usa 5 department of pathology, the johns hopkins university school of medicine, baltimore, md, usa   ** these authors contributed equally to this manuscript corresponding author: charles eberhart, md, ph.d. · department of pathology · the johns hopkins university school of medicine · 1800 orleans st. · sheikh zayed tower · baltimore, md 21287 · usa ceberha@jhmi.edu submitted: 21 april 2021 accepted: 17 june 2021 copyedited by: calixto-hope lucas published: 21 june 2021 https://doi.org/10.17879/freeneuropathology-2021-3334 keywords: astroblastoma, brainstem, fusion, methylation, neoplasms, neuro-oncology abstract the majority of astroblastoma occur in a cerebral location in children and young adults. here we describe the unusual case of a 38-year-old man found to have a rapidly growing cystic enhancing circumscribed brainstem tumor with high grade histopathology classified as astroblastoma, mn1-altered by methylome profiling. he was treated with chemoradiation and temozolomide followed by adjuvant temozolomide without progression to date over one year from treatment initiation. astroblastoma most frequently contain a mn1-bend2 fusion, while in this case a rare ewsr1-bend2 fusion was identified. only a few such fusions have been reported, mostly in the brainstem and spinal cord, and they suggest that bend2, rather than mn1, may have a more critical functional role, at least in these regions. this unusual clinical scenario exemplifies the utility of methylome profiling and assessment of gene fusions in tumors of the central nervous system. introduction astroblastomas are rare central nervous system (cns) neoplasms that most frequently occur in cerebral locations in children and young adults. here we describe the unusual case of a 38-year-old man with a brainstem tumor with an integrated diagnosis of malignant neoplasm consistent with astroblastoma with mn1 alteration following methylome profiling, and found to have a non-canonical ewsr1-bend2 fusion. this unusual clinical scenario exemplifies the utility of methylome profiling and assessment of gene fusions in tumors of the cns. case report a 38-year-old male with a past medical history of melanoma in situ of the trunk, status post excision, and no family history of cancer, presented with subacute onset of progressively worsening dysesthesia first involving his upper extremities and progressing to affect his right chest and leg over two weeks. mri of the brain and cervical spine demonstrated a well-localized cystic enhancing anterior medullary-cervical lesion measuring 1.3 x 1.1 x 1.7 cm, with t2 hyperintensity of the lesion and medullary pyramids (fig.1a, 1b, 1c). he was admitted to the neurology service for an expedited evaluation of infectious, rheumatologic, neoplastic, and inflammatory etiologies. mri of the spine and whole-body pet/ct revealed no other lesions. serum studies were unremarkable, and lumbar csf contained 1 wbc, 3 rbc, glucose 55, protein 57 (ref 15-45), no oligoclonal bands and negative infectious studies, flow cytometry, and cytopathology. he was treated with high dose iv methylprednisolone for five days and discharged home on a steroid taper. he had progression of symptoms, and a repeat mri two months later showed growth of the lesion to 3.3 x 1.7 x 1.5 cm (fig.1d, 1e, 1f). subsequently, he underwent a suboccipital c1 laminectomy and subtotal surgical resection of the mass. figure 1. radiographic findings. coronal (a, d), sagittal (b, e), axial (c, f) post-contrast t1 mri of the brain. the top row (a-c) shows mri imaging of the brain at presentation, and the bottom row (d-f) is two months later before tumor resection. pathology microscopic evaluation showed a cellular tumor with compact growth. large, pleomorphic to epithelioid cells predominated in some regions (fig.2a), while in other areas prominent perivascular growth was noted. this included scattered cells with a somewhat astroblastic phenotype, exhibiting stout processes extending to the surface of blood vessels (fig.2b). necrotic foci without pseudopalisading were present. cellular regions of tumor had 1 to 5 mitotic figures per high power field, and the ki67 proliferation index was moderate to high, up to 20-30% (fig.2c). s100, olig2 and ema were strongly positive on immunohistochemical analysis (fig.2d, e and data not shown), and gfap was focally positive, supporting glial differentiation. in contrast, markers of melanocytic (melana, sox10, hmb45), epithelial (cytokeratin ae1/ae3), and neuronal (synaptophysin) differentiation were all negative (fig.2f and data not shown). figure 2. histopathology. the tumor included more pleomorphic regions (a), as well as neoplastic cells with thick, short processes (arrow, b) arrayed around proliferating blood vessels (asterisks, b). the tumor was quite proliferative on ki67 immunostain (c), and diffusely positive for olig2 (d) and s100 (e), while blood vessels did not express these glial markers (asterisks, e). melana was not expressed in the tumor (f). (original magnifications: a-e 400x, f 200x). molecular diagnostics next-generation sequencing (ngs) found a tumor mutation rate of 0.88 mutations per megabase, no known pathologic variants, and several variants of unknown significance, including adgra2 (p.g407s), ar (p.a646d), blm (p.r643h), epha5 (p.l907v), and prkdc (p.i1013v), all with allele frequencies of 46% or higher. no alterations in tp53, atrx, braf, h3f3a or hist1h3b were detected on ngs. dna methylation profiling was consistent with a “high-grade neuroepithelial tumor with mn1 alteration”, with a calibrated score of 0.994 (fig.3a). copy number evaluation using data from the methylation array demonstrated alterations in chromosome 22 and x (fig.3b). subsequent gene fusion testing found a ewing sarcoma breakpoint region 1/ews rna binding protein 1 (ewsr1) ben domain containing 2 (bend2) fusion between loci on chromosomes 22 and x (fig.3c). the integrated diagnosis was malignant neoplasm consistent with astroblastoma with mn1 alteration. figure 3. methylation and copy number profiling of astroblastoma with an ewsr1-bend2 fusion. (a) the lesion (blue box) was plotted on an x–y coordinate graph (red dot to the lower left of the x–y intersection), where closer proximity to other dots indicates greater similarity of the index tumor’s genomic cpg methylation pattern to existing cases in the library. (b) copy number profiling demonstrates the location for ewsr1-bend2 fusion at chromosome 22 and x (blue arrow). (c) fusion between exon 7 of ewsr1 and exon 5 of bend2 genes with breakpoints at genomics positions chr22:29683123 and chrx:18234853 respectively. the red arrow represents the direction of the gene specific primary utilized by the archer assay to enrich the amplicons for ewsr1 fusion events. legend: mb,g3, medulloblastoma, subclass group 3; epn, pf a, ependymoma, posterior fossa group a; epn, pf b, ependymoma, posterior fossa group b; subepn, pf, subependymoma, posterior fossa; mb, shh chl ad, medulloblastoma, subclass shh a (children and adult); mb, shh inf, medulloblastoma, subclass shh b (infant); contr, cebm, control tissue, cerebellar hemisphere; lipn, cerebellar liponeurocytoma; cns nb, foxr2, cns neuroblastoma with foxr2 activation; sp-epn-mycn, mycn amplified spinal cord ependymoma; epn, yap, ependymoma, yap fusion; hgnet, mn1; high grade neuroepithelial tumor with mn1 alteration; gbm, g34, glioblastoma, idh wildtype, h3.3 g34 mutant; gbm, mes, glioblastoma, idh wildtype, subclass mesenchymal; gbm, mid, glioblastoma, idh wildtype, subclass midline; gbm, mycn, glioblastoma, idh wildtype, subclass mycn; gbm, rtk i, glioblastoma, idh wildtype, subclass rtk i; gbm, rtk ii, glioblastoma, idh wildtype, subclass rtk ii; gbm, rtk iii, glioblastoma, idh wildtype, subclass rtk iii; hgnet, bcor, cns high grade neuroepithelial tumor with bcor alteration; ptpr, a, papillary tumor of the pineal region group a; ptpr, b, papillary tumor of the pineal region group b; contr, pons, control tissue, pons; contr, wm, control tissue, white matter; contr, hypthal, control tissue, hypothalamus; contr, hemi, control tissue, hemispheric cortex; cn, central neurocytoma; subepn, spine, subependymoma, spinal; lgg, myb, low grade glioma, myb/mybl1; lgg, dnt, low grade glioma, dysembryoplastic neuroepithelial tumor; lgg, gg, low grade glioma, ganglioglioma; ihg, infantile hemispheric glioma; lgg, pa/gg st, low grade glioma, rosette forming glioneuronal tumor; lgg, rgnt, rosette forming glioneuronal tumor; contr, react, reactive tumor microenvironment; dlgnt, diffuse leptomeningeal glioneuronal tumor; ana pa, anaplastic pilocytic astrocytoma; pxa, (anaplastic) pleomorphic xanthoastrocytoma; lympho, lymphoma; dmg, k27, diffuse midline glioma h3 k27m mutant; lgg, pa pf, subclass posterior fossa pilocytic astrocytoma; lgg, pa mid, midline pilocytic astrocytoma; contr, inflam, control tissue, inflammatory tumor microenvironment; epn, rela, ependymoma, rela fusion; atrt, tyr, atypical teratoid/rhabdoid tumor, subclass tyr; chgl, chordoid glioma of the third ventricle; lgg, sega, subependymal giant cell astrocytoma. subsequent clinical course the patient was treated with fractionated radiation (5040 cgy in 28 fractions) with concurrent daily temozolomide (75 mg/m2) followed by adjuvant temozolomide (150-200 mg/m2, 5 days-on/23 days-off) with a partial response. at the time of writing, the patient has not progressed since initiation of treatment over one year ago. clinically the patient was able to return to work full time with continued central neuropathic pain of the left face and right-side extremities and trunk managed with gabapentin. discussion astroblastoma has historically been a controversial entity since its introduction in the 1924 classification of cns brain neoplasms by cushing and bailey.1 mn1-altered astroblastomas arise primarily in cerebral locations in pediatric patients and young adults.2 in addition to the presence of a ewsr1-bend2 fusion, our case is unusual compared to other described mn1-altered astroblastoma in that the patient is near his fourth decade of life and with tumor located in the brainstem and upper cervical spine, rather than the cerebrum. including the case presented, there are three other described cases of cns tumors with a ewsr1-bend2 fusion (tab. 1).3–5 aside from one case that did not provide additional information, these tumors occurred in males and were infratentorial, involving the spinal cord and sometimes the brainstem.3–5 although non-specific, mri of astroblastomas have been described to enhance, as well as appear well-demarcated, cystic, and lobulated, which is consistent with our case and similar to other cases of ewsr1-bend2 fused astroblastoma tumors (fig.1).3,4,6 comparable to conventional mn1-altered astroblastomas and other cases of ewsr1-bend2 fused tumors, our case showed perivascular growth and immunohistochemistry was positive for s100, olig2, ema and gfap.2–4 ultrastructural observations in astroblastomas suggest a relationship to tanycytes, an ependymal cell subtype, but it is unclear if this applies also to mn1-altered and ewsr1-bend2 fused astroblastomas.1,2 molecular profiling has had a huge impact on the diagnosis of “astroblastic” tumors and has helped clarify subtypes of astroblastoma. in 2016, sturm et al. identified a group of 41 tumors with a common methylation profile and mn1 fusions, designating them ‘cns high-grade neuroepithelial tumor with mn1 alteration’ (cns-hgnet-mn1).7 notably, the majority of these contained astroblastic or ependymal perivascular pseudorosettes, although a number of other histopathological appearances were also represented. subsequent studies of mn1-altered brain tumors have confirmed that many, but not all, have ependymal or astroblastic features.8 based on these and other studies, the consortium to inform molecular and practical approaches to cns tumor taxonomy (cimpact-now) has proposed designating these tumors “astroblastoma, mn1-altered”.2 early case series of astroblastoma reported prior to these newer molecular tools likely contained other tumor subtypes, as exemplified by a recent study in which molecular profiling of 14 adult tumors with histologic feature of astroblastoma found they were pleomorphic xanthoastrocytomas or high grade gliomas with alterations activating the mitogen-activated protein kinase pathway, and none had clear evidence of a mn1-bend2 fusion, or clustered with that group on methylation profiling analysis.9 the mn1 alteration is usually a fusion between mn1 and bend2.2 while half or more of microscopically defined astroblastomas harbor an mn1 alteration, a significant number do not.3,10 our patient is unique due to the presence of a rare non-canonical ewsr1-bend2 fusion between chromosomes 22 and x. interestingly, early molecular analyses of astroblastomas identified deletions in chromosomes 22q and x.1,2 including our case, there are at least four known cases of cns tumor with ewsr1-bend2 fusions in the literature (table 1).3–5 one case was a 3-month -old male with tumor spanning the lower medulla to the c4 spinal cord level,3 with a second in a 36-year-old male patient with a thoracic spinal “ependymoma” with ewsr1-bend2 fusion.4 a third case was found in a series of molecularly characterized pediatric tumors which did not provide clinical information.5 remarkably, in at least two of these cases, as in ours, methylation clustering led to the tumor falling into the category of astroblastoma/hgnet with mn1 alteration.3,4 these reports highlight the possibility of astroblastic or ependymal tumors with a methylation profile consistent with “astroblastoma, mn1-altered” being driven by fusions in genes other than mn1, and also suggest that it may be bend2, rather than mn1, which has a more critical functional role. supporting this notion, there is one reported case of an neuro-epithelial tumor with an mn1-patz1 fusion found with rna sequencing a supratentorial mass in a 1-year-old girl that suggest a similar pathogenic role as a chimeric protein.11 however, this case lacked astroblastomatous rosettes and did not have epigenetic similarities with cns hgnet-mn1, but did have similarities to patz1-sarcomas.11 table 1: known cases of astroblastoma with an ewsr1-bend2 fusion in the literature the grading and clinical behavior of astroblastomas is not well understood. as noted by the 6th cimpact-now update and other case series, a significant proportion of tumors in the methylation class astroblastoma/cns-hgnet-mn1 do not conform to astroblastoma histologically, and it is unclear if disparate histologic patterns have biologic relevance aside from high grade features.1,2 the 2016 who, completed before identification of the mn1-altered molecular group, does not assign grades, but observed that astroblastic neoplasms generally fall into two general categories: well-differentiated or anaplastic/malignant.1,12 case series that predated molecular evaluation of astroblastomas likely included other tumor subtypes, but found that in histologically defined astroblastomas, an elevated proliferation rate is associated with worse outcomes.1,12 a case series of 14 neuroepithelial tumors with mn1 alterations, including three spinal cases, two of which were the oldest (14.6 and 36 years old) and only males in the series, describes heterogeneous treatments consisting of focal or cranial spinal radiation without chemotherapy.8 the event-free survival ranged considerably from 6 to 100 months, with some cases resulting in metastasis in the cns, arguing that intermittent monitoring with completed neuroaxis imaging is warranted.8 a meta-analysis of 73 patients with mn1-altered neuroepithelial tumors (astroblastomas) found a 5and 10-year progression-free survival of 38% and 0%, and 5and 10-year overall survival of 89% and 55%, respectively.13 the natural history of astroblastoma with ewsr1-bend2 fusion has not been well described in the literature, and it is unclear if the clinical behavior is significantly different from astroblastomas with a mn1 alteration. the case we present showed anaplastic microscopic features, as well as aggressive clinical behavior with rapid growth over two months histologic features and clinical behavior was similar to other described cases of astroblastoma with a ewsr1-bend2 fusion.3,4 given the rarity of these tumors, there is no defined standard established treatment. based on studies of non-molecularly characterized astroblastoma, resection remains the cornerstone of therapy to improve outcomes, as supported by an analysis of 116 patients that found that gross total resection improved outcome with a 5-year progression-free survival of 83% versus 55% in those with subtotal resection.1 regarding radiation, a systemic review of 95 histologic astroblastoma patients did not show a survival benefit with radiation therapy; however, the series had a broad age range from 1 to over 61 years of age and did not compare survival differences in astroblastoma with and without anaplastic features.14 review of the literature found that a 20-year-old woman with a spinal cord mn1-altered astroblastoma, and a 36-year-old man with a spinal astroblastoma with ewsr1-bend2 both had a reduction in tumor size after radiation, temozolomide, and bevacizumab.4,15 in another case, a 3-month old boy with ewsr1-bend2 astroblastoma had progressive disease after five days of temozolomide and etoposide.3 given the significant growth of the presented patient's tumor in two months (fig.1) and dramatically elevated ki67 proliferation index, he was treated with concurrent radiation with temozolomide followed by adjuvant temozolomide based on the “stupp” protocol and this appears to be a viable option, as he has had a partial response control of his tumor over one year later at the time of writing.16 this case exemplifies the use of advanced molecular testing, including the evaluation of chromosome fusions and methylation profiling, to accurately diagnose rare neoplasms of the cns. through these technologies, improved diagnosis accuracy provides clinically impactful insights that enhance the understanding of tumor biology. funding this work was supported by the national institutes of health t32 research training grant. authors’ contributions m.a.s case report concept, design, and critical revision of content. z.a., k.d.a, m.q., m.k.r., f.j.r., c.g.e and l.j.l. provided critical revision of content. acknowledgments the authors thank rust turakulov at the nih for help with formatting the methylation plot for publication. the authors also thank the patient and his caregivers. references 1. brat, d. j., hirose, y., cohen, k. j., feuerstein, b. g. & burger, p. c. astroblastoma: clinicopathologic features and chromosomal abnormalities defined by comparative genomic hybridization. brain pathology 10, 342–352 (2006). 2. louis, d. n. et al. cimpact-now update 6: new entity and diagnostic principle recommendations of the cimpact-utrecht meeting on future cns tumor classification and grading. brain pathol bpa.12832 (2020) doi:10.1111/bpa.12832. 3. yamasaki, k. et al. spinal cord astroblastoma with an ewsr1-bend2 fusion classified as a high-grade neuroepithelial tumour with mn1 alteration. neuropathol appl neurobiol 46, 190–193 (2020). 4. tsutsui, t. et al. path-23. adult spinal cord astroblastoma with ewsr1-bend2 fusion. neuro-oncology 22, iii429–iii429 (2020). 5. ramkissoon, s. h. et al. clinical targeted exome-based sequencing in combination with genome-wide copy number profiling: precision medicine analysis of 203 pediatric brain tumors. neuro oncol 19, 986–996 (2017). 6. sener, r. n. astroblastoma: diffusion mri, and proton mr spectroscopy. comput med imaging graph 26, 187–191 (2002). 7. sturm, d. et al. new brain tumor entities emerge from molecular classification of cns-pnets. cell 164, 1060–1072 (2016). 8. baroni, l. v. et al. treatment response of cns high-grade neuroepithelial tumors with mn1 alteration. pediatr blood cancer 67, (2020). 9. boisseau, w. et al. molecular profiling reclassifies adult astroblastoma into known and clinically distinct tumor entities with frequent mitogen-activated protein kinase pathway alterations. oncologist 24, 1584–1592 (2019). 10. mhatre, r. et al. mn1 rearrangement in astroblastoma: study of eight cases and review of literature. brain tumor pathol 36, 112–120 (2019). 11. burel-vandenbos, f. et al. a polyphenotypic malignant paediatric brain tumour presenting a mn1-patz1 fusion, no epigenetic similarities with cns high-grade neuroepithelial tumour with mn1 alteration (cns hgnet-mn1) and related to patz1 -fused sarcomas. neuropathol appl neurobiol 46, 506–509 (2020). 12. bonnin, j. m. & rubinstein, l. j. astroblastomas: a pathological study of 23 tumors, with a postoperative follow-up in 13 patients. neurosurgery 25, 6–13 (1989). 13. chen, w. et al. central nervous system neuroepithelial tumors with mn1-alteration: an individual patient data meta-analysis of 73 cases. brain tumor pathol 37, 145–153 (2020). 14. sughrue, m. e. et al. clinical features and post-surgical outcome of patients with astroblastoma. j clin neurosci 18, 750–754 (2011). 15. yamada, s. m. et al. primary spinal cord astroblastoma: case report. journal of neurosurgery: spine 28, 642–646 (2018). copyright: © 2021 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. reflection on my 37 years of practice as a neuropathologist: home alone. feel free to add comments by clicking these icons on the sidebar free neuropathology 1:30 (2020) reflections reflection on my 37 years of practice as a neuropathologist. home alone. mara popović institute of pathology, faculty of medicine, university of ljubljana corresponding author: mara popović · mara popović · institute of pathology · faculty of medicine · university of ljubljana · korytkova 2, 1000 ljubljana · slovenia mara.popovic@mf.uni-lj.si submitted: 20 october 2020 accepted: 27 october 2020 copyedited by: nima sharifai published: 2 november 2020 https://doi.org/10.17879/freeneuropathology-2020-3062 additional resources and electronic supplementary material: supplementary material keywords: reflections, neuropathology early life and education i was born 70 years ago in the small town of sinj, dalmatia, croatia, yugoslavia. when i was 5 months old, my father was sent to prison for two years on goli otok (bare island), an ill-famed prison for politically inappropriate people at that time. at 24 years old, he could not understand that stalin, who was an idol for young communists, had become an enemy overnight after the informbiro resolution, by which the yugoslav communist party was expelled from the international communist party led by stalin. it was a price for living free on the outer side of the iron curtain, between east and west, where yugoslavia remained until the end of 1990, when the balkan wars started. my father has stuck with communist ideas until today, almost 95 years old (figure 1). under his influence, i entered the communist party in high school, but very quickly realized that politics is not something i would like to practice. figure 1. my father and our late dog maša 15 years ago. after primary school, i attended classical gymnasium “natko nodilo” in split, croatia, where, in addition to other high school subjects, i learned latin and ancient greek. at that time, my life consisted of studying, taking care of my two younger brothers, and helping my mother in everyday domestic jobs. after gymnasium in 1969, i decided to study medicine, mainly because it was not available in split at that time. i wanted to distance myself from my primary family to escape my mother’s control. i wanted to be free. my mother supported me in my decision (figure 2). i finished faculty of medicine at the university of zagreb in january 1975. after one year of obligatory practice, my first employment was in the emergency clinic in karlovac, where i worked for three years alternating between twelve-hour day and night shifts, as i traveled from my residence in zagreb. it was a very stressful job for a young, inexperienced m.d., and i was terrified of the possibility of doing something wrong. i was looking for a residency in pathology in zagreb, but the salary for a beginner was so small that i could not pay rent for my small apartment. i therefore moved to a small town in slovenia, ravne na koroškem, which has a large iron factory and where i in cared for factory workers’ health. i received an apartment from the health centre, only having to pay a small rent. figure 2. my mother in her youth in the national costume of continental dalmatia, which she was wearing just for the photo. my family i met my husband srđan during my third year of medical faculty. he had just entered the faculty of mechanical engineering. he was a city boy from zagreb, full of energy, honest and kind. he introduced another dimension into my life – fun and entertainment. every weekend we spent together, we joined his friends and enjoyed life. three years later, i completed my study of medicine. we married in 1978, just before he was conscripted into the army for one year. figure 3. me and my husband long ago on the top of one of the slovene hills we had reached by bike. after military service of one year, my husband joined me in the small town in slovenia, but he was not very happy there. he missed the greater opportunities of city life. our daughter saša was born in 1980, and our son marko in 1983. in 1982 i obtained a residency in pathology in the nearby small town of slovenj gradec, and went to study pathology at the institute of pathology, faculty of medicine, university of ljubljana (ip fm ul). after two years of residency, the institute offered me a staff position, and i accepted. my husband was very happy. he obtained a good position at an engineering company in ljubljana and we have remained in ljubljana to this day. my husband has supported me throughout my career, taking care of our children, but asking that the weekends must be ours (at least sundays). so throughout our life together we spent our weekends practicing many sports: tennis, jogging, hiking, mountain climbing, cycling. during the summer we would wind-surf as well, and during the winter we would ski (and later cross country skiing). we became serious mountain bikers, practicing twice a week for years (figure 3). last year, for my 69th birthday, i switched to an e-mtb bike. i use only eco power, the smallest of five levels of support, but it helps me a lot and i no longer suffer. our daughter saša is an architect, single, loves cats and especially loves her dog cloe, a young beagle with a particularly stubborn character (figure 4). our son marko is a happily married father of two sons (figure 5). figure 4. our daughter saša and her dog cloe. figure 5. our son marko with family. my mother (91) and my father (almost 95) live in split, in their own home, with no sign of dementia and without any medication except vitamin b12 for my mother, who looks after the two of them (figure 6). my father goes to a nearby café every day to drink coffee and read the newspaper, and my mother goes every day to the market across the street to buy fresh vegetables, fish, fruit and other things. last year, they celebrated 70 years of marriage. figure 6. my parents, 94 and 90 years old, last year. neuropathology in the third year of my residency in pathology, in 1984, prof. danilo tavčar, the pathologist who established the department of neuropathology (np) at the ip fm ul in 1972, invited me to join the department and become his assistant. i was also invited by prof. nina gale, who was head of the department of head and neck pathology. i needed some days to decide which invitation to accept. prof tavčar was almost 70 years old, and i would very soon remain alone in a quite unknown field of pathology. nina gale was only a little older than me, a very successful and prominent head and neck pathologist. it would be much easier to accept her offer, but the unknown field of np attracted and challenged me more. so i accepted prof. tavčar’s offer, and i did not regret the decision, even though it was not easy. i worked with prof. tavčar for two years parallel to my residency in pathology, which i completed in 1986. in the meantime, i attended postgraduate study for neurology in zagreb, which lasted four months during the academic year 1984/1985. my working days were in zagreb, and i spent the weekends with my family in ljubljana. my husband took care of our 4-year-old daughter and our 1.5-year-old son spent the working days with my husband’s parents in zagreb, and travelled with me for weekends to ljubljana. prof. tavčar took me to the international congress of neuropathology in stockholm in 1986 where we met srečko pogačar (1929 to 2018) (figure 7), a slovene neurologist and the first slovenian neuropathologist to have gone to rhode island medical centre in the usa to study neuropathology, in 1965. srečko had been the first to establish a np lab at the clinic of neurology in ljubljana in the early sixties. when he came back from the usa to ljubljana, the np lab no longer existed. disappointed, srečko decided to leave slovenia and continue his career in the usa practicing psychiatry and neuropathology, especially brain cuttings. he was a lecturer on neuropathology and neurology at harvard and brown universities [1]. figure 7. international congress of neuropathology stockholm 1986. from the left: prof. danilo tavčar, myself, prof. tavčar’s wife maca, and srečko pogačar. prof. tavčar undertook further training in np in massachusetts general hospital, boston, under dr. e.p. richardson, for six months in 1974. he shared all his knowledge of np with me. in the first half of the 1980s, immunohistochemistry started to become a useful tool in pathology. it was a very attractive challenge for me. prof. tavčar left me to do that job. after we returned from stockholm, he experienced an epileptic attack in his left leg – jackson’s epilepsy for the first time. he knew what it meant. after a ct-scan, which showed a focal lesion in the right parietal lobe, on which he was operated, he did not want to see the biopsy. he worked until spring 1987, going to radiotherapy from his office, and did not want to take any of the alternative therapies that his wife had asked him to. he died in december 1987, twelve months after his first epileptic attack. i stayed in contact with srečko pogačar, who offered to organize a six-month fellowship for me at rhode island hospital (rih) in providence, where i could learn neuropathology under the mentorship of the now deceased dr. mary ambler, a neurologist and neuropathologist. it was a great opportunity that i had to seize. i reached an agreement with the current chief of ip mf ul, prof. dušan ferluga, and with my family. my daughter was 9-years-old and my son 6-years-old. my husband had a good job in ljubljana and could not drop everything to go with me. i was enthusiastic and happy for the opportunity to be more confident in np. i remember the early morning in zagreb when my husband drove me to the airport. my children stayed with their grandpa and grandma. i had a cramp in my epigastrium from the time i boarded the airplane in zagreb until i landed six months later at the same airport and hugged my children and my husband. at that moment the cramp disappeared. professionally and otherwise, i had a good time in the usa, but i missed my family a lot. no skype, no facebook, no sms, no whatsapp, no e-mail. only atlantic surface mail and expensive telephone calls once a week with my husband. figure 8. six months training in neuropathology in rhode island hospital. a. mentor mary ambler, me and rosemarie guglielmi. b. mary ambler organized a leaving party for me. dr. mary ambler was a great person and teacher, and her technician rosemarie guglielmi still remains my dear friend (figure 8). while in the usa, i spent one week in the department of pathology at cleveland university hospital, where i worked with another slovene, uroš roessmann, whom i met at icnp in stockholm in 1986 (figure 9). he gave me the opportunity to see more child brain tumours, which rih did not have. uroš became a good friend and, after he retired in 1991, he visited slovenia twice, spending three months each time at the ip mf ul as a visiting professor. it was another valuable opportunity to learn more from an experienced neuropathologist. figure 9. international congress of neuropathology stockholm 1986. from the left: maca and danilo tavčar, milči and uroš roesmann, and me. before going to the usa, i had joined the research group of janez sketelj (figure 10), pathophysiologist, eminent researcher and head of the institute of pathophysiology mf, ul. we had planned research on the regeneration of the rat sciatic nerve after various types of injury, which i continued to pursue after coming back to ljubljana. this research resulted in my msc in 1992, phd in 1996 and four articles [2-5]. figure 10. janez sketelj, my mentor for msc and phd. i was left without a mentor or colleague at the institute who could help me in cases of rare brain tumours and brain pathology. in september 1987, i attended the danube neurology symposium in innsbruck, where i met herbert budka, a well-known neuropathologist and head of the institute of neurology in vienna (figure 11). figure 11. herbert budka, who i met for the first time at the danube symposium of neurology in innsbruck in 1987. throughout almost my entire career in np, until his retirement in 2011, he and his staff were a great help to me and to slovene np. i visited them several times and spent three months with them in 2007. it was an excellent way of learning, through a daily microscopic session with herbert and all the others around a multiple head microscope. i remember asking herbert why they had to look at every meningioma. “you have to see a lot of ordinary meningiomas to recognize a peculiar one” herbert said, and he was right. in 1995, herbert budka invited slovenia to join a cjd surveillance project led by him and financed by the eu. i have very pleasant memories of the annual meetings in baden near vienna, with excellent scientific and social programs, where i learned a lot about prion diseases and had an enjoyable time with scandinavian neuropathologists (figure 12). as a result of that program, ip mf ul established a slovene cjd surveillance system together with the national institute of public health and slovenian neurologists [6, 7]. ip mf ul became the referral centre for cjd in slovenia. to date, we have confirmed 88 scjd cases, one genetic cjd, two cases of gerstmann-sträussler-scheinker syndrome (gss, a a mother in 2007 [8] and her son in 2020). a case of clinically obvious fatal insomnia with a new n181s prnp mutation, without family history, was recognized in 2015. despite regulations surrounding this project by slovenian law in 2001, some clinically suspected cjd cases and the case of fatal insomnia escaped autopsy. fifty-three clinically suspected cjd cases were disproved by autopsy. a prion laboratory with containment level 3 was established in 2000, supported by the ministry of health and stimulated by the work of vladka čurin šerbec and her team from the centre for transfusion medicine in ljubljana, who produced an anti-prp antibody named v5b2 with the same quality as other commercially available anti-prp antibodies. four phds and several valuable papers have been produced in that lab [9-12]. thanks to herbert and the cjd surveillance system, the annual incidence of scjd increased in slovenia from 0,5 per 1 mil (in the period 1985-2000) to 1,5 per 1 mil (in the period 2001-2020). in the last ten years, specifically, the incidence reached 2,5 per 1 mil [13, 14]. slovenia is still a member of the cjd surveillance project, which is now lead by the united kingdom. we have not identified any variant cjd (vcjd) cases in slovenia, however a case of scjd, mv2a molecular subtype, had clinical, radiological and pathological similarities with vcjd [15]. figure 12. scandinavian neuropathologists, from the left: sverre mork, me, peter stubbe teglbjaerg and henning laursen. excellent company at the annual cjd meetings in baden by vienna. my collaboration with the institute of neurology at akh in vienna was also fruitful in tumour pathology. herbert budka, christine haberler, johannes hainfellner and ellen gelpi were of great help in my rare and difficult brain tumour cases, which resulted in several papers [16-18]. ellen gelpi and gabor g kovacs recognized one of our cases of brainstem tauopathy [19], to probably be a new entity of anti-iglon5-related tauopathy [20]. figure 13. a. the late john kepes, an excellent neuropathologist and person. b. his letter announcing that he would no longer be available for consultation. another neuropathologist from the usa also provided me with great help, the late john kepes, who was a very nice person. not only would he send me his opinions on cases i had sent him for consult, but he also sent microphotographs to point out the main changes important for the case diagnosis. i was very touched when he sent me a letter with an apology that he was unable to help any more (figure 13). i developed my academic career at the ip mf ul and became full professor of pathology in 2011. even though it is my duty to research, i am not a researcher at heart. i don’t like statistics but i really enjoy studying every brain, especially neurodegenerative ones. during my career i have attended all european and some international congresses of np, presenting these cases (my cv in supplement). i would like to highlight some of which i am proud. i identified and published neuropathological changes in four family members with a p364s mapt mutation. the mutation was very new and had been described by italian researchers two years previously [21]. they described the clinical presentation of the patient, made recombinant tau with the mutation, and showed that under in vitro conditions “p364s tau revealed a remarkable increase in aggregation rate with respect to wt tau, displaying mostly protofibrils and some short fibrils after 24 hours, and a consistent presence of both straight and twisted long fibrils after 5 days. the propensity of p364s tau to aggregate was even higher than p301l tau used as well-known mutated control.” figure 14. composite neuronal tau inclusion (cnti) in a family with a p136s mutation in mapt. a. h&e, b. gallyas silver staining displays only the peripheral part of the inclusion, which is 4rtau positive, while the central part is only 3rtau positive in double immunohistochemistry on 3r and 4r tau (in c). i recognized that this mutant tau produced in all affected family members a new neuronal tau inclusion consisting of two components (figure 14). we designated it composite neuronal tau inclusion (cnti) [22]. additionally, i discovered that in one family member’s brain, in addition to cnti, all other neuronal tau inclusions known so far were present [23]. another interesting result of brain research was a case of a young male with paranoid schizophrenia, who developed bulbar symptoms and died in his sleep. a few months before death, he was diagnosed by mri as having multiple sclerosis. the brain cutting revealed that he had light chain deposition disease (lcdd) brain vasculopathy with multifocal hypoxic brain injury, especially prominent and fatal in the medulla (figure 15). when we submitted the manuscript to the journal human pathology, it was the first case of lcdd restricted to the brain [24]. by the time of publication, however, the first published case of lcdd restricted to the brain had been published by fischer et al. [25] figure 15. severe light chain deposition disease vasculopathy (lcddv) of medulla. h&e, original magnitude 40x. my most exciting and successful research in brain pathology was a case of zika virus-induced microcephaly, with the first confirmation of causality displaying the virus in the foetal brain by pcr, immunohistochemistry using maternal sera, and by electron microscopy [26]. several papers were produced on this case thanks to our young resident at that time, jernej mlakar, who performed a perfect brain autopsy (figure 16) [27-29]. jernej is now a general pathologist involved in np together with three other fields of pathology. i was lucky and privileged only having done np throughout my career as a pathologist. even though i was a solitary neuropathologist for most of my np career, several valuable people were included in np, without whom my work would not have been possible. two excellent np technicians, dori jazbec and marija zupančič; two administrators, marija blejc and mojca keber; immunohistochemical masters, daniel velkavrh, majda dimnik, miša omerzel and ajla hajrlanović; geneticists alenka matjašič, ema boštjančič and andrej zupan (the most invaluable people in degenerative and especially in tumour brain pathology today) [8, 15, 22, 23, 30-32], and brain cutting assistants, janez caserman, srđan čekić and damir novak. last but not least, i should mention the numerous students and residents of pathology, neurology and neurosurgery to whom i have tried to impart all my knowledge of np. i have also learned a lot from them. many of the residents prepared clinico-pathological conferences, which we had almost every fourth wednesday of the month before the coronavirus pandemic. figure 16. me (actual) and jernej mlakar, future head of department of neuropathology at the institute of pathology, faculty of medicine, university of ljubljana, slovenia. so i am approaching the end of my almost 37-year career in np. i have never regretted my choice, and even though i was the only neuropathologist in slovenia most of the time, i have always favoured mondays to fridays. i know that after the 1st july 2021, when my retirement will start, my life will not be the same. i will miss my beloved np a lot. acknowledgment i am deeply grateful for technical support to our it team, metod perme and miha juvan and our researcher, nina hauptman, who have always been available when my pc did not obey. new technology is great and very useful, but never makes mistakes, which is unbearable. references 1. pogacar, s. and m. popovic, dr. srecko pogacar: from a castle in slovenia to a clinic in ri. r i med j (2013), 2013. 96(10): p. 38-40. 2. popović, m., m. bresjanac, and j. sketelj, regenerating axons enhance differentiation of perineurial-like cells involved in minifascicle formation in the injured peripheral nerve. j neuropathol exp neurol, 1994. 53(6): p. 590-7. 3. sketelj, j., m. bresjanac, and m. popović, rapid growth of regenerating axons across the segments of sciatic nerve devoid of schwann cells. j neurosci res, 1989. 24(2): p. 153-62. 4. popović, m., m. bresjanac, and j. sketelj, role of axon-deprived schwann cells in perineurial regeneration in the rat sciatic nerve. neuropathol appl neurobiol, 2000. 26(3): p. 221-31. 5. popović, m., j. sketelj, and m. bresjanac, changes of schwann cell antigenic profile after peripheral nerve injury. pflugers arch, 1996. 431(6 suppl 2): p. r287-8. 6. popović, m., et al., creutzfeldt-jakob disease in slovenia from 1985 to 2003. wien klin wochenschr, 2004. 116(15-16): p. 524-9. 7. budka, h., concern about mad cow disease: end of the beginning, or beginning of the end? wien klin wochenschr, 2004. 116(15-16): p. 505-7. 8. kojović, m., et al., de novo p102l mutation in a patient with gerstmann-sträussler-scheinker disease. eur j neurol, 2011. 18(12): p. e152-3. 9. curin serbec, v., et al., monoclonal antibody against a peptide of human prion protein discriminates between creutzfeldt-jacob’s disease-affected and normal brain tissue. j biol chem, 2004. 279(5): p. 3694-8. 10. colja venturini, a., et al., anti-idiotypic antibodies: a new approach in prion research. bmc immunol, 2009. 10: p. 16. 11. lukan, a., et al., regional distribution of anchorless prion protein, prp226*, in the human brain. prion, 2014. 8(2): p. 203-9. 12. vranac, t., et al., a single prion protein peptide can elicit a panel of isoform specific monoclonal antibodies. peptides, 2006. 27(11): p. 2695-705. 13. čakš jager, n., et al., analysis of 22 years of surveillance for prion diseases in slovenia, 1996 to 2017. zdr varst, 2018. 57(4): p. 227-33. 14. rus, t., et al., high incidence of sporadic creutzfeldt-jakob disease in slovenia in 2015: a case series. dement geriatr cogn dis extra, 2018. 8(1): p. 42-50. 15. bošnjak, m., et al., a case of mv2k subtype of sporadic creutzfeldt-jakob disease with florid-like plaques: similarities and differences to variant creutzfeldt-jakob disease. neuropathology, published online april 5, 2020. 16. gelpi, e., et al., pleomorphic xanthoastrocytoma with anaplastic features presenting without gfap immunoreactivity: implications for differential diagnosis. neuropathology, 2005. 25(3): p. 241-6. 17. filbin, m.g., et al., developmental and oncogenic programs in h3k27m gliomas dissected by single-cell rna-seq. science, 2018. 360(6386): p. 331-35. 18. clarke, m., et al., infant high-grade gliomas comprise multiple subgroups characterized by novel targetable gene fusions and favorable outcomes. cancer discov, 2020. 10(7): p. 942-63. 19. pretnar-oblak, j., et al., isolated bulbar paralysis in a patient with medullar tau pathology: a case report. j neurol neurosurg psychiatry, 2010. 81(8): p. 847-9. 20. gelpi, e., et al., neuropathological criteria of anti-iglon5-related tauopathy. acta neuropathol, 2016. 132(4): p. 531-43. 21. rossi, g., et al., new mutations in mapt gene causing frontotemporal lobar degeneration: biochemical and structural characterization. neurobiol aging, 2012. 33(4): p. 834.e1-6. 22. popović, m., et al., tau protein mutation p364s in two sisters: clinical course and neuropathology with emphasis on new, composite neuronal tau inclusions. acta neuropathol, 2014. 128(1): p. 155-7. 23. štrafela, p., et al., familial tauopathy with p364s mapt mutation: clinical course, neuropathology and ultrastructure of neuronal tau inclusions. neuropathol appl neurobiol, 2018. 44(6): p. 550-62. 24. popovic, m., et al., light chain deposition disease restricted to the brain: the first case report. hum pathol, 2007. 38(1): p. 179-84. 25. fischer, l., et al., a 19-year-old male with generalized seizures, unconsciousness and a deviation of gaze. brain pathol, 2006. 16(2): p. 185-6, 187. 26. mlakar, j., et al., zika virus associated with microcephaly. n engl j med, 2016. 374(10): p. 951-8. 27. onorati, m., et al., zika virus disrupts phospho-tbk1 localization and mitosis in human neuroepithelial stem cells and radial glia. cell rep, 2016. 16(10): p. 2576-592. 28. štrafela, p., et al., zika virus-associated micrencephaly: a thorough description of neuropathologic findings in the fetal central nervous system. arch pathol lab med, 2017. 141(1): p. 73-81. 29. vesnaver, t.v., et al., zika virus associated microcephaly/micrencephaly-fetal brain imaging in comparison with neuropathology. bjog, 2017. 124(3): p. 521-25. 30. matjašič, a., et al., a novel ptprz1-etv1 fusion in gliomas. brain pathol, 2020. 30(2): p. 226-34. 31. matjasic, a., et al., expression of loc285758, a potential long non-coding biomarker, is methylation-dependent and correlates with glioma malignancy grade. radiol oncol, 2017. 51(3): p. 331-41. 32. matos, b., et al., dynamic expression of 11 mirnas in 83 consecutive primary and corresponding recurrent glioblastoma: correlation to treatment, time to recurrence, overall survival and mgmt methylation status. radiol oncol, 2018. 52(4): p. 422-32. copyright: © 2020 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. studies on inflammation and stroke provide clues to pathomechanism of central nervous system involvement in covid-19 feel free to add comments by clicking these icons on the sidebar free neuropathology 1:16 (2020) opinion piece studies on inflammation and stroke provide clues to pathomechanism of central nervous system involvement in covid-19 ádám dénes 1, stuart m. allan 2, tibor hortobágyi 3,4, craig j. smith 5 1 laboratory of neuroimmunology, institute of experimental medicine, szigony u. 43. 1083, budapest, hungary 2 division of neuroscience and experimental psychology, lydia becker institute of immunology and inflammation, the university of manchester, manchester academic health science centre, av hill building, manchester, m13 9pt, uk 3 department of pathology, faculty of medicine, university of szeged, szeged, hungary 4 mta-de cerebrovascular and neurodegenerative research group, department of neurology, university of debrecen, debrecen, hungary 5 division of cardiovascular sciences, lydia becker institute of immunology and inflammation, the university of manchester, manchester academic health science centre, manchester centre for clinical neurosciences, salford royal nhs foundation trust, salford, m6 8hd, uk corresponding author: ádám dénes · laboratory of neuroimmunology · institute of experimental medicine · szigony u. 43. 1083 · budapest · hungary denesa@koki.hu submitted: 26 may 2020 accepted: 28 may 2020 copyedited by: calixto-hope g lucas, jr. published: 05 june 2020 https://doi.org/10.17879/freeneuropathology-2020-2818 keywords: covid-19, sars-cov-2, neuro-covid, stroke, neuroinflammation, neuropathology recent data, including a number of controversial findings from clinical covid-19 studies, have initiated an intense debate regarding central nervous system (cns) involvement in sars-cov-2 infection-associated pathologies and overall outcomes. in our opinion, involvement of the brain may be an important contributor to the highly complex pathophysiology caused by sars-cov-2 and lessons from studies on stroke and systemic inflammation provide important clues. numerous cns symptoms, including loss of smell and taste, headache, dizziness, nausea, seizures and respiratory distress have been reported in covid-19. several neurological syndromes, such as stroke, encephalitis, epilepsy, and guillain-barre syndrome have been associated with covid-19. more recently, large-vessel occlusive stroke has been described in younger patients without typical covid-19 symptoms1-3. in addition, history of stroke is associated with increased severity of covid-194. these observations highlight the brain as an important target of this multiorgan disease. while neuroinvasion of sars-cov-2 has been associated with cerebral thrombosis, hemorrhagic infarction, demyelinating lesions and encephalopathy (termed as neuro-covid)5,6, it has also been suggested that some respiratory symptoms in patients with covid-19 could indicate neurological involvement7. post-mortem examination of a series of patients with positive polymerase chain reaction testing for covid-19 in pleural effusions revealed negative testing in all cerebrospinal fluid (csf) samples along with no signs of encephalitis or cns vasculitis8. this data may suggest that brain involvement in covid-19 does not play a major role in the disease pathogenesis. however, it appears difficult to draw firm conclusions from these observations. sars-cov-2 might not be detectable in the csf due to low viral load, increased clearance, or the sensitivity of detection5, while macroscopic analysis may not be sufficient to reveal the presence of infection in the brain tissue. nevertheless, other arguments also indicate that neurological symptoms reported to date may be nonspecific and not necessarily imply cns disease, while respiratory failure alone does not suggest cns invasion by sars-cov-29. however, in the absence of comprehensive neuropathological analysis, the extent and anatomical distribution of sars-cov-2 infection in the cns remain unanswered. the robust inflammatory and prothrombotic response directly and indirectly affecting the cns could explain some of the major neurological complications, which may be complemented by effects of possible sars-cov-2 infection in the brain. coronaviruses have long been recognised as potentially neurovirulent microorganisms10. neuroinvasiveness of severe acute respiratory syndrome coronavirus (sars-cov) and middle east respiratory syndrome coronavirus (mers-cov), has been previously reported with pronounced infection in brainstem nuclei involved in respiratory and cardiovascular control2,11. based on these data, sars-cov-2 might also reach the cns via multiple routes. in viraemia, sars-cov-2 binds to the angiotensin-converting enzyme-2 (ace2) receptor on the endothelium and, after crossing the blood-brain barrier (bbb), also binds to neurons expressing the receptor12. one of the currently available case reports with post-mortem neuropathology limited to electron microscopic examination in a frontal lobe sample demonstrates the virus in endothelial cells with features suggestive of transit of the virus towards the neuropil, and neuronal ‘viral-like’ particles in cytoplasmic vacuoles13. via the olfactory route, the virus infects the olfactory epithelium, enters the nervous system across the cribriform plate through axons of olfactory bulb neurons, and then infects the sustentacular cells that maintain the integrity of olfactory sensory neurons. sars-cov-2 may also enter and spread via the cerebral lymphatic (glymphatic) drainage system since the virus can infect the endothelial cells of the olfactory lymphatic system which connect to the brain12,14-16. in addition, similar to herpesviruses or the avian influenza virus, sars-cov-2 could reach the brain via peripheral nerves, possibly via retrograde transport and trans-synaptic spread17-19. deficient systemic immune response due to older age, chronic disease or immunosuppressive therapy, altered ace2 expression in diabetic or hypertensive patients, cerebrovascular disease (major risk factors for covid-19-associated mortality)1,4, as well as vascular inflammation or impaired blood-brain barrier (bbb) function in aged or comorbid patients could also increase the risk and severity of infection. if cns infection occurs, the outcome largely depends on the ability of the cns immune system to control the spread of the virus. studies on neurotropic virus infection suggest that microglia, the main inflammatory cells of the cns, are important in controlling both the spread of the virus and shaping the cerebral inflammatory response. this key role of microglia has been experimentally demonstrated in coronavirus, herpesvirus, theiler’s virus and vesicular stomatitis virus infection among others20-24. importantly, lack of normal microglial function not only increases viral spread in the brain, but is also associated with markedly worsened neurological symptoms (motor function deficits, neuronal injury, brain oedema, seizures, etc.) and increased mortality in animal models20-24. microglial phenotype is heavily influenced by age, comorbidities and systemic inflammation25. therefore, it is likely that compromised microglial function is an important contributor to poor outcome in neuro-covid, especially if brain areas involved in respiratory, cardiovascular and neuroendocrine control are affected by sars-cov-2. effective antiviral drug delivery through the bbb may therefore be important in the management of neurological complications. irrespective of whether cns sars-cov-2 infection occurs, the cerebral effects of systemic inflammation associated with “cytokine storm” and prothrombotic state have profound impact on outcome in severe covid-19 cases. high serum levels of inflammatory cytokines, such as il-6, predict poor outcome26 and may contribute to cardiacand respiratory arrest, coma and multiorgan failure through complex mechanisms that include microcirculatory deficits, hypotension, oedema and thrombosis. circulating inflammatory cytokines stimulate the autonomic nervous system and the hypothalamic-pituitary-adrenal (hpa) axis with major impact on blood pressure and flow, respiration and neuroendocrine function. the autonomic nervous system and hpa axis also play an important role in the regulation of immune cell responses, cytokine production, cell trafficking and cell death via both humoral and neural mechanisms. the sustained increase in serum adrenaline, noradrenaline and cortisol levels due to prolonged autonomic and hpa axis activation eventually lead to dysregulation of inflammatory responses. this, in line with excessive cytokine production, results in insufficient elimination of infectious agents. the lymphopenia due to apoptosis and impaired lymphopoiesis shifts immune cell balance towards excessive monocyte and granulocyte load, further increasing the production myeloid cell-derived proinflammatory cytokines. the excessive systemic inflammatory response also contributes to the neurological complications via direct and indirect actions. findings from clinical and experimental stroke studies with preceding or post-stroke infection may help to shed light on the mechanisms of covid-19-related neurological impairment. stroke in apparently healthy youngand middle-aged people with rapid formation of thrombi in the cerebral circulation and high mortality of older patients with chronic inflammatory disease collectively suggest a high impact of altered coagulation in patients with covid-1927,28. infections in general (e.g. seasonal flu) increase stroke incidence29. as known from studies of viral and bacterial sepsis and from comorbid models of stroke, increased systemic inflammatory burden promotes vascular inflammation, platelet activation and alters coagulation cascades, leading to a procoagulant state leading to thrombosis and disseminated intravascular coagulation. such coagulopathy, predictive of worse clinical outcome, has been reported in covid-1930,31. this promotes thrombus formation in both veins and arteries – a known feature of severe covid-19 with thromboembolic complications reported in around one third of infected patients32. the cerebrovascular endothelial cells have particularly high sensitivity to proinflammatory cytokines and sars-cov-2 infection could further boost the expression of adhesion molecules and increase vascular permeability. brain injury and mortality are generally far more severe in patients with additional stroke risk factors (e.g. in obese, diabetic, hypertensive patients or after infection), similarly to that seen in experimental stroke models. as an example, localized infection of the lungs by influenza virus or streptococcus pneumoniae leads to marked increases in circulating proinflammatory cytokines, endothelial activation (indicated by increased levels of adhesion molecules) and platelet aggregation. these infection-driven changes are associated with augmented brain inflammation and leukocyte recruitment, leading to increased neuronal injury and worse neurological outcome33-35. targeted blockade of proinflammatory cytokines (e.g. il-6, il-1 or tnf) or platelet-endothelial interactions attenuated infection-induced brain injury in experimental models33,34,36-38, while the potential efficacy of il-6 receptor antagonist tocilizumab to reduce mortality in severe covid-19 cases has been suggested39. recent reports have described patients with ischaemic stroke complicating covid-19 infection, often manifesting as large-vessel infarcts occurring in multiple territories and associated with features of prothrombotic coagulopathy3,40,41. however, these small case series may not be representative of wider clinical practice, and no causal relationship has yet been established between covid-19 infection and stroke. other mechanisms may also be relevant, such as destabilisation of atheromatous plaques resulting in thrombosis and cerebral atheroembolism, atrial fibrillation in critically ill patients causing thromboembolism of cardiac origin, or haemorrhage secondary to microangiopathy and cerebral vasculitis. importantly, patients may also acquire sars-cov-2 infection following stroke. suppression of both innate and adaptive immune response is well-documented after stroke, driven by autonomic nervous system failure and activation of the hpa axis, which could exacerbate subclinical infection, or increase susceptibility to nosocomial infection. an important issue to address is whether neurological manifestations (reported in up to one third of cases with different severity of infection1,42) result from systemic effects on the brain, direct cns infection by sars-cov-2, or both, and if the latter is a major contributor to covid-19-related severe illness and mortality. firm conclusions cannot be drawn at this stage due to limited data availability, but it is likely that the impact of cns-related effects on disease outcome is considerable. most neurological manifestations appear to occur early in the illness, preceding severe respiratory distress and the need for mechanical ventilation, while several patients are admitted to the hospital merely based on neurologic manifestation with no respiratory symptoms1. at this time, data are largely from single case reports. for example, occurrence of neurological symptoms such as fever, anosmia, dysgeusia, headache and possible seizure in line with respiratory distress and severe ventilator asynchronies were found in a patient where autopsy later confirmed the presence of sars-cov-2 infection in the brain. findings included widespread tissue damage involving the neurons, glia, nerve axons, and myelin sheath, progressively more severe from the olfactory nerve to the gyrus rectus and to the brainstem43. a recent report showed that after positive diagnosis for sars-cov-2, a patient developed complete anosmia and dysgeusia, with mri showing signs of bilateral olfactory bulb oedema, followed by normalization of both sensory symptoms and mri signal by day 1444. another report found that 44% of patients admitted to the intensive care unit with covid-19 and neurological symptoms showed cns abnormalities on mri, which included cortical (frontal, parietal, occipital, temporal, insular) and deep white matter flair signal abnormalities, in the absence of sars-cov-2 in the csf (50% of cases tested). thus, while focal neuropathologies appear to be frequent in severe cases, the extent of associated brain infection remains unclear presently. of note, the incidence of epileptiform discharges, seizure-like events and new onset encephalopathy is more than two-fold higher in acutely ill covid-19 patients with neurological symptoms compared to non-infected patients, but it is not known if this is through direct or indirect actions on the cns45. while sars-cov-2 infection shares many similarities with bacterial sepsis, the inflammatory response was considered more modest (e.g. lower il-6 levels), and progressive and profound suppression of adaptive immunity was noted in covid-19 relative to sepsis46. therefore, further studies are required to assess the nature of systemic inflammatory changes and their impact on neurological symptoms and disease outcome. in conclusion, the available evidence strongly indicates that the brain is an important target of sars-cov-2 and the 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investigated for sars-cov-2/covid-19: a small case series preliminary report. epilepsia open. 2020; https://doi.org/10.1002/epi4.12399 46. remy ke, brakenridge sc, francois b, daix t, deutschman cs, monneret g, jeannet r, laterre pf, hotchkiss rs, moldawer ll. immunotherapies for covid-19: lessons learned from sepsis. lancet respiratory medicine. 2020;s2213-2600(20)30217-4. doi:10.1016/s2213-2600(20)30217-4 copyright: © 2020 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. enteric synucleinopathy: from trendy concept to real entity. feel free to add comments by clicking these icons on the sidebar free neuropathology 1:26 (2020) opinion piece enteric synucleinopathy: from trendy concept to real entity. adrien de guilhem de lataillade 1,2, thibaud lebouvier 3, wendy noble 4, laurène leclair-visonneau 1, pascal derkinderen 1,2 1 université de nantes, inserm, tens, the enteric nervous system in gut and brain diseases, imad, nantes, france 2 chu nantes, department of neurology, nantes, f-44093, france 3 univ. lille, inserm urm_s1172, chu lille, distalz, licend, f-59000 lille, france 4 king’s college london, institute of psychiatry, psychology and neuroscience, department of basic and clinical neuroscience, maurice wohl clinical neuroscience institute, 5 cutcombe road, camberwell, london. se5 9rx, uk corresponding author: pascal derkinderen · inserm u1235 nantes · 1 rue gaston veil · 44035 nantes · france · tel: +33(0)240165202 · fax: +33(0)240165203 pascal.derkinderen@chu-nantes.fr submitted: 29 july 2020 accepted: 23 august 2020 copyedited by: aivi t. nguyen published: 28 august 2020 https://doi.org/10.17879/freeneuropathology-2020-2920 keywords: synucleinopathies, tauopathies, enteric nervous system, aggregates, alpha-synuclein, parkinson’s disease abstract an accumulating body of literature has emerged in the past 25 years to show that parkinson’s disease (pd) is not only a disorder of the brain but also of the gastrointestinal tract and more generally of the gut-brain axis. gastrointestinal symptoms occur in almost every pd patient at some point and in nearly every case examined pathologically autopsy studies find alpha-synuclein deposits, the pathological hallmarks of pd, in the enteric nervous system. this concept of ‘enteric synucleinopathy’ led to the hypothesis that the enteric nervous system might play a pivotal role in the initiation and spreading of pd. although this hypothesis opens up interesting perspectives on the pathogenesis of neurodegenerative disorders, some important questions are still pending. the present opinion paper describes and compares the physiological and pathophysiological properties of alpha-synuclein in the brain and the enteric nervous system. we conclude that the existing data supports the existence of pathological alpha-synuclein species in the gut in pd. we also discuss if gut-brain interactions are important in other neurodegenerative disorders. abbreviations ad alzheimer’s disease, ens enteric nervous system, gi gastrointestinal, lb lewy bodies, ln lewy neurites, pd parkinson’s disease, pmca protein misfolding cyclic amplification, psp progressive supranuclear palsy the enteric nervous system (ens) is an intricate neural network embedded within the gastrointestinal (gi) tract and distributed from the lower oesophagus to the rectum. compared to other components of the peripheral nervous system, the ens shows some unique features that closely resemble the cns and therefore it is sometimes referred to as ‘the brain-in-the-gut’ or the ‘second brain’ [1] (figure 1). this close homology between the cns and ens suggests that a disease process affecting the cns could also involve its enteric counterpart. parkinson’s disease (pd) is the best example of this assumption. pd is the most common synucleinopathy (or synuclein proteinopathy), a group of neurodegenerative disorders characterized by a common pathological lesion composed of aggregates of alpha-synuclein in selectively vulnerable neuron populations in the cns [2]. although pd has traditionally been considered a disease of dopaminergic neurons in the substantia nigra, analyses of gi samples from pd patients have consistently found neural pathology, with the presence of alpha-synuclein deposits being detected in the ens in nearly every pd patient examined [3–11]. this has led to the emerging concept of enteric synucleinopathy. although these observations open up interesting perspectives on the pathogenesis of neurodegenerative disorders, some important questions are still pending, among which are: what are the biochemical and pathological characteristics of alpha-synuclein deposits in the ens? are they similar to those observed in the cns? is there any neuronal loss in the ens in pd? besides pd, are gut-brain interactions important in other neurodegenerative disorders? here, we first provide a brief overview of the normal expression profiles of alpha-synuclein in the ens, before discussing these questions and the arguments for and against the existence of enteric synucleinopathies. figure 1. anatomical organisation and local reflexes of the enteric nervous system. the ens is a neuronal network embedded within the gi tract and distributed from the lower oesophagus to the rectum. it is organized in two major ganglionated plexuses, the myenteric (mp or auerbach’s plexus) mainly involved in the control of smooth muscle activity, and the submucosal (smp or meissner’s plexus), which regulates secretion (sn, secretory neurons) and microvasculature. compared to other sections of the peripheral nervous system, the ens shows unique features that closely resemble some of the cns: it contains a variety of functionally distinct enteric neurons along with a vast repertoire of neurotransmitters and intercellular messengers which are the basis for enteric neurotransmission. it also harbours a prominent component of glial cells (egc for enteric glial cells) which, like astrocytes in the cns, contribute to support, protection and maintenance of the neural networks. local distention of the intestinal wall and chemical contents in the gut lumen activate intrinsic primary afferent neurons (ipan) located in both the smp and mp. the ipan projects both in oral and anal directions to synapse with interneurons (in) and motor neurons (mn). cholinergic (ach) mn in red are excitatory while nitrergic (no) mn in green are inhibitory. the peristaltic reflex includes an ascending excitatory reflex mediated by cholinergic mn and elicits contraction of the circular or longitudinal smooth muscles located orally to the site of stimulation. the descending inhibitory reflex involves inhibitory nitrergic mn that elicit relaxation of the circular muscles and longitudinal muscles located anally to the site of stimulation. alpha-synuclein is physiologically expressed by enteric neurons alpha-synuclein was first isolated in 1988 from the electric organ of the pacific electric ray torpedo californica. in addition to its strong presynaptic localisation, maroteaux et al., also identified alpha-synuclein in the nucleus, thus accounting for the name ‘synuclein’ (synapse + nucleus) [12]. several studies have documented the physiological function of alpha-synuclein in modulating synaptic vesicle release [13]. human alpha-synuclein, 140 amino acids in length, is mainly expressed by cns neurons and erythrocytes and is composed of 3 different functional regions (figure 2a) [13]. there is still open debate about the physiological structure of brain alpha–synuclein as some research groups concluded that it occurs as a helically folded tetramer [14], while others counterclaim that it exists primarily as a disordered monomer [15]. alpha-synuclein has a natural propensity to aggregate as amyloid structures in a nucleation-dependent process in which monomers assemble via oligomers into fibrils [16] (figure 2b). figure 2. alpha-synuclein structure and molecular mechanism of its oligomerisation and fibrillogenesis. (a) schematic representation of alpha-synuclein, which is composed of 3 distinct regions : (i) an n-terminal domain (amino acids 1–60) that binds lipids and contains the lysines that are ubiquitinated (ii) a central domain known as the non-amyloid component (nac) (amino acids 61–95) which is involved in aggregation, and (iii) a c-terminal acidic tail (amino acids 96–140) accountable for most interactions with other proteins and small molecules and that contains most of the phosphorylation sites including serine 129. some of the truncated c terminal forms of alpha-synuclein are also shown. (b) illustration of the molecular steps involved in alpha-synuclein oligomerisation and fibrillogenesis leading to lewy body formation. soluble alpha-synuclein is natively unstructured and monomeric. under pathological conditions, soluble α-synuclein forms β-sheet-like oligomers (protofibrils), which convert into amyloid fibrils and eventually deposit in lewy bodies. alpha-synuclein is also expressed by enteric neurons. in rodents, guinea-pigs and human alpha-synuclein positive neurons are present in the two plexuses of the ens and along the entire digestive tract [5, 17, 18] (figure 3). using amine-reactive cross-linking, we showed that, unlike brain neurons, alpha-synuclein exists primarily as a monomer in enteric neurons [19]. as far as we know, the expression of alpha-synuclein by enteric glial cells, the enteric counterpart of cns astrocytes, has not been evaluated. detailed immunohistochemical characterisation showed that alpha-synuclein immunoreactive neurons are mostly cholinergic [17, 18] (figure 1) and that alpha-synuclein is closely associated with the vesicular apparatus [20]. although the role of alpha-synuclein in the ens is still mostly unknown, a recent study suggested that it is involved in the development and electrophysiological properties of enteric cholinergic neurons [21]. figure 3. physiological expression of alpha-synuclein and phospho-alpha-synuclein histopathology in the ens. left: anti-alpha-synuclein antibody syn-1 was used to detect alpha-synuclein in the myenteric ganglia (colon) in a control subject devoid of neurodegenerative disorders; βiii tubulin antibody was used to label the neuronal network. scale bar is 50 µm. right: the colon from a pd patient was microdissected into mucosa, whole mount of submucosa and myenteric plexus. each part was stained with an antibody specific for the phosphorylated form of alpha-synuclein. phospho-alpha-synuclein histopathology with a ln-like pattern is observed in the mucosa (arrow, the asterisk is for the crypt) and submucosa, while a lb-like structure is found in the myenteric plexus. scale bar is 40 µm. does the ens contain pathological aggregated forms of alpha-synuclein? lewy bodies (lb) and lewy neurites (ln) are the defining neuropathological characteristics of pd. lb typically appear in neuronal somata as eosinophilic, rounded inclusions while ln are strand-like structures observed in axons. in 1997, it was demonstrated that lb and ln isolated from pd brain were highly immunoreactive for alpha-synuclein, thereby suggesting that this protein was one of the main components of lewy pathology [22]. subsequent work showed that alpha-synuclein in lb and ln was hyperphosphorylated at serine residue 129 [23] (figure 2). because alpha and phospho-alpha-synuclein immunohistochemical staining has a much greater sensitivity than hematoxylin and eosin staining for the detection of lb [24], this method quickly became the method of choice for the neuropathological diagnosis of pd [25]. using this approach, several neuropathology laboratories demonstrated that lb and ln-like structures were observed in the ens (in both the myenteric and submucosal plexus) in the vast majority of pd patients [3–7] (figure 3). in some of these studies, proteinase k [3] or alkaline protease [7] pre-treatments were used primarily to unmask antigens and enhance immunolabeling, but such treatments might also allow protease-resistant misfolded, aggregated and hyperphosphorylated alpha-synuclein to be distinguished from soluble forms of the protein. on the whole these findings suggest that pathological aggregated alpha-synuclein is present within the gi tract of pd subjects. two key issues should however be borne in mind. first, the mere detection of alpha-synuclein phosphorylated at serine 129 is not synonymous with aggregation since soluble alpha-synuclein is also physiologically phosphorylated at this residue [26]. secondly, immunohistochemical approaches are not sufficient to show that alpha-synuclein is misfolded/aggregated; biochemical confirmation of altered solubility is required. indeed, in the cns, a comprehensive biochemical characterisation of alpha-synuclein forms in lb has already been carried out. using one and two-dimensional immunoblot analysis with modification-specific synuclein antibodies and mass spectroscopy, anderson et al. confirmed that the predominant modification of alpha-synuclein in lb was phosphorylation at serine 129. they also found a set of additional characteristic modifications including ubiquitination at amino-terminal lysine residues and specific carboxy-terminal truncations [27] (figure 2). an additional property of alpha-synuclein aggregates found in diseased brains is their seeding ability: the amyloid fibrils formed by alpha-synuclein aggregates can act as templates for the conversion of physiological alpha-synuclein, resulting in the growth of the fibrils and spread of alpha-synuclein pathology [28]. how about the biochemical characterisation of alpha-synuclein in diseased ens? there are some existing data but it is definitely less complete and robust than those available for the cns. using one and two-dimensional analysis of colonic biopsies, we were unable to detect any differences in the expression levels, phosphorylation or aggregation status of alpha-synuclein between controls and pd specimens [29]. these negative findings might however be explained by the relative sparsity of neuronal structures and/or alpha-synuclein inclusions in the gi samples that were used; due to a shortage of samples, only 2 routine colonic biopsies per subject were pooled and analysed. however, when we had the opportunity to analyse 4 pooled biopsies per subject, we were able to detect acidic and high molecular weight alpha-synuclein species in the gi tract of pd patients but not controls, which likely represent hyperphosphorylated and aggregated forms of the protein, respectively (lebouvier-derkinderen unpublished results, figure 4). such a pattern is reminiscent of that observed in pd brain [27] (figure 4). the limitations of the two-dimensional immunoblotting technique prompted us to use more sensitive approaches, based on the seeding efficiency of alpha-synuclein. using an assay inspired by the protein misfolding cyclic amplification (pmca) assay, we showed that gi biopsies from pd patients (2 to 4 biopsies per subject taken from the upper or lower gi tract) seeded alpha-synuclein aggregation in 10 out of 18 cases [30]. more recently, viviane labrie and patrik brundin’s group focused on the vermiform appendix, a structure which is particularly enriched in alpha-synuclein [31]. using mass spectrometry, they identified full-length alpha-synuclein together with a set of 10 truncated forms in the healthy human appendix [32]. additional experiments performed with an in vitro shaking assay (also inspired by the pmca approach), showed that appendix lysates from either control or pd subjects seeded aggregation and truncation of alpha-synuclein. these intriguing and provocative findings suggest that aggregated and truncated alpha-synuclein are consistently found in the human healthy appendix, thereby supporting the assumption that the appendix may act as a reservoir for pathogenic forms of alpha-synuclein [31, 32]. figure 4. high molecular weight and post-translationally modified alpha-synuclein species in the gi tract of pd patients. immunoblots of total mesencephalon lysates (a and b) from a control (a) and a pd patient autopsy sample (b), and immunoblots of colonic biopsy lysates (c and d) from a control (c) and pd patient (d) were resolved by two-dimensional page using ph 3–10 ief gradients. immunoblots were probed with the alpha-synuclein antibody syn-1. white asterisks mark full-length synuclein. boxes highlight differences between pd patients and controls: acidic modifications of full-length monomer (solid boxes), and high molecular weight species (dotted boxes) are present in pd patients and absent in controls, in both substantia nigra and colonic biopsies (n=2); pi values and positions of molecular weight standards are indicated. an equivalent amount of protein was loaded in a-b and c-d, respectively. it has also been shown that pathological alpha-synuclein obtained from pd brain has the ability to trigger alpha-synuclein pathology in the cns of rodents and non-human primates [33, 34]. for example, the intranigral inoculation of alpha-synuclein-containing lb extracts obtained from pd brains leads to widespread alpha-synuclein pathology together with dopaminergic neurodegeneration in mice and monkeys [35]. for the sake of comparisons between brain and enteric alpha-synuclein, it is important to know if pathological alpha-synuclein obtained from the gi tract shares the same pathogenic capacity. although the precise answer to this question remains unknown, a recent study evaluated the effects of alpha-synuclein aggregates from post-mortem pd stellate ganglia (pooled from 3 patients) injected into mouse brain [36]. stellate ganglion is a paravertebral ganglion, which like the gut, exhibits marked lewy pathology in almost all pd subjects [4, 37]. in contrast to the findings obtained with substantia nigra-derived alpha-synuclein, no pathological effects were observed when peripheral aggregates from stellate ganglion were injected into the brain of wild-type mice, at least up to 6 months following injection [36]. this intriguing observation, which casts doubt on the pathogenicity of peripheral alpha-synuclein, requires replication using extracts from other peripheral organs affected by lewy pathology, such as the gi tract or the salivary glands [3, 38]. is there any enteric neuronal loss in pd? in the substantia nigra of pd subjects, the presence of alpha-synuclein aggregates is accompanied by severe neuronal loss and clinicopathological findings strongly suggest that the classical pd motor symptoms, including bradykinesia and hypertonia, are driven primarily by neuronal loss rather than only the aggregation of alpha-synuclein [39]. as such, the quantitative evaluation of enteric neuron populations is important to determine the pathological underpinnings of gi symptoms, which are so frequently observed in pd [40]. with that said, and despite the publication of proposed guidelines and consensus techniques, the quantification of submucosal and myenteric neuron number remains challenging [41], mainly because of the fishnet-like architecture of the ens plexus. in the most comprehensive post-mortem study to date, annerino et al. used formalin-fixed paraffin embedded sections to compare myenteric neuron density along the length of the gi tract in 6 patients with pd and 12 controls. there were no differences in total myenteric neuron density between controls or patients in any segment examined [42]. similarly, when whole mounts of submucosa obtained from colonic biopsies were analysed by immunohistochemistry, no major decrease in neuronal density was observed in pd relative to controls in two independent studies that included a total of 58 pd and 30 control cases [8, 43]. on the whole, these data strongly suggest that enteric neuron loss is not a feature of pd. besides alpha-synuclein, are other aggregated proteins found in the ens? the observation that alpha-synuclein deposits are a feature of pd gut logically leads to speculation that such a phenomenon might also occur in other neurodegenerative disorders such as tauopathies. like alpha-synuclein, tau is physiologically expressed by enteric neurons. however, in contrast to the cns neurons that express all six tau isoforms, adult human ens primarily express only two tau isoforms [44]. tau aggregates found in tauopathies generally contain tau in an elevated state of phosphorylation that is often aberrantly cleaved [45]. in a preliminary report, we analysed by western blot colonic biopsies from 5 patients with probable progressive supranuclear palsy (psp) using 2 different phospho-tau antibodies and one antibody specific for caspase-cleaved tau [44]. the phosphorylation and truncation patterns of tau in psp were indistinguishable from those in controls [44]. in a subsequent study, brittany dugger et al. performed a comprehensive immunohistochemical analysis of the ens in tauopathies. using formalin fixed paraffin embedded sections, they examined the sigmoid colon in 26 psp, 21 ad and 19 controls using one antibody for total tau and two phospho-tau antibodies [46]. no differences in the staining pattern were observed between colonic specimen from tauopathy patients and controls [46]. so, enteric synucleinopathy: myth or reality? even if they are still preliminary, recent findings and in particular the ones obtained with ultrasensitive amplification techniques such as pmca, convincingly showed that alpha-synuclein aggregates with seeding capacity are found in the ens [30, 32]. it therefore seems justified to use the word ‘enteric synucleinopathy’. that said, some outstanding questions remain: (i) existing findings suggest that enteric neuron loss is not a feature of pd but there could be more subtle morphologic changes [47] that could be evaluated in the future using computer-assisted analysis of the enteric neurons [48] (ii) a comprehensive inventory of alpha-synuclein forms present the ens from patients with pd still need to be carried out, as has already been performed for the cns [27] (iii) the results obtained with pmca need to be independently replicated and confirmed by others amplification techniques such as rt-quic (real-time quaking-induced conversion) [49]. in addition, it still remains to be determined if pathological alpha-synuclein purified from the ens is capable of promoting alpha-synuclein pathology when intracerebrally inoculated, as already demonstrated for cns-derived alpha-synuclein aggregates [50]. this is a critical issue with regard to pd pathogenesis as it has been speculated 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candelise n, baiardi s, et al (2020) ultrasensitive rt-quic assay with high sensitivity and specificity for lewy body-associated synucleinopathies. acta neuropathol 140:49–62. https://doi.org/10.1007/s00401-020-02160-8 50. arotcarena m-l, dovero s, prigent a, et al (2020) bidirectional gut-to-brain and brain-to-gut propagation of synucleinopathy in non-human primates. brain 143:1462–1475. https://doi.org/10.1093/brain/awaa096 51. beach tg, corbillé a-g, letournel f, et al (2016) multicenter assessment of immunohistochemical methods for pathological alpha-synuclein in sigmoid colon of autopsied parkinson’s disease and control subjects. j parkinsons dis 6:761–770. https://doi.org/10.3233/jpd-160888 copyright: © 2020 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. top ten discoveries of the year: neuroinflammation feel free to add comments by clicking these icons on the sidebar free neuropathology 1:3 (2020) review top ten discoveries of the year: neuroinflammation hans lassmann center for brain research, medical university of vienna, vienna, austria corresponding author: hans lassmann · center for brain research · medical university of vienna · spitalgasse 4 · a-1090 wien · austria hans.lassmann@meduniwien.ac.at submitted: 25 december 2019 accepted: 08 january 2020 published: 11 january 2020 https://doi.org/10.17879/fnp-2020-2612 keywords: neuroimmunology, brain inflammation, lymphocytes, microglia, autoantibodies, multiple sclerosis abstract ten neuropathological studies, published in 2019, are discussed, which address important aspects of neuroimmunology and inflammatory brain disease. they include topics related to new mechanisms of inflammation and immune mediated neurodegeneration, which are relevant for multiple sclerosis (publications 1 to 4) and discuss the role of specific autoantibodies against myelin oligodendrocyte glycoprotein or aquaporin 4 in neuromyelitis optica spectrum disorders (publications 5 and 6). other studies highlight the discovery of new virus induced diseases of the nervous system and their relevance for clinical neurology and diagnostic neuropathology (publications 7 and 8). finally, very interesting studies are discussed dealing with microglia and immune mechanisms in neurodegeneration (publication 9) and the neuropathological long-term outcome of aß vaccination in alzheimer’s disease (publication 10). all these studies highlight the central role of neuropathology in neurological disease research. introduction there is a common belief that it is modern technology in molecular neurobiology, immunology and genetics, which drives progress in the understanding, diagnosis and therapy of human diseases of the nervous system, while neuropathology is sometimes considered an old fashioned and descriptive discipline which has little to add. however, very important new insights for human disease, achieved during the last years, came from neuropathological studies. in the field of neuroim-munology this included the discovery of new disease entities, new insights into the mechanisms of immune surveillance of the brain, of brain inflammation and of inflammation induced tissue damage in the nervous system. although all these discoveries were based on a combined multidisciplinary approach, the interpretation of the structural changes in the brain tissue in human disease and experimental models, based on neuropathological competence and experience, was essential to draw the correct conclusions. here i will discuss some recent work published during the last months, which contributed to a shift in the understanding of disease mechanisms. 1. neuronal vulnerability and multilineage diversity in multiple sclerosis (schirmer et al 2019) this study is based on a potentially revolutionary technical development. up to now gene expression was performed either on homogenized tissue, which did not discriminate spatial, temporal or cellular changes during lesion development, or by in situ hybridization, which generally only provided information on the expression of single genes. recently new technologies of single nucleus rna sequencing together with multi-color or multi-channel-based methods of in situ hybridization and immunohistochemistry have been developed, which promise to overcome some of the above listed limitations. the study by schirmer et al (2019) is one of the first to apply such technology for the analysis of multiple sclerosis (ms) brain tissue and combined this investigation with detailed confirmation by in situ hybridization and immunohistochemistry. the study supports the importance of meningeal inflammation with high b-lymphocyte content for cortical lesions, the heterogeneous astrocyte and microglia activation in the lesions, the loss of oligodendrocytes, the phagocytosis of myelin debris in macrophages during lesion activity and the importance of oxidative injury as a mechanism of neuronal injury. the most innovative finding is that a distinct subpopulation of excitatory neurons is dominantly affected in subpial cortical lesions of ms patients. thus, this study impressively shows that this new technology can be applied on autopsy material of the human brain, and that the results reliably confirm what has been described with conventional technology before (mahad et al 2015, reich et al 2018). even the main finding of a selective loss of a subset of excitatory neurons in the cortex is not surprising, since these cells are dominantly located in the outer cortical layers, which have been described before to show the most severe neuronal loss in subpial lesions (magliozzi et al 2010). a similar approach has been used recently to determine oligodendrocyte heterogeneity in normal human controls and multiple sclerosis patients (jäkel et al 2019). this study, too, showed that this new technology is suitable for the analysis of post mortem tissue of human inflammatory brain diseases and it reveals a new dimension regarding the complexity of cell phenotypes already in the normal tissue, being even more complex in multiple sclerosis lesions. however, to draw firm conclusions related to disease pathogenesis such studies have to be performed on much larger samples of patients and lesions and on the basis of much more elaborate selection and characterization of different lesion types. thus, in future studies such technologies have to be applied on cases and lesions, which have been very carefully selected according to the specific research question. unfortunately, availability of such material is very limited when the technology requires the use of fresh frozen tissue. thus, major efforts are necessary to adapt these new technologies to formaldehyde fixed and paraffin embedded archival material. 2. epstein-barr virus specific cd8 t cells selectively infiltrate the brain in multiple sclerosis and interact locally with virus-infected cells: clue for a virus driven immunopathological mechanism (serafini et al 2019) even in the absence of an overt inflammatory disease of the central nervous system there is a small to moderate number of t-lymphocytes present in the brain and spinal cord, while infiltration of the tissue by t-cells and b-cells is massively increased in inflammatory brain diseases. from experimental data in normal mice and mouse models of neurodegenerative diseases or autoimmune encephalomyelitis it was suggested that these cells are mhc class ii restricted cd4+ t-cells, which enter the brain in the course of immune surveillance and, when directed against autoantigens of the cns, may exert both, disease-promoting as well as regulatory or protective functions. these concepts however, were difficult to harmonize with previous observations that the majority of t-cells in the normal human brain and in the cns of patients with neurodegenerative disease are cd8+ mhc class i restricted t-cells, displaying the phenotype of tissue resident memory cells (smolders et al 2018). similarly, in the ms brain cd8+ t-cells with a phenotype of tissue resident memory cells prevail, where they show focally restricted activation in particular in active lesion (van nierop et al 2017, machado santos et al 2018; figure 1c). so far, such tissue resident memory cells have been found, defined and characterized in solid tissues, including the brain, and in models of virus infection, where they form a very effective barrier against re-infection by the same agent (steinbach et al 2018). thus, a key question is, what target antigen is recognized by the cd8+ tissue resident t-cells in the ms brain. multiple sclerosis is regarded by immunologists as an autoimmune disease, driven by t-lymphocytes directed against myelin anti-gen(s). however, attempts to identify an ms-specific autoimmune response have so far failed (hohlfeld et al 2016). even when the investigation specifically focused on activated cd8+ t-cells in the ms lesions, no reactivity was found against the classical candidates of myelin directed autoimmunity, but a substantial number of isolated t-cells recognized epitopes from epstein barr virus (ebv; van nierop et al 2017, serafini et al 2019). furthermore, serafini et al (2019) showed that these ebv reactive t-cells form close contacts with b-lymphocytes expressing epitopes of ebv related proteins, and these t-cells display cd 107 on their surface, suggesting the release of the content of their cytotoxic granules. overall these data suggest that the chronic inflammatory response in the ms brains may in part be driven by a t-cell response against ebv (serafini et al 2019). however, the question whether there is productive ebv expression in the ms brain is still controversial. it is described by some groups, while other groups using similar techniques in material from comparable disease phenotypes and stages failed to confirm the data (van nierop et al 2017). the reason for these discrepancies is still unresolved (lassmann et al 2011). another recent study (konjevic sabolek et al 2019) describes the interaction of activated cd8+ cytotoxic cells with local macrophages, but not with b-cells, which are the potential target for ebv infection. thus, the specific antigenic target of t-cells in ms lesions is still unresolved, but recent data indicate that the concept of autoimmun-ity against myelin or other cns antigens may be wrong. figure 1: one well established and several new mechanisms triggering or propagating inflammation in the central nervous system (cns) a: the basic well-established mechanism of immune surveillance and inflammation of the central nervous system is that t-cells, activat-ed in the peripheral immune system, are able to pass the blood brain barrier and enter the cns in the process of immune surveillance. when they encounter their specific antigen, presented in the meninges, the perivascular space or the cns parenchyma they get re-activated, produce proinflammatory cytokines and attract additional immune cells into the lesion. red cells: activated t-cells, red ring cells: resting t-cells; star like cells: macrophages and microglia involved in antigen presentation; the blue circles indicate the endotheli-um of the blood brain barrier and the glia limitans. b: an alternative way to start inflammation in the cns (as described by publication 3) is the local presence of damaged tissue or foreign antigens from infectious agents (red dots); they interact with microglia and induce inflammasome activation and the production of pro-inflammatory cytokines (green triangles). these cytokines activate endothelial cells at the blood brain barrier and allow the recruitment of t-cells into the focal site of tissue injury. in case these t-cells (red circles) find their cognate antigen within the lesion, they become activated (red cells) and trigger the full-blown inflammatory lesion. c: in conditions of chronic brain inflammation, such as for instance in multiple sclerosis (as discussed in context with publication 2), high numbers of tissue resident memory t-cells (green circles) are present within the brain and lesions and they are mainly in an inactivated stage. when there is re-appearance of their cognate antigen (for instance a foreign antigen or a modified self-antigen) presented by perivascular b-cells (blue circles) or by macrophages / microglia (blue stars), they get re-activated and propagate the chronic inflammatory process. d: a new mechanism of brain inflammation is outlined in publication 6. when high titers of antibodies against aquaporin 4 are present in the circulation, the very low amount of antibodies which can pass the normal blood-brain barrier, is sufficient to induce astrocyte dysfunction and their pro-inflammatory activation (green cells). this leads to the production of pro-inflammatory cytokines (green triangles), which activate the endothelium and massively disturb the blood brain barrier. this allows more antibodies together with leukocytes (red circles) and complement to enter the perivascular space, to destroy the astrocytes at the glia limitans and to promote inflammation. 3. microglia nodules provide the environment for pathogenic t cells in human encephalitis (tröscher et al 2019) a key question in the pathogenesis of inflammatory brain diseases is, why and how t-cells enter the brain and initiate the lesions. one mechanism is that they are activated in the peripheral immune system and get access to the brain in the course of immune surveillance (wekerle et al 1986; figure 1a). the other possibility, which may be particularly important in conditions of virus infection or neurodegeneration, is that local cells in the brain, such as for instance microglia, sense focal tissue injury and provide signals which drives the initial recruitment of cd8+ t-cells into the cns (figure 1b). rasmussen’s encephalitis is a rare inflammatory seizure disorder mediated by cytotoxic t-cells which attack neurons and astrocytes (bauer et al 2012). in the study by tröscher et al (2019) it is shown that the initial stage of lesion formation is characterized by microglia activation and the formation of microglia nodules, which occurs before the first cd8+ t-cells have entered the brain. the activated microglia initially show inflammasome activation and up-regulation of endosomal toll-like receptors, which is followed by the production of chemokines, the expression of major histocompatibility complex class i antigen, the activation of interferon signaling pathways and the recruitment of cd8+ t-lymphocytes. these data suggest that the initial trigger of inflammatory lesions in rasmussen’s encephalitis is not the passage of activated t-cells through the blood brain barrier in the course of immune surveillance, but a local cell infection or tissue damage, which creates a focal pro-inflammatory environment through inflammasome activation. subsequently, a subset of these recruited t-cells has to recognize their cognate antigen within the cns to propagate the inflammatory reaction. such a scenario may play a major role in the induction of inflammation in viral diseases of the nervous system, but similar microglia activation and the formation of microglia nodules is also seen around active lesions in the ms brain (burm et al 2016). 4. post mortem multiple sclerosis lesion pathology is influenced by single nu-cleotide polymorphism (fransen et al 2020) irrespective of the specific antigen, recognized by the inflammatory cells in ms lesions, the incidence and severity of brain inflammation appears to be influenced by the genetic background of the patients. this is now well established from genome wide association studies (gwas), which define ms as a disease with a highly polygenic background (international multiple sclerosis genetics consortium 2019). more than 200 gene loci have been found associated with disease incidence, each of them with only very little individual impact. the global interpretation of these findings is that ms, as described before by pathology, is a chronic inflammatory disease of the nervous system, and its likelihood to affect a patient is influenced by multiple genes which in their interaction may enhance pro-inflammatory immune mechanisms. it is likely that such genetic polymorphisms also modify the pathological evolution of ms lesions, but considering their low impact on the disease in the general ms population, it was unexpected that such an effect may become apparent in autopsy cohorts. fransen et al (2020) addressed this question on a material of 179 ms brains, containing a very high number of very well staged and characterized individual ms lesions. on this basis they were able to show that certain variants of immune related genes, which were before shown to be associated with disease severity, were also associated with the proportion of active lesions. the respective genes were in part related to apoptosis (fas), t-cell activation (ctla4) or are playing a role both in inflammation and neurodegeneration (clec16a). this study provides a first hint on how the genetic background of ms patients may affect pathological outcome of the lesions. however, there are still controversial issues regarding the statistical design for such investigations. performing genotype/phenotype correlations with multiple potential risk associ-ated gene polymorphisms and multiple different pathological outcomes requires enormous stringency related to multiple testing, and there is some doubt that this can ever be reached with the limited autopsy material available. 5. characterization of human myelin oligodendrocyte glycoprotein antibody response in demyelination (tea et al 2019) pathological studies performed during the last decade suggest that immunological mechanisms responsible for the induc-tion of ms-like inflammatory demyelinating lesions are heterogeneous between patients, in particular at the early stages of the disease and in patients with aggressive disease course (lucchinetti et al 2000). one subtype of lesions was defined by immunoglobulin and complement deposition in active lesions with initial stages of demyelination. this was interpreted as evidence for the involvement of pathogenic autoantibodies, as it is reflected in models of experimental autoimmune encephalomyelitis, in which demyelination is triggered by antibodies against myelin oligodendrocyte glycoprotein (mog) on the background of a t-cell mediated inflammatory reaction in the brain and spinal cord. new technologies, using trans-fected cell lines, are now available to identify potential pathogenic autoantibodies and using these techniques it was re-cently possible to identify a subset of patients with inflammatory demyelinating disease with a systemic antibody response against mog (borisow et al 2018). identification of such demyelinating anti-mog antibodies is difficult, since they are directed against complex conformational epitopes, while antibodies directed against conventional linear mog epitopes are not pathogenic. the study by tea et al (2019) describes the results of a detailed epitope mapping, which provides new information on the relevant target epitopes, recognized by potentially pathogenic mog auto-antibodies. this is important for diagnosis of mog antibody associated inflammatory demyelinating disease and for further analysis of pathogenetic mechanisms involved in antibody mediated demyelination. the identification of pathogenic mog-antibodies is a major breakthrough in multiple sclerosis research, since current data suggest that the presence of anti-mog antibodies defines an inflammatory demyelinating disease distinct from ms. it presents with clinical features of acute or relapsing disseminated encephalomyelitis or neuromyelitis optica and shows a different response to current anti-inflammatory treatments (jarius et al 2016). these new findings further suggest that the experimental models of autoimmune encephalomyelitis more closely reflect mog antibody associated disease than multiple sclerosis itself. 6. circulating aqp4-specific auto-antibodies alone can induce neuromyelitis optica spectrum disorder in the rat (hillebrand et al 2019) another disease, which originally has been classified as a variant of multiple sclerosis, is neuromyelitis optica (nmo). however, using similar techniques of auto-antibody detection as described above, it became clear that the majority of patients with nmo have auto-antibodies against the astrocytic water channel aquaporin 4 (aqp4). further comprehensive clinical characterization of patients with aqp4 auto-antibodies enlarged the clinical phenotype, now designated as neuro-myelitis optica spectrum disorders (nmosd), which are pathologically characterized by a primary inflammatory astro-cytopathy with secondary demyelination (fujihara et al 2019). experimental studies showed that patient derived auto-antibodies induce tissue damage in vitro or after direct injection into the brain, or when systemically injected into animals with t-cell mediated autoimmune encephalomyelitis. the study by hillebrand et al (2019) shows for the first time that circulating aqp4 directed autoantibodies alone can induce nmosd like disease in rats, provided that the injection resulted in a very high titer of circulating antibodies with very high specificity and affinity. circulating antibodies reached the cns tissue not only through circumventricular organs but also by the physiological leakage through cerebral veins (figure 1d). thus, a very low degree of aqp4 antibody leakage through the intact blood brain barrier may target perivascular astrocytes at the perivascular glia limitans and may augment further blood-brain barrier damage in the course of antibody mediated damage of the astrocytic process. this property is unique for aqp4 auto-antibodies and not seen with other antibodies such as those directed against mog. the study further shows that brain damage by the leakage of the antibody in circumventricular organs is very limited and not associated with complement activation, possibly by the expression of complement inhibitory proteins. in contrast, when present in the perivenous space aqp4 antibodies induce profound blood-brain barrier injury, leakage of complement, recruitment of granulocytes and the activation of macrophages, and this gives rise to the full pathological spectrum of nmosd lesions. this study, thus, provides some explanation for the heterogeneous spectrum of pathology in different brain regions of nmosd patients. 7. the neuropathology of fatal encephalomyelitis in human borna virus infection (liesche et al 2019) accurate diagnosis of non-purulent lymphocytic encephalitis in humans is a challenge. when no positive identification of a specific virus infection is possible, a diagnosis of encephalitis of autoimmune or probable infectious cause is assigned (venkatesan and murphy 2018, seilhean 2019). the study by liesche et al (2019) defines a new entity of lymphocytic scle-rosing panencephalomyelitis, which is caused by infection with borna disease virus 1 (bodv-1). although the disease may follow generalized immunosuppression, in the majority of cases it developed spontaneously. the study provides a clear account of the pathological features of this new disease, consisting of mononuclear cell infiltrates, edema, microglia acti-vation and the presence of eosinophilic spherical inclusion bodies. virus rna was detected in neurons, ependymal cells, astrocytes and oligodendrocytes, but not in lymphocytes, macrophages or microglia. the distribution of the lesions in the brain and spinal cord was variable ranging from a dominant brain stem encephalitis, to prominent affection of the hippo-campus or a multifocal or diffuse pathology in other cases. in addition, infection may also involve the peripheral nervous system. this study adds borna virus infection to the list of potential causative agents driving an acute or subacute inflam-matory disease of the central and peripheral nervous system, which has to be included in the spectrum of clinical differen-tial diagnoses. how frequent this condition is in the global population of patients with encephalitis has to be determined in the future. 8. zika virus replicates in adult human brain tissue and impairs synapses and memory in mice (figueiredo et al 2019) another recently discovered disease of the nervous system is associated with zika virus infection. although originally be-lieved to disturb brain development in the course of intrauterine infection (counotte et al 2019), it became clear in the last years that it may also infect the adult human nervous system and give rise to inflammatory diseases of the central and peripheral nervous system (da silva et al 2017). the underlying mechanisms were up to now not defined. in this study the authors show that zika virus can infect neurons and replicate not only in the human, but also in the mouse brain tissue in vitro. this allowed to study in more detail the consequences of zika virus brain infection. in the mouse it mainly accumu-lates in memory associated brain regions, such as for instance the hippocampus, where it induces disturbances of long-term potentiation and impairs memory. these effects are associated and possibly mediated by microglia activation with the up-regulation of pro-inflammatory cytokines and the complement components c1q and c3. in line with this view it was also found that blockade of microglia activation or neutralization of tnf-α prevented synapse and memory dysfunction. these findings suggest that analysis of cognitive disturbances in zika virus infected patients should be performed in a more systematic manner. however, it has also to be noted that similar mechanisms of cytokine and microglia activation associated synapse and memory disturbance occur in many different inflammatory conditions of the central nervous system, including even experimental autoimmune encephalomyelitis (musella et al 2016). it is, thus, rather a general consequence of brain exposure to pro-inflammatory cytokines. whether the mechanisms inducing synaptic dysfunction are different and specific in zika virus infected brain has to be clarified in the future by direct comparison. figure 2: microglia activation in neurodegeneration and brain inflammation the normal brain is populated by so called resting or homeostatic microglia. when they sense neurodegeneration, they become activat-ed through the trem2/apoe pathway. in this activation state there are phagocytic cells responsible for the removal of tissue debris, which is essential to limit further injury and to allow tissue repair. when these activated microglia are further exposed to inflammatory cytokines, they differentiate into immunological effector cells, which are actively involved in tissue damage. thus, both, an impairment of the initial step of microglia activation as well as the proinflammatory activation are detrimental for the brain. 9. impact of trem2 risk variants on brain region-specific immune activation and plaque microenvironment in alzheimer’s disease patient brain samples (prokop et al 2019) for many years our views on the pathogenesis of alzheimer’s disease focused on protein misfolding and deposition of aß and tau in the extracellular space or in the neuronal cytoplasm. in recent years, however, evidence accumulated that ele-ments of the innate immune system may play a role in promoting the cognitive disturbance in patients and the neuropa-thological damage in the brain. in particular, recent genome wide association studies identified polymorphisms of genes involved in microglia activation to be associated with the disease (sims et al 2017). one of these genes is the trem2 re-ceptor (johnsson et al 2013), which when activated by neurodegeneration triggers initial microglia activation through the trem2 / apoe pathway (krasemann et al 2017). although the role of these molecules in microglia activation in general and in experimental models of neurodegeneration is well understood, it is important to determine the neuropathological effects of the respective gene variants in alzheimer’s disease patients. this question was addressed by the study of prokop et al (2019). the authors describe that carriers of the respective trem2 risk variants had lower numbers of plaque-associated microglia and this was associated with a higher degree of axonal injury and tau pathology in comparison to carefully matched alzheimer’s disease patients without these trem2 variants. in addition, microglia senescence was found to be enhanced and some global decrease in microglia activation in trem2 variant ad cases was apparent. the study clearly underlines the difficulty of such an investigation in human autopsy material, since such differences only became apparent when exactly matched lesion stages were compared in patients with similar disease severity. this is due to the fact that these gene polymorphisms do not enable or prevent ad pathology, but just modify its speed of development. overall, the data suggest that impairment of microglia activation not only enhances the progression of cognitive impairment, but also promotes axonal degeneration and tau pathology. whether this is due to a reduced neuroprotective function of microglia or a reduced clearance of misfolded proteins due to impaired phagocytosis remains to be determined in the future. 10. persistent neuropathological effects 14 years following amyloid-ß immunization in alzheimer’s disease (nicoll et al 2019) following promising results, documenting the removal of amyloid plaques in mice after active immunization with aß, a first clinical trial had been initiated almost 20 years ago, which was followed by several other trials using immunization strategies or the systemic application of specific aß-antibodies (wisniewski and goni 2014, 2015). the first trial was stopped due to the appearance of autoimmune inflammatory disease of the central nervous system as a complication of the vaccination strategy. a detailed analysis of the neuropathology of patients who died in the years after the trial suggested that this vaccination resulted in clearance of aß-deposits from the brain in association with the development of an aß antibody response, but that this had no significant effect of the progression of dementia (holmes et al 2008). the present study by nicoll et al (2019) in detail describes the neuropathology in relation to the clinical outcome seen in 22 patients of this trial, who died within 14 years after the immunization. this comprehensive analysis provides important insights related to the design and outcome of aß directed immunotherapies in alzheimer’s disease. only 77% of the patients included in this cohort had alzheimer’s disease, while dementia in the others was due to different diseases. in the ad patients with aß vaccination the vast majority provided evidence for plaque removal and the extent for amyloid deposition correlated inversely with the titer of antibodies directed against the n-terminal region of aß, documenting the sustained effect of aß vaccination reducing amyloid load. despite a significantly lower extent of neurofibrillary pathology in patients with low amyloid load, there was still profound neurofibrillary tangle pathology in all cases. in addition, severe cerebral amyloid angiopathy was noted in the majority of patients, irrespective of the effect on aß plaque removal. progression of dementia was seen in most patients, even in those with extensive plaque removal. in conclusion, this study impressively documents the value of systematic neuropathological studies to monitor clinical dementia trials. it shows that alzheimer’s disease patients can remain plaque-free even for 14 years after active immunization, but that this has little effect on tau pathology and the further development of dementia. conclusion in this review a number of recent neuropathological studies are presented, which contributed to the dynamic change in our understanding of the pathogenesis of neuroinflammatory diseases. they highlight and confirm that the field of neuro-pathology occupies a central position in human disease research. experimental models provide important insights into basic and molecular mechanisms, but their relevance for the disease in humans has to be determined by studying the disease in patients. correct interpretation of the changes occurring in damaged 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(2019) microglial nodules provide the environment for pathogenic t cells in human encephalitis acta neuropathol 2019; 137:619-635 van nierop gp, van luijn mm, michels ss, et al. phenotypic and functional characterization of t cells in white matter lesions of multiple sclerosis patients. acta neuropathol 2017; 134:383-401 venkatesan a, murphy oc. viral encephalitis. neurol clin 2018; 36:705-724 wisniewski t, goni f. immunotherapeutic approaches for alzheimer’s disease. neuron 2015; 85: 1162-1176 wekerle h, linington c, lassmann h, meyermann r (1986) cellular immune reactivity within the cns. trends neurosci 1986; 9:271-277 wisniewski t, goni f. immunotherapy of alzheimer’s disease. biochem pharmacol 2014; 88: 499-507 copyright: © 2020 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. optimizing filter trap assay for the detection of aggregated alpha-synuclein in brain samples feel free to add comments by clicking these icons on the sidebar free neuropathology 1:14 (2020) letter optimizing filter trap assay for the detection of aggregated alpha-synuclein in brain samples thibauld oullier 1*, alice prigent 1*, guillaume chapelet 1,2, michel neunlist 1, franck letournel 3, pascal derkinderen 1,4 1 université de nantes, inserm, tens, the enteric nervous system in gut and brain diseases, imad, nantes, france 2 chu nantes, clinical gerontology department, nantes, france 3 chu angers, neurobiology and neuropathology laboratory, angers, france 4 chu nantes, department of neurology, nantes, france * these authors contributed equally to this work. corresponding author: pascal derkinderen · inserm u1235 nantes · 1 rue gaston veil · 44035 nantes · france · tel: +33(0)240165202 · fax: +33(0)240165203 pascal.derkinderen@chu-nantes.fr ; derkinderenp@yahoo.fr submitted: 04 april 2020 accepted: 26 april 2020 copyedited by: christian thomas published: 28 april 2020 https://doi.org/10.17879/freeneuropathology-2020-2749 additional resources and electronic supplementary material: supplementary material keywords: alpha-synuclein, parkinson’s disease, filter trap, dot blot, aggregates over the past two decades, the progress in molecular genetics and neuropathology enabled a better understanding of the pathogenesis of neurodegenerative disorders. the observation that abnormal protein accumulation is characteristic of a particular disease has led to a neuropathological classification according to the composition of the abnormal protein aggregates [1]. several methods have been developed for the detection of these insoluble protein aggregates including, for instance, dye binding assays and immunohistochemistry with proteinase k pretreatment [1]. filter retardation assay is a simple and rapid method which detects protein aggregates formed either in vivo or in vitro, including amyloid-beta/tau aggregates from alzheimer’s disease (ad) [2–4] and polyglutamine expansion from huntington’s disease [5,6]. in this assay, ‘lysis buffer’-resistant protein aggregates, but not soluble monomeric species, are retained by a cellulose acetate membrane and subsequently detected by immunoblotting. as loading control, a ‘classical’ dot blot with either polyvinylidene fluoride (pvdf) or nitrocellulose membranes is run in parallel with the same lysates. in contrast to polyglutamine expansion, tau and beta-amyloid, there are only few studies on using filter retardation assays for the detection of alpha-synuclein aggregates, which are characteristic of parkinson’s disease (pd) and dementia with lewy bodies (dlb) [7]. in addition, a full characterization of any filter-based assay for alpha-synuclein, including the effect of membrane composition, lysis procedure and antibodies has not yet been reported. we therefore set out the current study in which we characterize a vacuum-based 96-well format filter assay for the detection of aggregated alpha-synuclein in human brain specimens. frozen brain samples from neuropathologically confirmed cases of dlb, progressive supranuclear palsy (psp) and ad were obtained from the neuropathology department of angers (dr franck letournel). brain samples were lysed with the “precellys 24™” tissue homogenizer (bertin technologies, cat# p000669-pr240-a, montigny-le-bretonneux, france) followed by a brief sonication (one minute, 5 seconds on, 5 seconds off, amplitude 70%; vcx130 ultrasonic processor with a 6 mm probe, sonics and materials, newton, ct, usa) either in (i) radioimmunoprecipitation assay (ripa) buffer (merck millipore, cat# 20-188, molsheim, france) containing 2 mm orthovanadate (sigma, cat# s6505, saint quentin fallavier, france), 1% (v/v) phosphatase inhibitor cocktail iii (sigma, cat# p0044) and a protease inhibitors cocktail (roche, cat# 14424700, meylan, france) at 4°c; (ii) ‘sodium dodecyl sulfate (sds) buffer’ containing 1% (w/v) sds, 2 mm orthovanadate (sigma, cat# s6505) and 1% (v/v) phosphatase inhibitor cocktail iii (sigma, cat# p0044); (iii) ‘urea buffer’ containing 7 m urea (sigma, cat# t7875), 2 m thiourea (sigma, cat# l3771), 4% (v/v) chaps (sigma, cat# c3023), 2 mm orthovanadate (sigma, cat# s6505), 1% (v/v) phosphatase inhibitor cocktail iii (sigma, cat# p0044) and a protease inhibitors cocktail (roche, cat# 14424700). lysates were centrifuged at 10,000 x g for 5 minutes; supernatants were recovered and protein concentration determined using the pierce bca protein assay kit (thermofisher scientific, cat# 23227, saint herblain, france) along with the varioskan™ multimode microplate reader (thermofisher scientific, cat# vl0l00d0); samples lysed in urea buffer were diluted 5 times for compatibility with the bca assay (https://assets.thermofisher.com/tfs-assets/lsg/application-notes/tr0068-protein-assay-compatibility.pdf). twenty µg of these supernatants were subjected to vacuum filtration through a 96-well dot blot apparatus (bio-rad, cat# 1706545, marnes-la-coquette, france) with 0.45 µm pore size cellulose acetate (sterlitech, cat# ca0453001, kent, usa), nitrocellulose (thermofisher scientific, cat# 77010) or pvdf membranes (thermofisher scientific, cat# 88518). the resultant membranes were blocked for one hour at room temperature in tris-buffered saline (tbs, sigma, cat# t5912) with 0.1% (v/v) tween-20 (sigma, cat# p1379) and 5% (w/v) non-fat dry milk, then incubated overnight at 4°c with the primary antibodies listed in table 1. bound antibodies were detected with horseradish peroxidase-conjugated anti-rabbit (life technologies cat# 31460, diluted 1:5,000) or anti-mouse antibodies (sigma, cat# a9044, diluted 1:5,000) and visualized by enhanced chemiluminescent detection (supersignal™ west pico plus chemiluminescent substrate, thermofisher scientific, cat# 34580). oligomeric aggregates of alpha-synuclein, which were obtained by incubating monomeric recombinant alpha-synuclein (sigma, cat# 575001) in phosphate-buffered saline under agitation at a concentration of 10 mg/ml at 37°c for one week, served as a control [8]. table 1. alpha-synuclein and phospho-alpha-synuclein antibodies used in the current study. when cellulose acetate membranes were used, recombinant oligomeric alpha-synuclein and dlb lysates were barely or not detected by two of the total alpha-synuclein antibodies (syn211 and syn-1), regardless of the lysis procedure (figure 1a). in contrast, mjfr1 antibody efficiently detected both oligomeric alpha-synuclein and aggregated alpha-synuclein from ripa-lysed dlb samples (figure 1a). no specific signal was observed with this antibody when dlb samples were lysed with sds or urea buffers (figure 1a). two of the phosphospecific antibodies (ep1536y and to a lesser extent psyn#64) detected aggregated alpha-synuclein in ripa-dlb lysates. no signal was observed when psp and ad lysates were filtered and analyzed with either mjfr1 or ep1536y antibody. these findings show that sample lysis with ripa sample lysis together with mjfr1 and/or ep1536y immunoblotting is the most specific and reproducible approach. we therefore used these two antibodies and ripa-lysed samples to perform additional dot blots with pvdf or nitrocellulose membranes, that could serve as a loading control. the results show that pvdf achieved better reproducibility and specificity for the detection of total and phosphorylated alpha-synuclein than nitrocellulose membranes (figure 1b). figure 1. results from the filter trap and dot blot assays. (a) recombinant oligomeric alpha-synuclein (oligo) and samples from dlb, psp and ad brains lysed in ripa, sds and urea buffers were subjected to vacuum filtration through a 96-well dot blot apparatus with an acetate cellulose membrane; immunoblotting (ib) was then performed with 3 different antibodies to total alpha-synuclein (mjfr1, syn-1 and syn211) and 3 antibodies specific for the phosphorylated form of the protein (ep1536y, psyn#64 and phospho s129). (b) recombinant oligomeric alpha-synuclein (oligo) and ripa-lysed samples from dlb, psp and ad brains were subjected to vacuum filtration through a 96-well dot blot apparatus with a pvdf or a nitrocellulose membrane ; immunoblotting (ib) was then performed with mjfr1 or ep1536y antibodies. results shown are representative of 3 to 5 independent experiments. the results presented here, although preliminary, provide the basis for reproducible and specific detection of pathological alpha-synuclein in diseased brains using a filter trap assay. we show that the selection of primary antibody and lysis buffer has important effects on the performance of the assay. as an optimized protocol, we suggest that brain homogenates should be lysed in ripa buffer and that immunoblotting should be performed with mjfr1 for the detection of total alpha-synuclein and ep1536y for the detection of the phosphorylated form of the protein (a step by step standard operating procedure is provided as supplementary material). owing to the high-throughput capability of the filter trap assay, such an approach is not only potentially interesting for detecting aggregated alpha-synuclein in post mortem brain samples but also in the peripheral nervous system of living pd patients in order to develop original biomarkers of the disease [9]. references 1. kovacs gg. molecular pathological classification of neurodegenerative diseases: turning towards precision medicine. int j mol sci. 2016;17:189. 2. bieschke j, cohen e, murray a, dillin a, kelly jw. a kinetic assessment of the c. elegans amyloid disaggregation activity enables uncoupling of disassembly and proteolysis. protein sci. 2009;18:2231–41. 3. boyé-harnasch m, cullin c. a novel in vitro filter trap assay identifies tannic acid as an amyloid aggregation inducer for het-s. j biotechnol. 2006;125:222–30. 4. chang e, kuret j. detection and quantification of tau aggregation using a membrane filter assay. anal biochem. 2008;373:330–6. 5. novoselova tv, margulis ba, novoselov ss, sapozhnikov am, van der spuy j, cheetham me, et al. treatment with extracellular hsp70/hsc70 protein can reduce polyglutamine toxicity and aggregation. j neurochem. 2005;94:597–606. 6. van waarde-verhagen mawh, kampinga hh. measurement of chaperone-mediated effects on polyglutamine protein aggregation by the filter trap assay. methods mol biol. 2018;1709:59–74. 7. recasens a, dehay b, bové j, carballo-carbajal i, dovero s, pérez-villalba a, et al. lewy body extracts from parkinson disease brains trigger α-synuclein pathology and neurodegeneration in mice and monkeys. ann neurol. 2014;75:351–62. 8. corbillé a-g, neunlist m, derkinderen p. cross-linking for the analysis of α-synuclein in the enteric nervous system. j neurochem. 2016;139:839–47. 9. lebouvier t, tasselli m, paillusson s, pouclet h, neunlist m, derkinderen p. biopsable neural tissues: toward new biomarkers for parkinson’s disease? front psychiatry. 2010;1:128. copyright: © 2020 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. quantitative proteomic profiling of white matter in cases of cerebral amyloid angiopathy reveals upregulation of extracellular matrix proteins and clusterin feel free to add comments by clicking these icons on the sidebar free neuropathology 1:28 (2020) original paper quantitative proteomic profiling of white matter in cases of cerebral amyloid angiopathy reveals upregulation of extracellular matrix proteins and clusterin antigoni manousopoulou1**, ho ming yuen2**, matthew macgregor sharp2, satoshi saito2, roxana aldea3, norman mazer3, spiros d. garbis4, roxana o. carare2* 1 beckman research institute, city of hope, duarte, ca, usa 2 faculty of medicine, university of southampton, southampton so16 6yd, uk 3 roche pharma research and early development, pharmaceutical sciences, roche innovation center basel, f. hoffmann-la roche ltd, basel, switzerland 4 proteome exploration laboratory, beckman institute, division of biology and biological engineering, california institute of technology, pasadena, ca, usa ** authors contributed equally corresponding author: prof roxana o carare · university of southampton · faculty of medicine · southampton general hospital · south academic block, mp806 · tremona road · southampton, hampshire · so16 6yd · united kingdom r.o.carare@soton.ac.uk submitted: 14 august 2020 accepted: 06 october 2020 copyedited by: biswa ramani published: 08 october 2020 https://doi.org/10.17879/freeneuropathology-2020-2955 additional resources and electronic supplementary material: supplementary material (zip) keywords: extracellular matrix, cell adhesion, clusterin, cerebral amyloid angiopathy, caa, white matter, proteomics abstract aims: cerebral amyloid angiopathy (caa) is the accumulation of amyloid beta (aβ) in the walls of cerebral arterioles, arteries and capillaries. changes in the white matter in caa are observed as hyperintensities and dilated perivascular spaces on mri suggesting impairment of fluid drainage but the pathophysiology behind these changes is poorly understood. we tested the hypothesis that proteins associated with clearance of aβ peptides are upregulated in the white matter in cases of caa. methods: in this study, we compare the quantitative proteomic profile of white matter from post-mortem brains of patients with caa and age-matched controls in order to gain insight into the cellular processes and key molecules involved in the pathophysiology of caa. results: our proteomic analysis resulted in the profiling of 3,734 proteins (peptide fdr p<0.05). of these, 189 were differentially expressed in caa vs. control. bioinformatics analysis of these proteins showed significant enrichment of proteins related to cell adhesion | cell-matrix interaction, mitochondrial dysfunction and hypoxia. upregulated proteins in caa included emilin2, col4a2, tln1, clu, hspg2. downregulated proteins included dsp, ide, hbg1. conclusions: the present study reports an in-depth quantitative proteomic profiling of white matter from patients with caa, highlighting extracellular matrix proteins and clusterin as key molecules in the pathophysiology of white matter changes in cases of caa. abbreviations caa cerebral amyloid angiopathy, wmh white matter hyperintensities, ipa ingenuity pathway analysis, col4a2 collagen iv alpha 2, hspg2 heparan sulfate proteoglycan 2, emilin2 emi domain endowed, tln1 talin1, clu clusterin, ide insulin degrading enzyme, dsp desmoplakin, ipad intramural periarterial drainage. introduction cerebral amyloid angiopathy (caa) is characterized by the deposition of amyloid proteins including amyloid beta (aβ), cystatin c, prion protein, abri/adan, transthyretin, gelsolin and immunoglobulin light chain amyloid (1) in the walls of leptomenigeal arteries and cortical arterioles (caa-type 2) (2) and in the walls of arteries or capillaries (caa-type 1), but rarely in the walls of venules (1-3). sporadic caa is a common feature of alzheimer’s disease (1-3) and reflects the failure of clearance of proteins and interstitial fluid from the ageing brain (4, 5). although caa is uncommon in the white matter, (6) there are radiological features in the white matter in cases of caa that include white matter hyperintensities (wmh) and dilated perivascular spaces (7, 8). the underlining pathophysiology is poorly understood, but the wmh are associated with small vessel disease (9). in addition to damage to nerve fibres particularly in the deep white matter, the wmh suggest that there is an increase in the volume of interstitial fluid (isf) in the affected white matter. pathologically, wmh are characterised by pallor of myelin staining in the affected white matter with morphological changes in astrocytes and axons and disruption of the distribution of aquaporin 4 in astrocyte processes (10). imaging studies in the early stages of wmh suggest that the lesions are due to altered interstitial fluid mobility and water content that may be reversible (11). interstitial fluid and aβ enter the basement membranes of capillaries and arteries to be cleared from the brain along the intramural periarterial drainage (ipad) pathways (12, 13). with increasing age, the structure of basement membranes changes and aβ accumulates in the walls of arteries as caa (14-16). our proteomic studies performed on leptomeningeal arteries from brains with caa demonstrated an upregulation of clusterin (apolipoprotein j) and tissue inhibitors of metalloproteinases 3 (timp3) (17). the aim of the present study was to compare the quantitative proteomic profile of white matter from patients with caa to the corresponding brain region of age-matched controls in order to gain insight in the pathophysiology of white matter changes in caa and to identify novel therapeutic targets. an overview of the present study workflow is presented in figure 1. figure 1: outline of experimental workflow. labelled peptides were analysed using two-dimensional liquid chromatography and tandem mass spectrometry. materials and methods tissue samples fresh frozen white matter from the occipital lobe of severe caa cases (n=6) and non-demented aged matched controls (n=4) was supplied by newcastle brain tissue resource (ethics rec 08/h0906/136) (table 1). all cases were diagnosed according to internationally-used criteria including neuritic braak stages (18), thal amyloid phases (19), and cerad scores (20) (table 1). table 1: header table 1: demographics of the cases used for this study. pm: post-mortem. the pm delay refers to the number of hours post-mortem until the tissue was dissected and frozen. tissue quantitative proteomics analysis samples of 1mg per case were dissolved in 0.5 m triethylammonium bicarbonate in 0.05% sodium dodecyl sulphate. these were then subjected to pulsed probe sonication (misonix, farmingdale, ny, usa) and lysates were centrifuged (16,000 g, 10 min, 4°c). supernatants were measured for protein content using the bicinchoninic acid assay (pierce bca protein assay kit, thermo fisher scientific, waltham, ma, us). 100 μg of protein was used per sample, adjusted to the highest volume. proteins were reduced with 4μl 50mm tris(2-carboxyethyl)phosphine hydrochloride at 60°c for 1 hour and alkylated using 2 μl 200mm methanethiosulfonate at room temperature for 10 min. proteins were enzymatically digested using trypsin overnight in dark at 37°c (sigma aldrich, st. louis, mo, usa). peptides from each sample were labelled using the 10plex tandem mass tag (tmt) reagent kit (thermo fisher scientific, waltham, ma, us). labelled peptides were mixed and analyzed using two-dimensional liquid chromatography and tandem mass spectrometry as reported previously (17). database searching unprocessed raw files were submitted to proteome discoverer 1.4 (thermo fisher scientific, waltham, ma, us) for target decoy search using sequest. the uniprotkb homo sapiens database which comprised 20,159 entries (release date january 2015) was utilized. the search allowed for up to two missed cleavages, a precursor mass tolerance of 10ppm, a minimum peptide length of six and a maximum of two variable (one equal) modifications of; oxidation (m), deamidation (n, q), or phosphorylation (s, t, y). methylthio (c) and tmt 6-plex (k, y and n-terminus) were set as fixed modifications. false discovery rate (fdr) corrected p value at the peptide level was set at <0.05. percent co-isolation excluding peptides from quantitation was set at 50. reporter ion ratios from unique peptides only were taken into consideration for the quantitation of the respective protein. quantitation per protein in each of the six caa patients was divided by each of the two matched same sex controls, which yielded 12 quantitative ratios per protein. these ratios, which represent the overall fold change in protein in the caa patients with respect to the controls, were median-normalized and log2 transformed. we then combined the two respective log2 ratios per caa patient when compared to the two controls by their mean which generated one observation per caa patient per protein (see supplementary table 1). due to small sample size, statistical analysis was performed per protein regardless of gender (n=6). based on a previous study (16), we set the cut point of log2 ratio higher than or equals to 0.6, or lower than or equals to -0.6 to identify differentially expressed proteins (deps) (equivalent to a 1.5-fold change), but this was considered too restrictive for complex structure such as the white matter (number of proteins included = 51). hence, we have narrowed the gap by halving the cut point to ±0.3 (equivalent to 1.2-fold change) instead in this study (number of proteins included = 189). proteins identified with at least two unique peptides were included in the analysis. one-sample t test was performed on these log2 ratios per protein, comparing to 0, to identify the proteins that were statistically significant and differentially expressed in caa patients comparing to controls (p <0.05). bioinformatics analysis ingenuity pathway analysis (ipa) (qiagen, hilden, germany) and metacore (clarivate analytics, philadelphia, pa, usa) were used to identify significantly enriched processes and pathways in the differentially expressed proteins of white matter from patients with caa vs. control. p values in the context of canonical pathway analysis reflect how significantly the pathway is over-represented in the dataset of deps in white matter from patients with caa vs. controls. significance was set at p < 0.05. results the global, untargeted proteomic analysis resulted in the profiling of 3,734 proteins (peptide fdr p<0.05) (supplementary tables 1, 2, 3). of these, 189 were differentially expressed in caa (supplementary table 4). bioinformatics analysis of the differentially expressed proteins (deps) using metacore showed significant enrichment of the cell adhesion | cell-matrix interaction process (figure 2). mapping on this process network, emilin-2 (emilin2), fibrillin-1 (fbn1), collagen alpha-2(iv) (col4a2) and the extracellular protein heparan sulfate proteoglycan (hspg2) were upregulated; inter-alpha trypsin inhibitor heavy chain h3 (itih3) was downregulated in caa vs. control. figure 3 is a bar graph showing the relative expression levels of key upregulated (emilin2, col4a2, talin-1 (tln1), clusterin (clu)) and downregulated (desmoplakin (dsp), insulin-degrading enzyme (ide), hemoglobin subunit gamma-1 (hbg1)) proteins in the white matter of patients with caa vs. controls (figure 3). figure 2: bioinformatics analysis using metacore showed significant enrichment of the cell-adhesion | cell-matrix interaction processes in the differentially expressed proteins of white matter from patients with caa vs. controls. upregulated proteins (emilin-2, fibrillin, collagen iv, perlecan) are highlighted with a red circle whereas downregulated proteins (itih3) with a blue circle. the p represents significance of enrichment (i.e. this pathway is significantly enriched in the list of differentially expressed proteins). figure 3: up-regulated (red) and downregulated (blue) proteins in white matter from patients with caa vs. controls. a positive log2ratio (ratio>1) reflects upwhereas a negative log2ratio (ratio<1) reflects downregulation of the respective protein in patients with caa vs. controls. metacore demonstrated dysregulation of the hypoxia-inducible factor 1alpha (hif1a) in caa with upregulation of diamine acetyltransferase 2 (sat2) and downregulation of heat shock 70kda protein 4 (hspa4), heat shock 70kda protein 1a/1b (hspa1a), 78kda glucose-related protein (hspa5), endoplasmin (hsp90b1), transitional endoplasmic reticulum atpase (vcp) and guanine nucleotide binding protein subunit beta-2-like-1 (gnb2l1), suggesting altered response to hypoxia in caa (figure 4). figure 4: negative regulation of hif1a was a significantly enriched pathway map in the differentially expressed proteins of white matter from patients with caa vs. controls. upregulated proteins are highlighted with a red circle whereas downregulated proteins with a blue circle. more specifically, sat2 was upregulated whereas hsp70, hspa4, hsp90, rack1 were downregulated in caa vs. control. the p represents significance of enrichment (i.e. this pathway is significantly enriched in the list of differentially expressed proteins). mitochondrial function also appeared altered in caa with upregulation of the mitochondrial fission 1 protein (fis1), a marker of mitochondrial and mitophagy (21) and reductions in proteins associated with the normal function of the mitochondria (mitochondrial succinate dehydrogenase flavoprotein subunit (sdha), mitochondrial aconitate hydratase (aco2), mitochondrial phospholipid hydroperoxide glutathione peroxidase (gpx4), mitochondrial atp synthase subunit beta (atp5f1b), mitochondrial thioredoxin reductase 2 (txnrd2), mitogen-activated protein kinase 9 (mapk9)). mitochondrial dysfunction was a significantly over-represented canonical pathway (figure 5). figure 5: mitochondrial dysfunction was a significantly enriched canonical pathway in the differentially expressed proteins of white matter from patients with caa vs. controls. upregulated proteins are denoted with red whereas downregulated proteins with blue. more specifically, fis1 was upregulated whereas sdha, aco2, gpx4, atp5f1b, txnrd2 and mapk9 were downregulated in caa vs. control. the p represents significance of enrichment (i.e. this pathway is significantly enriched in the list of differentially expressed proteins). discussion the present study reports a comprehensive global proteomic profile of post-mortem white matter samples from patients with caa compared to the corresponding brain region of age-matched controls. out of 189 proteins identified as differentially expressed with both metacore and ipa, several proteins associated with mitochondrial dysfunction, hypoxia, and clearance of aβ are significantly upor downregulated. the pathways reflecting hypoxia and mitochondrial dysfunction have been observed in previous analyses of the white matter in alzheimer’s disease (22). hypoxia-inducible factor-1 (hif1) regulates oxygen homeostasis and thus has a major role in the control of energy/metabolism and angiogenesis. diamine-acetyltransferase-2 (sat2), guanine nucleotide binding protein subunit beta-2-like 1 (gnb2l1) and heat shock proteins are involved in the degradation of hif1 (23, 24) and they were expressed at altered levels in the white matter of caa cases (figure 4). in the present study there was an increase in fis1, a marker of mitochondrial and mitophagy (21), and a reduction in proteins associated with the normal function of the mitochondria (sdha, aco2, gpx4, atp5f1b, txnrd2, mapk9), suggesting an overall dysregulation of the functions of the mitochondria in the white matter from cases of caa (figure 5). most of the novel proteins showing substantial changes in the present study of the white matter in caa were proteins associated with the extracellular matrix constituents and clearance of aβ. proteins with increased expression collagen iv alpha 2 chain (col4a2) is a highly conserved protein across species and is present in almost all vascular basement membranes (bm) (25). a common variation in the col4a2 gene is associated with patients with intracerebral haemorrhage (26), which is one of the most frequent caa-related cerebrovascular complications. consistent with the current proteomic results, previous studies have reported increased collagen iv expression in cerebral microvessels in patients with alzheimer’s disease (27) and increased collagen iv immunostaining in the leptomeningeal arteries in hereditary cystatin c amyloid angiopathy (28). a recent proteomic study of the grey matter in ad with caa type 1 (predominantly capillary caa) has identified collagen alpha-2(vi) (col6a2) as increased in the grey matter of caa type 1(29). perlecan is a heparan sulfate proteoglycan 2 (hspg2), also a component of bm and provides dynamic flexibility to the bm, while promoting the aggregation of aβ (30, 31). pathological studies have reported increased expression of hspg2 in alzheimer’s disease (32) with a significant association of hspg2 polymorphism in apolipoprotein ε4 allele carriers (33). the upregulation of col4a2 and hspg2 have been observed in other studies in the grey matter, but their increase in the white matter of caa cases in the present study demonstrate the widespread changes in the extracellular matrix in the brain, reflected in a failure of intramural periarterial drainage (ipad). emilin-2 was expressed at higher levels in the white matter from brains with caa vs. controls. emilin-2 is part of the eden (emi domain endowed) spectrum of proteins (34), with roles in angiogenesis, as well as cell adhesion/migration (34, 35). emilin-2 possesses the gc1q domain, which interacts with α4β1 integrin, which in turn binds to fibronectin, a major component of bm (36). recent studies demonstrated a reduction in fibronectin in post-mortem brains with white matter hyperintensities as well as on cerebrovascular smooth muscle cell cultures exposed to hypercapnia as a model of hypoperfusion (37). although the expression of emilin-2 in brain is relatively small compared to other organs and the physiological role of emilin-2 is unclear (38), its association with bm (39) suggests that the upregulation of emilin-2 reflects a compensatory upregulation of angiogenesis and ipad in the face of the hypoperfusion and altered extracellular matrix characteristics of the white matter in caa (15, 40). talin-1 (tln1) is a heavy protein that contains a c-terminal flexible rod domain which binds to vinculin and actin while the n-terminal head binds to integrin cytoplasmic tails (41-43). binding of the talin head to integrin is a key step required for integrin activation (44, 45). integrins are essential for cell adhesion and α5β1 is anti-apoptotic and a receptor for aβ (46). as talin also promotes axon growth (47), it is possible that it is upregulated as a compensatory effect of axonal and synapse destruction in white matter lesions (22). in the present study, the expression of clu (clusterin) was higher in the white matter of caa cases compared to controls. our own prior proteomic study of leptomeningeal arteries in caa revealed a high expression of clu, and a recent study on the proteomic expression of grey matter in caa also revealed an increased expression of clu (17). genome-wide association studies identified variations in clu expression associated with alzheimer’s disease (48, 49). clusterin may act as a chaperone molecule that facilitates intramural periarterial drainage of soluble aβ (50). increased expression of clu may be a compensatory mechanism to the cerebrovascular accumulation of aβ in these cases. proteins with decreased expression ide (insulin-degrading enzyme) was significantly decreased in our study. ide is a key enzyme that degrades aβ in the extracellular spaces (51). in diabetes mellitus there is inhibition of aβ degradation directly associated with a reduced activity of ide (52-54). overexpression of ide ameliorates aβ pathology (55). ide is expressed within the vessel walls and its activity is decreased in caa (56). the decreased expression of ide may reflect a failure of the proteolysis and consequent increased toxicity of aβ. the expression of hbg1, the γ(a)-globin subunit of fetal haemoglobin, was also decreased in our study. hbg1 is capable of strongly binding to aβ (57). an animal model of caa exhibited impaired aβ transcytosis from brain to blood, resulting in decreased level of aβ in the blood (58). decreased expression of hbg1 may result from reduced level of the aβ and hbg1 complex in the blood in cases of caa. desmoplakin (dsp) is a desmosome-associated transmembrane glycoprotein and maintains the integrity of the epidermis and myocardium (59). the physiological role of desmoplakin in the brain remains unknown. interestingly, it was reported that desmoplakin is a specific marker of lymphatic vessels in human tongue (60). since ipad represents the lymphatic drainage pathways of the brain parenchyma, (61) a decreased level of desmoplakin may simply reflect impaired ipad. the present study has some limitations: firstly, the sample size is low due to the difficulty in obtaining fresh frozen white matter. this has also prevented us from performing an analysis by sex or any other stratification. secondly, it was not possible to specifically select areas of wmh from the frozen samples of brain and to study them separately. nevertheless, our study has revealed generalised changes in the white matter in cases of caa. thirdly, we have not performed validation of the results by immunocytochemistry for the proteins involved. however, we have provided evidence from other studies that support our findings and provide a working hypothesis. while previous proteomic studies in the white matter in alzheimer’s disease have identified modifications in proteins that are involved in cell survival and cell adhesion (22), in our study, we highlight that in the white matter of severe cases of caa there is upregulation of the proteins that mark cell-matrix interaction, hypoxia, mitochondrial dysfunction and intramural periarterial drainage of soluble aβ. the study from castano et al in 2013 used ad cases with a very low caa score (2 out of a maximum of 12) and identified gfap, tropomyosins, calmodulin, annexin 1 and alpha-internexin as significantly upregulated, while ubiquitin carboxyl-terminal esterase and fascin-1 were down-regulated (22). the upregulated proteins are part of the cytoskeleton maintenance, calcium metabolism and cell survival pathways. we also have identified annexin and ubiquitin carboxyl-terminal esterase modified in the same pattern. the different results between the two studies are likely to be due to the very different caa status of the cases; the present study points to pathways that are involved in the clearance of aβ along the vessel walls. our results suggest a working hypothesis that there is upregulation of the intramural periarterial drainage (ipad) pathways in the white matter as a compensatory mechanism to the failure of drainage of interstitial fluid and solutes from the white matter and the grey matter that is a key pathogenetic factor in caa. acknowledgments the authors thank the newcastle brain tissue research facility, the patients and their families. ethical approval newcastle brain tissue resource (ethics rec 08/h0906/136). author contributions antigoni manousopoulou performed the proteomic experiments, analysed data, interpreted data and wrote manuscript; roxana o. carare and spiros d. garbis designed the study; ho ming yuen assisted with statistical analysis; matthew macgregor sharp, satoshi saito, norman mazer and roxana aldea had important intellectual contributions to the design of the study and analysis. references 1. jellinger ka, attems j. prevalence and pathogenic role of cerebrovascular lesions in alzheimer disease 1. j neurolsci. 2005;229-230:37-41. 2. banerjee g, carare r, cordonnier c, greenberg sm, schneider ja, smith ee, et al. the increasing impact of cerebral amyloid angiopathy: essential new insights for clinical practice. j neurol neurosurg psychiatry. . 2017;88(11):982-94. 3. schreiber s, 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makino s, totsuka y, et al. desmoplakin as a specific marker of lymphatic vessels. microvasc res. 2001;61(1):40-8. 61. engelhardt b, carare ro, bechmann i, flugel a, laman jd, weller ro. vascular, glial, and lymphatic immune gateways of the central nervous system. acta neuropathol. 2016;132(3):317-38. copyright: © 2020 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. taylor’s focal cortical dysplasia revisited: history, original specimens and impact feel free to add comments by clicking these icons on the sidebar free neuropathology 2:11 (2021) flashback taylor’s focal cortical dysplasia revisited: history, original specimens and impact burkhard s. kasper epilepsy center, department of neurology, friedrich-alexander university erlangen-nuremberg, germany corresponding author: burkhard s. kasper · epilepsy center · department of neurology · friedrich-alexander university erlangen-nuremberg · schwabachanlage 6 · 91054 erlangen · germany burkhard.kasper@uk-erlangen.de submitted: 01 april 2021 accepted: 18 april 2021 copyedited by: jerry lou published: 23 april 2021 https://doi.org/10.17879/freeneuropathology-2021-3324 additional resources and electronic supplementary material: supplementary material keywords: dysplasia, epilepsy, history, taylor, fcd original paper:   focal dysplasia of the cerebral cortex in epilepsy. dc taylor, ma falconer, cj bruton, j.a.n. corsellis, journal of neurology, neurosurgery and psychiatry 1971; 34: 369-387   free full text link at jnnp: https://dx.doi.org/10.1136/jnnp.34.4.369 abstract 50 years ago back in 1971, david c. taylor and colleagues from england reported on a small series of surgical epilepsy cases proposing a new type of tissue lesion as a cause of difficult-to-treat focal epilepsy: a localized malformation of cerebral cortex. the lesion is now known as focal cortical dysplasia (fcd) type ii or taylor’s cortical dysplasia. fcd ii is not rare, and today is a frequent finding in neurosurgical epilepsy specimens. medical progress has been achieved in that the majority of fcd ii is diagnosed non-invasively by magnetic resonance imaging today. detailed studies on fcd revealed that the lesion belongs to a spectrum of mtor-o-pathies, thereby confirming the authors´ initial hypothesis of a relationship to tuberous sclerosis. here, selected original materials from taylor´s series are presented as virtual slides, supplemented by original clinical records, in order to give a first-hand impression of this milestone finding in neuropathology of epilepsy. what exactly did the authors report in this study? tissue findings from surgical brain specimens from 10 epilepsy patients were presented in detail [1], illustrating intriguing but formerly un-appreciated abnormalities of neocortical architecture. as core findings, neurons of bizarre size and shape were described throughout cortical layers ii-vi (today known as dysmorphic neurons) causing laminar disruption, in most cases accompanied by another pathological cell type, named grotesque cells of probable glial origin (today known as balloon cells). disorganization of cortical architecture and these cellular components were defined as pathognomonic features and suggested a new type of malformative cns lesion, i.e. focal dysplasia, today known as focal cortical dysplasia (fcd) type ii or taylor’s dysplasia [2]. why is this a milestone paper? the authors’ synopsis was groundbreaking since it defined a new category of circumscript lesion responsible for intractable focal epilepsy, adding to the spectrum of better-known epileptogenic lesions such as hippocampal sclerosis and benign neoplasms. epilepsy surgery was a rather young discipline back then. most causal epileptogenic lesions could be detected ex-post by histopathology only, since other supporting diagnostic modalities were unavailable at the time. dc taylor, a psychiatrist (!), recognized the similarities between these few cases out of several hundreds of cases just by reading through the reports, un-aided by any data such as imaging techniques [3]. these lesions were largely invisible to naked eye macroscopic examination in the operating theater or during tissue preparation [1]. by now, fcd has become a classic cause of epilepsy, ranking among the top 3 structural causes in epilepsy surgical series and in the majority is detected pre-surgically by magnetic resonance imaging (mri) [2, 4] what had been known before this seminal contribution? the authors in 1971 stated that “no reports of closely similar observations had been traced thus far” [1]. findings with some similarity to focal dysplasia had been sporadically reported under different names, such as “abortive tuberous sclerosis” [5], “infantile cerebral gliosis with giant nerve cells” [6] or “localized cerebral gliosis with giant neurons” [7]. maybe the earliest reference to a dysplasia-like pathology from epilepsy brains goes back to 1896 [8], but reports were overall scarce before the era of epilepsy surgery. which of the authors central hypotheses or conclusions turned out right or wrong? the histological reminiscence to tuberous sclerosis (ts) was noted and pointed out [1]. corsellis extensively searched for hints towards ts in both the patients and their families (see supplements 2-6). in a preceding conference presentation, the term “cryptic tuber” had already been used [9]. however, absence of any other signs of ts or suspicious familial traits was emphasized [1]. meanwhile, fcd ii has been identified as closely relating to genetic alterations within the mtor-signalling-cascade and belonging to the spectrum of “mtor-o-pathies” together with tuberous sclerosis and other malformations [10, 11]. fcd was correctly proposed to be a solitary lesion in most cases, its removal likely predicting seizure improvement or freedom. the authors proposed that dysplasia of their type would directly underlie the electrical and clinical manifestations of certain epilepsies. this has been strikingly verified later, since fcd turned out as intrinsically epileptogenic, i.e. the dysmorphic neurons themselves show the abnormal firing behavior, as proven by many techniques today such as in-vivo invasive recordings, electrocorticography, non-invasive measurements and in-vitro cellular recordings in fcd (for reference, see [4]). while taylor and the group did not comment on the presumed frequency of fcd, it is remarkable, that they found their 10 cases in a cohort of 300 epilepsy surgeries corresponding to 3%, while nowadays fcd ranks among the top pathologies in surgical series [2], see also below. at what stage of the authors´ career was the paper published? three out of four authors were well-known authorities in their fields, since murray falconer had established his fine epilepsy surgical series at the maudsley cooperating with the experienced neuropathology service at runwell, provided by j.a.n corsellis and clive joseph bruton. david taylor had been a research fellow at the beginning of his thirties and had started researching the psychiatric aspects of epilepsy surgery at the maudsley, giving rise to several publications from 1967 on (see also a letter by him in suppl. 7). after recognizing the commonalities in the tissue reports, he and the co-authors meticulously collected all available data leading to this seminal paper. how was the paper received over the time? more than 800 citations have cumulated since publication up to 2020 with about 20 citations per decade in the seventies and eighties and >200 citations per decade since then, 75% citations from clinical neurology [12]. the paper was translated early, e.g. to german, french and russian abstracts [13] and was widely recognized. “taylor’s dysplasia” as a term was coined by montreal epileptologist fred andermann (personal communication, see [3]). it was discussed later that the microscopic features were not strictly unique to taylor’s dysplasia, but may be seen in cases of hemimegalencephaly, for example [14]. dysplasia still was estimated to account for a minority of 2-5% of epilepsy surgery specimens as late as 1992 [14], while numbers have raised in modern series up to 30% [2], which relates to largely improved imaging and recording techniques. what happened to the original histological slides until today? starting early after 1950 j.a.n. corsellis built a large collection of brain specimen recruited from his neuropathology service including both surgical and autopsy tissues [15]. this collection came to be known as the corsellis collection later on [3]. it was the first of its kind and located in runwell hospital/wickford essex for years [15,16], and later moved to london (held by the west london mental health nhs trust), after runwell hospital closed down. of note, corsellis in addition to meticulously performing macroand microinvestigations documented in great detail the clinical patient findings and obtained many clinical notes (see supplemental material). his activities were funded by the medical research council as early as 1953 [16], he became a reference pathologist consulted by international colleagues [16], and after his death in 1994 his activities were continued by clive bruton. the original slides of focal dysplasia remained undiscovered as part of the corsellis collection brain bank in london for decades. an incomplete but significant part of slides, paraffin blocks and accompanying material including microphotographs and patient notes were found there and saved during research on the life and work of j.a.n. corsellis [3] and given to bs kasper by the corsellis collection to act as official custodian of this material when it became clear that the collection would be dismantled. did the re-discovery of this material elicit any surprising insights? vast material accompanying the tissue specimen impressively illustrates the working strategy at the institution led by corsellis. clinical notes and findings not only were systematically searched for, collected and saved (see suppl. 1-6), but also valued and integrated into the reports and discussions. there was a close exchange between the pathology group and the clinicians involved, not only the active neurosurgeons, but also the hospital and external neurologists, doctors and caregivers of the patients, as well as possible. this included postoperative years also. corsellis insisted on postsurgical cognitive tests, for example, and with taylor even visited the patient at home. no challenge seemed too difficult to overcome for the sake of obtaining further important information, as exemplified by correspondence between corsellis and neuropathologist professor h. orthner from göttingen/germany (see suppl. 4) which showed the length to which corsellis labored to obtain details about this patients mother who had died and had autopsy (see suppl. 6) as well as about the patients son, in order to check for any manifestations of tuberous sclerosis. what can be seen on the original slides? we present case 2 from the taylor paper for this re-appreciation. original slides in various stains were found preserved (he, van gieson, bielschowsky, ptah, nissl, oil-red, marchi). these slides correspond to figure 6 from the 1971 paper (for orientation see original figure and compare to macro-photographs and drawings in suppl. 1). exploring the original slides in h&e and nissl stains as well as new stainings obtained from the original paraffin blocks (see figure) nicely illustrates the presence of dysmorphic giant neurons, especially found at the bottom part of a sulcus. you are invited to explore the interactive, virtual microscopy slides in detail, just as taylor and corsellis did back then. this lesion nowadays would be classified as focal cortical dysplasia type iia, since balloon cells (then described as grotesque cells of probable glial origin) were not found, as confirmed by review. however, the majority of specimens in taylor´s series did contain balloon cells, i.e. focal cortical dysplasia type iib, representing the most common correlate of “fcd” or “taylor type dysplasia” as defined by imaging today. remember that in 1971 no magnetic resonance imaging (mri) was available. figure 1. original (historical) slides from taylors series (nissl, he) and new slides cut from the original paraffin blocks (evg, gfap, vimentin, nestin). clicking into the respective picture will lead you to the full virtual slide. clicking into the respective picture will lead you to the full virtual slide https://doi.org/10.57860/min_dts_000013 supplementary material supplements 1-6 as well as their content appear in chronological order. to follow, click on the links. free full text taylor 1971: https://dx.doi.org/10.1136/jnnp.34.4.369 suppl. 1: tissue workup incl. notes, macroand microphotographs by j.a.n. corsellis suppl. 2: clinical notes on the patient’s surgery: presurgical investigations, intraoperative electrocorticography, early postsurgery eeg and summary suppl. 3: documentation relating to corsellis’ inquiries for clinical data and early follow up suppl. 4: correspondence with prof. orthner, göttingen, germany suppl. 5: documents illustrating the patient’s long term follow up suppl. 6: medical history of the patient´s mother suppl. 7: a personal letter from david c. taylor the notes and records presented here accompanied the preserved patient’s material, as collected, stored and delivered by the corsellis collection. the full original material (photos & notes) were digitized in high quality to jpg-format at erlangen university. information enabling for identification of the patient or her mother was eliminated by image editing using acdsee pro7 software (bsk). acknowledgement this contribution, 50 years after the original publication in jnnp, is dedicated to the memory of professor david c. taylor (1933-2021) [19] and professor j.a.n. corsellis (1915-1994) [17,18,3], two great men of neuroscience, who were the right people at the right place in the right time back then. thanks to dr. thomas zobel from the wwu imaging network for support and hosting the omero image server. thanks to christian thomas, münster, for producing the virtual slides. thanks to frank bittner, erlangen, for excellent digitizing service and to thomas sebille (fotomas) for help with image editing. references 1. taylor dc, falconer ma, bruton cj, corsellis jan. focal dysplasia of the cerebral cortex in epilepsy. journal of neurology, neurosurgery and psychiatry 1971; 34: 369-387. 2. blümcke i, thom m, aronica e, armstrong dd, vinters hv, palmini a, jacques ts, avanzini g, barkovich aj, battaglia g, becker a, cepeda c, cendes f, colombo n, crino p, cross jh, delalande o, dubeau f, duncan j, guerrini r, kahane p, mathern g, najm i, ozkara c, raybaud c, represa a, roper sn, salamon n, schulze-bonhage a, tassi l, vezzani a, spreafico r. the clinicopathologic spectrum of focal cortical dysplasias: a consensus classification proposed by an ad hoc task force of the ilae diagnostic methods commission. epilepsia 2011; 52: 158-174. 3. kasper bs, taylor dc, janz d, kasper em, maier m, williams mr, crow tj. neuropathology of epilepsy and psychosis: the contributions of j.a.n. corsellis. brain. 2010;133: 3795-3805. 4. kasper bs, rössler k, hamer hm, dörfler a, blümcke i, coras r, roesch j, mennecke a, wellmer j, sommer b, lorber b, lang jd, graf w, stefan h, schwab s, buchfelder m, rampp s. coregistrating magnetic source and magnetic resonance imaging for epilepsy surgery in focal cortical dysplasia. neuroimage clin. 2018;19: 487-496. 5. yakovlev pi. congenital morphologic abnormalities of the brain in a case of abortive tuberous sclerosis. arch neurol psychiat (chic) 1939; 41: 119-139. 6. crome l. infantile cerebral gliosis with giant nerve cells. journal of neurology, neurosurgery and psychiatry 1957; 20: 117-124. 7. cravioto h, feigin i. localized vertebral gliosis with giant neurons histologically resembling tuberous sclerosis. j neuropathol exp neurol 1960; 19: 572-579. 8. roncoroni l. la fine morfologia del cervello degli epilettici e dei delinquenti. arch psich scienze penali antropol criminale 1896; 17: 92-116. 9. corsellis jan. “cryptic tubers” as a cause of focal epilepsy. proceedings of the society of british neurosurgeons (abstract) journal of neurology, neurosurgery and psychiatry 1971; 34: 104-105. 10. d'gama am, woodworth mb, hossain aa, bizzotto s, hatem ne, lacoursiere cm, najm i, ying z, yang e, barkovich aj, kwiatkowski dj, vinters hv, madsen jr, mathern gw, blümcke i, poduri a, walsh ca.somatic mutations activating the mtor pathway in dorsal telencephalic progenitors cause a continuum of cortical dysplasias. cell rep. 2017;21: 3754-3766. 11. baldassari s, ribierre t, marsan e, adle-biassette h, ferrand-sorbets s, bulteau c, dorison n, fohlen m, polivka m, weckhuysen s, dorfmüller g, chipaux m, baulac s. dissecting the genetic basis of focal cortical dysplasia: a large cohort study. acta neuropathol. 2019;138(6):885-900. 12. https://apps.webofknowledge.com (searched aug 13 2020). 13. corsellis jan, bruton cj, taylor dc, falconer ma. lokalisierte dysplasie der hirnrinde auf grund von gewebeuntersuchungen, die durch neurochirurgische eingriffe bei epileptikern gewonnen wurden. psychiatr. neurl. med. psychol (leipz) 1973; 17-18: 43-53. 14. janota i, polkey ce. cortical dysplasia in epilepsy a study on material from surgical resections for intractable epilepsy. recent advances in epilepsy 1992; 5: 37-49. 15. schoefert, anna kathryn. research on the human brain post mortem in britain, c.1950-1980: constituting the corsellis collection. m phil thesis, cambridge university, 2011. 16. metters js. isaacs report. tso, may 2003; page 235-239 see: http://image.guardian.co.uk/sys-files/society/documents/2003/05/12/isaacs_report.pdf (last looked up 16.9.2020). 17. janota i. obituary j.a.n corsellis (1915-1994). neuropathology appl neurobiol 1995; 21: 168. 18. crow tj. obituary j.a.n corsellis (1915-1994). psychiatric bulletin 1996; 20: 508-509. 19. kasper bs. in memoriam: david c. taylor. epilepsy behav 2021 apr 15; 118:107960 copyright: © 2021 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. neurooncology: 2021 update feel free to add comments by clicking these icons on the sidebar free neuropathology 2:5 (2021) review neurooncology: 2021 update pieter wesseling1,2 1 department of pathology, amsterdam university medical centers, location vumc, brain tumor center am-sterdam, amsterdam, the netherlands 2 laboratory for childhood cancer pathology, princess máxima center for pediatric oncology, utrecht, the netherlands corresponding author: pieter wesseling, md, phd · department of pathology · amsterdam university medical centers/vumc · de boelelaan 1117 · 1081 hv amsterdam · the netherlands p.wesseling@amsterdamumc.nl submitted: 26 february 2021 accepted: 09 march 2021 copyedited by: deanna fang published: 17 march 2021 https://doi.org/10.17879/freeneuropathology-2021-3271 keywords: brain tumor, molecular diagnostics, glioma, medulloblastoma, tumor microenvironment, covid-19 abstract this article briefly presents 10 topics that were selected by the author as ‘top 10 discoveries’ published in 2020 in the broader field of neurooncological pathology including neurosciences as well as clinical neurooncology of interest for neurooncological pathology. the selected topics concern new information on the molecular characteristics of gliomas (infratentorial idh-mutant diffuse astrocytomas, pediatric low-grade gliomas, infant-type high-grade gliomas, hypermutation in gliomas), the immunological aspects of the brain tumor microenvironment (tme), the impact of the tme on preclinical glioma models, and the importance of lymphatic drainage on brain tumor surveillance. furthermore, important papers were published on two ‘new’ genetic syndromes predisposing to medulloblastoma, on liquid biopsy-based diagnosis of central nervous system (cns) tumors, and on the ‘microbiome’ in glioblastomas (and other cancers). in the last part of this review, a dozen of papers are given as examples of papers that did not make it to the top 10 list of the author, underscoring the subjective component in the selection process. acknowledging that 2020 will be remembered as the year in which the world changed because of the covid-19 pandemic, some of the consequences of this pandemic for neurooncological pathology are briefly discussed as well. hopefully, this review forms an incentive to appreciate the wealth of information provided by the papers that were used as building blocks for the present manuscript. introduction even though, in 2020, the covid-19 pandemic had a quite dramatic, worldwide impact on the way we interacted (e.g. wearing of face masks, social distancing, travel restrictions, or even complete lockdowns), the publication of articles in international, peer-reviewed scientific journals seemed to continue more or less as usual. the author of this review was asked to contribute a review on the ‘top 10 discoveries’ in neurooncology published in 2020. as a search for candidate papers in pubmed using ‘brain & tumor & 2020’ or ‘spinal cord & tumor & 2020’ as key words resulted in over 10,000 hits, it took some time before the selection process for the ‘top ten discoveries’ was finished. here follows the list of the discoveries that emerged because they can be expected to have substantial implications for neurooncological pathology: infratentorial idh-mutant astrocytomas are different [1] molecular landscape of pediatric low-grade gliomas [2] gene fusions in infant-type high-grade gliomas [3] hypermutation in gliomas [4] immunological aspects of the brain tumor microenvironment [5-7] lymphatic drainage enables brain tumor surveillance [8, 9] impact of tumor microenvironment on preclinical glioblastoma models [10] newly recognized medulloblastoma predisposition syndromes [11, 12] liquid biopsy diagnosis of cns tumors [13] the glioblastoma microbiome [14] the order in this list is not based on any ranked importance of the findings but reflects an attempt to create some flow in this review. furthermore, acknowledging that authors did their best to optimally summarize the essence of their findings in (especially the abstracts of) the papers, in this review several phrases were copied and pasted from the original papers, always accompanied by adding the reference. of course, there is quite a subjective component in the selection of papers presented in this review. for example, the selection was not per se solely based on articles in the high(est)-impact journals but also aimed for a somewhat broader blend of topics. ergo, other colleagues would probably have selected other papers, but, as far as i am concerned, that is all in the game (‘de gustibus non est disputandum’). to make up for this situation to some degree, a few papers that just did not make it to my top 10 list will be briefly mentioned in the discussion. because of the extraordinary impact of the covid-19 pandemic in 2020 on our (professional) lives, some covid-19 aspects related to neurooncological pathology are very briefly discussed as well. topic 1. infratentorial idh-mutant astrocytomas are different [1] in the world health organization (who) 2016 classification, three main groups of adult-type diffuse gliomas are listed: astrocytomas, idh-mutant; oligodendrogliomas, idh-mutant and 1p/19q-codeleted; and astrocytomas, idh-wildtype (with glioblastoma idh-wildtype as by far the most frequent representative in this last group) [15]. in the upcoming 5th edition of the who classification (to be published in 2021), some changes can be expected in the definitions and nomenclature of idh-mutant astrocytomas. this is in line with what has been proposed by different updates of the consortium to improve molecular and practical approaches to cns tumor taxonomy (cimpact-now). cimpact-now update 5 suggested to grade the idh-mutant astrocytomas as who grade iv if molecular analysis reveals the presence of homozygous cdkn2a/b deletion and not only if the tumor histologically shows features of the highest malignancy grade [16]. furthermore, cimpact-now update 6 proposed to group the who grade ii-iv diffuse idh-mutant astrocytic tumors under a single name (“astrocytoma, idh mutant”) with arabic numerals assigned for the grade. the most malignant form in this group would then be ‘astrocytoma, idh-mutant, grade 4’ (rather than ‘glioblastoma, idh-mutant’), facilitating discrimination from the much more frequent and more aggressive ‘idh–wildtype glioblastoma’ [17]. in these considerations, the location of the idh-mutant astrocytic tumors is not taken into account. indeed, in the vast majority of patients presenting with a diffuse idh-mutant astrocytoma, the tumor is located in a cerebral hemisphere. banan r et al (with hartmann c and reuss d as corresponding authors) published in acta neuropathologica the clinical and pathological characteristics of a series of 42 primary infratentorial idh-mutant astrocytic tumors [1]. the mean age of the patients in this series was 37 years (similar to the patients with supratentorial tumors), the male:female ratio was 1.8:1, and approximately 30%, 40% and 30% were considered to be who grade ii, iii and iv, respectively. interestingly, only 1 out of 4 of these infratentorial idh-mutant tumors had the idh1 r132h mutation (in supratentorial tumors this is > 80%), with idh1 r132c/g and idh2 r172s/g as the most frequent ‘other’ idh mutations. also, atrx loss and mgmt promoter methylation were significantly less frequently found in the infratentorial compartment. of note, gene panel sequencing revealed two samples with idh1 r132c/h3f3a k27m co-mutations in this series. overall, the clinical outcomes of patients with infratentorial idh-mutant astrocytomas were significantly better than for patients with diffuse midline gliomas, h3k27m-mutant but were significantly worse than for patients with supratentorial idh-mutant astrocytomas when the two cases with idh1/h3k27m co-mutation were included. after exclusion of these two cases, only a trend toward worse outcome for infratentorial idh-mutant astrocytomas was seen. this study shows that diffuse gliomas in the brainstem and cerebellum are easily missed as being idh-mutant in cases where only immunohistochemistry for idh1 r132h mutant protein is performed and that h3k27m mutations co-occur in a small subset of these tumors and signify worse prognosis. topic 2. molecular landscape of pediatric low-grade gliomas [2] ryall s et al (with tabori u and hawkins c as co-senior authors) published a paper in cancer cell reporting the results of combined clinical, morphological and molecular profiling of > 1000 well-annotated, pediatric low-grade gliomas (plggs) with extensive clinical follow-up. the vast majority of the cases that could be adequately analyzed at the molecular level were found to harbor a driver alteration leading to activation of the ras/mapk pathway, while those without an identified alteration often showed upregulation of this pathway as well. based on the type of molecular alteration, plggs could be broadly classified in different classes: rearrangement-driven/fusion-positive tumors versus tumors with a single-nucleotide variant (snv). tumors in the former category were diagnosed at a younger age, enriched for who grade i histology, progressed infrequently, and rarely resulted in death as compared to tumors in the latter category. correlation of type of molecular alteration with outcome (incidence of disease progression or death) allowed for stratification of plggs into three risk categories: low risk (braf fusions, nf1 alterations, fgfr2 fusions, myb/mybl1 rearrangements, fgfr1 tyrosine kinase duplications, fgfr1-tacc1 fusion); intermediate risk (braf p.v600e, idh1 p.r132h, fgfr1 snv, met snv); and high risk (braf p.v600e + cdkn2a deletion, h3.3 p.k27m). additionally, a small, remaining group of ‘unknown risk’ was found with fusions of or snvs in other genes. importantly, while most plggs in children with the genetic pre-disposition disorder nf1 occurred as optic pathway glioma and had a good prognosis, nf1 plggs arising outside the optic pathway had significantly worse overall and progression-free survival. also, the most common kiaa1549-braf fusion, i.e. 16:09 (involving exon 16 of kiaa1549 and exon 9 of braf), was found to be associated with cerebellar location and good prognosis. in contrast, the kiaa1549-braf 15:09 fusion was the only fusion type seen in hemispheric tumors, the primary fusion found in tumors with ‘extensive dissemination’, and associated with a worse progression-free survival. these findings indeed provide very helpful guidance for the prognostication and clinical management of patients with plggs (e.g. ‘watch & wait’ for low-risk tumors versus a more aggressive approach for intermediate and high-risk tumors). obviously, for individual patients, such information should be evaluated in the context of the clinical situation, other molecular findings, and the exact who diagnosis (figure 1). fig. 1. kiaa-braf1549 duplication (and fusion) as seen in the copy number variation (cnv)-profile of a pilocytic astrocytoma (a) and a diffuse leptomeningeal glioneuronal tumor (dlgnt) (b). a tandem duplication of chromosome 7q34, indicative of the kiaa1549-braf fusion, is a frequent event in pilocytic astrocytomas and dlgnts. such a duplication is generally clearly visible as a relatively high position for this region (indicated by arrows in a and b) compared to the rest of chromosome 7 in the detailed cnv profiles that can be obtained using e.g. methylome profiling [55]. ryall s et al propose disease stratification of pediatric low-grade gliomas (plggs) based on the type of molecular alteration(s) in the tumor, with most kiaa1549-braf fusion-positive tumors belonging to the low-risk category [2]. it is important to put such information in perspective of clinical and other molecular findings and of the who diagnosis of the individual patient. for example, all dlgnts typically show loss of chromosome 1p. the cnv profile of the dlgnt depicted in b additionally shows gain of chromosome arm 1q and of chromosome 8, two features that fit very well with the more aggressive ‘methylation class 2’ of these tumors [56]. interestingly, rt-pcr analysis of this dlgnt revealed the 15:09 kiaa1549-braf fusion, i.e. the fusion that, according to ryall s et al, is the primary one seen in plggs with ‘extensive dissemination’ and that is associated with worse progression-free survival. topic 3. gene fusions in infant-type high-grade gliomas [3] in 2019, guerreiro stucklin as, ryall s, et al (with tabori u and hawkins c as last authors) published a study of 171 glioma samples from 150 infants in nature communications in which they identified three main subgroups of infant gliomas: 1) hemispheric rtk-driven tumors, including those showing fusion involving alk, ros1, ntrk, or met, enriched for high-grade gliomas and with an intermediate clinical outcome; 2) hemispheric ras/mapk-driven tumors, characterized by excellent long-term survival post-surgery; and 3) midline ras/mapk-driven tumors, enriched for low-grade glioma with braf alterations and a relatively poor outcome, even after conventional chemotherapeutic approaches [18]. in 2020, clarke m, mackay a, ismer b, et al (last author jones c) published in cancer discovery the findings of a thorough histological and molecular analysis of 241 tumors diagnosed as high-grade and/or diffuse gliomas in children < 4 years of age at the time of diagnosis [3]. after excluding non-gliomas and gliomas matching known subtypes, 130 infant gliomas remained that seemed to be part of an "intrinsic" spectrum of disease specific to the infant population. these included those with targetable mapk pathway alterations. a large proportion of remaining cases harbored gene fusions; these tumors typically occurred in very young children (age at diagnosis less than < 1 year), were located in the cerebral hemispheres, and (like the ‘group 1 tumors’ as reported by guerreiro stucklin as et al) frequently carried fusions involving alk (n = 31), ntrk1/ntrk2/ntrk3 (n = 21), ros1 (n = 9), or met (n = 4) as their driving alterations. importantly, compared to the fusion-negative tumors in this group, these fusion-positive tumors had significantly better outcomes. also, they would be good candidates for targeted therapy. this study is a next example of how in-depth molecular profiling of pediatric brain tumors not only greatly helps to improve prognostication for the children suffering from these neoplasms, but also to discover actionable targets that have the potential to substantially improve their prognosis [19]. topic 4. hypermutation in gliomas [4] while only a limited number of newly diagnosed gliomas are characterized by an inherited mismatch repair (mmr) defect and/or a ‘hypermutator’ phenotype, recurrent gliomas more often show such a phenotype, especially after alkylating chemotherapy [20]. the combination of low tumor mutational burden (tmb) and a highly immunosuppressive microenvironment in most newly diagnosed diffuse gliomas renders (at least so far) effective immunotherapy for these tumors quite challenging. assuming that the hypermutator status leads to an increased expression of neoantigens, gliomas/glioblastomas with a hypermutator phenotype could be better candidates for immune checkpoint blockade. in a paper in nature, touat m and li yy, et al (with beroukhim r, bandopadhayay p, bielle f, and ligon kl as supervising authors) report the results of their comprehensive analysis of the molecular determinants of mutational burden and signatures in 10,294 gliomas [4]. the gliomas were classified into molecular subgroups according to histopathology as well as idh1/idh2 and 1p/19q codeletion status. the median tmb in all samples was 2.6 mutations per mb (range 0.0–781.3). 558 (5.4%) tumors that were designated as being hypermutated (median tmb 50.8 mutations per mb, range 8.8–781.3) were analyzed more in depth. the majority of de novo hypermutated gliomas harbored mutational signatures associated with defects in the mmr pathway (cosmic signatures 6, 15, 26 and 14) or the dna polymerase pole (10 and 14) (2/3 and 1/3 of the samples, respectively), implying that constitutional deficiency in mmr or pole was likely to be the underlying genetic cause of hypermutation. in contrast, almost all gliomas showing high tmb post-treatment had a mutational signature associated with temozolomide exposure (signature 11), and half of these samples showed a co-existing minor mmror pole-deficiency signature component, suggesting that defective dna repair and mutagen exposure cooperate to drive hypermutation in these recurrent gliomas. the observation that bulk analyses of such post-treatment hypermutated glioma did not readily detect microsatellite instabilities (msis), while single-cell whole-genome sequencing analysis did identify microsatellite mutations, can be explained by intra-tumor heterogeneity for this characteristic and a lack of sufficient evolutionary time to select subclonal msi populations. while the therapy-induced snv mutations might not readily elicit effective antitumor responses (because of, for example, the quality of the mutations and/or the subclonal nature of their associated neoantigens), longer treatment exposure or combinatorial strategies may improve the efficacy of checkpoint blockade for such tumors. importantly, and acknowledging that acquired mmr deficiency occurs in the tumors that are considered as being the most temozolomide-sensitive, it is not yet clear whether the acquired mmr deficiency outweighs the positive effects of temozolomide in gliomas. the finding that mmr-deficient cells retain sensitivity to ccnu supports the hypothesis that hypermutation reduces cellular fitness and tolerance to dna-damaging agents other than temozolomide. one of the conclusions of the authors is that longitudinal, molecular analysis of diffuse gliomas can help improve therapeutic management and, ultimately, prognosis as well. topic 5. immunological aspects of the brain tumor microenvironment [5-7] in two papers published back-to-back in cell in june 2020, the authors report on the abundance and heterogeneity of tissue-resident and peripherally recruited leucocytes in glial and metastatic brain tumors [5, 6]. klemm f et al (last author joyce j [5]) comprehensively characterized the tumor microenvironment (tme) of gliomas and brain metastases using flow cytometry, rna sequencing, protein arrays, culture assays, and spatial tissue characterization. they found that ‘education’ of immune cell types in the tme depends on tumor origin and idh mutational status, with pronounced differences in proportional abundance of tissue-resident microglia, infiltrating monocyte-derived macrophages, neutrophils, and t cells. friebel b, kapolou k, et al (last authors neidert mc, becher b [6]) mapped the leukocyte landscape of brain tumors using high-dimensional single-cell profiling (cytof) and found a heterogeneous composition of tissue-resident and invading immune cells within the tme, allowing for a clear distinction between gliomas and brain metastases. gliomas typically showed tissue-resident, reactive microglia, whereas tissue-invading leukocytes accumulated in brain metastases. tissue-invading tumor-associated macrophages showed a distinctive signature trajectory, revealing tumor-driven instruction along with contrasting lymphocyte activation and exhaustion. indeed, these integrated analyses further elucidate the multifaceted immune cell activation within brain tumors and are instrumental for a rational design of more efficacious, targeted immunotherapy strategies. in the study published in cancer discovery, bayik d et al (last author lathia jd) used mouse models to study the presence of monocytic versus granulocytic myeloid-derived suppressor cells (mmdscs/gmdscs) [7]. mdscs are known to be elevated in blood and tumor tissue of patients with glioblastoma and for blocking antitumor immunity. bayik d et al observed that, in their models, mmdscs were enriched in the male tumors, whereas gmdscs were elevated in the blood of females. furthermore, depletion of gmdscs extended survival only in female mice. using gene-expression signatures coupled with network medicine analysis, the authors demonstrated that mmdscs could be targeted with antiproliferative agents in males, whereas gmdsc function could be inhibited by il1β blockade in females. analysis of patient data confirmed that proliferating mmdscs were predominant in male tumors and that a high gmdsc/il1β gene signature correlated with poor prognosis in female patients. interestingly, there is a quite striking sex disparity for glioblastomas at other levels as well. for example, the incidence of glioblastoma is significantly higher in males than in females (incidence rate ratio 1.59:1) (figure 2), and there is a survival advantage for females with glioblastoma independent of treatment, age, karnofsky performance status (kps), or idh mutation status [21, 22]. the work of bayik et al may explain some of such sex differences in patients with glioblastoma and indicate that (immuno)therapeutic approaches should be adapted to the patient’s gender. fig. 2. incidence rate ratios by sex for primary brain tumors. the incidence of most gliomas and embryonal brain tumors is significantly higher in males. for glioblastomas the male:female ratio is 1.59:1 [21]. furthermore, there is a female survival advantage for patients with glioblastoma independent of treatment, age, karnofsky performance status (kps), or idh mutation status [22]. the work of bayik et al may provide support for the hypothesis that differences in immune system functions contribute to these sex differences and that (immuno)therapeutic approaches should be adapted to the patient’s gender. figure based on ostrom qt et al (2020) [21]. * = p<0.05. topic 6. lymphatic drainage enables brain tumor surveillance [8, 9] the central nervous system (cns) has long been considered as lacking lymphatic drainage, a situation which could then contribute to the generally limited immunological response in glioblastomas and other brain tumors. later on, however, it became clear that lymphatic vessels are present in the intracranial meninges along the dural sinuses. these lymphatics drain brain-derived soluble waste to deep cervical lymph nodes, thereby directly connecting the brain with the peripheral immune system. recent studies also demonstrated the presence of meningeal lymphatics along the spinal cord and even suggested that the cerebrospinal fluid (csf) is primarily drained via lymphatics, rather than into the dural sinuses [23]. in a study published in nature, song e et al (last authors thomas jl and iwasaki a) report that in mice orthotopically injected with glioblastoma cells, prophylactic injection of the lymphangiogenesis-promoting protein vegfc in the csf resulted in enhanced priming of cd8 t cells in the draining deep cervical lymph nodes, migration of such cells into the tumor, rapid clearance of the glioblastoma and a long-lasting anti-tumor memory response [8]. transfection of a vegfc-expression mrna construct was found to work synergistically with checkpoint blockade therapy to eradicate existing glioblastoma. furthermore, hu x, deng q, ma l, et al (last author luo j) also published a study last year on the role of meningeal lymphatic vessels in mice with intracranial gliomas or metastatic melanomas [9]. disruption of dorsal meningeal lymphatics alone impaired the dissemination of tumor cells and dendritic cells from the brain tumors to deep cervical lymph nodes, while such trafficking of dendritic cells was found to be increased in mice with enhanced dorsal meningeal lymphangiogenesis. also, disruption of dorsal meningeal lymphatics alone (without affecting basal or nasal meningeal lymphatics) significantly reduced the efficacy of combined anti-pd-1/ctla-4 checkpoint therapy in striatal tumor models, while tumors overexpressing vegfc displayed a better response to such combination therapy. these studies suggest that the immunosurveillance-promoting capacity of vegfc may be exploited to increase the efficacy of immunotherapeutic approaches for brain tumors. topic 7. impact of tumor microenvironment on preclinical glioblastoma models [10] a wide variety of preclinical models for human glioblastomas serve as an important tool for studying these brain tumors. however, no model is perfect, and it is important to determine if/how different models recapitulate different aspects of human glioblastomas. to address this issue, pine ar, cirigliano sm, et al (last author fine ha) performed a complete transcriptomic characterization of tumor cells from 5 patients across four patient-specific glioblastoma–derived model types: 1) glioma spheres (gss), 2) tumor organoids (tos), 3) glioblastoma cerebral organoids (glicos), and 4) patient-derived xenografts (pdxs). they found that glicos and pdxs more closely recapitulated the invasive growth of human glioblastomas. also, the results obtained with bulk and single cell rna sequencing analysis of these models more closely resembled the human tumors. compared to the other models, glicos were enriched for a neural progenitor-like cell subpopulation, showed lower percentages of mesenchymal cells, showed retention of neural and oligodendrocyte progenitor cell populations and recapitulated the cellular states and their plasticity found in the corresponding human tumors best. although glicos thus had a profile most similar to that of the parental glioblastomas, the similarity between glicos and the original glioblastomas was reduced when glico cells were replated in two-dimensional culture conditions. this work underscores the critical impact of the microenvironment in glioblastoma models on the degree to which cellular states, as found in human glioblastomas, are recapitulated. thereby, this study serves as a kind of reality check for those who exploit such preclinical models for elucidation of the pathobiology of and identification of therapeutic approaches for glioblastomas in clinical practice. topic 8. newly discovered medulloblastoma predisposition syndromes [11, 12] medulloblastomas are by far the most frequent of the embryonal cns tumors, most commonly present in childhood and display considerable biological heterogeneity, with distinct molecularly defined groups listed in the who classification. a recent review reports that pathogenic germline variants in established cancer predisposition genes can be identified in about 5% of patients with medulloblastoma [24]. this includes syndromes with a germline defect in sufu or ptch1 (nevoid basal cell carcinoma syndrome/gorlin syndrome), tp53 (li–fraumeni syndrome), apc (familial adenomatous polyposis), crebbp (rubinstein–taybi syndrome), nbs1 (nijmegen breakage syndrome), palb2, and brca2. in 2020, two additional germline mutations were reported that predispose to pediatric medulloblastoma. waszak sm, robinson gw, et al (last authors korbel jo, northcott pa and pfister sm) published a study in nature of 1022 patients with medulloblastoma from whom blood samples and tumor samples were analyzed for germline mutations in 110 cancer predisposition genes [11]. they identified rare germline loss-of-function variants across the elongator complex protein 1 (elp1) gene in 14% of pediatric patients with the sonic hedgehog medulloblastoma subgroup (mbshh). elp1 was the most common medulloblastoma predisposition gene and increased the prevalence of genetic predisposition to 40% among pediatric patients in this mbshh subgroup. parent-offspring and pedigree analyses identified two families with a history of pediatric medulloblastoma. most elp1-associated medulloblastomas also exhibited somatic alterations in ptch1. tumors from patients with elp1-associated mbshh were characterized by universal biallelic inactivation of elp1 owing to somatic loss of chromosome arm 9q alterations. the tumor cells were found to show changes consistent with loss of protein homeostasis due to elongator deficiency. the authors conclude that such a genetic predisposition to proteome instability may be a determinant in the pathogenesis of other (pediatric) cancers as well and may provide new targets for therapeutic interference. in another study, begemann m, waszak sm, et al (last authors pfister sm, kontny u, kurth i, and published in the journal of clinical oncology) investigated families with childhood medulloblastoma to identify predisposing germline mutations [12]. initial findings were extended to genomes and epigenomes of 1,044 medulloblastoma cases. the authors identified in six patients with infant-onset medulloblastoma a heterozygous germline mutation in the g protein-coupled receptor 161 (gpr161) gene, which is located on chromosome 1q. gpr161 mutations were exclusively associated with the mbshh subgroup and accounted for 5% of infant cases in these cohorts. molecular profiling revealed loss of heterozygosity (loh) at gpr161 in all affected mbshh tumors without additional somatic driver events. additionally, analysis of 226 mbshh tumors revealed somatic copy-neutral loh of chromosome 1q as a characteristic hallmark and the primary mechanism for biallelic inactivation of gpr161 in the affected mbshh tumors. obviously, the diagnosis of an underlying predisposition syndrome as reported in these papers is important because of its implications for the management of the patients and their families. topic 9. liquid biopsy diagnosis of cns tumors [13] circulating tumor dna (ctdna) in blood is considered to be an easily accessible source of diagnostic, prognostic and/or predictive information that may be very helpful for improving the management of cancer patients. so far, however, for patients with primary brain tumors, the use of csf appeared to be a more promising biosource because of the limited abundance of ctdna in blood of these patients. at the same time, approaches that go beyond dna sequence information, such as analysis of epigenetic signatures in ctdna, can help to boost the use of blood for liquid biopsy diagnostics [25]. in the study published in nature medicine, nassiri f, chakravarthy a, feng s, et al (last authors zadeh g and de carvalho dd) used cell-free methylated dna immunoprecipitation and high-throughput sequencing (cfmedip-seq) to recover and profile methylated dna fragments from plasma [13]. plasma samples of patients with diffuse (idh-wildtype and idh-mutant) gliomas, of patients with other intracranial and extracranial tumors, and of healthy controls were used. these samples were split into training and test sets, and random forest classifiers were trained using the top 300 differentially methylated regions for the different tumor classes. indeed, the classifier was able to accurately distinguish different tumors based on the differences in dna methylation profiles. the potential of such a methylome-based liquid biopsy approach has also already been reported for non-cns tumors and can be applied to other biosources (e.g. urine) as well [26, 27], underscoring the potential of analysis of methylated plasma ctdna to facilitate blood-based diagnosis and monitoring of patients with cancer. topic 10. the glioblastoma microbiome [14] tumor formation involves the co-evolution of neoplastic cells together with extracellular matrix, tumor vasculature and immune cells. evaluating tumors as complete ‘organs’, and not simply as masses of transformed tumor cells, is of paramount importance because heterologous cell types within tumors can actively influence therapeutic response [28]. bacteria were first detected in human tumors more than 100 years ago, raising the possibility that the tumor microbiome may be an additional player in the complex tumor ecosystem. however, the existence of the tumor microbiome remained an issue of debate because of the suspicion of sample contamination. in their study published in science, nejman d, livyatan i, fuks g, et al (last authors shental n, straussman r) characterized the microbiome of 1010 tumor samples from seven human cancer types (breast, lung, ovary, pancreas, melanoma, bone, and brain/glioblastoma) as well as of 516 normal samples (including normal tissue adjacent to the tumor from the same patients) [14]. the authors took multiple measures to minimize and control for contamination. they used pcr sequencing techniques to gain species-level resolution as well as multiple visualization methods and ‘culturomics’ for identification of bacteria. the authors report the presence of bacteria in each tumor type, including in tumors that have no direct connection with the external environment such as glioblastomas. the detection rate ranged from 14.3% in melanomas to >60% in breast, pancreatic, and bone tumors. breast tumors had a richer and more diverse microbiome than all other tumor types tested, and live bacteria could be cultured from these tumors. the bacteria were found to be predominantly localized intracellularly in both cancer cells and immune cells, with different tumor types showing a distinct microbiome composition. the authors also noted correlations between intratumor bacteria and the smoking status and/or the response to immunotherapy of the patients. obviously, these findings immediately raise additional questions, for example: what (if any) is the role of intratumoral bacteria in the development of cancer? and how does the tumor microbiome affect the immune tumor microenvironment and the response to immune therapy? the answers to such questions are crucial for further elucidation of the existence and the importance of the tumor microbiome in cancer [29]. discussion hopefully, this review indeed provides concise, easily digestible information on the topics selected by the author as the top 10 discoveries in the year 2020. of course, during the selection process, quite a few papers were noted that didn’t make it to the top 10 list but that are certainly of great interest as well. for example, it is reassuring to learn that the use of molecular markers for the diagnosis of glioblastoma, idh-wildtype as suggested by cimpact-now update 3 was corroborated in an independent study [30, 31], but it is also good to know that some caution may be warranted for histologically low-grade idh-wildtype diffuse astrocytic tumors with an isolated tert promoter mutation [32]. furthermore, some new tumor types were recognized that will make it to the new who classification of cns tumors (especially diffuse glioneuronal tumor with oligodendroglioma-like features and nuclear clusters (dgonc) and desmoplastic myxoid tumor of the pineal region, smarcb1-mutant [33, 34]), and new insights were obtained in areas such as the oncogenesis of h3.3g34-mutant gliomas [35], posterior fossa group a (pfa) ependymomas [36], subependymal giant cell astrocytomas [37], and in the methylation characteristics of germline-driven replication repair-deficient high-grade gliomas (‘unique hypomethylation patterns’) [38]. very interesting new information was published on how glioblastomas interact with neurons [39], and a roadmap was presented for shaping the emerging field of cancer neuroscience [40]. in depth (epi)genetic analysis of single (brain tumor) cells is ‘hot’ nowadays and indeed provides an enormous amount of new information. a novel tool for exploiting single-cell rna sequencing data, the single-cell tumor-host interaction tool (scthi), was published in a journal with the impressive name gigascience [41]. although not included in the top 10 list in this review, much has been discovered in 2020 about and because of the covid-19 pandemic, with some consequences for neurooncology as well. because of the very infectious and often serious character of the disease, governments had to take drastic actions such as enforcing the wearing of face masks, installing social distancing measures and limiting the free movement of citizens. combined with the finding that cancer patients have an increased risk of dying because of covid-19 infection, this has caused increased stress and anxiety amongst brain tumor patients and their caregivers [42, 43]. neurooncologic care needed to be delivered in an adapted form, minimizing exposures for patients and addressing risks and benefits of all therapeutic interventions, while at the same time ensuring the health of the multi-disciplinary neurooncology workforce as much as possible [44]. interestingly, it appeared that some diagnostic (neuro)pathology services can be reliably provided while working from home when using platforms for whole slide images (wsis). of course, some activities (e.g. tissue sampling, processing, cutting, staining, molecular analyses) still need to be done in the institutes [45-47]. as an emergency response to the pandemic, the uk royal college of pathologists provided guidance for remote reporting of digital pathology slides and the us food and drug administration (fda) granted a waiver for the use of readily available consumer monitors at home [48, 49]. furthermore, the covid-19 pandemic has boosted the exploitation of present-day technology for teaching of medical students and pathology residents (e.g. using digital lectures, videoconferencing, and online resources utilizing collections of wsis) [50, 51] and has drastically changed the way scientific communities interact now that meetings cannot be organized in their traditional format. however, the ‘human touch’ is easily missed in such virtual encounters. finding the right balance between attending web-based meetings, getting (the rest of) our daily job done from home, and maintaining a healthy personal life can be challenging [52]. also, it remains to be seen what the long-term impact of the pandemic is on cancer research [53]. last but not least, ignorance of one’s own ignorance is worrisome, especially so because not infrequently it is combined with overconfidence, a phenomenon called the dunning-kruger effect [54]. improving skills and metacognitive competence helps one to move on from conscious incompetence to a phase of conscious competence that is more in balance with the level of confidence (figure 3). although a review like this only allows for ‘scratching the surface’ of the selected discoveries, it hopefully does help one to move further away from unconscious incompetence, especially so if the review indeed works as an incentive to read and appreciate the original papers that were used as building blocks for the present manuscript. fig. 3. the dunning-kruger effect: a remarkable relationship between competence and confidence. in 1999, david dunning and justin kruger described the phenomenon that people tend to initially hold overly favorable views of their abilities in many social and intellectual domains [54]. indeed, people not infrequently share strong opinions on particular topics without being hindered by their lack of competence (in other words, they’re on top of ‘mount stupid’ without knowing it). improving skills and metacognitive competence helps to move on via the ‘valley of despair’ (conscious incompetence: knowing that you only have very limited knowledge and/or understanding of a particular topic) to the ‘slope of enlightenment’, where competence and confidence are more in balance. acknowledging that competence is often a matter of continuing education and professional development, i doubt if the ultimate goal in medicine is really to strive for the level of unconscious competence (hence the question mark behind that term). meanwhile, 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human brain tumours using methylation array-based copy number and gene panel sequencing data. neuropathol appl neurobiol, 2020. 56. deng, m.y., et al., molecularly defined diffuse leptomeningeal glioneuronal tumor (dlgnt) comprises two subgroups with distinct clinical and genetic features. acta neuropathol, 2018. 136(2): p. 239-253. copyright: © 2021 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. top ten discoveries of the year: neurooncology feel free to add comments by clicking these icons on the sidebar free neuropathology 1:8 (2020) review top ten discoveries of the year: neurooncology pieter wesseling1,2 1 department of pathology, amsterdam university medical centers, location vumc, brain tumor center amsterdam, de boelelaan 1117, 1081hv amsterdam, the netherlands 2 princess máxima center for pediatric oncology, heidelberglaan 25, 3584 cs utrecht, the netherlands corresponding author: pieter wesseling, md, phd · department of pathology · amsterdam university medical centers/vumc · de boelelaan 1117 · 1081 hv amsterdam · the netherlands · tel: +31-20-4444979 p.wesseling@amsterdamumc.nl submitted: 30 january 2020 accepted: 23 february 2020 published: 26 february 2020 https://doi.org/10.17879/freeneuropathology-2020-2671 keywords: brain tumor, neuropathology, molecular diagnostics, glioma, medulloblastoma, methylome analysis abstract this article briefly discusses 10 topics that were selected by the author as top 10 discoveries published in 2019 in the broader field of neuro-oncological pathology (so including neurosciences as well as clinical neuro-oncology but with implications for neuro-oncological pathology). some topics concern new information on immunohistochemical and molecular markers that enable improved diagnosis of particular tumors of the central nervous system (cns) and information on a refined classification of medulloblastomas. subsequently, several papers are discussed that further elucidate some pathobiological aspects of especially medulloblastomas (histogenesis, molecular evolution) and diffuse gliomas (mechanisms involved in cns infiltration, role of cancer stem(-like) cells, longitudinal molecular evolution). the remaining topics concern progress made in vaccination therapy for glioblastomas and in using cerebrospinal fluid for liquid biopsy diagnosis of gliomas. clearly, substantial, and sometimes even amazing progress has been made in increasing our understanding in several areas of neuro-oncological pathology. at the same time, almost every finding raises new questions, and translation of new insights in improving the outcome for patients suffering from cns tumors remains a huge challenge. introduction looking back from time to time is good. the request of the editor-in-chief of this journal to contribute a review on ‘top 10 discoveries’ in neuro-oncology published in 2019 was the reason for the author to look back in a somewhat more structured way in order to select which topics could/should qualify for such a label. from the start it was clear that there are multiple ways to shape such a selection process. to cut a long story short, keeping the readership of free neuropathology in mind and with some help of the editor-in-chief of this journal, the choice was made to select topics based on original papers published in 2019 from a broad range of journals, important criteria being that indeed relevant progress was made and that in one way or another the findings can be expected to have substantial implications for neuro-oncological pathology. furthermore, it was decided to not just focus on articles in the high(est)-impact journals and to aim for a somewhat broader blend of topics. some topics presented in this review were selected based on just one article, while the information of two or three papers was combined for the discussion of other topics. the 10 topics that emerged from this (quite subjective!) selection process are:   1. lack of h3k27me3 staining is a promising surrogate marker for oligodendrogliomas [1] 2. improved molecular diagnosis of rare cns tumor types [2, 3] 3. refined molecular classification of non-wnt/non-shh medulloblastomas [4] 4. histogenesis of posterior fossa tumors in children [5-7] 5. longitudinal molecular analysis of diffuse gliomas [8] 6. notch1-sox2 signaling controls glioma cell invasion in white matter tracts [9] 7. ‘synaptic cooption’ in glial and metastatic cns tumors [10-12] 8. the role of cancer stem(-like) cells in glioblastomas [13] 9. personalized (neo)antigen vaccination therapy for glioblastoma [14, 15] 10. liquid biopsy diagnosis of gliomas using cerebrospinal fluid (csf) [16, 17]   of note, the ranking in this list is not based on importance of the findings, but rather on an attempt to create a flow in this review (from information that may quite readily be implemented in daily diagnostic practice via more hard-core neurobiology/neuroscience findings to some future perspectives). also, there is a bias in the selection of the topics in this list towards diffuse gliomas and embryonal tumors/medulloblastomas. obviously, other colleagues might have presented a quite different list of ‘top 10 discoveries’, also because many more very good and interesting papers with implications for neuro-oncological pathology were published in 2019. for example, the study of paramasivam et al [18], providing novel information on mutational patterns and regulatory networks of subgroups of meningiomas would be a good alternative for a topic. having said that, it is hoped that this review not only provides easily digestible information on the top 10 discoveries in neuro-oncological pathology as selected by this author, but will also be used as a stepping stone to appreciate the much more detailed information in the original papers that were used as building blocks for this review. topic 1. lack of h3k27me3 staining is a promising surrogate marker for oligodendrogliomas [1] unequivocal histological recognition of (anaplastic) oligodrogliomas, idh-mutant & 1p/19q-codeleted (‘canonical oligodendrogliomas’) has been hindered by the lack of specific immunohistochemical markers for these tumors [19]. in a study analyzing an epigenetically well-defined cohort of diffuse gliomas in adults (idh-mutant & 1p/19q-codeleted oligodendrogliomas, n = 26; idh-mutant astrocytic tumors, n = 34; idh-wildtype glioblastomas, n = 101), filipski et al report that 25 out of the 26 oligodendrogliomas showed lack of nuclear h3k27me3 staining, in the remaining case h3k27me3 staining was interpreted as non-conclusive [1]. interestingly, in an otherwise ‘nucleonegative’ oligodendroglioma of a female patient only dot-like nuclear staining was seen. this phenomenon has previously been described to represent the inactivated x chromosome, with h3k27me3 functioning as a transcriptional silencing mechanism via chromatin remodeling [20] (fig. 1). the vast majority of idh-mutant and idh-wildtype astrocytic tumors showed clear nuclear h3k27me3 staining. based on these findings in combination with the results of immunohistochemistry for atrx, idh1r132h and h3k27m, filipski et al report that in their cohort of h3k27-wildtype diffuse gliomas the tumors showing lack of nuclear h3k27me3 staining, retention or non-conclusive nuclear atrx staining, and idh1r132h mutation are oligodendroglioma, idh-mutant & 1p/19q codeleted with a predicted probability of 0.9678. ideally, in case of h3k27me3and h3k27m-nucleonegative diffuse gliomas that in addition show retained or non-conclusive atrx staining but no idh1r132h-mutant protein staining, sequencing analysis is performed to demonstrate presence/absence of an idh1 or idh2 mutation. obviously, the results of immunohistochemistry should be interpreted with caution in especially biopsies that are small and/or only show low tumor cell percentage. the use of an antibody panel including h3k27me3, h3k27m mutant protein, atrx, and idh1r132h may thus greatly facilitate recognition (anaplastic) oligodendrogliomas, idh-mutant & 1p/19q-codeleted, especially so in a situation where molecular testing is not readily available/possible. the pathobiology underlying the global lack of h3k27me3 in tumor cell nuclei and 1p/19q codeletion needs further elucidation. of note, while in diffuse gliomas in adults lack of h3k27me3 staining may thus indicate canonical oligodendroglioma with a relatively favorable prognosis, in several other tumors of the (central) nervous system (e.g. posterior fossa ependymomas, malignant peripheral nerve sheath tumors, meningiomas) such a lack of nuclear staining is associated with worse prognosis [21-23]. fig. 1. loss of nuclear h3k27me3 immunohistochemical staining; a surrogate marker for oligodendroglioma, idh-mutant and 1p/19q-codeleted. in the past, the diagnosis oligodendroglioma was mainly based on histology in hematoxylin and eosin (h&e) stained sections, the ‘fried egg’ phenotype of the tumor cells being an important clue for this diagnosis (a); nowadays, demonstration of idh-mutant status and (complete) 1p/19q codeletion is required for the diagnosis of ‘canonical’ oligodendroglioma (b; result of methylome analysis showing (with almost perfect calibrated score of 0.99) suggested diagnosis of oligodendroglioma, idh-mutant and 1p/19q-codeleted, as well as copy number profile based on methylome analysis with loss of chromosome arms 1p and 19q, and hypermethylated status of mgmt promoter); filipski et al report that immunohistochemistry can be used to recognize ‘canonical’ oligodendrogliomas as well, the combination of lack of nuclear h3k27me3 staining, retention or non-conclusive nuclear atrx staining, and positive staining for idh1r132h mutant protein in h3k27-wildtype diffuse gliomas being highly suggestive for this diagnosis (c; while the nuclei of non-neoplastic cells in the microvessel wall show strong h3k27me3 staining (arrowheads), the tumor cell nuclei (arrows) are largely negative; in this female patient, the remaining positive dots in the tumor cell nuclei represent the inactivated x chromosome [20]). topic 2. improved molecular diagnosis of rare cns tumor types [2, 3] only recently, genome-wide dna methylation analysis has been introduced as a very helpful tool for improved diagnosis of cns tumor types and subtypes [24-29]. in 2019, two papers were published by the ‘heidelberg team’ + coauthors from other centers/countries demonstrating the power of methylome analysis. sievers et al [2] report the results of in-depth analysis of 30 tumors initially identified through methylome analysis as a separate group in a heidelberg cohort of > 25,000 tumors. most tumors in this group were histologically diagnosed as rosette-forming glioneuronal tumor (rgnt), and all revealed fgfr1 hotspot mutations, with in about two-thirds of the cases co-occurrence of a pik3ca mutation and in one third of the tumors an additional loss-of-function mutation in nf1. in contrast to most low-grade gliomas, rgnts displayed co-occurrence of two or even all three of these mutations. these highly recurrent combined genetic alterations affecting both mapk and pi3k signaling pathways may offer potential therapeutic targets for rgnts [2]. in a study of hou et al [3], 17 of the 28 tumors initially histologically diagnosed as papillary glioneuronal tumor (pgnt) showed methylation profiles typical for other tumor entities (mostly dysembryoplastic neuroepithelial tumor and pilocytic astrocytoma). the remaining 11 cases exhibited a unique profile and were considered as a distinct pgnt methylation class. three additional tumors in the heidelberg cohort clustered with this methylation class as well but were originally not diagnosed as pgnt. in all 12 cases of this methylation class of which material was available for further analysis, fusions involving prkca were identified (slc44a1–prkca, n = 11; notch1-prkca, n = 1), whereas such fusions were not found in the tumors belonging to other methylation classes. both the study of sievers et al and of hou et al thus provide information on molecular characteristics that can be used for improved recognition of such rare cns tumor types and again illustrate the power of methylome analysis in this context. topic 3. refined molecular classification of non-wnt/non-shh medulloblastomas [4] the 2016 who classification of cns tumors recognizes four molecular variants of medulloblastoma: wnt-activated, shh-activated and tp53-mutant, shh-activated and tp53-wildtype, and non-wnt/non-shh. the non-wnt/non-shh subgroup, accounting for about 65% of all medulloblastomas, encompasses the group 3 and group 4 molecular variants of medulloblastoma. however, these groups have heterogeneous clinical characteristics and survival outcomes, and their biology remained less clear. studies published in 2017 suggested the existence of 4, 6 or 8 subgroups within the overarching non-wnt/non-shh group [30-32]. in an attempt to provide clarity and a basis for further biological studies, sharma et al analyzed the number and nature of subtypes that could be identified in a cohort of 1501 genomically characterized non-wnt/non-shh medulloblastomas [4]. in this study, rather than advocating one single analytical approach or method, multiple class-definition approaches were used and equal weight was given to each analytical technique. in a lower complexity analysis group 3 and group 4 were identified again, but more complex analysis strongly supported the existence of eight robust group 3/group 4 subtypes (i-viii). these subtypes could generally be recognized based on their dna-methylation profiles and showed differences in enrichment for specific driver gene alterations, cytogenetic events, and ages of incidence with mostly unimodal age distribution. cytogenetic signatures, chromosomal copy-number aberrations and information on one gene or a set of genes in isolation were found to be insufficient for recognition of subtypes i-viii. the relevance of these subtypes is supported by differences in survival, with e.g. subtype iv being associated with low-risk clinical behavior, and with relatively frequent late relapses in patients with a subtype viii medulloblastoma. these findings can be expected to improve risk stratification, therapy and thereby the outcome for patients with non-wnt/non-shh medulloblastomas. of note, although the bimodal age distribution that was found for subtypes v and vii may indicate that some further refinement of the classification is still possible, the authors report that they did not find strong evidence for more than eight molecular subgroups in the non-wnt/non-shh category. subtyping of non-wnt/non-shh medulloblastomas into subtype i-viii is now available for the community by performing methylome analysis as described by capper et al [24, 25] and using an extension of the heidelberg brain tumor classifier (https://www.molecularneuropathology.org/mnp/classifier/7). topic 4. histogenesis of posterior fossa tumors in children [5-7] for quite some time it is clear that different subtypes of medulloblastomas have different developmental origin [33]. in fact, cerebellar tumors may be the result of a disorder of early cerebellar development. in an attempt to further elucidate cerebellar development at the single cell level, vladiou et al performed large-scale single-cell rna sequencing (scrna-seq) of more than 60,000 cells from the developing mouse cerebellum [5]. these analyses allowed them to reconstruct the cellular hierarchy in development at various points in time, with many of the normal mouse cerebellar cell populations only being present for a restricted time period during the fetal or very early postnatal period. subsequently, this transcriptome information was compared to that of pediatric cerebellar tumors: medulloblastomas, posterior fossa (pf) ependymomas, and pilocytic astrocytoma. the authors find that (different molecular subgroups of) these tumors indeed mirror the transcription of cells from distinct, temporally restricted cerebellar lineages: shh medulloblastomas, group 3 and group 4 medulloblastomas were found to transcriptionally resemble respectively the granule cell hierarchy, nestin+ stem cells, and unipolar brush cells, while pf type a/pf type b (pfa/pfb) ependymomas and cerebellar pilocytic astrocytomas resembled prenatal gliogenic progenitor cells. these findings indicate that each of these cerebellar tumor types arises from a particular cell type. however, across the medulloblastoma subgroups (shh, group 3, group 4), as well as pfa ependymoma and pilocytic astrocytomas, the scrnaseq data also demonstrated high levels of single-cell heterogeneity, with evidence of multiple lineages of differentiation and tumor cells matching different time points in the differentiation hierarchy [5]. in another study, hovestadt et al performed single-cell transcriptomics to investigate intraand intertumoral heterogeneity in 25 medulloblastomas spanning all molecular subgroups. they find that wnt, shh and group 3 tumors comprise subgroup-specific undifferentiated and differentiated neuronal-like populations of malignant cells, whereas group 4 tumors consist of differentiated neuronal-like neoplastic cells. the tumor cells in shh medulloblastomas closely resembled granule neurons of varying differentiation states that correlated with patient age. using cross-species transcriptomic analysis, it appeared that distinct glutamatergic populations are the putative cells-of-origin for shh and group 4 medulloblastomas. furthermore, the tumor cells of group 3 and group 4 medulloblastomas exhibited characteristics ranging from primitive progenitor-like to more mature neuronal-like cells, with the relative proportions of these cells distinguishing these subgroups [6]. in a third study, based on single-cell transcriptome analysis of >65,000 cells of the embryonal pons and forebrain, jessa et al derived signatures for 191 distinct cell populations of these regions [7]. bulk transcriptome analysis of wnt-activated medulloblastomas revealed a match with the mossy fiber neuronal lineage of the rhombic lip. embryonal tumors with multilayered rosettes (etmrs) appeared to be derived from a neuronal lineage as well, but especially the tyr and myc subgroups of atypical teratoid/rhabdoid tumor (at/rt) seemed to originate from non-neuroectodermal cells. all in all, these studies provide strong evidence that pediatric cns tumors are the result of a disorder of early development, although the possibility that they arise from more mature cells undergoing de-differentiation at later time points or from trans-differentiation of other cell lineages cannot yet be completely ruled out. topic 5. longitudinal molecular analysis of diffuse gliomas [8] genomic characterization efforts such as the cancer genome atlas (tcga) have greatly increased our understanding of glioma biology [34-37]. based on these findings three major, clinically relevant subgroups of diffuse gliomas in adults were introduced in the who 2016 classification of cns tumors: (1) idh-mutant and chromosome 1p/19q codeleted (idh-mutant-codel); (2) idh-mutant without codeletion of chromosome 1p/19q (idh-mutant-noncodel); and (3) idh-wildtype. so far, however, such studies were generally limited to analysis of tumor tissue as obtained by first operation, and how the genetic landscape of these gliomas evolves over time and in response to therapy remained largely unknown. in december 2019, the glioma longitudinal analysis (glass) consortium published the results of analysis of 222 diffuse glioma patients (25 idh-mutant-codel, 63 idh-mutant-noncodel, and 134 idh-wildtype) with high-quality data on mutations and chromosomal copy numbers in samples of at least two time points [8]. the study revealed that the driver genes detected in the initial sample were retained in the recurrent tumor, and there was little evidence of recurrence-specific gene alterations. also, current standard of care therapies (chemotherapy, irradiation) generally did not seem to coerce the tumors down predictable paths. idh-mutant-noncodels were found to be most sensitive to developing a hypermutator phenotype after therapy with alkylating agents, a phenomenon that has been reported before [38, 39]. importantly, no differences were found in overall survival between hypermutators and non-hypermutators independent of age, subtype and mgmt promoter methylation status. in line with a recent study demonstrating that homozygous cdkn2a loss is a marker for high-grade malignancy in idh-mutant-noncodels [40], in the glass study recurrent idh-mutant-noncodels were enriched for homozygous cdkn2a deletions and this was associated with shorter survival compared to patients without these alterations. no differences in the levels of immunoediting were found between initial and recurrent gliomas. the glass study thus indicates that the strongest selective pressure in these three major subgroups of diffuse gliomas occurs during early glioma development and that current therapies shape this evolution in a largely stochastic manner. hopefully, such information on how diffuse gliomas evolve over time and in response to therapy will help to design more efficacious therapeutic strategies for these tumors. topic 6. notch1-sox2 signaling controls glioma cell invasion in white matter tracts [9] one of the main reasons that patients with diffuse gliomas so far cannot be cured is the diffuse infiltrative growth of the tumor cells along white matter tracts. one would hope that unraveling the mechanisms underlying this phenomenon may help to identify novel therapeutic targets. in an analysis of human glioma tissue samples, wang et al found that at the invasive front, cd133+ ‘glioma stem cells’ (gscs) were preferentially located along white matter tracts [41]. also, these tracts showed significant swelling and seemed to have discontinuous myelin, indicating that some gscs in the glioma-brain interface are preferentially located adjacent to unmyelinated white matter tracts. based on their findings the authors postulate that glioma invasion along these tracts occurs along axons inside myelin sheaths rather than along the outer surface of such sheaths, and that glioma-associated edema may play a role in local destruction of white matter fibers and thereby pave the way for direct interaction of glioma cells and axons. additionally, based on their findings in preclinical experiments that nearly all cd133+ gscs were also notch1+ and that nerve fibers at the invasive frontier all expressed jagged1, wang et al postulate that the interaction between jagged1 and notch1 may be an important determinant for the distribution of gscs. indeed, axonally expressed jagged1 was found to activate the notch signaling pathway in gscs and to subsequently promote the transcription of sox2 via sox9. conversely, sox2 upregulation decreased the methylation of the notch1 promoter to reinforce the high expression of notch1 in gscs and facilitate their white matter-tract tropism, while inhibition of notch signaling was found to attenuate this tropism. the findings of wang et al indicate that the notch1-sox2 positive-feedback loop is an important determinant of gsc invasion along white matter tracts, and that molecules in this loop may be exploited as therapeutic targets. however, the authors acknowledge that the mechanisms underlying glioma cell invasion along white matter tracts are probably more complex, and may also involve mechanisms as discussed in the next topic (topic 7). topic 7. ‘synaptic cooption’ in glial and metastatic cns tumors [10-12] after reporting earlier that neuroligin-3 secreted by active neurons promotes glioma growth [42, 43], in a recent study published in nature, venkatesh et al found that electrochemical communication exists between neurons and glioma cells through bona fide ampa (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) receptor-dependent synapses between these cells [10]. also, neuronal activity was found to evoke non-synaptic activity-dependent potassium currents that were amplified by gap junction-mediated tumor interconnections and resulting in an electrically coupled network. depolarization of glioma membranes was found to promote proliferation, whereas blocking electrochemical signaling inhibited the growth of glioma xenografts in mice. intraoperative electrocorticography in glioma patients revealed increased cortical excitability in the glioma-infiltrated brain areas. based on these findings the authors suggest that glioma growth is promoted by synaptic and electrical integration into neural circuits, with glioma-induced increase in neuronal excitability and activity-regulated glioma growth as elements of a positive feedback loop. in the same issue of nature, venkaratamani et al also report that functional synapses between neurons and glioma cells exist [11]. previously, these authors already demonstrated that many tumor cells in astrocytic tumors including glioblastomas extend ultra-long membrane protrusions (microtubes) that are used for brain invasion and proliferation, and that interconnect over long distances [44] (fig. 2). in their recent study, venkaratamani et al show the presence of ‘neurogliomal synapses’ in human glioma samples and in different disease models. such synapses were found to be located on tumor microtubes and produce postsynaptic currents that are mediated by glutamate receptors of the ampa subtype. in their experiments, neuronal activity led to synchronized calcium transients in tumor-microtube-connected glioma networks. furthermore, perturbation of ampa receptors and use of an ampa receptor antagonist reduced calcium-related invasiveness of tumor cells and of glioma growth. intriguingly, in a third study published in that same issue of nature, zeng et al demonstrate the existence of ‘pseudo-tripartite synapses’ between breast cancer cells and glutamatergic neurons and show that such cancer cells in the brain can co-opt a neuronal signaling pathway involving activation by glutamate ligands of n-methyl-d-aspartate receptors (nmdars) [12]. these three studies thus suggest very peculiar brain tumor-microenvironment interactions in the form of direct, biologically relevant synaptic communication between neurons and tumor cells with potential therapeutic implications. fig. 2. human glioma cells forming a mycelium-like network in mouse brain. based on findings in especially two-dimensional histological slides, tumor cells in the periphery of diffuse gliomas have been considered as ‘guerilla warriors’ that move as single cells into the preexistent cns tissue [41]. more recent findings, however, show that such cells in diffuse astrocytic tumors form a network of microtubes. this figure shows such network formation by human glioblastoma cells 28 days after implantation into a mouse brain (yellow: glioblastoma cells labeled by human-specific anti-vimentin antibody; magenta: mouse astrocytes labeled by anti-gfap antibody; blue: cell nuclei labeled by dapi; figure kindly provided by pavel gritsenko and peter friedl and based on work recently published [61]). in addition to such a mycelium-like network formation, studies discussed under topic 7 now report that direct, biologically relevant synaptic communication exists between neurons and tumor cells as well. topic 8. cancer stem cells in glioblastomas; do they exist? [13] it is been proposed that in glioblastomas cancer stem cells (cscs) or stem-like cells reside at the top of a hierarchical organization, are able to (re)create intra-tumoral heterogeneity by generating more differentiated offspring, and are relatively resistant to therapy and thus contribute significantly to tumor recurrence [45]. expression of particular cell membrane antigens (cd133, cd15/ssea, cd44, and/or a2b5) has often been used for identification of the csc subset in glioblastomas. however, it is increasingly clear that no single marker allows for unequivocal identification of a csc population in glioblastomas. moreover, there is still controversy if a bona fide csc population exists at all (and if so, if it concerns a quiescent or proliferative subpopulation). in this context, dirkse et al addressed the question whether glioblastoma cells expressing csc-associated cell membrane markers are indeed a defined entity at the apex of a hierarchical organization. they found that, like in patient biopsies, csc markers were heterogeneously expressed in patient-derived glioblastoma xenografts and stem-like cell cultures. more precisely, all facs-sorted subpopulations of tumor cells of glioblastomas were able to self-renew over multiple passages without significant differences between each other. this suggests that phenotypically heterogeneous glioblastoma cells can adapt to a variety of environmental changes and acquire similar stem cell properties in vitro. dirkse et al also found that accelerated reconstitution of heterogeneity provided a growth advantage in vivo, suggesting that tumorigenic potential is linked to intrinsic plasticity rather than csc multipotency. the study of dirkse et al thus provides strong evidence that cscs do not constitute a defined cellular entity, but rather a cellular state adapting to microenvironmental cues. the capacity of any given tumor cell to reconstitute heterogeneity in glioblastomas cautions against therapies targeting only a small subpopulation of tumor cells with stem cell(-like) characteristics. meanwhile, inherent cancer cell plasticity emerges as a novel relevant target for treatment [13]. topic 9. personalized (neo)antigen vaccination therapy for glioblastoma [14, 15] in patients with e.g. melanomas, high tumor mutational load (tml) is associated with increased frequency of neoantigens and improved response to checkpoint inhibition. administration of personalized neoantigen vaccines has been shown to successfully recruit t cells to the tumor and can lead to tumor regression. unfortunately, also after the introduction of immunotherapies, so far the very poor survival rates for patients with glioblastoma have not improved much. of note, glioblastomas often have a relatively low mutational burden and are considered as immunologically ‘cold’. recent studies suggest that for patients with glioblastoma, a personalized molecular approach is needed to improve the benefit of treatment with programmed cell death protein 1 (pd-1) inhibitors nivolumab or pembrolizumab, and that the neoadjuvant administration of pd-1 blockade may represent a more efficacious approach for these tumors [46, 47]. two recent studies published in nature explored the feasibility of vaccination therapy using tumor (neo)antigens for patients with glioblastoma. keskin et al performed a phase i/ib multi-epitope, personalized neoantigen vaccination study [14]. patients who did not receive dexamethasone generated circulating polyfunctional neoantigen-specific cd4+ and cd8+ t cell responses that were enriched in a memory phenotype. after vaccination, neoantigen-specific t cells were found to migrate from peripheral blood into the glioblastoma, suggesting that they may favorably alter the immune milieu of the tumor [14]. hilf et al studied vaccination using both unmutated tumor antigens and neoepitopes for more effective immunotherapy of glioblastomas, including those with a low mutational load [15]. highly individualized vaccinations with both types of tumor antigens were integrated into standard care for patients with newly diagnosed glioblastoma. fifteen patients were first treated with a vaccine with a previously constructed library of non-mutated antigens that are over-represented in glioblastomas (apvac1), followed by a second vaccine (apvac2) targeted against mutated neoantigens or non-mutated antigens that were not present in apvac1. the vaccines were personalized by analysis of mutations and of the transcriptomes and immunopeptidomes of the individual tumors. both vaccines showed favorable safety and elicited t cell responses against the proteins in the vaccine, with apvac1 inducing a sustained cd8+ t cell response, and apvac2 both cd4+ and cd8+ t cell responses [15]. topic 10. liquid biopsy diagnosis of gliomas using csf [16, 17] cancer cells release nucleic acids, vesicles, proteins, and other components into the blood stream and other body fluids. already for quite some time, circulating tumor dna (ctdna) in blood is considered as an easily accessible source of potentially very useful diagnostic, prognostic and/or predictive information that can be used to improve the management of cancer patients [48]. so far, however, liquid biopsy diagnosis for detection and monitoring of patients with gliomas has not yet entered the clinic. part of the problem may be that, compared to other cancers, gliomas release relatively limited amounts of ctdna into the bloodstream [49]. sequencing of ctdna from the cerebrospinal fluid (csf) may provide an alternative way to diagnose glial and other cns tumors. indeed, in 42 out of 85 adult patients with diffuse glioma (49.4%), miller et al detected ctdna in csf that was obtained by lumbar puncture [16]. presence of ctdna was associated with disease burden and adverse outcome. the glioma genomes detected in csf closely resembled those in matched tumor samples. alterations occurring early in gliomagenesis (idh1/idh2 mutation, 1p/19q codeletion) were shared in all matched ctdna-positive csf-tumor pairs, whereas growth factor receptor signaling pathways showed considerable evolution. most patients with ctdna-positive csf did not have detectable malignant cells in the csf, and no significant association was found between ctdna-positive csf and glioma grade, disease duration or prior therapy. of note, in this study of miller et al the csf samples were derived from patients relatively late in their disease course (median disease duration before csf collection for patients with idh-wildtype glioblastomas about a year, and for patients with idh-mutant lower grade gliomas over 5 years), and all patients already had treatment for their glial tumor. in contrast, pan et al explored the potential of ctdna analysis in csf obtained prior to surgical manipulation in a cohort of 57 patients with brainstem glioma [17]. over 90% of these csf samples were obtained intraoperatively. pan et al report that alterations were identified in the csf ctdna in 36/37 cases (97.3%) in which the primary tumors harbored at least one mutation, while in 31/37 of cases (83%) all primary tumor alterations were detected in the csf. in these patients, mutation detection using plasma ctdna was found to be much less sensitive than sequencing the csf ctdna. these studies thus suggest that indeed csf may be a much more promising biosource for liquid biopsy diagnostics of gliomas than blood. combination of such an approach with analyses going beyond dna sequence information (e.g. analysis of epigenetic and immune signatures in cell free dna) may further boost the exploitation of liquid biopsy diagnostics in patients with (glial) cns tumors [49]. discussion the papers discussed in this review as top 10 discoveries illustrate the substantial and sometimes even amazing progress that has been made in increasing our understanding of particular topics in neuro-oncological pathology. hopefully, this review not only provides easily digestible information on the topics selected by the author, but also works as a stepping stone to read and appreciate the original papers that were used as building blocks for the present manuscript. obviously, most of the findings presented here immediately elicit next questions. for example, are the findings presented by filipski et al already ‘mature’ enough to be used as surrogate markers for the diagnosis of (anaplastic) oligodendroglioma, idh-mutant and 1p/19q-codeleted in clinical practice? and while wang et al report that the notch1-sox2 positive-feedback loop is an important (and possibly targetable) determinant of invasion of glioma cells along white matter tracts, the authors acknowledge that other mechanisms as discussed in topic 7 may be involved as well. but then again, is synapse formation between neuronal and glioma cell processes especially relevant for the invasive front? or also for proliferation of tumor cells in the highly cellular center of glioblastomas where neurites can be expected to be (much) more scarce? what about the presence of ‘pseudo-tripartite synapses’ in the ‘bulky’ areas of metastatic breast cancer in the cns? and (see topic 8): do bona fide glioma stem cells exist? nowadays, a rapidly increasing number of diagnostic and prognostic molecular markers can be used for an improved, ‘histo-molecular’ diagnosis of cns tumors [50]. multiple assays have been developed for this purpose, ranging from single gene tests to high-throughput, integrated techniques enabling detection of multiple genetic aberrations in a single workflow [51]. immunohistochemistry is a helpful, relatively inexpensive alternative for further molecular characterization of particular cns tumors. within a few years after its introduction, genome-wide methylation profiling has already had a revolutionary impact on cns tumor classification [24, 25, 52]. the papers discussed in topic 2 (‘improved molecular diagnosis of rare cns tumor types’) provide examples of the strength of this platform. however, such methylome analysis is relatively expensive. obviously, a test providing clinically very valuable information at low costs will much more easily be accepted as a routine diagnostic tool compared to an expensive test with limited clinical benefits. unfortunately, a clear framework for assessment of cost-effectiveness of molecular testing of cns (and other) tumors is lacking [53]. it is important to realize though that molecular diagnostics of (cns) tumors is relatively inexpensive compared to e.g. neuro-imaging and chemotherapy, and that a diagnosis that is ‘on target’ will not only have substantial clinical benefit for the patients but may also help to avoid unnecessary health care costs [52, 54]. meanwhile, since the publication of the who classification of cns tumours in 2016, the consortium to improve molecular and practical approaches into cns tumor taxonomy (cimpact-now) has already published several updates with suggestions on how exactly particular molecular markers can be used for improved diagnosis in clinical practice (see for summary of round 1 updates [55]). the findings described in topic 3 (‘refined molecular classification of non-wnt/non-shh medulloblastomas‘) and topic 4 (‘histogenesis of posterior fossa tumors in children’) may soon be incorporated into a more refined next who classification of these tumors (5th edition expected to be published within a year from now!). when shaping a who classification, one may indeed follow such a more progressive approach (i.e. introduce the latest tools and findings and include e.g. the most refined subclassification of non-wnt/non-shh medulloblastomas), or be somewhat more conservative in an attempt to better preserve long-term correlations, to avoid major disruption of patient management, and/or because of limited availability of (often expensive) diagnostic tools. obviously, finding the right balance is key, as there are many ‘strings attached’ (fig. 3). fig. 3. ‘strings attached’ to designing a next who classification of (cns) tumors. finding the right balance between a conservative and progressive attitude with regard to incorporating the latest findings into a next who tumor classification is key but can be challenging, not only because there are many strings attached, but also because of different aspects of the underlying dynamics. figure based on input of dr. david n. louis. next to diagnostic and prognostic markers, clinical neuropathologists are increasingly asked to perform testing for predictive biomarkers, e.g. for assessment of the likelihood of response of gliomas to particular immunotherapeutic approaches. only a limited number of newly diagnosed gliomas is reported to have an (occasionally inherited) mismatch repair (mmr) defect and/or a ‘hypermutator’ phenotype. recurrent gliomas more often show such a phenotype, especially so after alkylating chemotherapy [8]. assuming that the hypermutator status leads to an increase in expression of neoantigens, these gliomas/glioblastomas with a hypermutator phenotype could be good candidates for immune checkpoint blockade. unequivocal scoring of immunohistochemical staining for pd1/pd-l1 (surface proteins on tumor-infiltrating lymphocytes and tumor cells, respectively, and involved in suppression of the immune system) can be challenging. alternatively, mmr deficiency, microsatellite instability (msi), dna polymerase epsilon (pole) mutations, and high tumor mutational load (tml) are being used in this context, but assessment of these biomarkers can be challenging as well. for example, mmr gene (mlh1, msh2, msh6, and/or pms2) mutations often, but not always lead to lack of immunostaining of tumor cell nuclei for the corresponding protein(s). also, the techniques and thresholds to be used for optimal assessment of high tml are not yet settled. while for non-small cell lung carcinoma ≥10 mutations per mb is considered as high tml, it is unclear if this threshold should be used for gliomas as well [56, 57]. of course, identification and optimization of biomarkers for response to immunotherapy only makes sense if at least some patients can be expected to significantly benefit from that particular therapy. furthermore, the studies reported under topic 9 (‘personalized (neo)antigen vaccination therapy for glioblastoma’) indicate that biomarker discovery in this context is a moving target. in conclusion, ‘the times they are a-changin’ and one can expect that there is much more change to come. liquid biopsy-based diagnoses may emerge soon as a clinically helpful source of information. furthermore, a very recently published study showed that the use of artificial intelligence outperformed radiologists in the mammogram-based prediction of breast cancer [58]. similarly, deep learning/machine learning approaches may increasingly be used for automated, histology-based brain tumor classification and/or for predicting molecular markers based on mri or other neuro-imaging modalities [59, 60]. such developments could have significant impact on the ‘core business’ of the (neuro)pathologist. however, to quote the editor-in-chief of free neuropathology: ‘as long as neuropathology continues to be creative and innovative at the forefront of classifying and diagnosing neurological disorders and revealing their molecular pathogenesis …, its future will be bright’ [52]. references 1. filipski, k., et al., lack of h3k27 trimethylation is associated with 1p/19q codeletion in diffuse gliomas. acta neuropathol, 2019. 138(2): p. 331-334. 2. sievers, p., et al., rosette-forming glioneuronal tumors share a distinct dna methylation profile and mutations in fgfr1, with recurrent co-mutation of pik3ca and nf1. acta neuropathol, 2019. 138(3): p. 497-504. 3. hou, y., et al., papillary glioneuronal tumor (pgnt) exhibits a characteristic methylation profile and fusions involving prkca. acta neuropathol, 2019. 137(5): p. 837-846. 4. sharma, t., et al., 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p. 97-107. copyright: © 2020 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. association of acute disseminated encephalomyelitis (adem) and covid-19 in a pediatric patient feel free to add comments by clicking these icons on the sidebar free neuropathology 2:19 (2021) case report association of acute disseminated encephalomyelitis (adem) and covid-19 in a pediatric patient liam chen department of laboratory medicine and pathology, university of minnesota medical school, minneapolis, mn, usa corresponding author: liam chen · department of laboratory medicine and pathology · university of minnesota medical school · c515 mayo memorial building · 420 delaware street · minneapolis, mn 55455 · usa llchen@umn.edu submitted: 8 june 2021 accepted: 8 july 2021 copyedited by: deborah mcintyre published: 12 july 2021 https://doi.org/10.17879/freeneuropathology-2021-3388 keywords: sars-cov-2, covid-19, acute disseminated encephalomyelitis (adem), acute hemorrhagic leukoencephalitis (ahle), acute necrotizing encephalopathy of childhood (anec), demyelinating abstract cases of acute disseminated encephalomyelitis (adem) and its hyperacute form, acute hemorrhagic leukoencephalitis (ahle), have been reported in coronavirus disease 2019 (covid-19) patients as rare, but most severe neurological complications. however, histopathologic evaluations of adem/ahle pathology in covid patients are extremely limited, so far having only been reported in a few adult autopsy cases. here we compare the findings with an adem-like pathology in a pediatric patient taken through a biopsy procedure. understanding the neuropathology may shed informative light on the autoimmune process affecting covid-19 patients and provide critical information to guide the clinical management. numerous evidence has confirmed that coronavirus disease 2019 (covid-19), caused by severe acute respiratory syndrome coronavirus 2 (sars-cov-2) [1], affects the nervous system [2]. most of the associated neurological dysfunctions are mild symptoms such as anosmia and dysgeusia [3] while severe debilitating neurological disorders such as stroke and meningoencephalitis are less frequent [4, 5]. these manifestations are likely caused by sars-cov-2 entering brain through direct infection of olfactory neuroepithelium [6], or transmitted by circulating lymphocytes and monocytes that are able to cross the blood-brain barrier [7]. within the brain and spinal cord, various cell types including endothelial cells, vascular pericytes and smooth muscle, neurons and glia all express angiotensin converting enzyme 2 (ace2) receptor [8, 9], the primary receptor for sars-cov-2 spike protein [10]. it is true that sars-cov-2 could be detected in olfactory epithelium [11], brain and cranial nerves by rt-pcr [12, 13], immunohistochemistry (ihc) [14], in situ hybridization (ish) [11] and electron microscopy [15]. in addition, neuropathological findings such as leptomeningeal and parenchymal lymphocytic inflammation, microglial nodules and neuronophagy are compatible with a diagnosis of viral meningoencephalitis [12, 16]. nevertheless, neither viral inclusions nor cytopathic changes have been observed on hematoxylin and eosin (h&e) stained slides. furthermore, in the few studies that have reported evidence of viral rna or protein in the cranial nerves or brainstem, the degree and distribution of neuropathologic changes have shown no correlation with the amount of virus in a given area of pathology, suggesting that the pathology is rather secondary to the systemic effects of viral infection. indeed, convincing evidence has elucidated that systemic hypercoagulability plays an important role in covid-19 related stroke process, as sars-cov-2 could induce cytokine storm and endotheliopathy [17, 18], which in turn lead to the histologic findings of microthrombi, hemorrhages and infarcts [19, 20]. in contrast, there is still considerable debate over whether parainfectious, autoimmune mediated process such as acute disseminated encephalomyelitis (adem), or its hyperacute form, acute hemorrhagic leukoencephalopathy (ahle), can truly be attributed to covid-19. one reason for this controversy is the lack of neuropathological descriptions of adem and ahle despite that a handful clinical and imaging case studies of covid-19 patients have suggested lesions characteristic of adem or ahle [21-31]. so far, there are only few case reports with neuropathological features of adem and ahle in adult patients who died from complications of covid-19 [32-34]. another reason for the controversy, as will be discussed further below, is whether the observed ademor ahle-like pathology is truly due to a primary demyelinating process or merely a secondary white matter injury of a comorbid vascular disease or a combination. r. ross reichard et al. reported the neuropathological findings of a 71-year-old patient who died from complications of covid-19 [32]. hemorrhagic white matter lesions were present throughout the cerebral hemispheres. luxol fast blue/ periodic acid–schiff (lfb/pas) identified loss of myelin, pas-positive macrophages, and fragmented axonal processes within these lesions. it has features of ahle, although necrotic blood vessels and perivascular inflammation were not identified within the hemorrhagic lesions. a second distinct pathology identified was characterized by small subcortical white matter pallor with a variable perivenular distribution, resembling an adem-like histological appearance. notably, prominent acute axonal injury was present in the regions of myelin loss, which is not characteristic of classic adem [32]. jamie m. walker et al. presented two cases of fatal covid-19 with severe neurologic sequalae [33]. one was a 51-year-old woman with an irregular pattern of demyelination centered primarily around veins and venules. both perivascular cd3+ t-lymphocytes as well as frequent perivascular and parenchymal cd68+ histiocytes and activated microglial cells were present, consistent with a diagnosis of adem. another autopsy examination of a 64-year-old demonstrated myelin pallor, perivascular cd3+ t-lymphocytes, axonal spheroids, as well as numerous ringand ball-hemorrhages throughout the white matter with central blood vessels showing fibrinoid necrosis, most consistent with a diagnosis of ahle. it should be noted that in all three aforementioned cases, global hypoxic-ischemic injury was present, in addition to microscopic cortical infarcts in the first case [32] and large areas of cerebral intraparenchymal hemorrhages in the third case [33]. thus, the neuropathological lesions should be interpreted with caution and the possibility of a cerebrovascular origin with secondary demyelinating pathology should be considered. adem and ahle usually affects children and young adults after an infection or vaccination [35]. however there are very few reported cases of adem in pediatric population with sars-cov-2 infection based on clinical features and mri findings [25, 36, 37]. this is consistent with the differing clinical presentations of covid-19 in children and adult patients. recently, we have examined a brain biopsy taken from an 8-year-old girl who was admitted with new-onset seizures. on hospital day 2, covid-19 spike igg antibody testing was positive. brain mri demonstrated extensive t2 hyperintensity centered at bilateral basal ganglia, extending to the frontal white matter, external and internal capsules, corpus callosum, thalami, insula, as well as the cerebellar hemispheres, brainstem and the spinal cord. interval mri imaging demonstrated persistent diffusion restriction in the affected white matter throughout much of the central white matter tracts, cerebral peduncles, corticospinal tracts and cerebellar white matter, suggesting ongoing demyelination, but no evidence to suggest hemorrhages. three weeks later, there was vacuolating necrosis in bilateral basal ganglia. ventriculoperitoneal shunt placement and stereotactic brain biopsy were performed on hospital day 48. neuropathological examination of the brain biopsy tissue revealed subcortical white matter pallor with perivascular lymphocytic infiltrates centered primarily around venules (figure 1a). immunohistochemical staining for sars-cov-2 spike protein was negative. no microglial nodules or evidence of neuronophagia was present. demyelination was confirmed by lfb stain (figure 1b) whereas axons were relatively preserved as shown by neurofilament immunostain (figure 1c). interestingly, app immunostain highlighted damaged, swollen axons in areas surrounding the vessels, a pattern seen in adult patient as well (figure 1d). the lymphocytes were composed predominantly of cd3-positive t cells (figure 1e) whereas cd68 stain highlighted numerous perivascular macrophages and the diffuse distribution of activated microglia (figure 1f). figure 1. neuropathological findings of a brain biopsy from a child post sars-cov-2 infection. a. h&e section of white matter pallor, reactive astrogliosis and perivascular lymphocytic infiltrates around small veins and venules. b. lfb stain demonstrates the perivascular myelin loss within the subcortical white matter lesion. c. neurofilament immunostain shows preservation of most axons in the region of myelin loss. d. app immunostain identifies axonal swellings adjacent to the perivascular areas. e. perivascular lymphocytes are predominantly cd3-positive t cell. f. cd68 immunostain confirms both perivascular and diffuse distributions of macrophages within the area of white matter pallor. scale bar, 100 μm in a-e. in this case, the absence of confounding concomitant cerebrovascular lesions makes the pathological interpretation relatively straightforward. these adem/ahle cases represent rare, but the most severe end of the covid-19 neuropathologic spectrum. consequently, adem/ahle should be a consideration during evaluation of patients, especially children with encephalopathy, seizures and/or focal neurologic deficits after recovering from covid-19. another intriguing development in this pediatric patient is the later mri findings of bilateral basal ganglia necrosis, suggestive an even rarer entity, acute necrotizing encephalopathy (ane), also referred as acute necrotizing encephalopathy of childhood. ane is characterized by multiple, symmetrical lesions in the thalami, striatum, cerebral white matter, and brain stem [38]. despite its association with viral infection, ane is not considered an inflammatory encephalitis in comparison to adem and ahle. in fact, it has been suggested that an intense surge of cytokines causes damage to the blood-brain barrier with necrosis as a secondary effect [39]. given sars-cov-2 is a cytokine storm trigger, it is not surprising to observe ane as a probable association of covid-19 [40, 41]. it would be important to understand whether these parainfectious demyelinating diseases reflect distinct pathological processes or a continuum of a single disease.  references 1. zhu, n., et al., a novel coronavirus from patients with pneumonia in china, 2019. n engl j med, 2020. 382(8): p. 727-733. 2. lou, j.j., et al., neuropathology of covid-19 (neuro-covid): clinicopathological update. free neuropathol, 2021. 2. 3. lechien, j.r., et al., olfactory and gustatory dysfunctions as a clinical presentation of mild-to-moderate forms of the coronavirus disease (covid-19): a multicenter european study. eur arch otorhinolaryngol, 2020. 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under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. inhibiting the mitochondrial pyruvate carrier does not ameliorate synucleinopathy in the absence of inflammation or metabolic deficits feel free to add comments by clicking these icons on the sidebar free neuropathology 1:33 (2020) original paper inhibiting the mitochondrial pyruvate carrier does not ameliorate synucleinopathy in the absence of inflammation or metabolic deficits wouter peelaerts1,2*, liza bergkvist1,#*, sonia george1, michaela johnson1, lindsay meyerdirk1, emily schulz1, jennifer a. steiner1, zachary madaj3, jiyan ma1, katelyn becker1, k. peter r. nilsson4, jerry r. colca5 and patrik brundin1 1 center for neurodegenerative science, van andel institute, grand rapids, mi, usa. 2 ku leuven, laboratory for neurobiology and gene therapy, dept. of neurosciences, leuven, belgium. 3 bioinformatics and biostatistics core, van andel institute, grand rapids, mi, usa. 4 department of physics, chemistry and biology, linköping university, linköping, sweden. 5 metabolic solutions development company, kalamazoo, mi, usa. # current affiliation: dept. of neurobiology, care sciences and society, karolinska institute, stockholm, sweden. * these authors contributed equally to this work. corresponding author: patrik brundin · center for neurodegenerative science · van andel institute · grand rapids, mi · usa patrik.brundin@vai.org submitted: 01 october 2020 accepted: 21 november 2020 copyedited by: jerry j. lou published: 25 november 2020 https://doi.org/10.17879/freeneuropathology-2020-3049 additional resources and electronic supplementary material: supplementary material keywords: parkinson’s disease, synuclein, neurodegeneration, metabolism, synucleinopathy abstract epidemiological studies suggest a link between type-2 diabetes and parkinson’s disease (pd) risk. treatment of type-2 diabetes with insulin sensitizing drugs lowers the risk of pd. we previously showed that the insulin sensitizing drug, msdc-0160, ameliorates pathogenesis in some animal models of pd. msdc-0160 reversibly binds the mitochondrial pyruvate carrier (mpc) protein complex, which has an anti-inflammatory effect and restores metabolic deficits. since pd is characterized by the deposition of α-synuclein (αsyn), we hypothesized that inhibiting the mpc might directly inhibit αsyn aggregation in vivo in mammals. to answer if modulation of mpc can reduce the development of αsyn assemblies, and reduce neurodegeneration, we treated two chronic and progressive mouse models; a viral vector-based αsyn overexpressing model and a pre-formed fibril (pff) αsyn seeding model with msdc-0160. these two models present distinct types of αsyn pathology but lack inflammatory or autophagy deficits. contrary to our hypothesis, we found that a modulation of mpc in these models did not reduce the accumulation of αsyn aggregates or mitigate neurotoxicity. instead, msdc-0160 changed the post-translational modification and aggregation features of αsyn. these results are consistent with the lack of a direct effect of mpc modulation on synuclein clearance in these models. introduction synucleinopathies are age-related neurodegenerative diseases characterized by α-synuclein (αsyn) protein misfolding, inflammation and metabolic deficits. people with parkinson’s disease (pd), dementia with lewy bodies (dlb) and multiple system atrophy (msa) present with peripheral and central synucleinopathies that coincide with a wide range of non-motor (e.g., hyposmia, constipation and rapid eye movement sleep behavior disorder) and classical motor symptoms1. the disease mechanisms underlying synucleinopathies are not completely understood, but evidence suggests that mitochondrial dysfunction, neuroinflammation and altered protein homeostasis all play a role, as reviewed in detail elsewhere2. epidemiological studies have indicated a link between pd and type 2 diabetes3-5. type 2 diabetes is associated with a 38% increased risk of developing pd3,4. on a molecular level, both people with pd and those with type 2 diabetes have similar metabolic abnormalities such as mitochondrial dysfunction and insulin resistance5. given these links between pd and metabolic dysfunction, clinical trials in pd were initiated to test drugs already approved for the treatment of diabetes. in a randomized, double-blinded placebo-controlled trial with the brain penetrant insulin sensitizing glucagon-like peptide 1 (glp1)-agonist exenatide, it was shown that exenatide treatment was associated with a reduced decline in motor dysfunction in pd patients6. specifically, once-weekly injections of the slow release form of exenatide for 48 weeks resulted in improvements in the mds-updrs (part iii)-defined primary endpoint with effects persisting after a 12-week wash out period6. in a post hoc analysis evaluating non-motor symptoms, secondary end points such as patient and observer-led scales assessing mood also showed positive effects from the treatment7. exenatide is currently in a phase iii trial in 200 pd patients who will be treated for 2 years, with the primary endpoint focused on disease modification (clinicaltrials.gov identifier nct0423296). a second insulin sensitizer that has been associated with a lower risk of pd is pioglitazone3,4. however, when evaluated in a phase 2 clinical pd trial, pioglitazone did not have any significant effect on disease progression as measured via msd-updrs (part iii)8. pioglitazone belongs to a class of anti-diabetic drugs, termed thiazolidinediones or tzds, and was believed to exert its anti-diabetic effects via activation of the transcription factor peroxisome proliferator-activated receptor-γ (pparγ). due to strong binding of pioglitazone to pparγ the drug has several negative side effects. as a result, pparγ-sparing tzds, including msdc-0160 were developed. interestingly, when compared to pioglitazone, msdc-0160 has similar anti-diabetic effects but it does not activate pparγ, indicating that tzds act on a different receptor to elicit effects. the primary target was later found on the inner membrane of mitochondria and was identified as the previously unknown mitochondrial pyruvate carrier (mpc)9. because of the link between tzds and a lowered associated risk of pd, msdc-0160’s limited side effects and its bioavailability in brain, we previously tested it in cell and animal models of pd and found that msdc-0160 is neuroprotective10. specifically, msdc-0160 protects against dopaminergic cell loss in the substantia nigra in neurotoxin-challenged mice and in a genetic mouse model with hemizygous loss of the transcription factor engrailed 1 (en1), which is believed to entail mitochondrial deficits11. we also observed a mitigation of neuron death of msdc-0160 treatment in caenorhabditis elegans (c. elegans) in which we over-expressed αsyn. taken together, we hypothesized that the beneficial effects of msdc-0160 were due to its anti-inflammatory effects and its ability to normalize metabolic deficits. the effects of msdc-0160 on αsyn aggregation in progressive, chronic pd models have previously not been investigated. in this study, we therefore evaluated msdc-0160 in two different α-syn based rodent models of pd; 1) a rat model with adeno-associated virus (aav) vector over-expression of human αsyn and 2) a mouse αsyn pre-formed fibril (pff) seeding model. unexpectedly, we found that msdc-0160 increased the levels of aggregated αsyn in the aav-overexpression model, possibly via increased oxidation of soluble αsyn. in the pff seeding model, msdc-0160 also increased αsyn burden transiently; a significant difference between the treatment groups was observed five weeks after pff injection, but it was not present at the later 13-week timepoint. we examined both models further for inflammatory and lysosomal markers, and found them both to be unaltered, even in the presence of robust αsyn pathology. we conclude that while neuroprotective actions of msdc-0160 may occur via restoring inflammatory and metabolic deficits, we could not demonstrate effects on protein aggregation or protein spreading in these rodent models of synucleinopathies. methods animals twelve-week-old male and female c57bl/6j wildtype (wt) mice were sourced from the jackson laboratory. eight-week old female rats were obtained from charles river. animals were housed with a maximum of four mice or two rats per cage under 12-h light/12-h dark cycles with free access to food and water. for a direct comparison of msdc-0160 and pioglitazone biodistribution of their metabolites in vivo, msdc-0160 and pioglitazone were administered via oral gavage for 3 days for a dose of 30mg/kg/day. collection of plasma, csf and brain mitochondria was performed after day 3. for the chronic administration of msdc-0160, mice and rats were fed a diet of chow formulated to deliver msdc-0160 (30 mg/kg) or control chow starting at 1 week after stereotactic surgery. mice were euthanized at two different time points: one and three months after pbs/pff injections. rat were euthanized four months after viral transduction. the housing of animals and all procedures were performed in accordance with the guide for the care and use of laboratory animals (united states national institutes of health) and were approved by the van andel research institute's animal care and use committee. pff production and ob injections mouse αsyn pffs were produced as described previously12. before surgery, pffs were prepared by the sonication of αsyn aggregates in a water-bath cup-horn sonicator for four min (qsonica, q700 sonicator, 50% power, 120 pulses 1 s on, 1 s off). mice were anesthetized with isoflurane/oxygen and injected unilaterally in the right olfactory bulb with either 0.8 μl of pff (5 μg/μl; n=36, 18 females/males) or 0.8 μl of pbs as a control (n=36, 18 females/males). coordinates from bregma: ap: + 5.4 mm; ml: +/0.75 mm and dv: 1.0 mm from dura. injections were made at a rate of 0.2 μl/min using a glass capillary attached to a 10 μl hamilton syringe. after injection, the capillary was left in place for three min, before being slowly removed. viral vector production and nigral surgeries raav2/5-cmvsyn-human-αsyn and raav2/5-cmvsyn-gfp production was performed as described previously13. genome copies were determined via qpcr and two viral vector titers were used for injection into the rat sn. nigral injections were performed under general anesthesia using an isoflurane/oxygen mixture. for the αsyn low dose and αsyn high dose a total of 1.2x1011gc and 2.5x1011gc were injected, respectively. as a control, 3x1011gc of gfp expressing viral vector or pbs were injected. coordinates from bregma: ap: 5.3 mm, ml: 2.0 mm and dv: 7.2 mm measured from dura14. a volume of 3 μl of viral vector was infused at a rate of 0.25 μl/min using a 30g needle and 10 μl hamilton syringe. msdc-0160 metabolite analysis mice and rats were allowed free access to chow containing either msdc-0160 (30 mg/kg) or placebo for up to four months. for mice, whole blood was collected five and 13 weeks post-surgery from the submandibular vein in 6 ml bd heparinized tubes. for rats, whole blood was collected in identical tubes via cardiac puncture four months after viral vector injection. the samples were centrifuged for 15 min at 4°c (2000 x g), followed by collection of the supernatant (plasma) which was snap frozen and stored at -80°c. for collection of brain mitochondria, brain was isolated and homogenized via dounce homogenization in a sucrose homogenization buffer (225 mm sucrose, 6 mm k2hpo4, 5 mm mgcl2, 20 mm kcl and 2 mm egta). samples were centrifuged at 750g to remove cell debris followed by a second step of centrifugation at 15,800g. the resulting pellet was resuspended in acetone to dry and collected in control plasma to store at -20°c for future analysis. frozen heparinized plasma samples were blind-coded and analyzed by charles river laboratories (mattawan, mi) for the concentrations of the msdc-0160 metabolite msdc-0037 in study protocol 1443-007b. in short, each 25 μl aliquot of standard, qc sample, or study sample was mixed with 10 μl of working internal standard solution (1,250 ng/ml in methanol/water [50/50, v/v]) and 140 μl of water. the samples were vortexed and transferred to an isolute sle plate and eluted with 1.0 ml of mtbe. the samples were evaporated and reconstituted with 100 μl of methanol followed by 100 μl of 5 mm ammonium formate in water. the samples were mixed and transferred to a clean 96-well plate. an aliquot was injected onto an lc ms/ms system for analysis. the liquid chromatography system used a macmod ace 3 c18 column, 2.1 x 50 mm (3 μm particle size) with a gradient flow consisting of 5 mm ammonium formate in water/methanol (75/25, v/v) and 5 mm ammonium formate in methanol/acetonitrile/water (72/18/10, v/v/v) at a flow rate of 500 μl/minute. the analyte, metabolite, and internal standard were detected using a sciex api 5000 triple quadrupole lc ms/ms system equipped with an esi (turboionspray®) ionization source operated in the positive ion mode. the multiple reaction monitoring transitions of the respective [m+h]+ ions were used to monitor msdc-0160 and msdc-0037 as have previously been described for clinical studies15. western blotting rats were euthanized with sodium pentobarbital (130 mg/kg; sigma) and transcardially perfused with pbs to remove blood. brains were isolated and the substantia nigra was dissected on ice using a rat brain matrix. isolated samples were snap frozen and stored at -80°c for later analysis. frozen tissue was weighed in equilibrated eppendorf tubes and homogenized in pbs 10% w/v with protease and phosphatase inhibitors (thermofisher). homogenization was performed by probe sonication at 4°c for 2 rounds of 15 s pulses at 0.5 hz with a 10% amplitude. the whole homogenates were centrifuged for 10 min at 6000 x g at 4°c. the pellet was discarded and supernatant was collected for analysis. to isolate insoluble αsyn, 20% sarkosyl was added to the whole homogenate to result in a 1% sarkosyl pbs sample that was incubated on a rotating shaker for 1 h at room temperature. after incubation, samples were centrifuged for 10 min at 6000 x g at 4°c to remove remaining cell debris. the cleared supernatant was centrifuged at 100,000 x g for 60 min and the resulting pellet was gently washed in pbs after which it was resuspended in 1% sarkosyl in pbs with protease and phosphatase inhibitors. a second centrifugation step at 100,000 x g for 10 min was performed to collect the 1% sarkosyl insoluble pellet. protein concentrations were estimated using a bca kit (thermo fisher). samples were prepared with laemmli buffer containing 2% sds, heated at 95°c for 10 min for denaturation and stored at -80°c. protein samples were separated via 4-15% sds page (biorad) and transferred to a pvdf membrane using the biorad turboblot system. pvdf membranes were blocked with 5% bsa in pbs during 30 min at room temperature. overnight incubation with primary antibody, iba-1 (1:500, wako), human αsyn (4b12, 1:1000, biolegend), pser129-αsyn (ps129, 1:5000, abcam), lamp1 (1:1000, abcam), caspase (1:500, abcam) was followed by incubation with hrp-conjugated secondary antibody (cell signaling technology). signal was detected by chemiluminescence (pico chemiluminescent substrate, thermo fisher) using a biorad imager. western blot bands were quantified using imagej software. behavioral analysis to monitor functional deficits related to nigral dopamine neuron dysfunction and death, rats were subjected to a cylinder test to evaluate spontaneous forelimb use. rats were placed in a clear glass cylinder and were filmed for a total of 30 contacts with the cylinder. percentage of forepaw use was expressed as the percentage of right forepaw touches over total number of touches. non-lesioned rats score around 50%. to examine olfactory function in mice, the buried pellet test was carried out as recently described by our laboratory in johnson et al.16 in short, the mice were fasted overnight starting three days prior to testing. throughout the experiment, their body weight was monitored and mice who lost more than 10% of their original body weight were excluded from further fasting and from the experiment. at the first day of testing, a surface test was performed, where the treat (bio-serv, fruit crunchies) was placed on top of the clean bedding. mice who were not motivated to eat the treat were excluded from further testing (all mice in this study were motivated by the treat and were thus included). for four consecutive days, the treat was buried at different locations 1 cm under the clean bedding and latency for mice to uncover it was recorded. a maximum time was set at 300 s, and if a mouse did not uncover the treat within that time frame, 300 s was recorded as its latency. the average latency to uncover the treat per animal is presented here, with nine to ten animals analyzed per group. immunohistochemistry mice and rats were euthanized with sodium pentobarbital (60 mg/ml; sigma) and transcardially perfused with room temperature 0.9% saline followed by ice-cold 4% paraformaldehyde (pfa) in 0.1 m phosphate buffer. brains were removed, post-fixed overnight at 4°c in 4% pfa and subsequently placed in 30% sucrose. brains were frozen and coronal sections of 40 μm were cut on a microtome (leica) and collected as serial tissue sections. for immunohistochemistry in mice, a series (every 240 μm) of coronal free-floating sections were stained using 1:10000 anti-pser129 αsyn primary antibody (abcam, ab51253), 1:1000 anti-neun primary antibody (millipore, ab377), 1:5000 anti-nitrated αsyn primary antibody (thermo fisher, 35-8300) or 1:800 anti-iba1 primary antibody (wako, 019-19741) over night. for immunohistochemistry in rats, a series (every 240 μm) of coronal free-floating sections of sn was stained using 1:10000 anti-pser129 αsyn primary antibody (abcam, ab51253), 1:5000 anti-nitrated αsyn primary antibody (thermo fisher, 35-8300), 1:2000 anti-th (abcam, ab137869), or 1:800 anti-iba1 primary antibody (wako, 019-19741) overnight. corresponding secondary antibodies (1:500 goat anti-rabbit/goat anti-mouse biotinylated secondary antibody (vector laboratories, ba-1000 and ba-9200) were added. the antibody signal was amplified using a standard peroxidase-based method (vectastain abc kit) and developed using a dab kit (vector laboratories). immunostained tissue sections were mounted onto gelatin coated glass slides. neunand th-immunostained slides were counter stained with cresyl violet (cv). after dehydration, slides were sealed by coverslip with cytoseal 60 mounting medium (thermo fisher scientific). for lamp2 immunostaining, using fluorescence to detect antibody binding, sections were incubated overnight with 1:1000 anti-lamp2 primary antibody (abcam, ab13524) overnight, followed by a 2 h incubation with a fluorescent secondary antibody (thermo fisher/invitrogen, alexa fluor 488). dapi was used to visualize cell nuclei. for the co-stain between h-ftaa and pser129, tissue sections were incubated with 1:1000 anti-pser129 αsyn primary antibody (abcam, ab51253) for 2h at rt followed by a 1h incubation with 1:200 secondary goat anti-rabbit antibody alexa 647 (thermo fisher, a32733) and dapi to visualize cell nuclei. finally, 0.5 μm h-ftaa was added and allowed to incubate with the tissue for 30 min at rt. fluorescently stained tissue was sealed by coverslip using hard-set antifade mounting media (vector laboratories, h-1400). a nikon a1plus-rsi laser scanning confocal microscope (nikon) was used to image these sections. pser129 quantification z-stacks of mounted αsyn pser129-stained tissue sections were captured at 20x magnification using a whole slide scanner (zeiss, axioscan z1) at a 0.22 μm/pixel resolution. extended depth focus (edf) was used to collapse the z-stacks into 2d images as they were collected. tissue thickness was set to 20 μm, z-stacks were collected at 3 μm intervals and the method used was contrast. the images were exported with 85% compression as .jpeg files. the digitized images were then uploaded aiforia™ image processing and management platform (aiforia technologies, helsinki, finland) for analysis with deep learning convolution neural networks (cnns) and supervised learning. a supervised, multi-layered, cnn was trained on annotations from digitized αsyn pser129-stained coronal slices to recognize total αsyn-positive staining. the algorithm was trained on the most diverse and representative images from across multiple pser129 datasets to create a generalizable ai model capable to accurately detecting αsyn pser129-stained profiles in slides collected by multiple investigators. ninety-one images constituted training data. the ground truth, or features of interest used to train the ai model, were annotated for each layer within the aiforia™ cloud platform and constituted input data for each cnn. the first feature layer was annotated using semantic segmentation to distinguish the total tissue from the glass slide. the second feature layer was annotated using semantic segmentation to distinguish total αsyn pser129-positive staining. features that were considered artifact (glass slide, debris) were annotated as background, and constituted additional input training data for the multi-layered cnn. the regions corresponding to the aon and the prh were outlined according to the allen mouse brain atlas (allen institute) and the cnn was used to quantify the area percentage that contained αsyn pser129-positive signal. for each brain region, the average area with αsyn pser129-staining is reported as a percentage of the whole area assayed. for the one-month time point, six animals per group were analyzed and for the three-month time point seven to eight animals per group were included in the analysis. stereology surviving dopamine neurons and total neuronal cell counts were quantified using unbiased sampling and blinded stereology. for the aav αsyn over-expression model, tyrosine hydroxylase (th), αsyn pser129 and nitrated αsyn-positive cells were counted in the snpc and snpr in a series of seven sections at an interval of six sections at 40 μm thickness (240 μm intervals). contours of the regions were drawn at 10x magnification and quantifications were performed at 60x using a 200 μm counting frame. dissector height was 12 μm with a 3 μm guard zone. the gunderson estimated m=1 error was less than 0.1 and a total of eight animals per group was counted. for the αsyn pff seeding model, neunand cv-positive cells were counted in the aon, which was outlined according to the allen mouse brain atlas (allen institute) using a 2.5x objective. the quantification of neun and cv-positive cells was performed on every 6th aon (240 μm) section using the optical fractionator probe in stereo investigator software (mbf bioscience). the counting was accomplished using a 60x oil objective, with the grid set to 200x200 and the frame set to 40x40. the dissector height used was 10 μm, with a 3 μm guard zone. the gunderson estimated m=1 error was less than 0.1 and a total of nine to ten animals per group were counted. assessment of microglia morphology microglia hydraulic radius (area/perimeter ratio) was investigated as previously described13. in short, color (rbg) images were generated using a 60x oil objective. following imaging, a custom matlab script assessed the morphology of the imaged microglia, calculating the area/perimeter ratio. for the pff seeding model, five to six animals per group were analyzed. microglia were imaged from every 6th aon section and a minimum of 30 individual microglia was analyzed per animal, with the average microglia area/perimeter ratio reported. quantification of fluorescent lamp2 signal images corresponding to the aon were captured using a 20x oil objective on a nikon a1plus-rsi laser scanning confocal microscope (nikon) in a blinded manner. the area percentage occupied by lamp2 positive signal was quantified using fiji software (nation institute of health) after an unbiased threshold was applied. the average area percentage calculated for each animal is reported here. for the pff seeding model, a total of six mice per group were analyzed. statistics all aav and pff mouse model data were analyzed using r v3.6.0 (https://cran.r-project.org/) and assumed two-sided hypothesis tests with a significance level <0.05 after benjamini-hochberg multiple testing corrections. in the pff model, neuronal cell count, iba1 and pser129 quantification were done in parallel with another treatment group (pt302, data not shown), meaning they share the same set of placebo treated animals; the multiple testing corrections take into account these additional comparisons. for experiments that analyzed both hemispheres of the brain independently (e.g. ipsilateral versus contralateral) the appropriate mixed-effects model was used (r package lme4 https://cran.r-project.org/web/packages/lme4/index.html unless otherwise noted). count data were analyzed using negative binomial regressions; mixed-effects negative binomial regressions were done using the r package glmmtmb and the ‘nbinom1’ family (https://cran.r-project.org/web/packages/glmmtmb/index.html). time-to-event data (e.g. the buried pellet test) were analyzed using cox proportional hazards regression. all other data types were analyzed via linear regression with log and square-root transformed outcomes as needed based on standard regression diagnostics (i.e. normality of residuals and homoscedasticity) (log: if αsyn pser129 and soluble αsyn; square-root: soluble αsyn pser129, insoluble human αsyn, insoluble αsyn pser129). cook’s distance was used to assess individual observations with high amounts of leverage (set as 4/(n-p-1)). for linear regressions with highly influential observations, robust regressions with mm estimation were used. zero variance data (i.e. dab αsyn pser129+, if αsyn pser129+, soluble human αsyn, and nissl+ measures across both naive and gfp injected animals), logistic regression with a firth correction was used to determine if the odds of detection versus no detection differed between groups. an equivalence interval of +/-10% was used to determine if groups had strong evidence for equivalence based on their 95% confidence intervals. results inhibiting the mpc in a viral vector-based model of human α-syn overexpression to determine the effects of inhibiting the mpc on αsyn aggregation, we used a slowly progressing viral vector-based model that overexpresses αsyn. we selected two doses of viral vector, which from here on will be referred to as ‘αsyn low’ (4.3x1013gc/ml) and ‘αsyn high’ (8.6x1013gc/ml). these two titers yield a 4and 6-fold overexpression of αsyn in rat snpc, respectively (fig. 1a, 3b). fig 1. msdc-0160 treatment does not influence nigral dopaminergic cell viability in a progressive αsyn overexpression model. a) experimental overview of the αsyn viral vector overexpression model in rat sn. b) th expression and nissl+ cells in rat sn for animals treated with placebo or msdc-0160 in control or experimental conditions. scale bar = 500 μm. c) stereological quantification of total dopamine neurons in rat snpc for ipsiand contralateral sides. the contralateral hemisphere had significantly more th cells than the ipsilateral in the αsyn high mice (p = 0.001, ~22.5% more cells ci = 10.3% 36.0%, negative binomial mixed-effect model, n = 8, s.e.m.). in the ipsilateral side, αsyn high had significantly fewer th cells than naïve, gfp and αsyn low (p = 0.031, 14.9% fewer ci = 4.8 – 23.9, *p = 0.05, 13.4% fewer 95% ci 3.1 22.6 and p = 0.0313, 15.6% fewer ci = 24.55.6, respectively, negative binomial mixed-effect model, n = 8, sem). d) stereological quantification of nissl+ cells in rat sn for ipsiand contralateral sides. for the αsyn high group, the ipsilateral side has ~ 13% fewer nissl+ cells across both treatment groups than the contralateral (p < 0.0001, 95% ci 6.4 20.1, negative binomial mixed-effect model, n = 8, sem). αsyn high shows significantly fewer nissl+ cells compared to animals injected with naive, gfp, or low amounts of αsyn (p < 0.001 for all 3, gfp: 15% fewer ci = 8% 21%, naive: 18.4% fewer ci = 12.0% 24.4%, αsyn low: 16% fewer ci = 22.2% 9.3%, negative binomial mixed-effect model, n = 8, sem). female rats were stereotactically injected in snpc at 8 weeks of age with raav2/5 cmv-syn-αsyn or raav2/5 cmv-syn-gfp expressing vector that served as a control (fig. 1a). one week post-surgery, the animals were allowed free access to chow containing either msdc-0160 (30mg/kg) or placebo for four months. in order to evaluate the uptake and distribution of msdc-0160, we analyzed the levels of exposure of msdc-0160, pioglitazone and its metabolites in rat plasma, csf and brain mitochondria 20 hours after administration (supplementary fig. 1). hydroxymitoglitazone, the active metabolite of msdc-0160, was found at higher levels, compared to pioglitazone metabolites, in plasma and csf. in brain mitochondria hydroxymitoglitazone was also found at higher levels then pioglitazone-oh or other metabolites, showing its interaction with brain mitochondria but also suggesting that the interaction of msdc-0160 metabolites with mitochondria is stronger compared to pioglitazone metabolites (supplementary fig. 1). during the time of chow administration, we did not observe any differences in animal body weight or chow consumption (supplementary fig. 2a, b). at the four-month timepoint, plasma was collected and the presence of hydroxymitoglitazone (msdc-0160 metabolite) was quantified (supplementary fig. 2c), showing the presence and metabolization of msdc-0160 at expected levels. motor behavior was examined four months after viral transduction using the cylinder test. no difference in motor behavior was apparent between any of the experimental groups, nor did we observe any statistically significant differences between placebo or msdc-0160 treated groups (supplementary fig. 3). when assessing the total number of thand nissl-positive neurons in the sn via stereology we find a significant decrease in thand nissl-positive cells between the αsyn high group and all other experimental treatment groups (fig. 1a-b), suggesting dose-dependent neurotoxic effects of αsyn expression. no cell loss was observed in the gfp group, showing that no toxic effects are due to the viral vector itself. in addition, when the snpc is separately analyzed for its anterior (bregma -4.8 mm) and posterior (bregma -6.2 mm) areas, th loss in the anterior is much more significant compared to the posterior region (supplementary fig. 4 a-d). this indicates that αsyn expression from the overexpressing αsyn vector is higher in the anterior snpc since it is adjacent to the injection site, and lower in the posterior portion, which again confirms vector-driven αsyn neurotoxic effects with no neurotoxicity observed for the gfp control group. next, we performed stereological counts of nigral cells positive for phosphorylated αsyn (αsyn pser129) (fig. 2a). unexpectedly, in the αsyn low group, animals treated with msdc-0160 show significantly more αsyn pser129+ cells compared to the placebo group (12132 versus 7918 cells, **p = 0.002, negative binomial regression with benjamini-hochberg multiple testing adjustments, sem, n = 8) (fig. 2b). no significant effect was detected in the αsyn high group between msdc-0160 and the placebo group. figure 2. a metabolic switch leads to increased αsyn pathology in rat sn. a) overview of pser129-αsyn positive cells in rat sn. scale bars for higher and lower magnification represents 450 μm and 100 μm, respectively. b) stereological quantification of αsyn pser129 positive cells in rat sn. treatment with msdc-0160 leads to a significant increase in phosphorylated cells in the αsyn low group compared to placebo (n = 8, sem, **p < 0.01 and ***p < 0.001, negative binomial regression with benjamini-hochberg multiple testing adjustments, sem, n = 8)). we further analyzed phosphorylated levels of αsyn via biochemical analyses and found that both soluble αsyn pser129 as well as 1% sarkosyl insoluble αsyn pser129 (1% p129-αsyn) are increased in the msdc-0160 treated animals compared to the placebo group (*p = 0.046, 95% ci 0.039 – 0.471, square-root linear regression, sem, n = 6 for soluble αsyn pser129 and *p = 0.013, 95% ci 0.129 – 0.691, square-root linear regression, sem, n = 6 for 1% sarkosyl insoluble αsyn pser129) (fig. 3e, f). this increase was observed in the αsyn high group between msdc-0160 and placebo animals whereas a similar trend was noticeable in the αsyn low group between treatment conditions (fig. 3e, f). this indicates that administration of msdc-0160 results in increased αsyn aggregation in the slowly progressing viral vector-based model that overexpresses αsyn. figure 3. αsyn aggregation is increased in rat sn after inhibition of the mpc. a) inhibition of the mpc has no effect on viral vector-mediated expression of gfp. b) detection of total levels of αsyn in naïve and experimental conditions. αsyn overexpression leads to a 4and 6-fold increase in αsyn low and high conditions compared to naïve rats. c,d) isolation of sequentially extracted soluble and 1% sarkosyl insoluble human αsyn (1% hu-αsyn) shows no significant differences in aggregated αsyn after treatment with msdc-0160. e,f) inhibition of mpc leads to significantly increased levels of soluble and 1% sarkosyl insoluble αsyn pser129 (1% p129-αsyn) in the αsyn high group (n = 6, sem, *p < 0.05 square-root linear regression, sem, n = 6). g) representative western blot figures for different conditions tested. αsyn has been shown to bind the membrane of mitochondria17. due to its membrane curvature and the presence of the negatively charged cardiolipin in the mitochondrial outer membrane, αsyn has a strong binding affinity for mitochondria18. aggregated αsyn can disrupt mitochondrial membrane integrity and lead to toxicity19. to examine why inhibiting the mpc results in more αsyn aggregation, we investigated if altering mitochondrial function, or changing oxidative environment via msdc-0160 affects αsyn binding or αsyn oxidation status. stereological quantifications of the snpc for nitrated ni-αsyn (ni-αsyn) shows that in the αsyn low group, inhibiting the mpc with msdc-0160 leads to more ni-αsyn (*p = 0.021, 95% ci 11.5% – 111.7% more after negative binomial regression with benjamini-hochberg multiple testing adjustments), indicating that inhibiting the mpc leads to increased oxidation of αsyn (fig. 4). since it has been shown that oxidation of αsyn can affect αsyn aggregation, it raises the possibility that in this αsyn overexpression model a metabolic switch promotes the formation of insoluble assemblies of αsyn by increasing αsyn oxidation. figure 4. ni-αsyn increases upon msdc-0160 treatment. stereological quantification of ni-αsyn positive cells in rat sn. treatment with msdc-0160 leads to a significant increase in nitrated cells in the αsyn low group compared to placebo (*p < 0.05 via negative binomial regression with benjamini-hochberg multiple testing adjustments, n = 8, sem) since our previous work has shown that inhibiting the mpc has strong anti-inflammatory and metabolic effects, we further investigated if our αsyn viral vector model was lacking these features, which could explain the absence of any apparent therapeutic benefits. we examined changes in microglial and lysosomal markers at the four-month timepoint and found no changes in microglial activation (fig. 5a-b, e) and no changes in markers of autophagy (lamp1 and cathepsin d) in any of the groups examined (fig. 5c, f-g). taken together, in absence of metabolic or lysosomal deficits and microglial changes (at least at these later time points), a metabolic switch, by inhibiting the mpc, does not have a beneficial effect on αsyn aggregation. figure 5. absence of inflammatory or autophagy deficits in αsyn overexpressing animals. a) overview of microglial morphology via iba-1 staining in rat sn shows no microglial activation in αsyn overexpressing conditions. scale bar = 50 μm. b) quantification of microglial hydraulic radius as a measure of microglial ramification and activation (n = 8, sem). c) representative western blot images of inflammatory and autophagy markers in sn. d) iba-1 and e) gfap markers show no signs of inflammatory activation (n = 6, sem). d) cathepsin-d and e) lamp1 expression is unaltered in αsyn overexpressing animals (n = 6, sem). inhibiting the mpc in a mouse pff seeding model next, to investigate the effects of inhibiting mpc on αsyn aggregation, we examined the effects of msdc-0160 on αsyn propagation. we used a previously established seeding model where αsyn pffs are unilaterally injected into the ob of 12-week-old wild type mice. over time, αsyn pathology will propagate via intraneuronal connections and cause pathology in upstream regions19,20. one-week post pff or pbs injection into the ob, the mice were allowed free access to chow containing either msdc-0160 or placebo (fig. 6a). during the time of chow administration, we did not observe any differences in animal body weight or chow consumption between groups (supplementary fig. 5a, b). at 5and 13-weeks post-surgery, plasma was collected and the presence of hydroxymitoglitazone (msdc-0160 metabolite) was quantified (supplementary fig. 5c), showing detectable levels of the metabolite in treated groups. figure 6. a metabolic switch increases αsyn pathology load and propagation in mouse aon and prh. a) experimental timeline; at 12 weeks of age c57bl/6j mice were injected with pffs or pbs and starting at one-week post-surgery they had free access to msdc-0160 or placebo containing chow until the 5or 13-week endpoint. quantification of αsyn pser129 positive signal in b) the aon and c) the prh after 5 or 13 weeks of msdc-0160 or placebo treatment. msdc-0160 treated mice had significantly more αsyn pser129 positive stain at 5 weeks post-surgery than the placebo group (1.8 times more; p=0.02, 95% ci 1.176 2.824, beta mixed-effects regression). there is also weak evidence that this persists at the 13-week timepoint (p=0.085, 95% ci 0.986-1.92, beta mixed-effects regression). c) quantification of αsyn pser129 positive signal in the prh after one and three months of msdc-0160 or placebo treatment. d) representative images of αsyn pser129 staining in d) the ipsilateral aon (scalebar = 130 μm) and e) the prh (scalebar = 100 μm). unilateral pff injections have previously been shown to impair olfactory function in mice20. olfactory function was thus examined 13 weeks post-surgery using the buried pellet test. no significant difference was observed between the pbs and pff groups, regardless of whether the mice had been treated with placebo or msdc-0160 (supplementary fig. 6a). when assessing the total number of neun and cv-positive cells in the aon 13 weeks post-surgery via stereological quantification, we found no significant differences between any of the experimental groups (supplementary fig. 6b & c). immunohistological assessment at 5and 13-weeks post-surgery revealed a significantly higher percentage area containing pathological αsyn (αsyn pser129) in the aon than the prh, regardless of time point and treatment group (8.5-16 times higher; all p ≤ 0.001) (fig. 6b-e). the same was seen for the ipsilateral side compared to the contralateral side (65-78% less on the contralateral side; p ≤ 0.001). from 5 to 13 weeks post-surgery, αsyn pser129 increased significantly for msdc-0160 and placebo treated animals, which both gained ~99.2% and 163.2% of their αsyn pser129 immunopositively stained area from week 5 to week 13, respectively in the contralateral aon (p = 0.016 and < 0.001, respectively, 95% ci = 19.2 233.3 and 55.8 344.4). in addition, placebo-treated animals had a significant increase of pathology in the ipsilateral aon from the 5 to 13-week timepoint (p=0.016, 95% ci 18.3% 200%). this was not observed for msdc-0160 treated mice. when comparing treatment groups, msdc-0160 treated mice had significantly more pser129 positive signal at 5 weeks post-surgery than the placebo group (1.8 times more; p=0.02 95% ci 1.16 2.90). there is also weak evidence that this persists at the 13-week timepoint (p=0.085, 95% ci 0.98-1.95). no ni-αsyn was detected in the pff seeding model at the 13-week timepoint (supplementary fig. 7). for detection of potential amyloid (β-sheet rich) structures in the pff seeding model and the aav overexpression model, we used a luminescent conjugated oligothiophene (lco). lcos are small molecules with a flexible thiophene backbone that upon binding to amyloid structures become fluorescent21. one such lco, which has previously been shown to bind aggregates in tissue samples from patients suffering from systemic amyloidosis, as well as a variety of other disease-associated protein aggregates, is h-ftaa21 in the aon at 13 weeks post-surgery we observed ring-like h-ftaa positive structures (fig. 7) in the pff model. these overlapped with αsyn pser129 antibody staining, indicating that the αsyn pathology detected in this model is β-sheet rich with amyloid properties. on the contrary, we did not find a h-ftaa positive signal in the αsyn high group for the aav overexpression model. this highlights differences in αsyn assembly states several weeks after surgery in the two pd rodent models. figure 7. h-ftaa positive protein aggregates detected in the pff seeding model but not the aav overexpression model. representative images of the aon and the sn for the pff model and the aav overexpression model, respectively. the h-ftaa positive signal (green) detected in the aon 13 weeks following surgery co-localizes with the αsyn pser129 signal (pink). dapi was used to visualize the cell nuclei (blue), scale bar = 10 μm and 3 animals per group were analyzed. given the absence of any therapeutic benefits in the pff model, we again investigated the presence of inflammation via measuring the hydraulic radius (area/perimeter ratio) of microglia in the aon 13 weeks post-surgery. when we compared the injected side with the non-injected side, we found no significant change in microglia morphology (fig. 8a, b). furthermore, we did not detect any significant difference between pbs and pffs groups or differences between animals treated with placebo or msdc-0160. in addition, by quantifying lamp2-positive staining we do not observe any lysosomal deficits in the aon 13 weeks post-surgery (fig. 8c, d). taken together, in absence of lysosomal deficits and microglial changes, a metabolic switch, by inhibiting the mpc, does not reduce accumulation of αsyn pser129 in mice injected with pffs in the olfactory bulb. figure 8. absence of inflammatory or autophagy deficits 13-weeks post pff surgery. a) representative images of iba-1 positive staining in the ipsilateral aon, scale bar = 50 μm. b) quantification of microglial hydraulic radius as a measure of microglial ramification and activation (n = 5-6/group, sem), shows no microglial activation following pff injection. c) representative images of lamp2 positive staining (green) in the ipsilateral aon. dapi (blue) was used to visualize the cell nuclei. scale bar = 10 μm d) fluorescent lamp2 quantification after adding adaptive triangle thresholding in the aon (n = 6, sem) discussion improving metabolism, reducing inflammation and inhibiting the aggregation or the spread of pathological αsyn are considered promising therapeutic targets for the treatment of pd. through preclinical and clinical work, it has been suggested that anti-diabetic drugs might hold this promise22. we have previously shown that msdc-0160, a type 2 diabetes insulin sensitizer, can ameliorate pathology in an acute (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, mptp) and slowly progressive, genetic (engrailed1+/-) model of pd and that these effects occurred via modulation of the mpc and its downstream effect involving the mtor pathway10. since the direct effects of inhibiting mpc in chronic models of αsyn aggregation had not been studied, we asked if msdc-0160 could lower the levels of αsyn in two different, chronic and progressive αsyn-based animal models of pd. in our first model, αsyn is expressed via viral vector delivery in the rat sn resulting in aggregation and progressive accumulation of insoluble αsyn. after four months, no significant motor abnormalities with moderate neurodegeneration were observed but, unexpectedly, animals that were treated with msdc-0160 exhibited increased levels of aggregated αsyn. in the second model, pffs were injected in the ob of mice and allowed to seed and spread to connected regions. five weeks after pff injection, we observed an increase in αsyn pathology burden when mice were treated with msdc-0160. this effect was transient and at the later, 13-week timepoint no significant difference between the two treatment groups could be observed. both the αsyn viral vector and the αsyn pff seeding models are based on the introduction of exogenous αsyn that leads to progressive accumulation of phosphorylated and misfolded αsyn over time. the viral vector model results in local accumulation of highly concentrated but soluble αsyn that assembles into insoluble αsyn, whereas the pff model is based on the introduction of aggregated, seeding-potent αsyn that relies on the endogenous protein to template and spread via anatomically connected regions. to date, there are no studies that have directly compared the assembly states of the αsyn in the intraneuronal inclusions that remain after several weeks in an overexpression versus a seeding model, but it appears that inhibiting the mpc has different outcomes in these two models. this tentatively suggests i) that the conformational state or aggregation state of αsyn might be different in the two models and ii) that the effects of inhibiting the mpc depends on the assembly state of the protein. we observed a disparity in αsyn assembly states between the viral vector-based and the seeding model in multiple assays. first, we used detection of nitrated αsyn to determine that αsyn is oxidized in the viral vector-based model. a previous study showed that αsyn can bind directly to the mitochondrial import protein tom20, which leads to increased mitochondrial respiration and ros production17. we now find that oxidized αsyn species are abundant in neurons in the viral vector model, but undetectable in the pff model. we postulate that by altering the tca cycle when treating mice overexpressing αsyn with msdc-0160, the oxidation of αsyn is increased. this suggests that alterations in mitochondrial metabolism might post-translationally modify αsyn and make it more aggregation prone, when the protein is highly concentrated even though it is not known how exactly a metabolic switch would take place in αsyn overexpressing neurons nor how it would affect αsyn aggregation during anaplerosis. furthermore, we propose that elevated levels of soluble αsyn are required for these changes to take place, since we did not observe similar effects in the pff seeding model where the endogenous levels of αsyn are normal and aggregates are formed via an induced seeding mechanism. since the conformational state of αsyn is closely linked to its function23, it is possible that elevated levels of monomeric αsyn lead to changes in assembly state or gain of post-translational modifications resulting in an aberrant interaction with mitochondria. the binding affinity of αsyn is higher for mitochondrial membranes due to the unique presence of cardiolipin in mitochondria18. lipid interaction is an important catalyst for soluble αsyn to convert into aggregated αsyn24 and this conversion has been reported to lead to release of mitochondrial nitric oxide and increased oxidative stress25. αsyn can disrupt membrane integrity and cause mitochondrial damage and degeneration of dopaminergic neurons17,26. recombinant nitrated species of αsyn injected in rat sn have been shown to be more toxic than unmodified αsyn27. in msdc-0160 treated mice, we did indeed observe greater loss of dopamine neurons in the anterior portion of the sn, which was the region where viral vector-mediated expression of αsyn was the highest. multiple molecules of nitrated αsyn have been shown to form soluble assemblies that are off pathway to aggregation and relatively unstructured28. in order to determine if structured amyloid forms of αsyn were present in our animal models, we used the conformation sensitive lco h-ftaa probe29. lcos are amyloid-conformation-specific dyes that specifically bind to amyloid αsyn in brain tissue and csf30. a positive signal therefore indicates the presence of well-structured, β-sheet rich aggregated αsyn. in addition, h-ftaa has been used to distinguish protein inclusion bodies in sporadic inclusion body myositis (s-ibm) and liver diseases21. upon treatment with h-ftaa, we observed a strong positive signal in the aon of animals injected with pffs but no signal in the snpc of animals overexpressing αsyn following viral vector injection. the fluorescent signal was localized to neurons and the morphology was lewy body-like with aggresomal structures that resembled the intracellular distribution we observed when staining for αsyn pser129. this illustrates that the two αsyn animal models also differ in that the αsyn assembly states in these models are distinct. these differences were recently discussed by gómez-benito and colleagues; αsyn species from the aav overexpression model lack seeding activity while the pff model recapitulates αsyn propagation with the accumulation of lewy body-like structures31. in summary, msdc-0160 does not reduce αsyn aggregation in the model where a viral vector is used to overexpress the protein in the nigrostriatal system. on the contrary, the inhibition of mpc results in a slight increase in αsyn protein content. in the model using injection of pffs to trigger a cascade of αsyn aggregate propagation in the olfactory system, msdc-0160 exhibits no effect on the progressive development of synucleinopathy in the olfactory system. in experimental paradigms that involve more acute neuroinflammation, we previously showed that msdc-0160 alleviates the inflammatory response in vitro and in vivo10. however, in the pff and viral vector αsyn models, we did not detect changes in inflammation or evidence for autophagy deficits at the survival times we studied. we conclude that attenuation of the mpc in rodent models that do not exhibit inflammation and limited autophagy does not result in a reduction of introduced αsyn. acknowledgements research reported in this publication was supported by the cure parkinson’s trust award number 42-40384-1 (p.b.). w.p. acknowledges a post-doctoral fellowship from fulbright, idt technologies and fwo flanders. we thank the staff of the vivarium of van andel institute for caring for the mice and rats used in this study. author contributions w.p. performed rat studies (stereotaxic surgery and behavioral, biochemical and immunohistochemical analysis) and wrote the manuscript. l.b. performed mouse olfactory testing, neun immunostaining and stereology, pser129 immunostaining and analysis, ni-αsyn staining, lamp2 immunostaining and analysis, iba-1 immunostaining and analysis, h-ftaa/pser129 co-immunostaining and imaging and wrote the manuscript. s.g. performed pilot studies leading to the final conceptualization of the aav overexpression model study. m.e.j. performed pser129 immunostaining. j.s. organized the manufacturing of the msdc-0160 chow, metabolic detection, plasma, csf and brain mitochondria collection and provided advice. l.m. performed unilateral pff surgery, mouse perfusions and plasma, csf and brain mitochondria collection. e.s. performed unilateral pff surgery, mouse perfusions, plasma, csf and brain mitochondria collection and ni-αsyn staining optimization. z.m. performed the statistical analysis. j.m. provided the pffs. k.b. performed pff manufacturing. j.r.c. provided the compound (msdc-0160), expertise and advice. p.b. conceptualized this study and provided expertise and advice. all authors reviewed and edited the manuscript. disclosure p.b. receives commercial support as a consultant from calico life sciences, curasen, idorsia pharmaceuticals, lundbeck a/s, abbvie, fujifilm-cellular dynamics international, and axial biotherapeutics. he has received commercial support for research from lundbeck a/s and roche. he has ownership interests in acousort ab. j.r.c. is the cofounder and significant owner of metabolic solutions development company (msdc), which is currently developing msdc-0160 as a potential treatment for alzheimer’s disease. msdc-0160 is protected by several patents, including us 8389556 b2 and ep 2001468 b1, which are assigned to msdc. all other authors declare no additional competing financial interests. references 1. stern, m. b., lang, a. & poewe, w. toward a redefinition of parkinson's disease. mov disord. 27, 54–60 (2012). 2. park, j.-s., davis, r. l. & sue, c. m. mitochondrial dysfunction in parkinson’s disease: new mechanistic insights and therapeutic perspectives. 1–11 (2018). doi:10.1007/s11910-018-0829-3 3. brakedal, b. et al. 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an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. dementia with lewy bodies – a clinicopathological update feel free to add comments by clicking these icons on the sidebar free neuropathology 1:7 (2020) review dementia with lewy bodies – a clinicopathological update jános bencze1,2, woosung seo3, abdul hye4,5, dag aarsland5,6,7, tibor hortobágyi2,6,7,8 1 department of pathology, faculty of medicine, university of debrecen, debrecen, hungary 2 mta-de cerebrovascular and neurodegenerative research group, department of neurology, university of debrecen, debrecen, hungary 3 department of surgical sciences, radiology, uppsala university, uppsala, sweden 4 maurice wohl clinical neuroscience institute, institute of psychiatry, psychology and neuroscience, king’s college london, london, uk 5 nihr biomedical research centre for mental health & biomedical research unit for dementia at south london & maudsley nhs foundation, london, uk 6 department of old age psychiatry, institute of psychiatry psychology and neuroscience, king’s college london, london, uk 7 centre for age-related medicine, sesam, stavanger university hospital, stavanger, norway 8 institute of pathology, faculty of medicine, university of szeged, szeged, hungary corresponding author: tibor hortobágyi md phd dsc frcpath efn · institute of pathology · faculty of medicine · university of szeged · szeged · állomás utca 1 · h-6725 · hungary hortobagyi.tibor@med.u-szeged.hu submitted: 31 december 2019 accepted: 06 february 2020 published: 18 february 2020 https://doi.org/10.17879/freeneuropathology-2020-2613 keywords: α-synuclein, biomarkers, diagnostic criteria, clinico-pathological correlation, dementia with lewy bodies abstract dementia is one of the major burdens of our aging society. according to certain predictions, the number of patients will double every 20 years. although alzheimer’s disease (ad), as the most frequent neurodegenerative dementia, has been extensively analysed, less is known about dementia with lewy bodies (dlb). neuropathological hallmarks of dlb are the deposition of intracellular lewy bodies (lb) and lewy neurites (ln). dlb belongs to the α-synucleinopathies, as the major component of these inclusions is pathologically aggregated α-synuclein. depending on the localisation of lbs and lns in the central nervous system cognitive and motor symptoms can occur. in our work, we will systematically review the possible etiology and epidemiology, pathological (both macroscopic and microscopic) features, structural and functional imaging findings, with a special emphasis on the clinico-pathological correlations. finally, we summarize the latest clinical symptoms-based diagnostic criteria and the novel therapeutic approaches. since dlb is frequently accompanied with ad pathology, highlighting possible differential diagnostic approaches is an integral part of our paper. although our present knowledge is insufficient, the rapid development of diagnostic and research methods provide hope for better diagnosis and more efficient treatment, contributing to a better quality of life. abbreviations aa, alzheimer’s association; ad, alzheimer’s disease; aβ, amyloid-beta; apoe, apolipoprotein e gene; bne, brainnet europe consortium; bold, blood oxygen level dependent; chat, choline-acetyltransferase; chat-i, acetyl-cholinesterase inhibitors; cr, creatinine; csf, cerebrospinal fluid; dlb, dementia with lewy bodies; dmn, default mode network; dsm, diagnostic statistical manual; fmri, functional mri; fp-cit, [123i] 2ß-carbomethoxy-3b-(4-iodophenyl)-n-(3-fluoropropyl) nortropane; gba, glucosylceramidase-beta gene; ilbd, incidental lewy body disease; lb, lewy body; lbda, lewy body dementia association; ln, lewy neurite; mapt, microtubule associated protein tau gene; mds, movement disorders society; mri, magnetic resonance imaging; naa, n-acetyl aspartate; ncd, neurocognitive disorder; nf, neurofilament; nia, national institute on aging; nmdar, n-methyl-d-aspartate receptor; pd, parkinson’s disease; pdd, parkinson’s disease dementia; pet, positron emission tomography; psd95, density protein 95; rem, rapid-eye-movement; scarb2, scavenger receptor class b member 2; snare, snap (soluble nsf attachment protein) receptor; snca, α-synuclein gene; spect, single-photon emission computed tomography; ssri, selective serotonin reuptake inhibitors; upr, unfolded protein response; wml, white matter lesions; znt3, zinc transporter 3 epidemiology and etiology dementia with lewy bodies (dlb) is the second most common primary neurodegenerative dementia. the etiology is mainly unknown, however, in certain cases there is strong evidence of genetic background. a few papers have reported families with accumulating occurrence of cognitive impairment throughout generations1. the vast majority of the familial cases are traced to α-synuclein (snca) gene alterations, particularly to e46k mutation2. five candidate genes, including apoe, gba, mapt, snca and scarb2, are considered as a significant risk factor for dlb, nevertheless, further genetic studies are needed3. although it mostly appears in older age, dementia is not strictly associated with aging. rarely it may occur under the age of 65 and even in young adulthood. surprisingly in the case report of a teenager, the clinical symptoms and post-mortem pathological findings were consistent with those of in dlb4. the prevalence of the disease is not clearly established. according to a comprehensive analysis of epidemiological data, the prevalence is 4.2% in community based and 7.5% in clinical studies, while the incidence is 3.8% and grows linearly with aging5 and the prevalence of dlb among patients with dementia is probably around 15%6. pathologic background 1. macroscopic observation many of the general pathologic features resemble those in parkinson’s disease (pd). the brain weight is often within the normal limits, mild cortical atrophy of the frontal lobe, neuromelanin pigment loss in the substantia nigra and locus coeruleus are noted. in the case of severe concomitant ad pathology, atrophy of the temporal and parietal regions is more evident. in contrast to ad, the general brain atrophy is less prominent in dlb along with the relatively preserved temporal lobe and hippocampus7. 2. microscopic feature  lewy bodies (lbs), the key pathological findings in dlb, were initially described by friedrich lewy in 19128. however, lbs are characteristic of other neurodegenerative diseases including pd. their typical appearance, stained with conventional hematoxylin-eosin, is a central spherical eosinophilic core surrounded by a peripheral halo situated intracellularly, causing dislocation of subcellular organs. these features are regularly found in brainstem predominant lb formation. the rest of the brain expresses rather irregular lbs without the typical peripheral halo (figure 1). figure 1: dementia with lewy bodies (dlb) specific pathological changes are shown with hematoxylin and eosin (he) staining (panel a) and α-synuclein immunohistochemistry (ich) (panel b). cortical-type lewy bodies (lbs) are eosinophilic intracellar neuronal inclusions which dislocate the nucleus (panel a, black star). α-synuclein ihc highlights lbs (white star, panel b) and lns (arrowhead, panel b). histopathologically, the dominant component of the central core is α-synuclein, while the peripheral halo consists of several ubiquitinated proteins9. lewy neurites (lns), as an additional hallmark of α-synucleinopathies, are abnormal thickened neurites containing filaments corresponding to those in lbs10. interestingly, in experimental mouse models mutant snca inoculation of wild type mouse causes lb/ln-like pathology, supporting the pathognomonic role of the protein11. the background of lb genesis is still undiscovered, although numerous hypotheses have been proposed trying to explain the proper mechanism. according to the aggresome hypothesis, lb formation is originally a neuroprotective process, facilitating the cells to remove harmful proteins12. however, failure of the aggresome formation may occur, leading to excessive lb development13. other authors presume that autophagy dysfunction is responsible for the neuronal loss14. a recent study suggests that the increased unfolded protein response (upr) activation has an important role in the lb pathology15. nevertheless, according to tompkins et al. neurons burdened with lbs are less apoptotic16. moreover, the downregulation of tyrosine hydroxylase enzymes protects against toxic products of dopamine oxidation17. these findings confirm the theory that α-synuclein is physiologically involved in the maintenance of cell homeostasis. the first standardized criterion assessing the connections between pathological findings and dlb was made by kosaka et al. in 1984. based on the anatomical distribution of pathology they determined three different subtypes: i) brainstem predominant lbs (commonly in pd) ii) limbic (transitional) lbs and iii) diffuse cortical lbs18. later the consortium on dlb international workshop has improved the original assignment and published a more detailed instruction emphasizing the importance of the diagnostic procedure. the widely used immunohistochemical markers, α-synuclein (the most specific) and p62 serve the accurate pathological diagnosis19. the latest mckeith diagnostic consensus criteria recommends a semiquantitative grading of 10 different brain regions (dorsal motor nucleus of vagus, locus coeruleus, substantia nigra, nucleus basalis of meynert, amygdala, transentorhinal and cingulate gyri, temporal-, frontaland parietal lobes) based on the lesion density instead of the previously used lb counting method. moreover, it suggests two additional categories, the amygdala-predominant and the olfactory bulb only dlb. according to the distribution of lb pathology on α-synuclein immunostained slides, the scoring system distinguishes four stages: 1 – mild (sparse lbs or lns); 2 – moderate (1< lbs in a low power field and sparse lns); 3 severe (4≤ lbs and scattered lns in a low power field); 4 – very severe (numerous lbs and numerous lns). finally, as a synthesis of score and localization it ranks the seen pathology into one out of the three previously mentioned subtypes20. besides the mckeith staging and subtyping, the other widely used evaluating technique is the braak staging21. the authors proposed to assess the severity of lewy-type pathology labelled by α-synuclein immunostaining in 13 different brain areas (dorsal motor nucleus of vagus, locus coeruleus, raphe, substantia nigra, ca2 region of hippocampus, nucleus basalis of meynert, transentorhinal-, cingulateand insular gyri, temporo-occipital-, temporal-, frontaland parietal lobes). considering that neither mckeith nor braak protocol could reach more than 80% inter-observer agreement; the original methods have been modified by leverenz et al.22 and müller et al.23, respectively. however, their results did not lead to a significant improvement in the scoring systems. thus, in 2009 the brainnet europe consortium (bne) revised their former assignments and suggested modifications, to reach a better inter-observer agreement. using the original mckeith and braak staging, but eliminating their major pitfalls and obstacles as well as introducing the amygdala predominant category, bne’s novel strategy resulted in above 80% agreement in both typing and staging of α-synuclein pathology24. beach et al. also published their unified staging system with the aim of dividing every subject with lewy-type α-synuclein pathology into a well-defined neuropathological group25. in 2009 they used mckeith26 and braak21 staging systems and failed to categorize individuals with olfactory bulb or limbic-predominant lb pathology. investigating 10 standard brain regions they could classify all patients with pd, dlb, incidental lewy body disease (ilbd) and ad with concomitant lb pathology into one of the following stages: i olfactory bulb only; iia brainstem-predominant; iib limbic-predominant; iii brainstem and limbic; iv neocortical. moreover, they found strong correlation between the progression through these stages and the severity of nigrostriatal degeneration, cognitive impairment and motor dysfunction. it should be noted that olfactory bulb only and amygdala-predominant subtypes are also included in the current mckeith criteria20. as mentioned above, ad pathology is frequently encountered in dlb. approximately 80% of patients have diffuse amyloid-beta (aβ) plaques and 60% have neurofibrillary tangles with varying severity in the entorhinal cortex and rarely in the neocortex. some of these cases meet the pathological criteria of ad27. in contrast, “pure” neuropathological form of dlb is less common. autopsy series suggest that the frequency of this entity is approximately 25% of all dlb cases28,29. theoretically, the likelihood to manifest dlb clinical syndrome is directly proportional to the severity of lb pathology and inversely proportional to the severity of ad pathology. the mckeith classification integrates the assessment of concomitant ad pathology by national institute on aging and alzheimer’s association (nia-aa)30, the braak criteria and the type of lb pathology20. table 1 shows the probability of pathological findings in relation to dlb clinical syndrome. it is likely that not only the α-synuclein pathology is responsible for the cognitive decline, but plaques and phosphorylated tau proteins also contribute to the overall deficit31. table 1: likelihood of dementia with lewy bodies (dlb) clinical syndrome resulting from the assessment of alzheimer's-type and lewy body pathology (lb= lewy body; dlb=dementia with lewy bodies; nia-aa = national institute on aging – alzheimer’s association) [modified from mckeith et al.20] cognitive impairment is also a common hallmark of dlb and parkinson’s disease dementia (pdd). these two neurocognitive disorders share several clinical and neuropathological features, i.e. they are both characterized by cortical and subcortical α-synuclein/lb, β-amyloid and tau pathologies32. according to jellinger et al., a common pathophysiology in dlb and pdd is synaptic dysfunction due to aggregation of α-synuclein in the presynapses. this results in disruption of axonal transport and neurotransmitter deprivation leading to neurodegeneration32. interestingly, there are some morphologic differences as well. dlb seems to show higher load of β-amyloid and tau in several brain regions primarily in striatum, cortex, claustrum, amygdala and putamen compared to pdd32. jellinger et al. also describes that α-synuclein distribution is different in dlb and pdd where α-synuclein load was highest in hippocampal subarea ca2 and in amygdala in dlb, whereas in pd it is highest in the cingulate cortex. moreover, nigral neuronal loss is more marked in pdd which ultimately results in dopaminergic upregulation32. a simple interpretation of above findings could support the current diagnostic criteria where dlb is associated with early cognitive impairment including memory problems, which differs from pdd with mainly motor impairment in the early phase. in reality, it is not easy to define dlb based upon only the histological findings, thus in uncertain cases, anamnestic clinical data has invaluable support for the diagnostics. it is important to highlight that the listed strategies are not absolute or perfect diagnostic criteria, but rather useful schemes to predict the clinical syndrome of dlb based on the observed pathological findings. as mentioned above, a major weakness of current guidelines is the low inter-observer agreement; therefore further research is needed to assess the accuracy of the current clinical diagnostic criteria versus neuropathology similar to studies assessing the 2005 criteria in this respect33. 3. synaptic alterations in connection with the dopaminergic neuronal loss of substantia nigra, dopamine transporter level is decreased in the striatum in dlb, although not to the same extent observed in pd34. the choline-acetyltransferase (chat) levels are lower than in patients with ad, who suffer from similarly severe dementia35,36. interestingly, dynamin-1, which takes part in the regulation of synaptic transmission, shows significantly decreased levels in the prefrontal cortex in parallel with the severity of cognitive decline37. in addition, the amount of zinc transporter 3 (znt3) and postsynaptic density protein 95 (psd95), are significantly reduced in dlb compared to aged-controls and patients with ad38,39. it should be noted that at the beginning of the disease, an upregulation of soluble nsf attachment protein receptors (snare) complex is observed, probably as a compensatory response to the synaptic loss. however, during the progression of dlb synaptotagmin, synapsin and synaptophysin, proteins involved in the snare complex steadily disintegrate, contributing further to synaptic dysfunction40,41. recent studies have revealed that more than 90% of α-synuclein aggregates are located at the presynapses leading to neurotransmitter deficits42,43. these findings suggest that degeneration of postsynaptic neurons may result from the loss of their inputs. this theory serves as a potential explanation for the dlb-specific clinical symptoms as well as raises the possibility of future curative treatments by pharmaceutical modification of neurotransmission. 4. incidental lewy-body disease (ilbd) in ilbd, patients have histologically detectable α-synuclein deposits in their brain, without presenting any clinical symptoms. researchers accept that ilbd, which appears in approximately 8-17% of the clinically normal 60+ years old patients, is the early stage of pd or dlb44,45. confirming this hypothesis, it is frequently observed in patients who represent the prodromes of pd, including olfactory dysfunction or bowel frequency46,47. the presence of neuronal loss is usually minimal in these cases contrary to pd or dlb48. distribution of α-synuclein pathology in ilbd is particularly predictive, i.e. the brainstem predominant subtype frequently results in pd, while the cortical predominant subtype often leads to dlb44. clinical symptoms the new diagnostic statistical manual (dsm-5) (published in 2013) created a new neurocognitive disorder (ncd) group, replacing the former ‘dementia, delirium, amnestic and other cognitive disorders’ category, that were used in the previous dsm-iv handbook. the definition emphasizes that ncd is a progressive, acquired decline; which implies that disorders occurring at birth or at the early stage of cognitive development are excluded from this category. as an advantage, the new handbook removed the stigmatizing debilitating term, dementia. within the ncd there are two subcategories: major and mild neurocognitive disorders based upon the severity of decline. for the diagnosis, six main fields are examined by dsm-5: complex attention, executive function, learning and memory, language, perceptual motor or social cognition49. in the case of dlb, patients show both cortical and subcortical progressive dementia symptoms. characteristic features comprise of attention and spatial perception disorder, dysexecutive syndrome, fluctuating cognitive performance lasting from minutes to days. among the psychiatric alteration the most frequent is the visual hallucination, although anxiety, apathy or organised delusions could also be detected50,51. interestingly, advanced cerebral amyloid angiopathy and small vessel disease are associated with psychosis in ad and not in dlb52. depression is more common in patients with dlb than in patients with ad. it may accompany with mild dementia, although more frequent in cases of advanced dlb53,54. the vast majority of the patients, from the beginning or during the progression, show extrapyramidal signs, such as action tremor, gait disturbances, rigidity or changes in facial expressions55. rapid-eye-movement (rem) parasomnia is frequently diagnosed in dlb, characterised by vivid, often frightening dreams and complex purposeful motor activity. since it often appears in different neurodegenerative disorders, rem parasomnia might be an early herald of these diseases56. in dlb the autonomic nervous system dysfunction is more severe than in ad. patients usually complain of dizziness, falls or loss of consciousness. orthostatic hypotension, cardioinhibitory carotid sinushypersensitivity and urinary incontinency often accompany with dlb57. pathognomonic feature is the neuroleptic hypersensitivity, triggered by even a small dose of drug, leading to severe parkinsonism20. a 5-year prospective cohort study published by rongve et al. has reported that the progression of cognitive decline from mild to severe stage is more rapid in patients with dlb than in patients with ad58. many clinical symptoms of dlb cannot be explained only by the intracerebral localization of lbs and lns33. neurotransmitter depletion, synaptic alterations, concomitant ad-type pathology and metabolic changes highly influence the clinical presentation of dlb, and correlate with the severity of cognitive decline6,42,59,60. moreover, a recent paper has reported association of serum potassium levels with cognitive decline in dlb, specifically in patients not using any medications that affect serum k+ levels61. reduced perfusion and dysconnectivity of the occipital lobe may contribute to visual hallucinations and disturbance of visuospatial orientation62,63. rem sleep behavior disorder rbd associates with striatal dopamine depletion64. vegetative dysfunctions (i.e. orthostatic hypotension) may result from the deposition of lbs in the autonomic ganglia65. decreased znt3 levels were also identified in patients with depression, providing a novel therapeutic target38,66. interestingly, several clinical symptoms of dlb are transient in nature, however lbs and lns permanently occur in the brain. probably, metabolic disturbances67, hormonal alterations68, circadian rhythm69 and comorbidities such as high blood pressure70 can affect the clinical appearance of dlb. radiologic feature besides the clinical symptoms, the most significant diagnostic tool in the identification of dlb is the rapidly developing imaging techniques. the numerous reachable modalities allow both structural and functional examination. magnetic resonance imaging (mri) examination regarding the structural changes, the literature is not consistent: some studies have noted significant atrophy of the insular, frontal, inferior parietal, temporal or occipital cortex71, whereas others have found only a minimal volumetric decrease in the frontal and parietal lobe, or in the territory of hypothalamus, basal forebrain and midbrain72. however, these alterations are not specific to dlb and might appear in ad. although differentiation from ad is usually based on the absence of medial temporal atrophy, its presence cannot rule out the diagnosis of dlb73. sabattoli et al. have found atrophic changes in the anterior ca1, ca2/3 hippocampus, subiculum and presubiculum in dlb. this pattern differs significantly from those in ad74. according to an mri study, the annual progression rate of cortical atrophy is approximately 2x higher in patients with ad72. the role of white matter lesions (wml) in dlb, including loss of myelin, axonal damage and gliosis is not consistent. presumably, wmls are implicated in vascular dementia rather than being specific feature of neurodegenerative disorders75. mri spectroscopy provides a chance to indirectly assess the neuronal and glial function by measuring the key metabolites. n-acetyl aspartate (naa) and creatinine (cr) are two widely used markers in the characterization of central nervous system metabolism. watson et al. have found that the naa/cr ratio is relatively preserved in dlb in comparison to in ad76. functional imaging although, task-related functional mri (fmri) has been performed in very few cases of dlb, a paper has reported reduced activity in response to movement activity compared with aged controls and patients with ad77. alternatively, researchers have examined the functional activity during rest. the resting-state network shows increased activity during rest and decreased activity during cognitive tasks. the most investigated resting-state network to date is the default mode network (dmn), including prefrontal cortex, posterior cingulate gyrus, medial temporal lobe and precuneus. in these areas the task-related deactivation during the colour and motion tasks were decreased in dlb, but it was not significantly different from ad78. a technique for evaluation of functional resting state connectivity is based on the functional alterations in blood oxygen level dependent (bold) activity. galvin et al. have noted increased resting state connectivity between precuneus seeding regions, inferior parietal cortex and putamen, with decreased connectivity between medial prefrontal, frontoparietal operculum and visual cortex79. taking into account that the results in dlb significantly differed from those of in ad, the method can be considered in the in vivo differential diagnosis. the real value of perfusion imaging techniques in the identification of dlb is not fully consistent. the vast majority of the authors described occipital hypoperfusion as a hallmark of the disease on single-photon emission computed tomography (spect) images, whereas the specificity of the method is variable depending on the study80. [123i] 2ß-carbomethoxy-3b-(4-iodophenyl)-n-(3-fluoropropyl) nortropane (fp-cit) could be useful to discover dlb in early stages before the full spectrum of clinical symptoms evolve, moreover it reliably identifies neurobiological changes in the dopaminergic system81. in dlb there is markedly reduced tracer uptake in the regions of caudate and putamen reflecting the severely affected dopaminergic system. strong connection has been found between striatal fp-cit uptake and certain clinical symptoms, such as depression, anxiety, apathy and daytime somnolence82. positron emission tomography (pet) is probably a more sensitive functional imaging technique than spect in different dementias. ishii et al. have compared the two methods and found that pet is more reliable in the detection of occipital and parietal lobe hypometabolism83. besides the dopaminergic system, cholinergic transmission also has a crucial role in the pathomechanism of dlb, for instance in the alteration of memory, attention or arousal. significant cholinergic neuronal loss and reduced chat activity are noted extensively in the cortical and subcortical regions, including both nicotinergic and muscarinergic systems, compared to ad36. shimada et al. have shown reduced chat enzymatic activity in the medial occipital cortex with relatively preserved temporal activity in dlb, in contrast to ad84. the fact that eeg is involved in the mckeith criterion further justifies its diagnostic relevance. compared to ad the detectable slower background activity and more diffuse slow-wave activity probably reflect the severe cholinergic deficit in dlb85. biomarkers it is difficult to find specific biomarkers of dlb that would allow us to discriminate neuropathologically ‘pure’ forms of the disease from cases with concomitant ad pathology86, as that majority of dlb cases show a combination of α-synuclein, tau and β-amyloid pathologies87,88. however, novel biomarkers such as reduced electroencephalography activity89 and the detection of rbd90 also have some value. recent dlb biomarker research has focused on targets that are primarily related to ad. as in ad, csf levels of aβ1-42 are decreased in dlb91, although tau seems to show an opposite relationship from ad92. that said tau has been shown to be higher in dlb when compared to pd and pdd87,93,94. dlb is considered α-synucleinopathy alongside with pd and pdd. when α-synuclein was discovered as a major component of lb this initiated a string of studies investigating α-synuclein as a biomarker in csf95,96. in general, some groups have reported reduced α-synuclein97,98 while others have shown contradictory results99,100. these discrepancies may arise from the nature of α-synuclein expression. α-synuclein appears in four isoforms, (α-syn98, α-syn112, α-syn126 and α-syn140), based on alternative splicing of exon 3 and 5, and the largest isoform, α-syn140, is the most abundant isoform in the brain101,102. nonetheless, to overcome this issue several groups have tried using a combination of antibodies to quantify ‘total’ α-synuclein103. in addition to this many are convinced that oligomeric α-synuclein is potentially a more pathogenic form104 and efforts have been made to measure this in both csf and in plasma105–107. in addition to α-synuclein and ad biomarkers, other potential biomarkers such as neurofilaments (nf) have been investigated108. nfs are components within a cell that assists in maintaining the structural integrity. it has been shown that nfs are associated with ad and other degenerative disorders109–111, however that is not the case with dlb. nfs seem to provide only a general hint of neuronal and axonal dysfunction without providing any differential value to separate dlb from other disorders. on the other hand, other isoforms of nfs112 do exist and would have to be further investigated in dlb. since there is greater involvement of the dopaminergic and serotonergic neurotransmitters in dlb compared to ad, several groups have investigated metabolites from these pathways. in combination with csf aβ1-42, reduced levels of 5hydroxyindolacetic acid and 3methoxy-4-hydroxyphenylethyleneglycol have been found in dlb compared with ad104,113. in summary, current studies suggest that dlb is intermediate to ad and pd, such that biomarkers from ad and pd have been tested in dlb with moderate success114. likewise, new diagnostic proteins may be discovered in the future with further proteomic studies which could provide a better differentiation of dlb from other closely related neurodegenerative disorders115–117. diagnostic criteria considering the heterogeneous phenotype and the frequently associated ad pathology, the clinical symptom-based diagnosis of dlb is rather difficult. to date, there is no consensus on the guidelines for assessing clinical symptoms of dlb. a recent paper58 recommends the following rating strategies: for evaluating cognitive decline – clinical dementia rating scale118; for estimating fluctuating cognition – clinician assessment of cognitive fluctuations119 or mayo fluctuation questionnaire120; for investigating rem parasomnia mayo sleep questionnaire121; for rating parkinsonism – unified parkinson’s rating scale122; for testing disability rapid disability rating scale-2123; for diagnosing visual hallucinations or other psychiatric disorders – neuropsychiatric inventory124; for measuring effects of comorbidities – cumulative illness rating scale118. additional difficulty is to make a distinction, if it exists, between dlb and parkinson’s disease dementia. the question, whether dlb and pdd are different entities or the same one, is still debated. although there are a few morphologic differences between them (e.g. cortical spreading of lbs or rate of neuronal loss in sn)125, differential diagnosis is rather based on the temporal sequence of symptoms. if dementia occurs 1 year after the onset of extrapyramidal motor signs, it is considered as pdd, otherwise if dementia has proceeded or presented within 1 year after movement disorder, the diagnosis should be dlb126. the latest mckeith diagnostic criteria define probable and possible dlb based on the clinical symptoms and biomarkers (figure 2)20. the presence of dementia, memory impairment and deficit of attention, executive functions and visuospatial orientation is essential for diagnosis. core symptoms are frequently observed in dlb clinical syndrome; in the lack of at least one core feature, the probable dlb diagnosis cannot be established. the development of diagnostic techniques revealed that rem sleep behaviour disorder is more characteristic of lbd than it was previously thought. therefore, mckeith et al. recategorized rbd from supportive to core features20. supportive features may help the clinicians; however, these symptoms are not specific to dlb and their presence is not required for the diagnosis of neither probable nor possible dlb. the refreshed criteria emphasize the importance of disease-specific biomarkers. one indicative biomarker itself is sufficient for possible dlb diagnosis. if one indicative biomarker associates with one core symptom, the diagnosis is probable dlb. figure 2: symptoms and biomarkers contribute to the diagnosis of probable or possible dementia with lewy bodies (dlb). essential symptoms are mandatory for diagnosis of dlb. core clinical symptoms are characteristic to dlb, while supportive symptoms can confirm the decision by clinicians. indicative biomarkers are frequently observed in dlb, while supportive biomarkers can facilitate the decision-making procedure. important to note that clinicians can not establish the diagnosis of probable dlb based on the biomarkers. (recategorized or newly added features are highlighted in blue colour; rem = rapid eye movement; dat = dopamine transporter; mibg = metaiodobenzylguanidine; psg = polysomnography; rbd = rem sleep behavior disorder; spect = single-photon emission computed tomography; pet = positron-emission tomography; eeg = electroencephalography; ↓ = decrease). [adapted from mckeith et al.20] therapy there are two different therapeutic approaches: pharmacologic and non-pharmacologic. although there is no curative treatment strategy, the adequate therapy may slow the disease progression with the chance of a better quality of life. non-pharmacologic interventions at the first signs of dlb or mild ncd, installation of the below mentioned interventions are recommended. changes in the dietary habit, mediterranean diet, and regularly performed social and mental tasks as well as physical activities could reduce the rate of progression127. personalized cognitive rehabilitation trainings with focus on the declined field, improve both quality of life and memory128. pharmacologic treatments the frequently noted extrapyramidal signs should be treated with the smallest effective dose of levodopa, to avoid the worsening of psychiatric symptoms129. selective serotonin reuptake inhibitors (ssris) are widely-used and considered as effective medications for depression130. in the case of rem sleep behaviour disorder, clonazepam and melatonin also have beneficial effect131 acetyl-cholinesterase inhibitors (chat-i) have benefits in the treatment of psychiatric disturbances such as visual hallucination, delusions, behaviour disorders or apathy. despite gastrointestinal side effects (i.e. vomiting, diarrhoea) these drugs are usually well tolerated20. if chat-is (rivastigmine, donepezil) are not effective, the use of atypical neuroleptic drugs (i.e. clozapine) might be inevitable, but typical neuroleptics should be avoided to minimize the possibility of severe neuroleptic hypersensitivity reaction132. chat-is are more effective in patients with dlb compared to patients with ad. these drugs improve the cognitive functions, reduce fluctuation, decrease the progression and in addition patients score better on neuropsychological tests133. n-methyl-d-aspartate receptor (nmda-r) antagonist memantine is also useful in the treatment of cognitive decline134. lucza et al. have suggested treatment with chat-is in mild or mid-severe dementia and memantine combined with high dose of rivastigmine (13.5 mg) in severe dementia135. summary despite the continuously growing incidence of dementia, which could be the most prevalent disease within 20 years in the industrialized world, our present knowledge is still insufficient to fully comprehend the underlying pathomechanism. this is particularly true for dlb, which is the second most common neurodegenerative dementia. etiology of the disorder, its connection with aging, underlying pathomechanisms of clinical signs and imaging findings are still in need for further elucidation. unfortunately, the definitive diagnosis is possible only by post-mortem histopathological examination and not by in vivo techniques (apart from a low percentage of cases). the concomitant alzheimer’s-type and vascular pathology raises issues regarding diagnostic clarity and accuracy136. future research should be multidisciplinary and should include pathological, proteomic, genetic and epigenetic approaches to identify the key factors of the disease and reveal the correlation between clinical symptoms, radiological alterations and pathological findings. development of imaging techniques brings the possibility of in vivo diagnosis, which implies adequate treatments in early stages, a crucial advancement to ensure better quality of life. longitudinal cohort studies are also required to provide detailed prognostic information that are essential for planning a more effective and cost-efficient treatment protocol. author’s contribution authors contributed equally to the work. funding supported by the únkp-19-3 new national excellence program of the ministry of innovation and technology and efop-3.6.3-vekop-16-2017-00009 (j.b.); ginop-2.3.2-15-2016-00043, hungarian brain research program (2017-1.2.1-nkp-2017-00002), nkfih snn 132999, szte áok-kka no. 5s 567 (a202) and de áok research fund (t.h.). 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unless otherwise stated. the definition and role of brain invasion in meningioma grading: still controversial after all these years feel free to add comments by clicking these icons on the sidebar free neuropathology 2:8 (2021) opinion piece the definition and role of brain invasion in meningioma grading: still controversial after all these years arie perry department of pathology, division of neuropathology, university of california san francisco (ucsf), san francisco, ca 94143, usa corresponding author: arie perry · department of pathology, division of neuropathology · university of california, san francisco (ucsf) · 505 parnassus avenue, m551, box 0102 · san francisco, ca 94143 · usa arie.perry@ucsf.edu submitted: 05 march 2021 accepted: 23 march 2021 copyedited by: nima sharifai published: 24 march 2021 https://doi.org/10.17879/freeneuropathology-2021-3276 keywords: meningioma, brain invasion, grading in their landmark 1938 monograph on meningiomas, cushing and eisenhardt stated that brain invasion, defined similarly to today by tumoral penetration of the pia, had long been considered a sign of malignancy1. although this was accepted as undisputed gospel over the following decades, the precise role of brain invasion in meningioma grading and how to best define it remains unanswered nearly a century later. for instance, in 1993, mclean and colleagues questioned the role of brain invasion in grading, since they found no additional prognostic significance of this finding within their 28 otherwise atypical and anaplastic meningiomas; unfortunately, they did not address the controversial issue of brain invasive otherwise benign (biob) meningiomas2. similarly, the second edition of the world health organization (who) classification scheme in 1993 highlighted that “some participants maintained that gross brain invasion, not only metastasis, qualifies a tumor for the designation malignant; such lesions do not invariably exhibit histological malignancy and their immunocytochemical features may be similar to those of ordinary meningiomas”3. i suspect that the term “gross” in this phrase meant well-developed or extensive, rather than the surgeon’s gross impression. of course, the significance of brain invasion is a topic near and dear to my heart, as it was one of the first questions i tried to tackle in my neuropathology career, first in a 1997 mayo clinic clinicopathology study of meningioma grading criteria and then in a followup 1999 series addressing the issue of brain invasion and other pathologically defined forms of potential malignancy4,5. along with my mentor, bernd scheithauer, scott stafford (radiation oncology), and other collaborators, we proposed a grading scheme based on statistically significant associations between histopathologic variables and estimated patient survival times (recurrence-free and overall). a fact that is often forgotten today is that these associations were specifically limited to the cohort of patients treated with gross total resection, given that subtotally resected meningiomas frequently recurred regardless of microscopic features. to a large extent, our grading criteria were adopted in the 3rd edition of the who scheme in 20006 and have remained fairly intact ever since. based on our data that biob meningiomas had recurrence and mortality rates similar to atypical meningiomas without brain invasion, it was emphasized that such cases are prognostically equivalent to who grade ii meningiomas. however, it was not until the revised 4th edition of the who in 2016 that brain invasion was adopted as a definitive criterion for the diagnosis of atypical meningioma, who grade ii7. figure 1. examples of well-developed and extensive brain invasion that would likely be associated with high interobserver reproducibility. a, b. note the tongue-like or finger-like protrusions into adjacent brain with no obvious leptomeningeal layer between the two. c. a gfap stain shows entrapped slivers of gliotic cns parenchyma deep within the main tumor mass (upper portion). d. this brain invasive otherwise benign (biob) meningioma showed benign histologic features, including extensive progesterone receptor expression. images c and d are courtesy of dr. robert schmidt, washington university, st. louis, mo, usa. given that we found brain invasion in 4% of cases in our 1997 series, and just 23% of brain invasive meningiomas were otherwise benign in our 1999 expanded series, i expected that this classification change in 2016 would only have a minimal impact on clinical practice (with an expected increase in the incidence of who grade ii meningiomas of 1% at best). in other words, this would be the estimate if biob meningiomas only account for a fourth of the roughly 4% of brain invasive tumors, assuming that everyone was previously diagnosing such cases as who grade i rather than ii, which is clearly not true since some were already designating such cases as grade ii. to my surprise, however, i have personally noticed a mini-epidemic in biob meningiomas in my personal consults and especially in a clinical trial for which i am serving as the central pathology reviewer. of interest, i previously served in a similar role for a pre-2016 rtog/nrg clinical trial that included meningiomas of all three grades; in this series we found a strong concordance rate of 92.4% between the home pathologist’s interpretations of brain invasion and my own (kappa statistic 0.76)8. however, in this post-2016 followup trial focused specifically on atypical meningiomas, there have already been quite a few cases that i have rejected because they initially qualified as grade ii based on focal brain invasion alone, but i disagreed with that call during my central review. admittedly, this is merely an anecdotal impression on my part rather than hard data, given that the study is still accruing cases and no formal analyses have been performed. nonetheless, it might not be surprising if people have loosened their minimal criteria for calling brain invasion after the 2016 who, since it is only human nature (especially among pathologists) to not want to miss even the tiniest example of something if it has a potentially important association with outcome. however, i would argue the opposite approach in favor of stricter criteria so that one does not dilute the potential impact of this prognostic marker with questionable examples. in other words, if brain invasion is the only reason to upgrade a tumor, then i would rather it to be irrefutable before assigning that higher grade. figure 2. a, b. most meningiomas with attached cns parenchyma can be clearly interpreted as being non-invasive, given the broad, linear interface and clear leptomeningeal layer between the tumor and adjacent brain parenchyma. additionally, the modified who 2016 criteria sparked a renewed interest in this controversy and some of the subsequent clinicopathologic series have failed to find any prognostic differences between their biob and non-invasive who grade i meningiomas9-11. in contrast, a 2016 study of 206 who grade ii meningiomas found brain invasion to be an independent negative prognostic variable on multivariate analysis12. another study of who grade ii meningiomas found that brain invasion was prognostic only if specifically combined with the presence of necrosis13, even though a non-invasive meningioma with necrosis only would normally be considered as otherwise benign. for a more detailed summary of the strengths and limitations of previously published studies on this topic, the reader is referred to the review of brokinkel, hess, and mawrin14. suffice it to say that there are still no large, highly powered studies of strictly defined, gross totally resected biob meningiomas with long clinical followup times, given the overall rarity of such cases. as such, this question remains unanswered and the upcoming 5th edition of the who (in press) will include the following text on the question of brain invasion: “brain invasion by meningioma is characterized by irregular, tongue-like protrusions of tumour cells into underlying gfap-positive parenchyma, without intervening leptomeninges. extension along perivascular virchow-robin spaces does not constitute brain invasion because the pia is not breached. such perivascular spread and hyalinization is most commonly encountered in children and can mimic meningioangiomatosis, but does not constitute true brain invasion15,16. brain invasion occurs most often in meningiomas with additional high-grade features. nonetheless, the presence of brain invasion in gross totally resected, otherwise benign-appearing meningiomas remains controversial, as it has been associated with recurrence rates similar to other who grade 2 meningiomas in some, but not all studies5,10,11; larger series with longer follow-up times may be needed to resolve this issue.” given the issues raised above, i decided to write this opinion piece and offer my personal views on what does or does not constitute brain invasion, recognizing that this is at best an educated guess and there is no guarantee that i am any more likely to be correct than authors with opposing viewpoints. as a reminder, most cases with brain invasion will qualify for a higher histologic grade using other criteria anyway, and cases with absolutely no other worrisome features are generally rare. nevertheless, when encountering a biob meningioma on routine histopathology, it is perhaps best at this time to: 1) obtain gfap and other immunostains (ihc) to clarify the brain invasion in borderline cases, 2) order molecular studies to look for other prognostically relevant biomarkers (e.g., specific chromosomal losses, cdkn2a homozygous deletion, tert promoter mutation, etc.), 3) spell out the remaining uncertainties over the clinical significance of these rare cases in the pathology report, and/or 4) suggest close clinical followup just in case the tumor does behave in a more aggressive fashion. figure 3. challenging cases that are more likely to yield discordant interpretations. a. although the interface is irregular rather than linear, there is a discernible leptomeningeal layer between meningioma and brain; as such, i would not call this brain invasion. b. there is a focally irregular interface in this case and it is difficult to determine whether or not there is intervening leptomeninges; a gfap stain could help push me in one direction or the other in such a case. c, d. gfap stains in this case highlight a gently undulating tumor-cns interface; however, in contrast to figure 1c, there is minimal if any definite gfap-positive tissue within the main tumor mass. if this was the only worrisome feature in this meningioma, i would err on the side of caution and not accept this as definite brain invasion. in terms of minimal criteria for brain invasion, a common question is whether or not the surgeon’s intraoperative impression can serve as a surrogate. to my knowledge, all studies published to date have shown a much higher frequency of brain invasion on gross impression than that which is confirmed under the microscope, possibly due to cases with simple adhesion to leptomeninges being overcalled macroscopically. more importantly, with only one exception17, none of the published studies have found a statistical association with grossly diagnosed brain invasion and subsequent recurrence-free survival. as such, the gross intraoperative impression of brain invasion should not be considered a reliable surrogate. that said, of course, the detection of brain invasion is highly dependent on sampling issues and it therefore could be especially useful for the pathologist if the surgeon labels the tumor-brain interface or areas most suspicious for invasion intraoperatively. the importance of appropriate sampling has also been emphasized by other authors and, not surprisingly, the frequency of finding brain invasion increases with greater sampling or perhaps with additional guidance from the surgeon, more targeted sampling. that said, the reported frequency of brain invasion in 33% of otherwise benign meningiomas, by pizem and colleagues in 201418, seems remarkably excessive to me, even in meningiomas that are extensively sampled. in any case, i believe that the original definition of brain invasion, which requires a breach of the pial barrier, is still the best method. in my experience, the two scenarios that are most often mistaken for (or do not show sufficient evidence of) microscopic brain invasion are: 1) an irregular interface with adjacent brain that nevertheless shows an intervening layer of leptomeninges/collagen and 2) perivascular spread along virchow-robin spaces. examples of these and less controversial scenarios are illustrated. additionally, one should be cautious of overcalling brain invasion in highly cauterized or otherwise poorly preserved fragments of tissue. figure 4. a. there is extensive tumoral spread along perivascular virchow-robin spaces in this example, which could easily be misinterpreted as either brain invasion or associated meningioangiomatosis. however, careful examination shows the perivascular arrangement and since this is not considered a breach of the pial barrier, it does not qualify as brain invasion. this type of spread is often associated with radial collars of spindled tumor cells and hyalinization, similar to that seen in meningioangiomatosis. b. a gfap stain fails to show any convincingly entrapped glial tissue within the main tumor mass (upper portion). c. a somatostatin receptor 2a stain highlights the onion-like radial layers of perivascular tumor cells; note that the brain is also normally positive but displays a fibrillar staining pattern distinct from the spindled meningioma cells. d. the mostly centrally placed blood vessels within the virchow-robin spaces are highlighted on this smooth muscle actin immunostain. although this pattern of spread is more often encountered in pediatric patients, this one was from an adult. based on the above, my sincere plea is that we all adopt strict rather than loose criteria for brain invasion going forward, so that if this variable really does provide independent prognostic information, we will be in a better position to detect it. to quote the haarlem white paper from 2014, “diagnostic entities should be defined as narrowly as possible to optimize interobserver reproducibility”19 (or in this case a grading parameter rather than an entity). references 1. cushing h, eisenhardt l. meningiomas: their classification, regional behaviour, life history, and surgical end results. springfield, il: cc thomas; 1938. 2. mclean ca, jolley d, cukier e, giles g, gonzales mf. atypical and malignant meningiomas: importance of micronecrosis as a prognostic indicator. histopathology. 1993;23(4):349-353. 3. kleihues p, burger p, scheithauer b. histological typing of tumours of the central nervous system. in: sobin l, ed. world health organization. international histological classification of tumours. 2nd ed. berlin: springer; 1993. 4. perry a, stafford sl, scheithauer bw, suman vj, lohse cm. meningioma grading: an analysis of histologic parameters. am j surg pathol. 1997;21(12):1455-1465. 5. perry a, scheithauer bw, stafford sl, lohse cm, wollan pc. "malignancy" in meningiomas: a clinicopathologic study of 116 patients, with grading implications. cancer. 1999;85(9):2046-2056. 6. louis d, scheithauer b, budka h, von deimling a, kepes j. meningiomas. in: kleihues p, cavenee w, eds. world health organization classification of tumours. 3rd ed. lyon: iarc press; 2000:176–189. 7. perry a, louis dn, budka h, et al. chapter 10: meningioma. in: louis dn, ohgaki h, wiestler od, et al., eds. who classification of tumours of the central nervous system (revised 4th edition). lyon: iarc; 2016:232-245. 8. rogers cl, perry a, pugh s, et al. pathology concordance levels for meningioma classification and grading in nrg oncology rtog trial 0539. neuro oncol. 2016;18(4):565-574. 9. spille dc, hess k, sauerland c, et al. brain invasion in meningiomas: incidence and correlations with clinical variables and prognosis. world neurosurg. 2016;93:346-354. 10. baumgarten p, gessler f, schittenhelm j, et al. brain invasion in otherwise benign meningiomas does not predict tumor recurrence. acta neuropathol. 2016;132(3):479-481. 11. biczok a, jungk c, egensperger r, et al. microscopic brain invasion in meningiomas previously classified as who grade i is not associated with patient outcome. j neurooncol. 2019; 145(3):469-477. 12. champeaux c, dunn l. world health organization grade ii meningiomas. acta neurochir (wien). 2016. 13. garcia-segura me, erickson aw, jairath r, munoz dg, das s. necrosis and brain invasion predict radio-resistance and tumor recurrence in atypical meningioma: a retrospective cohort study. neurosurgery. 2021;88(1):e42-e48. 14. brokinkel b, hess k, mawrin c. brain invasion in meningiomas-clinical considerations and impact of neuropathological evaluation: a systematic review. neuro oncol. 2017;19(10):1298-1307. 15. giangaspero f, guiducci a, lenz fa, mastronardi l, burger pc. meningioma with meningioangiomatosis: a condition mimicking invasive meningiomas in children and young adults: report of two cases and review of the literature. am j surg pathol. 1999;23(8):872-875. 16. perry a, kurtkaya-yapicier o, scheithauer bw, et al. insights into meningioangiomatosis with and without meningioma: a clinicopathologic and genetic series of 24 cases with review of the literature. brain pathol. 2005;15(1):55-65. 17. behling f, fodi c, gepfner-tuma i, et al. cns invasion in meningioma-how the intraoperative assessment can improve the prognostic evaluation of tumor recurrence. cancers (basel). 2020;12(12). copyright: © 2021 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. top ten discoveries of the year: neurotrauma feel free to add comments by clicking these icons on the sidebar free neuropathology 1:10 (2020) review top ten discoveries of the year: neurotrauma daniel p. perl j. edward hebert school of medicine, “america’s medical school”, uniformed services university, bethesda, md, usa corresponding author: daniel p. perl · j. edward hebert school of medicine · “america’s medical school” · uniformed services university · 4301 jones bridge road, room b-3138 · bethesda, md 20814 · usa daniel.perl@usuhs.edu submitted: 12 february 2020 accepted: 22 march 2020 copyedited by: nicole schwab published: 30 march 2020 https://doi.org/10.17879/freeneuropathology-2020-2662 keywords: neurotrauma, tbi, chronic traumatic encephalopathy, cte, tau, mri, pet, cryo-em abstract neurotrauma represents a major public health problem and is one of the leading causes of death and disability worldwide. despite its high prevalence, there are major gaps in our understanding of the underlying pathophysiology leading to the substantial morbidity and mortality associated with this problem. here, ten studies published in 2019 are reviewed that addressed issues related to the acute and long-term effects of neurotrauma. these studies can be broken down into three separate categories, namely, the importance of neurotrauma-based damage to the cerebrovascular unit, white matter damage following neurotrauma, and research related to the long-term neurodegenerative consequences of repeated head trauma, especially chronic traumatic encephalopathy. the advances highlighted here indicate that progress has been made. however, major gaps in knowledge remain which will require additional neuropathologic studies of clinical specimens, as well as the development and investigation of a wide range of relevant pre-clinical models. further efforts in this field are clearly needed if there are to emerge better clinical outcomes for the numerous patients that suffer neurotrauma each year as well as those currently suffering from its long-term effects. abbreviations chimera closed-head impact model of engineered rotational acceleration, cryo-em cryo electron microscopy, crash clinical randomisation of an antifibrinolytic in significant haemorrhage, ct computerized tomography, cte chronic traumatic encephalopathy, gcs glasgow coma scale, gfap glial fibrillary acidic protein, mri magnetic resonance imaging, mtbi mild traumatic brain injury, nfl national football league, pet positron emission tomography, tbi traumatic brain injury, wt wild type. introduction according to pubmed, in 2019 there appeared 2,508 publications which included traumatic brain injury (tbi) as a keyword. this compares to 886 papers with tbi as a keyword published in 2009. each year, over the past decade, there has been a steady increase in publications, addressing this topic (see figure 1). this almost three-fold increase in published reports over the past decade reflects, i believe, increasing awareness of the importance of the health issues related to neurotrauma, opportunities for neurotrauma research, as well as increased funding for research on the topic. however, as will be pointed out, despite this increased research attention, there is much more to be done. figure 1: no. of tbi papers published (source: www.ncbi.nlm.nih.gov/pubmed) according to the centers for disease control and prevention, in the united states, in 2014 there were 2.87 million tbi-related emergency room admissions, 288,000 hospital admissions and 56,800 deaths (155 per day) (see www.cdc.gov/traumaticbraininjury). that number of annual deaths is greater than occurs each year related to other, more widely recognized, health issues such as breast cancer (42,170 deaths, data from the american cancer society), colon and rectal cancer (53,200 deaths, data from the american cancer society), pancreatic cancer (47,050 deaths, data from the american cancer society) and influenza (51,376 deaths, data from 2014-15 from the cdc) (see www.cancer.org and www.cdc.gov/flu as sources of data on death rates). a further reason for increasing attention to neurotrauma has been concerns about the long-term effects of tbi on the brain and the possible link to the subsequent development of neurodegenerative disorders. there has also been increasing interest among the us department of defense and veterans administration in both the acute and the long-term effects of impact and blast tbi among the more than 2 million military service members who have been deployed to the middle east to fight the war against terrorism. finally, there has been particular concern about the long-term effects of participation in contact sports, such as american football, with the occurrence of repeated concussive and subconcussive impact tbis and a risk of developing chronic traumatic encephalopathy (cte). all of this has combined to promote increasing attention to neurotrauma research. obviously i was not able to review all 2,508 papers appearing in the past year in making my selections of the most important contributions to this field. i readily admit that there is a degree of personal bias to those i have chosen, however, the following is a list of what i consider to be the most influential papers to have appeared in the past year, plus comments on how i believe they have influenced both clinical and pre-clinical concepts. role of the vasculature in tbi pathophysiology in 2019, there have been several reports that highlighted the importance of the cerebrovascular unit in the pathophysiology of tbi. in 2019, this was emphasized by the positive results of a very large clinical trial (crash 2019), the use of magnetic resonance imaging (mri) and subsequent neuropathology correlation in tbi patients (griffin, turtzo et al. 2019) and a pre-clinical model of sub-concussive blast exposure in the rat (gama sosa, de gasperi et al. 2019). over the past several years, unfortunately, there have been a large number of failed clinical trials seeking to improve outcomes of acute victims of tbi. however, in 2019, a randomized placebo-controlled clinical trial on the effects of treatment by tranexamic acid on death and disability in patients with acute tbi (crash 2019) was successful. the rationale for this approach is that tranexamic acid reduces bleeding by inhibiting the enzymatic breakdown of fibrin blood clots. fibrinolysis, with increased concentrations of fibrinogen degradation products, is commonly seen in the brains of acute tbi patients and predicts the development of further intracranial hemorrhagic expansion (zhang, he et al. 2018). the report involved an extremely large trial, consisting of a total of 12,737 hospitalized patients with acute tbi. enrollment criteria included adults with tbi who were within 3 hours of injury, had a glasgow coma scale (gcs) of 12 or lower or showed evidence of intracranial bleeding on ct examination. the study clearly demonstrated that administration of tranexamic acid within 3 hours of head injury significantly reduced the risk of death. the authors stressed that early treatment was essential to elicit this effect and that patients with a gcs score of 3 or less or who had non-reactive pupils at baseline failed to show a reduction in death-rate. in the tranexamic acid-treated group, excluding patients with a gcs of 3 or with bilateral unreactive pupils at baseline, the risk of head-injury-related death was 12.5% versus 14.9% in the placebo group (485 vs 525 events; rr 0·89 [95% ci 0·80–1·00]). in addition, their study demonstrated that tranexamic acid administration in the tbi patients had a very good safety record. the risks of serious vascular occlusive events (deep vein thrombosis, pulmonary embolism, stroke or myocardial infarction) or of seizures were not increased in the tranexamic treated patients when compared to the placebo group. importantly, tranexamic acid is a widely available drug which is quite inexpensive. indeed, the total cost of this medication, as given in the trial, was less than $10/patient! tbi is not just a problem in rich industrialized countries, but is seen worldwide, especially in developing countries. tbi is a leading cause of death and disability worldwide, with an estimated 10,000,000 cases being hospitalized or dying each year (roberts, belli et al. 2018). the low cost and demonstrated safety of this treatment makes it ideal for use throughout the world and, according to the authors, promises a dramatic impact on the survival of large numbers of patients. this important paper reports what i believe is the first clinical drug trial to ever show significantly reduced mortality from tbi. it is promising for changing clinical practice and saving numerous lives, worldwide. for neuropathologists, this paper emphasizes the importance of bleeding and vascular ischemic lesions as important factors leading to fatal outcomes following head trauma. these aspects have been poorly documented and understood in the neuropathology literature and will require further investigation, both in a clinical setting and in pre-clinical models. an example of this approach is my next chosen publication which involves mri and correlative neuropathology in the evaluation of acute tbi patients (griffin, turtzo et al. 2019). full disclosure requires that i identify myself as a participant in, and an author of this study which reports on the identification and characterization of microbleeds in the work-up of acute patients with tbi (griffin, turtzo et al. 2019). nevertheless, the results reported are sufficiently important to include it on my list of important contributions to the field of neurotrauma. within the initial clinical workup of patients coming into an emergency department following tbi is the use of ct examination looking for evidence of major sites of intracranial bleeding that might require surgical intervention. some research groups have now begun to employ mri imaging as a means of providing the more detailed morphologic spatial resolution inherent to this technology to the information available for acute tbi clinical management. in doing so, it has been noted that the brains of many tbi patients being evaluated in this way show the presence of multiple small, often punctate or linear hypointensities on t2-weighted imaging (huang, kuo et al. 2015, chiara ricciardi, bokkers et al. 2017). mri lacks a cellular level of resolution leading to conjecture as to what the underlying pathology of these lesions might represent. some have interpreted them to be traumatic microbleeds, while others have suggested them to represent the sites of hemorrhagic axonal injuries. importantly, whether such mri findings may be used as a biomarker for predicting successful, partial, or poor recovery remains an unanswered question. the data in this study (griffin, turtzo et al. 2019) is from a large number of civilian tbi victims who were clinically evaluated in several urban trauma centers in the washington, dc area. eligible patients were entered into an mri screening protocol along with further longitudinal clinical follow-up. a total of 439 patients met inclusion criteria for the initial neuroradiologic analysis. most of those enrolled were determined to have mtbi (83%), with a mean gcs at the time of initial evaluation of 15. importantly, approximately one-third of these mild patients showed evidence of punctate and/or linear microbleeds on initial mri exam, indicating that such lesions are not uncommon following mild and mostly asymptomatic head trauma. the presence of traumatic microbleeds was shown to be an independent predictor of subsequent disability in the cohort (p >0.05; odds ratio = 2.5). one of the patients who had received mri within 48 hours of injury and showed the presence of multiple punctate and linear intraparenchymal hemorrhages, died 7 months following the accident and was subjected to post-mortem examination with brain donation for use in research. using the clinical mri exams, supplemented by ex vivo imaging of the formalin fixed brain specimen, a correlational neuroimaging/neuropathology investigation was performed on the specimen in an attempt to identify the histopathologic nature of the punctate/linear hemorrhages that had been identified. using co-registration of the clinical and ex vivo mri images with histopathologic stained sections, the post-traumatic microhemorrhages showed the presence of iron-laden macrophages, predominantly within perivascular spaces. in these colocalized histopathologic slides, evidence of axonal injury was not observed in conjunction with the microhemorrhages. this study highlights the importance of damage to the cerebral vasculature as a consequence of mtbi and its effect on clinical outcome. this result, combined with the findings of the previously discussed paper on the effectiveness of tranexamic acid, underscores the importance of studying the effects of tbi on the integrity of the cerebrovasculature unit. we are far from knowing how best to use such mri findings for a more rational therapeutic approach to patients. however, additional correlational neuroimaging / neuropathology studies of this type will clearly lead to important information that will, in the future, refine clinical diagnostic and treatment guidelines. in a further investigation of the role of the cerebrovascular unit in the pathophysiology of tbi, gama-sosa and colleagues reported findings in a pre-clinical rat model of repetitive mild blast tbi (gama sosa, de gasperi et al. 2019). using a blast tube facility, the dose of blast exposure to the rats was mild and determined to represent 74.5 kpa (equivalent to 10.8 psi) and animals were exposed to one blast per day, over three consecutive days. exposed rats were sacrificed at 6 weeks, 8 months and 10 months following blast exposure. this model was considered to be a low level of blast exposure, comparable to those endured by military service members in training activities and combat exercises (thus emphasizing the clinical relevance of this model), and exposed animals failed to show evidence of generalized neuronal pathology at either the light or electron microscopic level. however, upon detailed examination of the neurovascular unit, significant pathology was identified. using purified vascular fractions, decreased levels of vascular-associated glial fibrillary acidic protein (gfap) and that of several neuronal intermediate filament proteins (α-internexin and low, middle and high neurofilament proteins) were identified 6 weeks following the blast exposure. electron microscopy showed evidence of damage to astrocytic end-feet associated with blood vessels. at 8 months following blast exposure, there was evidence of alterations of the vascular smooth muscle layer. these findings indicate that despite the low dose of blast exposure, significant chronic vascular damage had been induced. these intriguing findings need to be further investigated and serve to add additional support to the importance of the above discussed papers. white matter involvement in tbi a consistent clinical finding in patients with tbi is slowing of information processing. damage to neuronal circuitry, with disruption of long axons, is thought to play a major role in this phenomenon. in an attempt to model this aspect of tbi, a group experimentally studied aspects of tbi damage using a direct impact concussion model in mice (marion, radomski et al. 2018) that has previously been shown to induce traumatic axonal injury to the corpus callosum (mierzwa, marion et al. 2015). animals were studied neurophysiologically and morphologically. following impact, compound action potential velocities along myelinated axons were slowed within 3 days of injury with partial recovery of function by 2 weeks. this suggested to the authors that partial demyelination, followed by remyelination was responsible for the changed action potential velocities. using electron microscopy, dispersed demyelinated axons and disorganized myelin attachments to axons at paranodes were noted within the corpus callosum of the injured mice. animals examined after longer periods following tbi exhibited an overall loss of axons at 6 weeks post-injury and observable corpus callosum thinning was seen by 8 weeks. white matter damage is a consistent finding in the brains of patients who survive tbi and have significant cognitive issues and conspicuous thinning of the corpus callosum is often noted in such patients (johnson, stewart et al. 2013). this paper provides a useful experimental model for this phenomenon and is promising for a better understanding of the pathophysiology of such white matter damage and a substrate to explore treatment options for patients with this important aspect of head trauma’s clinical effects. the year 2019 has, among other things, witnessed increasing attention to the metoo movement, recognizing the high prevalence and importance of sexual harassment and violence against women. i was intrigued to find a publication which identified the importance of intimate partner violence as a substrate for tbi, as evidenced by its effects on the integrity of white matter (valera, cao et al. 2019). the authors point out that it is estimated that in the united states, about 42,000,000 women over age 15 have experienced physical or sexual abuse and that 75% of women with a history of intimate-partner violence have sustained at least one partner-related tbi, while 50% have sustained repeated tbis. despite this staggering number of victims, there has been almost no study of the long-term functional and structural consequences of such violence. the valera, et al preliminary study attempted to address this gap in knowledge by recruiting 20 women who had sustained such intimate partner assaults, based on a detailed semi-structured interview. participants underwent detailed study, including 3t mri neuroimaging. the neuroimaging aspects of the study revealed evidence of abnormalities in fractional anisotropy in the posterior and superior corona radiata as well as the posterior thalamic radiation. however, this was a preliminary study with limited power and lacked a control population for comparison. nevertheless, this paper identified a potentially very large group of women who are at-risk for significant tbi and has been largely overlooked. their preliminary results indicate that the degree of neurotrauma in women who sustain tbi through intimate partner violence is sufficient to produce changes in white matter integrity visible with 3t mri neuroimaging. as the authors point out, the number of women that could be affected vastly overwhelms that of professional contact sport athletes or even those with non-professional participation in such sporting activities. the underlying neuropathologic nature of damage related to this form of neurotrauma remains unstudied and is an issue that clearly needs further investigation. advances in the investigation of the effects of repeated impact tbi on the development of neurodegeneration (chronic traumatic encephalopathy, cte) there has been considerable interest in the subsequent development of neurodegeneration following repeated impact tbi, particularly in the form of cte. in specific, this has involved concerns about how prevalent the disorder is among professional contact sport athletes and, more broadly, about possible risks to the general public following much lesser extents of tbi exposure. in 2019, these concerns have been addressed in a number of ways. currently, cte can only be diagnosed by neuropathologic examination of the brain at autopsy. clinically, there is a considerable need to identify an approach by which patients at-risk for this condition can be evaluated and confidently diagnosed during life. for the most part, this effort has involved the use of a variety of positron emission tomography (pet)-scan ligands directed against tau deposits in the brains of at-risk subjects (dickstein, pullman et al. 2016, marquie, normandin et al. 2017). the use of similar pet ligands directed at beta-amyloid has been employed with considerable success in the diagnostic work-up of patients thought to be suffering from alzheimer’s disease and this paper (stern, adler et al. 2019) represents the use of similar approaches for cte, a form of tauopathy. in a 2019 study (stern, adler et al. 2019), 26 former national football league (nfl) players and 31 controls were evaluated using both flortaucipir and florbetapir pet ligands, reagents directed against tau and beta-amyloid, respectively (wong, rosenberg et al. 2010, marquie, siao tick chong et al. 2017) . the former football players had a minimum of 2 years playing in the nfl (mean 9.1 years ± 3.0) and a minimum of 12 years total football playing experience (mean 18.8 years ± 4.6). all participants reported to be suffering from cognitive, mood, and behavioral symptomatology. the controls were free of a history of tbi or evidence of cognitive impairment. the former football player cohort showed significantly elevated uptake by the tau pet ligand in the three neuroanatomic regions that had been examined (bilateral superior frontal, bilateral medial temporal and left parietal areas) when compared to that of the control group. there was no difference between groups in the beta-amyloid directed pet results. as the authors point out, the differences noted were based on collective group findings and the results “do not inform the use of testing in individual patients to determine cte pathology during life.” at the time of publication, all of the participants in this study are presumably still alive and thus there was no opportunity to validate the findings with post-mortem assessment. this is an important first step but validation using a much large sample will be necessary to determine the specificity and sensitivity of this approach to the diagnosis of at-risk individual patients. neuropathologists will need to be actively involved in such studies, particularly in attempts to identify individuals with cte and distinguish them from patients with other forms of neurodegenerative disorders, especially other forms of tauopathies. most studies to date have depicted cte in patients who participated in boxing or american professional football. in 2019, an epidemiologic study appeared documenting evidence of an increased incidence of neurodegenerative disorders among individuals who had been former professional soccer players (mackay, russell et al. 2019). the authors used mortality data derived from the death certificates of 7,676 former scottish professional soccer players and compared them to 23,028 controls that were drawn from the general population. further, they compared prescription information on the use of medications prescribed for the treatment of dementia between the two groups. the results indicated that scottish professional soccer players had a lower all-cause mortality rate than the general population but showed a significantly increased tendency to die of all forms of neurodegenerative disease. indeed, significantly higher mortality rates from 1) dementia, not otherwise specified, 2) alzheimer’s disease, 3) non-alzheimer’s dementias, 4) motor neuron disease and 5) parkinson’s disease were all seen in the former soccer players when compared to general population controls. recognizing the weakness of possible errors in death certificates’ stated causes of death, the authors turned to the prescription data for drugs used to treat dementia, showing a higher rate of use among the former soccer players. obviously, there was no opportunity to verify by autopsy examination the indicated causes of death on the death certificates. however, errors in ascribing such deaths would be expected to be roughly equal among the athletes and the controls. this study expands the growing epidemiologic literature supporting the concept that repeated head trauma has a correlation with the subsequent development of neurodegenerative diseases and dementia. the nature of that association and, in particular, whether it speaks to the induction of cte and/or alzheimer’s disease, parkinson’s disease or amyotrophic lateral sclerosis, will require additional study and neuropathologic investigation of incident cases. this paper points to the importance of such studies in the future. as an additional point, the authors remind their readers that soccer is played in over 200 countries worldwide with more than a quarter billion participants. while this study could not address the risks of more casual amateur play, it does raise further concerns regarding the long-term consequences of repeated head trauma, a subject that is likely to attract increasing interest and research in the coming years. attempts to model chronic traumatic encephalopathy (cte) in rodents chronic traumatic encephalopathy (cte) is a progressive neurodegenerative disease that has been closely linked to repeated head trauma, primarily in the setting of participation in contact sport athletics. the neuropathologic features of cte include deposition of abnormally phosphorylated tau in neurons and astrocytes in a unique and distinguishable pattern (mckee, cairns et al. 2016) (see figure 2). despite considerable efforts, no laboratory has been able to produce a consistent and appropriate experimental model of cte. in 2019, two papers (gangolli, benetatos et al. 2019, mouzon, bachmeier et al. 2019) described efforts to do so using differing impact rodent models and both failed to induce intraneuronal tau deposits in their injured animals. figure 2. characteristic tau neuropathology features of chronic traumatic encephalopathy (cte), including: a. depth of sulcus involvement, b,c. perivascular foci with involvement of neurons and astrocytes, and d. neurofibrillary tangles in substantia nigra, pars compacta neurons (arrows). hyperphosphorylated tau immunohistochemistry (at-8) in a former collegiate american football player. first and foremost, one needs to recognize that rodent tau is different from human tau and there has been an almost universal failure, under any experimental condition, to induce tau aggregates comparable to human neurofibrillary tangles in wild-type mice. accordingly, both groups used a humanized mouse transgenic model with knock out of the expression of the native mouse tau, in the hopes that this maneuver would enhance their chances of inducing such changes. in the gangolli et al. (gangolli, benetatos et al. 2019) experiment, head trauma was produce using the closed-head impact model of engineered rotational acceleration (chimera) approach. chimera is a relatively newly developed technology that is capable of producing both concussive and subconcussive injury to experimental animals and also produces a significant rotational acceleration component (namjoshi, cheng et al. 2017). mice were exposed to 20 daily impacts for 20 consecutive days and the energy used for the chimera impact was controlled to yield animals with either a subconcussive or concussive level of injury. all animals underwent behavioral testing at 3 months and 12 months post-injury and were sacrificed for morphologic and biochemical studies at the later time period. behavioral abnormalities were seen in both the subconcussive and concussive-exposed animals (more prominent following the higher energy impact model) and this was accompanied by evidence of white matter disruption. importantly, there was no evidence of tau pathology in any of the injured animals. clearly, these experiments demonstrated that repeated exposure to head trauma through the chimera approach could induce white matter damage, important in and of itself, but this failed to induce a model that could be used in studying the underlying tau-related pathophysiology of cte. the mouzon, et al. paper (mouzon, bachmeier et al. 2019) reports exposing humanized transgenic mice to closed-head impact injury with either a single blow or 5 successive blows over a ten day interval (48 hours apart). the animals were then allowed to survive for either 24 hours or 12 months, post-injury. evaluations included behavioral testing and, on sacrifice, morphologic and biochemical assays. the behavioral testing showed persistent and progressively worsening deficits in visuospatial learning from 2 to 12 months and significant deficits in visuospatial memory consolidation at 12 months post-injury. neuropathologic evaluation at both 24 hours and 12 months revealed evidence of axonal injury, thinning of the corpus callosum and activation of microglia and astrogliosis in the white matter. tau immunohistochemistry and elisa studies showed an increase in phosphorylated tau in the cortex at the site below the impact point and in the ca1/ca3 region of the hippocampus of both the single and repeated injury mice. however, this response was transitory in the immediate post-injury (24 hours) phase and was not detected at the 12-month survival time period. no neuronal tau aggregates were detected in the injured animals. the authors point out that the “post-tbi neuropathology in htau and wt mice was otherwise comparable, with essentially similar levels of axonal injury and chronic neuroinflammation in both study groups.” they further suggested that the observation of transient tau hyperphosphorylation at acute time periods post-injury “may be a normal physiologic response of the brain to mtbi.” both articles failed to show that the use of humanized tau mice with repeated impact tbi was able to develop a rodent model of cte and thus their results could not be used to mimic the human disease. in my opinion, despite considerable effort, the neurotrauma field has yet to identify a consistent and valid animal model of cte. characterization of tau filaments in chronic traumatic encephalopathy cases by cryo-electron microscopy cryo-electron microscopy is a technology that is capable of imaging the three-dimensional structure of complex biomolecules at the single digit angstrom level of resolution. this newly developed approach is so powerful, that its developers, drs. jacques dubochet, joachim frank and richard henderson, were honored with the 2017 nobel prize in chemistry for their work. tau filaments isolated from the brains of patients with alzheimer’s disease (fitzpatrick, falcon et al. 2017) and pick’s disease (falcon, zhang et al. 2018) have been reported using this technique and have been shown to form differing configurations. in 2019, falcon, et al. (falcon, zivanov et al. 2019) reported a cryo-em study of tau filaments isolated from three cases of cte (a former american football player and two former boxers). the configuration of the filaments isolated from each of the cte cases were identical and were distinctly different than that isolated from the alzheimer’s disease and pick’s disease brains. in the cte-derived tau filaments, the β-helix region of the molecule displayed a distinct hydrophobic cavity that was not seen in tau filaments from alzheimer’s or pick’s disease specimens. this cavity contained an additional density whose identify has yet to be identified but was thought to be a non-proteinaceous co-factor. these exciting findings serve to define cte as representing a distinct and different neurodegenerative process from alzheimer’s disease. in addition, the identity and function of the associated co-factor represents a potentially exciting therapeutic target for patients with cte. conclusions in this review a number of studies are presented, some involving clinically relevant issues while others use preclinical models to investigate pathophysiologic mechanisms related to neurotrauma. the studies discussed here have relevance to neuropathologists with an interest in aspects of neurotrauma, whether they be involved in clinical diagnostic issues or preclinical models. despite the clinical importance of neurotrauma and its acute and long-term consequences, there exist major knowledge gaps throughout the field that require further investigation. i hope the studies discussed here will help to stimulate neuropathologists, especially those newly entering the field, to become active in this important and fertile area of investigation. disclosure statement the opinions expressed herein are those of the author and are not necessarily representative of those of the uniformed services university of the health sciences, the united states department of defense or of the united states army, navy or air force. references chiara ricciardi, m., r. p. bokkers, j. a. butman, d. a. hammoud, d. l. pham, s. warach and l. l. latour (2017). 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"prognostic role of d-dimer level upon admission in patients with traumatic brain injury." medicine (baltimore) 97(31): e11774 copyright: © 2020 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. neuropathology as a life-task feel free to add comments by clicking these icons on the sidebar free neuropathology 1:29 (2020) reflections neuropathology as a life-task werner jänisch address for correspondence: werner jänisch · heinrich-heine-str. 13 a · 14547 fichtenwalde · germany werner.jaenisch@googlemail.com submitted: 12 september 2020 accepted: 13 october 2020 copyedited by: henry robbert and biswarathan ramani published: 16 october 2020 https://doi.org/10.17879/freeneuropathology-2020-3052 additional resources and electronic supplementary material: supplementary material keywords: neuropathology, personal reflections, gdr, charité 1. prologue neuropathology as a special field in medicine saw its development not until the second half of the twentieth century. its roots are in neurology and pathology. its significance has risen because of the emergence of neurosurgery. the validity of neuropathological diagnoses greatly increased especially because of the application of immunohistochemistry and genetics. this may have been the rationale for the editor of this journal to have neuropathologists’ paths of life delineated for future generations under the heading “reflections.” this includes the various pathways and detours to neuropathology and the ways research findings have been generated, which may eventually prove important or useless. it has been the desire of the editor that authors not only reflect on their professional activities and experiences, but also present themselves as persons. this paper should not be intended as an autobiography, but the development and experiences of the author be presented in appropriate length. i think of this as a good approach, which i will attempt to pursue. 2. social origin and education i was born in 1932 in the german reich, specifically in the industrial city of chemnitz, saxony. my parents married in 1928 and lost employment shortly after due to a hard-hitting economic crisis. even as my father successfully found work as a truck driver the income of my parents was so low and the living conditions so confined that i remained their only child. my childhood was overshadowed by the reign of national socialism in germany. in 1938, i entered the volksschule (i.e., the prevalent type of elementary school at that time). in the beginning, i felt enthusiastic about a young, skilled teacher. when he did not return after the summer break of the year 1939, i and the other kids were very disappointed. the teacher had been drafted as a soldier by the wehrmacht. he was replaced by an old teacher who approached school lessons by using a cane for whipping and punching, thus taking away our joy in studying. in the volksschule we learned how to read, write, basic math and adolf hitler’s curriculum vitae. many classes were replaced by foot drills in the schoolyard. they were directed by a teacher who had suffered a brain injury in the beginning of the war, disqualifying him from military service. by the end of 1943, when i, a mediocre student, had reached sixth grade, classes were suspended in the larger cities of the german reich. this was due to the alleged danger posed by anglo-american air strikes. my father was drafted as an ordinary soldier at the beginning of the war in 1939, finally became an unteroffizier (i.e., roughly on the level of a sergeant) and died as a prisoner of war in a military hospital in september of 1946. my hometown chemnitz was spared from air strikes until the beginning of 1945, but was largely destroyed on march 5, 1945 by anglo-american bomber squadrons. our two-room apartment, which was located in a big apartment complex, was affected by that, as well. my mother and i survived, became homeless and possessed only what we had on our bodies. we were placed in an emergency shelter in the small town of mittweida, 18 km north of chemnitz. in mid-april of 1945, this area was occupied by american troops without a fight. these troops left after about two weeks and a few days later troops of the red army took over the town, because it was part of the designated soviet occupation zone. a soviet military administration had control over this zone. one of its first actions was the order to vaccinate the entire population. this was a life-saving measure because of the aftermath of the war and the refugee streams infectious diseases such as typhus, diphtheria and tuberculosis were spreading out quickly. for me personally there was another order of the soviet military administration that was of great importance: it was said that beginning on october 15, 1945, classes were to resume. this meant that i, because of my age at that point, was able to get into eighth grade of the volksschule. together with 44 other boys, i joined class led by a teacher who was an elevator installer but had been prepared for teaching in a crash course. my knowledge gaps were vast, which was also the case with other students who went through similar experiences as i had. my teacher tried to make up for this with remarkable patience and a lot of empathy. however, it should be said that it helped that most students were very eager to study. our thirst for knowledge was insatiable. i am grateful to this teacher for providing us with a solid base of knowledge and teaching us important basic principles in ethics. unfortunately, eighth grade was the last grade of the volksschule. graduates were instructed in a profession, or they entered professional life without any instruction. as a child, i wanted to become engine driver of a steam locomotive. the requirement for this was the professional instruction in a railroad facility. a few weeks before graduation, i applied for such an apprenticeship and got a rejection. deeply disappointed, i started looking for an alternative. mittweida was part of the textile industry. workers were able to work in spinning mills, weaving mills and sewing rooms without instruction (unlike their superiors). these places also offered no apprenticeships. the only choices i had were to become instructed as a painter, carpenter, metal worker, chimney sweeper, baker or butcher. none of these prospects was desirable. in this situation suddenly a new perspective opened up due to another order of the soviet military administration: it was said that skilled graduates of the volksschule were able to enter the oberschule (roughly equal to high school). until then parents were only granted to put their children into oberschule (which are called gymnasium nowadays) after they had finished fourth grade – but only in case they were able to pay tuition. this was not the case for me. my teacher made arrangements so that i could continue my studies without having to pay tuition! my mother and i were thrilled. i initially joined a class for students who had to adapt, which means that lessons were conceptualized as to make sure students could gain the knowledge the other students had been building on since fifth grade of the oberschule. i was an enthusiastic student. latin, biology, chemistry, history, german literature and russian became my favorite subjects. math was not one of those, although we had a good teacher. i also encountered problems in english. i was not aware that english words were pronounced differently as could be assumed from the way they were written (for example, it was not clear to me how to pronounce the “u” in a word like “must” the way it sounds in english as opposed to a “u” in german). the english teacher, a former protestant minister, never imparted this knowledge to us. hence, i eagerly studied the vocabulary from the book but still had bad grades. after a couple of months the teacher left the school, so english lessons were suspended because there were not many english teachers left in the soviet occupation zone. because unemployment was widespread in the other occupation zones in the years 1946–1948, workers came to the soviet occupation zone, including english teachers. one of them came to our school and with him his unconventional methods. we have been studying walter scott’s novel “ivanhoe” for a full school year. he distributed copies of the novel’s german translation, told us to read it chapter by chapter, and demanded summaries and interpretations of its content in german. this became the basis for our grades. this way i was able to study an important work of english literature and got good grades. however, i did not learn a single word of english and i was aware of this. i became friend with a student of a higher grade who had english classes prior to 1945. he agreed to teach me some of his knowledge. this way i was able to improve my pronunciation of the vocabulary and learn some basic grammar. but still my english skills remained utterly mediocre. i was able to read and understand simple texts, but i was lacking an active command of the language. because i took great joy in studying, my time in the oberschule went by in a flash. a few months before the abitur (i.e., the high school diploma), we were asked to decide if we planned to continue studying, which subject we wanted to major in, and at which university we would try to apply. since i had been an eager student of biology in school and extended my knowledge reading zoological books and spending time in nature, i knew early that i wanted to study biology. but suddenly doubts arose: what would i do if biology would not meet my expectations, if chemistry and physics were more interesting to me? this led to the idea of studying medicine. it includes morphological subjects, microbiology, genetics, biochemistry, pharmacology, applied radiophysics and i could choose which scientific area i wanted to pursue later in my career! the next question was concerning the place i wanted to study. then, in the gdr (german democratic republic, ddr), there were medical schools at the universities in berlin, leipzig, jena, rostock and greifswald. because of my interest in biology i was leaning towards jena early on, even though i had never visited the city. the reputation of the biologist ernst haeckel and the anatomist christian loder made that university so attractive to me, which was why i applied to this school. since i graduated from the oberschule with the final grade “sehr gut” (i.e., the best overall grade) in 1951, i was promptly enrolled as a student of medicine and received a monthly stipend of 260 mark. it should be noted that in the gdr every student could apply for a scholarship, which was not a loan and did not have to be paid back later. the sum of the stipend was dependent on the final grade in high school and the grades a student got at the university and could vary between 180 and 260 mark. the sum of the stipend i received was sizable for me personally (my mother, who was full-time employed, earned much less than that). it should be noted that i paid my landlady 20 mark per month, that 1 kg of bread cost 30 pfennige (pennies) and one meal in the dining hall 70 pfennige. after my first year i completed my exams in physics, zoology and general botany with the best grade (“sehr gut”) and was ordered to the dean’s office. i was offered the possibility to continue my studies at a university in the soviet union. i was selected because of my academic performance so far, and my high school grade in russian. i asked for some time to think because i wanted my mother to agree. this was necessary, because she would be by herself for a long time if i went to study abroad. studying in jena, i was able to visit her for a weekend every two months. without any hesitation she recommended me to take the offer. so in october of 1952 i went to moscow with about 200 other students of various disciplines and from there went to leningrad to study medicine. there were three major civil medical schools with different instructional foci and a military medical school. the different foci of the schools can be explained by the fact that most graduates are sent to different parts of the country without receiving a specialist medical instruction. in remote places of the country (siberia, north of the polar circle, etc.) it was not uncommon for them to be the solely responsible physician. there was no telemedicine back then. i arrived at a medical school for future pediatricians. today this is the saint petersburg academy of pediatrics and maternity. its students came from all republics of the soviet union and many foreign countries. all special areas of medicine were taught, as was the case in german universities as well. what was special about this medical school was that there was a special focus on children. this concerned infectious diseases, nutritional disorders, and skin diseases, among others. but this also concerned theoretical subjects such as physiology, pathology (pediatric pathology was taught very extensively) and even forensic medicine. the instruction of the students took top priority, was very thorough and frequently individualized. interested students were offered “academic student circles” in different special areas and could thus learn about and participate in professors’ research activities. i took part in this as well, first in anatomy and later in pathology. in 1958, my studies concluded with a state exam “with distinction” (“ausgezeichnet”), and i returned to the gdr. fig. 1: in the reading hall of the dormitory in 1953. 3. memories of leningrad after arriving in leningrad, i was lodged in a dorm room. the dormitory’s construction began before world war ii and was finished after the end of the war, partly with the help of german prisoners of war. it was clean and cozy. on the ground floor there was a library with a reading room offering literature relevant for studies and the belles lettres that could be borrowed and studied (fig. 1). all newly arrived foreign students could take part in a tour of the city. since the city underwent restorations shortly before, the historical buildings were shiny and the parks well maintained. we were shown the memorials of tsar peter the great and tsar catherine the great and various cultural sites. i used to go to the theater frequently to enjoy the works of pushkin and chekhov in the beauty of their language, to opera performances and the ballet of leningrad, and occasionally, symphonies, as well. the latter was directed by kurt sanderling, who during the reign of national socialism emigrated to the soviet union and later acted as chief conductor in the gdr. leningrad presented itself as a vibrant big city with a dense net of buses, trolleybuses, and streetcars. back then underground railways did not exist yet. as a german student i was warmly welcomed and offered help by the university and the general population. never was i or, as far as i know, any other students from the gdr blamed for the drastic crimes that happened during the german blockade of leningrad. we received a monthly stipend of 800 rubles. (later the ruble, and the stipends, was devalued 10 to 1.) the dorm room cost 15 rubles per month. it should be noted that the monthly salary of our professors were between 1,200 and 1,600 rubles. hence, we could dedicate ourselves to our studies without having any material worries. my fellow students welcomed me with friendliness. they were eager to get to know how we were living in germany and how we experienced the war. the first question i was asked was: “is hitler still alive?” because of my imperfect command of the russian language, i thought i misunderstood that question, because for us germans it was clear without a doubt that hitler was dead. until some back and forth it became clear to me that even in 1952 the people still worried that hitler might have escaped and could become dangerous again. i asked many questions, too, and got to know a lot about the reality of everyday life and life during the war. successively i learned to understand the russian people’s way of thinking. one example from the first days of my stay might make this clear: i asked a russian student, whom i shared a dorm room with the question: “valentin, where is it you are from?” he replied: “prigorod”, which translates to suburb, i.e. close to leningrad. later i found out that the distance between the two places was 800 km. i got further insight when i was invited to the homes of university staff. i was introduced to russian hospitality and their difficult living conditions. one of the families, which consisted of two parents and two small daughters, lived in a room of about 40 m² in what was called “communal quarter”. these were large apartments consisting of eight or nine rooms in houses spanning four stories constructed during the reign of the tsar. because of the scarcity of housing, these large apartments were divided and rented roomwise. the former kitchen that was accessible to all families was equipped with a big boiling pot for everyone and a small gas stove for each family. at the apartment’s entrance, there was a doorbell and a list with the family names of the renters accompanied by a number of ringtones. my hosts had the number seven. when the ringtone occurred, somebody in the family answered the door. during my stay with them and during intense conversations the doorbell rang frequently. when i said, “this is for you” they replied, calmly, “no, it was only six times” and continued talking to each other. i mention this because this situation was unusual to me and gloomy, but they showed to me that there are more important things, namely practicing hospitality, which they did extensively. this had an influence on me, so for my wife and me it was clear that we were hospitable to visiting students and fellow researchers, regardless from which country they came. i also feel deep gratitude for my professors and their assistants. they trained me how to do my first pathological/anatomical dissections. they introduced me to the great public library of leningrad and showed me how to navigate the scientific catalogs. with their support i learned how to make microscopic preparations and use for my first academic publications. saying farewell to leningrad was hard for me. 4. becoming a pathologist and my journey to neuropathology in medical school i was especially interested in morphological subjects: first anatomy, then pathology. as a student i conducted more than 100 autopsies by myself, as well as writing the dissection reports and participating in clinicopathological conferences. this was an occasion for me to delve deep into scientific literature. for example, performing a postmortem of a patient with an oligodendroglioma motivated me to buy and study k. j. zülch’s book on brain tumors (23) in 1957. back then, i was already intent on becoming active as a physician in the field of pathological anatomy. it was my desire to get instructed in this field at a university institute. in order to be granted a full medical license in germany, one had to have practiced as an assistant in a clinical subject for at least one year. therefore, in 1958 i applied to the medical academy erfurt to be instructed for six months each in the clinics for internal medicine and surgery. in the clinic for surgery i was working in the department of neurosurgery, which was headed by professor usbeck. the department mostly treated patients with brain tumors and trigeminal neuralgia. at the end of this training professor usbeck offered me to train me as a neurosurgeon. i declined by explaining that i had decided to receive my specialized training at the institute for pathological anatomy of the medical academy erfurt. to this, he responded: “then go ahead and take care of histopathological diagnostics of glioma. almost every diagnosis i receive from the pathologists is astrocytoma. this cannot be right, because since baily and cushing (1) we know more about glioma and need exact diagnoses for our treatment strategies.” i took this suggestion seriously. first, because i knew from my own experience that at most german facilities for pathological anatomy – regardless if university or municipal – neuropathological questions did not get much attention. it was mostly neurologists and psychiatrists who were dealing with neuropathology. secondly, i had realized that the field of pathology was so extensive and diverse that it was not possible to gain profound knowledge in every subfield. furthermore, i desired to do research. therefore i decided to pay special attention to the area of neuropathology during my training in pathology. this began with in-depth studies of the literature. the institute for pathology at the medical academy erfurt, founded in 1954, had a well-equipped special library. also, the academy had a central library, which allowed to order any missing scientific literature from all major libraries in the country and abroad for free. thus, there was nothing in the way of further specialization. however, i needed professor harry güthert to agree to my endeavors (fig. 2). he became prosector of the municipal clinic in erfurt in 1946. he became the director of the newly built institute for pathology when the medical academy erfurt was founded in 1954. the institute also had a large barn for test animals. the institute’s staff comprised about 40 people, including two chief physicians (oberärzte) and nine assistant physicians (assistenzärzte). about six months after starting this activity i suggested to the director that i wanted to specialize in neuropathology. after a couple of minutes of thinking he replied: “agreed. i will send at the university of leipzig for a couple of weeks. there you can become familiar with the basics of neuropathology and study histopathological preparations.” he had his secretary immediately connect him to the director of that institute to arrange a date for my visit. i used to see again and again that this kind of rapid and focused reaction was typical of professor güthert’s style of leadership: when something was suggested to him that furthered the development of the staff and the institute he got behind those suggestions with all means that were at his disposal. fig. 2: professor harry güthert, director of the institute of pathological anatomy of the medical academy erfurt in the year of his retirement. after returning from this stay i suggested to open a neuropathological laboratory at the institute for pathology in erfurt. in leipzig i was able to get to know the advantages of microscopic large sections for brain research and the meticulous photographic documentation of medical findings. therefore, i asked professor güthert to acquire a microtome for large sections. back then in germany, the only producer of such devices was the company jung in heidelberg. the devices and the necessary equipment for preparing paraffin sections were rather expensive. since the universities in the gdr were wholly financed by the state, the necessary foreign currency (west-german deutsche mark) had to be provided by the ministry of education. this did not pose a major challenge for the director, either. in the fall of 1960, he sent me to heidelberg to the company jung so i could select and order a device tailored to our needs. furthermore, he arranged for me to get to know how to use the device at the institute of neuropathology at the university of gießen during the same trip. he also granted me the support of a photographer (who was on the institute’s staff) whenever i needed informative macro shots of neuropathological findings to be produced with a plate camera. hence, already at the beginning of my neuropathological activities, scientifically valuable documentations of medical findings could be generated in black and white, which i could later use for publications in books and journals. back then, many diseases of the central nervous system (e.g., tumors, aneurysms, abscesses) were not surgically operated, because the diagnostics and, in part, surgical procedures had not been developed well enough (there were no special imaging techniques except for pneumencephalography and arteriography, and no microsurgical methods). hence, at that time pathological findings of the brain without iatrogenic changes were not rare. furthermore, in the gdr there was an obligation to autopsy people who died with suspected or evidenced malignant tumors, infectious diseases, stillborn and babies, people who died without explainable cause of death, suspected unnatural death, or when physicians had a scientific interest in the autopsy findings. hence, autopsies could be conducted relatively often. for example, the staff of the institute for pathology at the medical academy erfurt conducted between 2,000 and 3,000 autopsies annually. the share of autopsies with neuropathological findings was accordingly large. i did not conduct every single of these neuropathologically relevant autopsies; however, the decision to photographically document the findings and the diagnostic treatment of neuropathological findings was reserved for me. this way, after a very short period of time i had a relatively large, diverse wealth of material at my disposal, which could be used for in-depth scientific analysis and the training of students. i was involved in the latter fairly early, first in courses and seminars, then in lectures on general pathology (tumors, inflammations) and neuropathology, as well. this material was also used in the regular clinico-neuropathological conferences with neurosurgeons and neurologists. my neuropathological training benefitted from the fact that professor usbeck regularly invited specialists from the federal republic of germany and other european countries, among them neuropathologists, to an annual symposium on neurosurgical issues. this way i got in touch with dr. jürgen peiffer in 1961 (later director of the institute for brain research at the university of tübingen), with whom i discussed subject-specific and societal problems. it has been decisive for my training as a pathologist that professor güthert made it possible for his staff to acquire knowledge outside the gdr. in 1961 i was allowed to take part in the annual conference of the german society of pathology in münster, which was succeeded by a two-week visit at the department of neuropathology at the university hospital hamburg-eppendorf. this was followed by multi-week study trips to university hospitals for nervous diseases in pécs (headed by the renowned neurologist, neurosurgeon and neuropathologist professor környey) and to the institute for pathology at charles university in prague with professor bednar, who also was a renowned researcher in the area of neuropathology. furthermore, i was offered the opportunity to take part with a convention of polish neuropathologists in krakow, as well as an international convention of neuropathologists in zurich. all these trips were funded by the state. this way i was able to acquire special knowledge and establish professional contacts to researchers abroad and neuropathologists active in the federal republic of germany. this led me, with dr. schnabel from the medical academy magdeburg and other interested physicians and scientists in the gdr, to found our own society of neuropathology. the history of the founding and activities of this society is subject of a doctoral dissertation, which was successfully defended by antonia stahl at the charité berlin in 2017. 5. neuropathological research endeavors my specific scientific interest was focused on tumors of the central nervous system, especially concerning their etiology. therefore questions about the experimental induction and the epidemiology of these tumors became the focus of my life’s work. 5.1 experimental neurooncology at the beginning of my engagement with the neuropathological literature i encountered statements about the etiology of glioma that i had doubts about. from general pathology i knew that the following factors may be responsible for the emergence of malignant tumors: ionizing radiation, chemical carcinogens, some virus types and chronic inflammation. the relevant publications said that this cannot apply to brain tumors, because the brain is protected by cranial bone from ionizing radiation, and that chemical carcinogens cannot reach the brain because of the blood-brain barrier. also, there were no cases documented where the emergence of brain tumors could be attributed to sequelae of chronic inflammation or trauma (2). instead, the hypothesis was that genetically determined hereditary dispositions were the main causal factor for the emergence of neuroectodermal tumors in the central nervous system. a prominent advocate of this hypothesis was berthold ostertag in his publications from 1936 and 1941. in that period of time in the german reich, forced sterilization of people with “sick genes” was conducted for ideological reasons. i had doubts about the validity of these arguments, which led to the decision to contribute to the solution of these etiological problems through intense studies of the literature and my own experimental research. as early as 1961 i was able to get dr. schreiber (fig. 3), an assistant physician who had just arrived at the institute, interested in neuropathology and research endeavors. the first result of our cooperation, which endured for decades, was the monography “experimental tumors of the central nervous system,” published in 1969 by veb gustav fischer verlag jena (7). its subtitle is “induction, morphology, transplantation and application,” which shows the range of its content. our aim was to consider all publications on the topic in the world, even if they were hard to access or not in one of the world languages. these studies of the literature offered various inspirations for our own subsequent examinations, which we critically discussed. soon the effort paid out. we were requested to have the book translated to english and published in the u.s. the publisher and us, the authors, agreed. professors d. d. bigner and j. a. swenburg arranged the translation and supplemented the text with new insights, especially in the area of viral tumor induction (8). our work on the topic continued when i was appointed full professor (ordinarius) for pathology at martin luther university in halle/wittenberg in 1970, where dr. schreiber followed with me as a docent. conducting further experiments, we were able to show that tumors in the central nervous system could be induced by nitroso compounds in various animal species regularly and in significant percentages, whereas other animal species turned out to be resistant. we were concerned with the question which of these two groups humans belong to, because if there were evidence showing that we are vulnerable, preventive measures would have to be introduced, for example regarding work life or through modifications of our eating habits. it goes without saying that experiments with humans are impossible, so we included rhesus monkeys into our research. it became apparent that intravenous injections of different doses of alkyl nitroso compounds did not lead to the emergence of tumors. this applied to adult, as well as to newborn animals. these results on rhesus monkeys could be regarded as indication, but not as evidence of a resistance of the human central nervous system against these substances. following this, we started looking for chemical differences between the brains of the different species. this work was conducted in a research group, which included dr. rath and dr. felicetti from the institute for pathology in halle. it showed that especially one enzyme, the zinc-activated acid phosphatase, is present in the normal brain tissue of different animal species in different quantities: in the brain of species where neuroectodermal tumors could be induced by alkyl nitroso compounds, it was high. in the brain of species that did not develop brain tumors, it was missing. in addition, this enzyme could not be found in human autopsy or biopsy brain tissue. we concluded that the danger of brain tumor induction by alkyl nitroso compounds is probably low for humans. however, this does not mean that other chemical substances, which have not been identified yet, could cause primary brain tumors in humans. fig. 3: professor dieter schreiber (second from left) in a seminar in madrid, 1980. 5.2 primary tumors of the central nervous system in fetuses and babies animal experiments showed that tumors in the central nervous system could also be induced transplacentally. this shifted our interest to brain tumors in human fetuses and babies. we derived from studies of the literature documenting that case studies existed, but that systematic examinations on this topic were yet missing. hence, we initiated a collection and analysis of as much material as possible. the aim was to find out which primary tumors occurred in this age group and which symptoms they exhibited. there were good conditions for this kind of research in the gdr: first, a mandatory national tumor register had been existing since 1952. all physicians were legally obligated to report suspected and evidenced malignant tumors. in 1956, this duty to report was extended to all tumors of the central nervous system, regardless of grade of malignancy. second, in the gdr there were special consulting centers for all pregnant women and women with babies. through these, all medical records on the pregnancy and birth including its development and diseases were accessible. third, autopsy of stillborns and dead babies was mandatory. this way we could access all necessary information and request pathology reports and microscopic preparations. additionally, our colleagues from abroad reported relevant cases to us and provided us with material to be examined. the support of this endeavor by our colleagues in poland and czechoslovakia deserves special thanks. on this basis dr. schreiber, dr. gerlach and i authored the monography “tumors of the central nervous system in fetuses and babies” (9). as a supplement to the morphological records, we obtained records on the development of the newborns and babies. we aimed to identify events that might have led to transplacental tumor inductions, and to detect the early symptoms of tumors in the central nervous system of newborns and babies. we intended to include this knowledge into the (continued) training of physicians. we achieved this (13), but the search for transplacental causes for tumors was suspended in 1990. the central tumor register of the gdr was not continued, and with it our access to new data. further inquiries into the data already collected was foiled by the changes in my professional career, leaving the emerging findings hypothetical and unpublished. 5.3 epidemiology of primary tumors of the central nervous system the fundamental works on the pathology of tumors of the central nervous system by zülch (23), folke henschen (2), and russell and rubinstein (19) give hints on the age and gender distribution of different types of tumors. these are composed of summaries of their own analyses and those of other researchers. however, they do not allow conclusions about the actual frequency in the population; they are not epidemiological findings. an epidemiology of tumors can only be realized if all cases in a predefined population group are recorded over time. statistics on the material analyzed in some institutions for pathology or some clinics do not amount to epidemiological findings. the existence of the central tumor register of the gdr described above provided us with the opportunity to generate epidemiological findings on primary tumors of the central nervous system in a population of 17 million people. with the help of dr. joanna haas, a well-trained epidemiologist from the u.s., and other staff from the tumor register we were able to generate robust findings. these were published in single publications (5, 10), as well as in books (9, 11). 5.4 neuroectodermal stem cell tumors studying tumors of the central nervous system of children sparked a scientific interest in a group of tumors that i summed up using the term “neuroectodermal stem cell tumors.” hart and earle (6) called them “primitive neuroectodermal tumors” (pnets). the term “stem cell tumor” is based on the idea that its cells are at the start of a process that will spawn highly differentiated cells through cell maturation. such maturation processes can be morphologically demonstrated (15). the cell maturation in stem cell tumors (neuroepithelioma, neuroblastoma, medulloblastoma, medullomyoblastoma etc.) can lead to a decrease of tumor growth and in some cases even to spontaneous recovery of the patients (11, 22). this has been seen with neuroblastoma in the sympathetic nervous system and very rarely also with stem cell tumors in the central nervous system. in many cases, the differentiation process remains on a low level and only gets to part of the tumor tissue, hence having no effect on the course of the disease without improving the prognosis. furthermore, together with my staff, i intended to research how these maturation processes are controlled, and to find out which therapeutic possibilities might emerge from this. this research produced first results, but had to be suspended in 1994, leaving the intended tasks unfinished. it is my hope that the next generation of researchers will take up this topic and perhaps win the nobel prize in medicine. fig. 4: participants of the international symposium on brain tumors in madrid, 1990. fig. 5: with professor paul kleihues at the rudolf virchow haus of the charité in berlin, 1991. 6. the importance of medical societies and personal contacts it is essential for every physician and professor involved in scientific research to keep up with research findings and the growth of knowledge in the field. this information can be learned from scientific writing, conventions and personal contacts. as a member of the international society of neuropathology i took part in several of the meetings and was able to gain many valuable inspirations for my own work; this was also the case for the congresses of the european confederation of neuropathological societies (euro-cns). i have been interested in informative presentations and panel discussions, as well as in the opportunity to get to know and discuss specific problems with renowned and next generation researchers from abroad. for example, at the 1970 international congress of neuropathology in paris, i met the well-known veterinary pathologist professor fankhauser, co-author of a work on comparative neuropathology (3). i used this opportunity to learn something from him about brain tumors in animals. he then invited me to a multi-week stay in bern where i had the chance to study his extensive collection of histological preparations of brain tumors of animals. there i also discussed problems of comparative neuropathology with him and professor frauchiger. but the exchange of scientific experiences and personal contacts were not limited to international events (fig. 4); the conventions of national societies of neuropathology and symposia proved valuable, as well. in the gdr, there were the annual conventions of the society of neuropathology and symposia with international attendance (20). the proceedings of these meetings were usually published in the journal “zentralblatt für allgemeine pathologie und pathologische anatomie” (21). several of these events were dedicated to tumors of the central nervous system. first they took place in erfurt, then in halle/saale. they offered neuropathologists in the gdr the chance to personally get in touch with researchers from many european countries and countries outside of europe. among the frequent guests were professor zülch, professor wechsler, professor kleihues (fig. 5), professor mennel from the federal republic of germany, professor cervos-navarro from west berlin, professor jellinger from vienna, professor mossakowski from warsaw, and dr. jablonowskaja from moscow, for instance. in exchange, my staff and i were invited to the national neuropathological events in their home countries. sometimes there were invitations from researchers that i had not met before. professor katsuo ogawa (fig. 6) from japan invited me together with lucy rorke (philadelphia) and umberto cravioto (new york) to the convention of the japanese society of neuropathology in okayama in 1985 (fig. 7). that society had more than 900 members back then (the society of neuropathology in the gdr had 34 members.) after that convention i had been invited to talks at different universities and research facilities in japan, among them tokyo, niigata, maebashi, nagasaki, and hiroshima. from this, professional and familial contacts with japanese pathologists and neuropathologists emerged. in 1992, on the occasion of the congress of neuropathology in niigata, my wife and i were invited to the house of professor ikuta (fig. 8), where we enjoyed japanese hospitality together with professor hirano (fig. 9). then again, my wife and i hosted many peers when they stayed in our hometown (figures 10 and 11). fig. 6: with professor katsuo ogawa in okayama, 1985. fig. 7: discussion with professor lucy b. rorke in okayama, 1985. 7. professorship in iraq in 1967, the government of the republic of iraq headed by president aref asked the government of the gdr to name qualified professors to be temporarily sent to work at iraqi universities. this was an unusual step, because at this time the gdr had not been internationally acknowledged as a sovereign state. the claim to sole representation by the federal republic of germany (hallstein doctrine) was still in place. it said that the federal republic of germany is the only successor state of the german reich and that foreign countries can have diplomatic relations to no other german state. any violations of this doctrine would be met with extensive sanctions, including the suspension of diplomatic relations and drastic trade restrictions. the only countries excluded from the hallstein doctrine were those in the socialist realm, which at this point already had the usual diplomatic and other intergovernmental relations with both the gdr and the federal republic of germany in place. the ministry of higher education of the gdr announced the iraqi government’s request to the country’s professors. i learned about this and expressed my interest in such an activity, however stating that my english skills at that point would not be sufficient for such an appointment. hence, i was invited to a language course that was offered in small groups for three months under the instruction of experienced simultaneous translators, which turned out to be very effective. in may of 1968, i flew to baghdad with my wife and kids. we were welcomed by the trade mission of the gdr. i was appointed to act as director of the chair for pathology at the university of mosul. the trade mission had rented a spacious house on the outskirts of the city, not far from ninive. since it was so far away from the medical school, the trade mission provided me with a car of the soviet brand lada. the advantage of this car was that it was very sturdy, and therefore always ready to be used in the summer heat and resistant to driving on desert roads. since this car was the only one of its type in the whole city everyone knew who was coming along when i was going somewhere in it. despite the lack of any traffic rules, i was never involved in an accident. the department of pathology was housed on the fourth floor of a new building for theoretical medical disciplines and had four large rooms and its own lecture hall for 150 students, but no room for conducting autopsies. the staff consisted of an assistant of kurdish nationality who had studied veterinary medicine, an english-speaking manager and four male staffers who only spoke in arabic. one of them was able to prepare paraffin sections and preparations with the usual aniline colorations. the other three assisted by fetching things, cleaning up and making tea. in an adjacent chamber, there was an electron microscope, which had been ordered by one of my predecessors, and it was still unused. there were no tools available for ultrathin sectioning. for the training of the students there were some commercial pathological/anatomical preparations of organs and histological preparations which had been purchased in england. these had been used in classes by the assistant physician. doing lectures was aided by a blackboard with chalk and a slide projector, but there were no lantern slides. fortunately, i had brought a large chunk of my collection of diapositives pertaining to general and specific pathology, which could be used to visualize the lectures. additionally, i demonstrated pathological findings to the students using native organ parts, which came from surgical operations. among them, echinococcus cysts from the dog tapeworm were frequent, which had been removed by surgeons from various regions of the body such as the liver, kidney and even from the ear lobe. echinococcus was a frequent disease in mosul, which was due to the breeding of sheep and the many stray dogs. there were no neurosurgeons active in mosul. therefore, i could demonstrate neuropathological findings to the students only in the autopsy room. however, the problem was that patients who had died did not undergo autopsy in the university hospitals. people who had died were usually picked up by their relatives shortly after death and buried before dusk. there was a small, simply equipped autopsy room in an adjacent building on the clinic’s premises, but this had only been used by forensic doctors. these autopsies had been ordered by the court and conducted against the will of the dead person’s relatives. the chair of forensic medicine was held by an iraqi who had been trained in england. i got in touch with him and we arranged that i would conduct autopsies if the circumstances of the death would allow it. in addition, there was a military hospital in mosul. they also sometimes requested me to do autopsies. every time, the building where the autopsy took place was guarded by the military in order to prevent relatives from entering the room, taking the dead person with them, or altercations. it was a problem for me that i could not schedule autopsies for demonstrations with the students, because they had to happen immediately. we solved this problem by sending out my staffers into the clinic building and informing randomly encountered students that there was going to be an autopsy in an hour. this information circulated quickly among the students, leading to crowds in the autopsy room. this way i was able to conduct and analyze more than 20 autopsies together with the students during my work at the university of mosul. among them there were heart attacks, fatal lung embolisms, but also neuropathological causes of death, such as hypertensive cerebral hemorrhage or a large tuberculoma in the cerebellum. i would like to stress that the students (nine female and 152 male) were disciplined, curious and grateful. fig. 8: break-time talk with professor ikuta, director of the institute for brain research at the university of niigata during the 33rd convention of the japanese society of neuropathology, 1992. fig. 9: staying at professor ikuta’s house. standing between my wife and me: mrs. ikuta and professor hirano (new york). 8. deputy health minister and who consultant back then, the minister of health of the gdr was a physician by the name of professor ludwig mecklinger. he had five deputies. in 1978, the government of the gdr was informed that the soviet union would meet its wish to drastically increase oil deliveries. energy generation through the unprofitable and environmentally harmful combustion of lignite could then be abandoned, leading to massive financial gains for the state. the government, headed by prime minister willy stoph, planned to allocate a large amount of these gains to medical research. mecklinger was ordered to lay out plans for this. he asked for a sixth deputy health minister whose main job would be the further development of medical research in the gdr. he wanted somebody who was a scientist himself and was experienced enough to evaluate research endeavors. this is how my name came into play. i declined to take this job because we were making great progress with our research at the department of pathology in halle. i also stated that i was lacking the necessary expertise in some fields of medicine. i was then assured that a “council for medical research” was to be founded and that renowned scientists from all fields of medicine would constitute this council in order to consult me. this reasoning as well as the significance of medical research for the further development of the health care system of the gdr convinced me to accept the assignment in 1979. i was not influenced by material incentives because even the salary of the minister of health of the gdr was below that of a university institute director. we started working, but by the end of 1980 were informed that the soviet union would eventually not deliver the amount of oil they had promised before. the power plants, which in the meantime had been adapted to the combustion of oil, had to be quickly reconfigured back to the combustion of lignite. this cost a lot of money, leading to not more but less investments in medical research. mecklinger was a smart and warmhearted person. he accepted that my position was not necessary anymore and let me leave the ministry in order to return to the university. this was how i came to the charité berlin in 1983. the “council for medical research” continued to exist and i belonged to it until its dissolution by the last minister of health, professor kleditzsch, in 1990. from 1986 to 1990, i was also acting as consultant to the who, where i was involved in the revision of the international classification of diseases (icd-10). therefore, i spent multiple months per year in geneva. this way i gained insight into the way a united nations agency works and got to know many professionals from several countries, which allowed me to expand my worldview. 9. the end of my academic career and research activities the integration of the gdr into the federal republic of germany brought along changes for many citizens of the gdr, and for me as well. in march of 1991 i was told that the senate administration for internal affairs in west berlin had ordered a vetting of all employees working in the public sector in east berlin (formerly gdr). it was to be decided who would be able to transition into the public sector of the federal republic of germany and who would have to be let go. the focus was on personal aptitude, and on whether or not somebody had worked for the ministry of state security (mfs). this also concerned the professors of the humboldt university. hence, i was asked to answer an extensive questionnaire. on august 27, 1991 i was informed that “no indications of formal or informal activities for the mfs were found and that for this reason no measures concerning employment law [were] to be taken.” i was allowed to continue my activities in the training of students, supervision of graduates and doctoral students, diagnostics of biopsies and autopsies, and heading the department of neuropathology. based on provisions in the unification treaty i was allowed to continue to bear the title “full professor” (“ordentlicher professor”), which i was awarded in 1970 by the ministry of higher education of the gdr. however, the title did not imply an according salary. in the federal republic of germany, professors were paid according to the salary groups “c3” or “c4”. my salary group equaled that of an assistant physician. it was my ethical belief as a professional that i would continue teaching and do my work in neuropathology until my retirement regardless of my salary. however, i was later informed that i would be able to apply as a professor. my position was advertised for applicants from the whole country under the heading “c3 professorship of pathology with emphasis on neuropathology.” i was allowed to apply to that, as well. i and four other applicants from west berlin and west germany were asked to give a talk in front of a commission. after that, we were interviewed in person. during my interview, the chairman of the commission, professor detlev ganden, was especially interested in my research on neuroectodermal stem cell tumors. i have never been notified about the result of the selection process. on december 2, 1993 i was notified by the personnel office of the charité that the selection process became unnecessary “because the advertised position was not part of the legally binding employment plan of humboldt university.” in the same letter, i was told that i would be terminated effective february 2, 1994, because “there [were] concerns regarding my personal aptitude for continued employment in the public sector.” i only received this letter after returning from vacation in indonesia that i had spent with my wife from december 1–22, 1993. i refrained from filing a lawsuit because i believed (and still do believe) that i would not force myself on anybody if my special knowledge and my efforts in the (continued) training of the next generation of physicians was not deemed necessary. i have explained this opinion to the institute’s staff when they tried to encourage me to take steps pertaining to employment law. apparently, the administrative director of the charité heard about this, too, because in mid-january of 1994 he offered me an annulment contract instead of a termination if i refrained from suing them. the benefit was that the contract would not be effective february 2, but february 28. this was indeed of great significance to me, because i had been storing large collections of scientific literature, photographic documentations, case studies and correspondence (i was still editor of the zentralblatt) at the institute. because i was given more time, i was able to rent a large storage facility, which my son helped me to store all the irreplaceable material in. we saved it from destruction, because the newly appointed director of the institute threatened to order the removing of everything in the building as soon as i had been dismissed. i was surprised that the annulment contract did not mention the “insufficient personal aptitude” as a reason for the threatened termination. fig. 10: professor jorge cervos-navarro, director of the institute of neuropathology at free university berlin, at our apartment, 2002. fig. 11: visitor from east asia in the rudolf virchow house of the charité berlin, 1992 (note the virchow bust in the background). 10. restarting my career shortly after the announcement of my separation from the charité the medical director of the landesklinik brandenburg offered me to become director of their department of neuropathology. it had five staffers, was well-equipped and had its own electron microscope (fig. 12). my main tasks would be to examine the brains of patients who had died (autopsies would be conducted at the institute of pathology of the municipal clinic brandenburg) and examining nerve and muscle biopsies. in addition, i was told that in a few months neurosurgery would be opened at the municipal clinic brandenburg and that we would be given tissue samples for examination. this was the reason for me to accept the job offer. then the prime minister of brandenburg converted the position of the head of department into that of a chief physician, thus putting it on the same level with the chief physicians of the clinics for neurology and psychiatry. one of the staffers in the department was dr. hermann, who had special skills in the area of morphological diagnostics of muscle and nerve biopsies. he undertook this part of the department’s tasks, which was good for me, as i did not have deep knowledge of this special area. working in this department and the cooperation between the clinics was good. clinicopathological conferences were introduced and occurred on a regular basis. physicians from the landesklinik as well as the municipal clinic for neurosurgery participated. the exchange of opinion that took place furthered the cooperation of the different disciplines to the advantage of the patients. since the chief physicians participated themselves in these conferences, they served as continued training for all participants. there also emerged a close cooperation with the brandenburg state institute for forensic medicine in potsdam. the forensic doctors asked for our help when examinations of the brain and spinal cord of patients who had died were of forensic significance. we were given these organs in toto, sectioned them, and photographically documented macroscopic findings. after microscopic examinations were finished, the forensic doctors received a detailed report of the findings, which included an epicritic evaluation. we were never present in court ourselves and only rarely corresponded with the prosecution. we also had neuropathological conferences involving the forensic doctors. when i reached retirement age the director of the landesklinik dismissed me, but shortly after dr. pauli, chief physician of the pathological institute at the municipal clinic brandenburg, offered me to work with him as a pathologist and neuropathologist. this way the cooperation with the neurosurgeons and forensic doctors could be continued. back when i was working at the landesklinik, an assistant at the institute for pathology in brandenburg who was in special training, dr. marlies günther (fig. 13), asked me to help her become acquainted with the field of neuropathology. hence, we could continue our cooperation until the point where she could conduct neuropathological diagnostics herself. i took pleasure in this cooperation because of her thirst for knowledge. my presence at the institute in brandenburg was usually limited to three days per week. therefore, it was possible for me to accept an offer to work at the institute of neuropathology at free university medical school berlin. the dean asked for my temporary help because the former director of the institute, professor cervos-navarro, had left after reaching retirement age, and his replacement, professor gisela stoltenburg-didinger, was on sick leave. i agreed to help on two days per week. the job was mainly concerned with the diagnostics of neurosurgical material and postmortal neuropathology. i was not asked to be involved with the training of students. a good cooperation with the director of neurosurgery, prof. brock, and his staffers quickly emerged. intraoperative frozen sections which were prepared in a room next to the operating room and diagnosed by me, fostered this cooperation. this way i was able to communicate and discuss the diagnosis with the surgeon instantly. he then demonstrated the intraoperative situs to me on a display and told me details that would be relevant for my decisions. fig. 12: working in the department of neuropathology of the landesklinik brandenburg, 1995. fig. 13: working with dr. marlies günther. 11. as a neuropathologist in thailand after my dismissal by the directors of the charité, i continued to receive invitations for conventions in the field of neuropathology. one of them became a special memory: it came from the medical school of the university in khon kaen in northeastern thailand. i was asked to organize a workshop for physicians consisting of presentations, autopsy seminars and case study discussions about tumors, inflammations and parasitoses in the central nervous system over the span of several days. the discussions were supposed to give participants the opportunity to present cases of their own choosing, request my opinion on them and discuss them together. i was also asked to give a lecture on neuropathology and answer medical students’ questions. the expenses of this ten-day journey were covered by a japanese sponsor of the university. the offer was appealing, but also somewhat risky as i did not have any information on how knowledgeable the participants would be, what their cases might be about, and if the students would even be interested in neuropathology. furthermore, the events was planned to take place in english and i was not sure if my presentations would be comprehensible. therefore, i prepared for the events using my extensive collection of diapositives because my previous teaching activity in mosul showed to me that language barriers could be overcome using illustrative and expressive diapositives. about 60 physicians took part with the workshops i offered for postgraduates. the physicians and students were attentive and engaged in sober discussions. the lecture hall was filled with students up to the last seat. my wife took a seat in the last row and later reported to me how attentively everyone was listening to my delineations. i felt this myself when, after a one-hour lecture, i invited listeners to ask questions. for more than two hours, i replied to profound inquiries, which were proof of the good knowledge basis and intellect of the thai students. this experience was of great joy to me and made the extensive preparations for these events pay off. 12. epilogue my activities in neuropathology teaching and research came to an abrupt halt in the spring of 1994. in 2012, i decided to conclude my activities as a pathologist and neuropathologist. as of this writing, i am living in the 89th year of my life. if someone asked me if would choose this career again, i would clearly reply “yes!” however, i have to advise younger readers that they cannot expect material wealth from working in this area. i am not the only person who had to make the experience of working for an hourly wage of two or three cents (co-)authoring neuropathological books (11, 12). i emphasized this in the prologue: this is not an autobiography. however, i still would like to briefly express my commitment to my philosophy of life: my childhood experiences made me a staunch enemy of national socialist ways of governing and the horrors of world war ii made me an opponent of war. i did not enroll in the army. the experiences of the japanese people taught me that nuclear armament poses a threat to civilization. therefore i joined the organization “international physicians for the prevention of nuclear war (ippnw)” in 1980 and worked for their cause. this worldview determines the way i live until this day. references 1. bailey p., cushing h.: gewebsverschiedenheit der hirngliome. gustav fischer-verlag, jena 1930 2. henschen, f.: tumoren des zentralnervensystems und seiner hüllen. handbuch der speziellen pathologischen anatomie und histologie, herausgeg von o. lubarsch, f. henke, r.rössle. band 13, teil 3; springer-verlag, berlin-göttingen-heidelberg, 1955 3. frauchiger e., fankhhauser r.: vergleichende neuropathologie des menschen und der tiere. springer-verlag, berlin-göttingen-heidelberg 1957 4. gerlach h., jänisch w., schreiber d.: perinatale teratome des zentralnervensystems. wiss. z. ernst-moritz-arndt-univ. greifswald 32: 112-114; 1983 5. haas j.f., jänisch w., staneczek w.: newly diagnosed primary intracranial neoplasms in pregnant woman: a population-based assessment. j. neurol. neurosurg. psychiat. 49: 874-800; 1986 6. hart m.n., earle k.m.: primitive neuroectodermal tumors of the brain in children. cancer 32: 890.897; 1973 7. jänisch w., schreiber d.: experimentelle geschwülste des zentralnervensystems. veb gustav fischer-verlag, jena 1969 8. jänisch w., schreiber d.: experimental tumors of the central nervous system. edited by d.d.bigner and j. swenberg; upjohn comp., kalamazoo/mich.; 1977 9. jänisch w., schreiber d., gerlach h.: tumoren des zentralnervensystems bei feten und säuglingen. veb gustav fischer-verlag, jena 1980 10. jänisch w.: zur epidemiologie der primären geschwülste des zentralnervensystems im ersten lebensjahr. arch. geschwulstforsch. 55: 489-494; 1985 11. jänisch w., schreiber d., güthert h.: neuropathologie (band 1), tumoren des nervensystems. veb gustav fischer-verlag, jena 1988 12. jänisch w., schreiber d., warzok r.: neuropathologie (band 2), pathomorphologie und pathogenese neurologischer krankheiten. veb gustav fischer-verlag, jena 1990 13. jänisch w.: hirngeschwülste bei säuglingen. z. allg. med. 65: 484-489; 1989 14. jänisch w: pathologie der geschwülste des nervensystems. in: klinische neuropathologie. herausgegeben von j. cervos-navarro u. r. ferszt: georg thieme-verlag, stuttgart-new york; 1989 15. jänisch w., grieshammer t.: expression von immunhistochemischen differenzierungsmarkern i n normalen und neoplastisch transformierten neuroektodermalen stammzellen. acta histochemica suppl.-band xlii: 139-142; 1992 16. jänisch w.: nocardiose. in: die entzündlichen erkrankungen des nervensystems, band 2; herausgeg. von h. henkes u. h.w. kölmel; ecomed-verlag, landsberg/lech 1993 17. jänisch w.: mykosen des zentralnervensystems. in: die entzündlichen erkrankungen des nervensystems, band 5; herausgeg. von h. henkes u. h.w. kölmel; ecomed-verlag, landsberg/lech 1993 18. paulus w., jänisch w.: clinicopathologic correlations in epithelial choroid plexus neoplasms: a study of 52 cases. acta neuropathol. 80: 635-641; 1990 19. russell d.s., rubinstein l.j.: pathology of tumours of the nervous system. edward arnold , london 1959 20. schreiber d. und jänisch w. (herausgeber): experimentelle neuroonkologie. wissenschaftliche beiträge der martin-luther –universität halle-wittenberg; heft 7, 1974 21. schreiber d., güthert h.: bericht über die 2. tagung der gesellschaft für neuropathologie der ddr am 1. und 2. oktober 1970 in erfurt. zbl. allg. path. 114: 276-285, 1971 22. warzok r., jänisch w., lang g.: morphology and biology of cerebellar neuroblastomas. j. neuro-oncol. 1: 373-379; 1983 23. zülch k.j.: die hirngeschwülste in biologischer und morphologischer darstellung. barth-verlag, leipzig 1956 copyright: © 2020 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. neuroinflammation: 2021 update feel free to add comments by clicking these icons on the sidebar free neuropathology 2:1 (2021) review neuroinflammation: 2021 update hans lassmann center for brain research, medical university of vienna, austria corresponding author: hans lassmann · center for brain research · medical university of vienna · spitalgasse 4 · 1090 wien · austria hans.lassmann@meduniwien.ac.at submitted: 18 december 2020 accepted: 08 january 2021 copyedited by: aivi nguyen published: 12 january 2021 https://doi.org/10.17879/freeneuropathology-2021-3166 keywords: brain inflammation, covid-19, multiple sclerosis, alzheimer’s disease, mogad abstract key requirements for the validity of a neuropathological study are the inclusion of large numbers of biopsy or autopsy cases and proper controls, the rigorous classification of the basic neuropathology and the selection of the most suitable technologies for investigation. whether the studies are performed with the fanciest, new, and state of the art technology or with rather conventional methodology is of minor importance. following these criteria, a spectrum of neuropathological studies has been published in 2020, which provides new insights on important questions related to neurological disease. they include the pathological substrate of brain disease in covid-19 infected patients, the nature of the adaptive and innate inflammatory response, or the type and mechanisms of tissue injury and repair in multiple sclerosis, and diagnostically relevant or mechanistic new insights into antibody-mediated diseases of the central nervous system. other studies describe in detail the dynamic changes of brain inflammation in patients with trisomy 21 as a disease model for alzheimer’s disease, or the presence and consequences of vascular comorbidities in a chronic inflammatory disease, such as multiple sclerosis. all these contributions have provided important, highly relevant clues for basic and translational neuroscience. 1) what was the brain pathology in patients who died during the course of covid-19? the covid-19 pandemic has been a major challenge for society and particularly for health care institutions during the last year. although covid-19 primarily affects the respiratory system and the major cause of death is pneumonia with respiratory failure, other organs, such as the renal, cardiovascular system, or the digestive tract, are affected (paterson et al. 2020). most relevant for neuropathology is that the nervous system, too, can be a target, reflected for instance by anosmia and ageusia, encephalopathy, focal ischemic stroke, encephalitis, meningitis, or polyneuritis (li et al. 2020, paterson et al. 2020, liu et al. 2020, hernandez-fernandez et al. 2020). to what extent these neurological deficits are due to direct sars-cov2 infection of the nervous tissue, immune-mediated brain damage in the course of a systemic cytokine storm, or are secondary complications of respiratory failure, the intensive care setting, or comorbidities is not clear (liu et al. 2020). neuropathology plays a critical role in answering these questions. the first approach to answer these questions was to analyze whether molecules, which are involved in cellular virus infection or propagation, are expressed within the nervous system. angiotensin concerting enzyme 2 (ace2) is one of the cellular receptors recognized by sars-cov2, when docking to the cell surface. thus, the presence of ace2 on brain cells, such as neurons, glia or cerebral endothelial cells suggests that brain infections is possible (kabbani and olds 2020, kanwar et al. 2020). another docking molecule for sars-cov2 is neuropilin 1 (nrp1) and a high expression of this molecule has been found in virus infected cells in the nasal cavity in covid-19 patients (cantuti-castelvetri et al. 2020). early neuropathological studies in the covid-19 pandemic were restricted to single case reports or investigations in very small patient cohorts, and they were generally restricted to some basic neuropathological investigations (kantonen et al. 2020, reichard et al. 2020, jensen et al. 2020, younger 2020, liu et al. 2020). the results were diverse and controversial, mainly providing evidence for brain damage that was not directly linked to sars-cov2 infection. the first systematic study on this issue appeared on october 2020 in lancet neurology (matschke et al. 2020), and this study combined classical neuropathology with virology and molecular studies on gene expression. in a first set of data the authors describe that different molecules, involved in virus docking and propagation, show a preferential expression in different cells of the central nervous system. for example, ace2 was mainly found in oligodendrocytes. the highest expression of transmembrane protease serine subtype 2 and 4 (tmprss2 / 4) was seen in neurons, and nrp-1 was mainly present in endothelial cells and astrocytes. these data suggest different mechanisms of infection in different brain cells. the neuropathological studies documented the presence of focal and diffuse ischemic lesions and diffuse astrogliosis. microglia activation with microglia nodules and inflammation were mainly seen in the lower brain stem. in about half of the patients, virus was found either by pcr or immunocytochemistry. however, the virus load was very low and by immunohistochemistry only a very small number of virus infected cells became apparent. this study is the first to describe the basic neuropathology seen in covid-19 patients and is, thus, very important and groundbreaking. however, it also has important limitations. it is based on autopsies of 43 patients, which is likely not sufficient to cover the entire neuropathological spectrum of the disease. another limitation is that it does not contain data regarding the neurological status of the patients. thus, it remains unclear, to what extent patients with specific neurological disease manifestations have been included. the third limitation is that it does not include a proper patient control group and, particularly, patients with similarly severe systemic immune activation. this particular question has been addressed in another study on covid-19 neuropathology, which revealed that the degree of inflammation and microglia activation seen in covid-19 patients is similar to that of patients who died under septic conditions (deigendesch et al. 2020), thus suggesting that it may at least in part be a secondary consequence of systemic immune activation. another study, also based on a large sample of autopsies, mainly focused on the possible routes of cns infection (meinhardt et al. 2020). it shows high virus load in the olfactory epithelium, including the olfactory sensory neurons, and in the olfactory pathways within the cns, supporting the view of a neuronal route of virus entry into the cns. additionally, however, virus antigen was also present in cerebral endothelial cells, associated with micro-thrombosis and cerebral microinfarcts. in summary, current pathological studies show that the central nervous system can be infected with sars-cov2, that local infection can be associated with nervous system damage, but the extent of infection is low. a large part of the neuropathological changes seems to be secondary to systemic immune activation and cytokine storm, critical-illness related encephalopathy, or hypoxia and comorbidities. 2) what new insights have emerged into phenotype and disease mechanisms of antibody mediated autoimmune diseases of the central nervous system? the discovery that autoantibodies against neuronal ion channels or neurotransmitter receptors are associated with a spectrum of acute and chronic neurological diseases, has revolutionized neurological disease research (höftberger and lassmann 2017). it has been shown that these diseases, which had before been regarded as functional diseases, neurodegenerative disorders or toxic conditions, are immune-mediated and can be successfully treated with immunosuppression. the number of diseases falling into this category has profoundly increased during the last years (höftberger and lassmann 2017). one reason, which may in part explain the increase in prevalence, is the introduction of immunological checkpoint inhibitor therapies in oncology. immunological checkpoints are critical steps in t-cell activation, which prohibit the development of auto-reactive t-cells and auto-antibodies. when these checkpoints are inhibited, immune reactions against antigens of malignant neoplasms can be triggered, but this may occur at the expense of autoimmunity, which is frequently directed against neuronal antigens (sechi et al. 2020). on this basis it is not surprising that auto-antibody-associated diseases of the central nervous system remained in the focus of research interest in 2020. the respective studies provided novel insights into the function of nmda and glycine receptor directed antibodies in relation to the clinical disease spectrum (matute et al. 2020, carceles-cordon et al. 2020, rauschenberger et al. 2020) or how autoantibodies against iglon 5 may trigger intracellular accumulation of tau-tangles (landa et al. 2020). here, i focus on one study, which deals with an interesting but also controversial aspect of these diseases. pitsch et al. (2020) identified antibodies against the postsynaptic actin binding protein drebrin in patients with severe epileptic seizures, which were associated with encephalopathy and neuropsychiatric symptoms, including depression and cognitive impairment. the antibodies, identified by selective binding to the neuropil and by western blot and sequencing, were specific for these patients, and the specificity for the target antigen was proven by the lack of binding in drebrin knock out mice. drebrin is an intracellular antigen, which is not exposed on the extracellular surface of neurons or synapses. for this reason, the antibodies themselves may not be pathogenic, but just represent a diagnostically useful marker for an autoimmune attack of cytotoxic t-cells, as it occurs in many classical paraneoplastic diseases. the pathology, described in a biopsy of one of the cases, is in line with this assumption, showing infiltrating t-cells in close contact with neurons. however, the study further describes neurophysiological experiments, which show that the antibodies induce altered neuronal activity patterns and increased firing and bursting rates in neuronal networks in vitro. these data suggest that auto-antibodies directed against a cytoplasmic protein in synapses may reach their specific target and induce functional changes. the authors suggest that extensive exocytosis and endocytosis, which takes place in active synapses, facilitates the entry of the antibodies into the intracellular compartment, but this proposed mechanism does not explain how the antibodies leave the endosomal compartment and access the cytosol. this important cell biological question with important disease relevance has to be clarified in future studies. 3) what is the pathological difference between mog antibody associated inflammatory demyelinating disease (mogad) and multiple sclerosis (ms)? focal plaques of demyelination with axonal preservation and reactive gliosis developing in the context of a chronic inflammatory reaction in the central nervous system has been regarded as the specific hallmark of ms pathology. however, a new disease entity has recently emerged, which is an inflammatory demyelinating disease associated with antibodies directed against myelin oligodendrocyte glycoprotein (reindl and waters 2019). not all anti-mog antibodies are pathogenic, but only those that are directed against a conformational epitope, which is accessible on the surface of native oligodendrocytes or on mog-transfected cell lines. using these diagnostic tests, it became clear that mog antibody-associated disease (mogad) differs from ms by its clinical manifestation and course as well as by its response to immunomodulatory treatment (fujihara and cook 2020). to explain these clinical differences, a neuropathological comparison of these diseases is mandatory, but respective knowledge on mogad was restricted to few biopsies with very limited tissue samples. this changed in 2020, with two studies describing the neuropathological changes in large cohorts of mogad patients (höftberger et al. 2020, takai et al. 2020). the lesions in mogad differ from those seen in ms in many aspects, including their topographical distribution in the cns, the type of demyelination, and the nature of the inflammatory response. in ms, new lesions are formed by simultaneous demyelination in large, focal areas or by the peripheral expansion of pre-existing lesions located throughout the brain and spinal cord. in contrast, mogad demyelination occurs by confluence of small perivenous lesions, generally resulting in a demyelination pattern similar to that seen in acute disseminated encephalomyelitis (figure 1). demyelination in mogad is associated with complement deposition at the site of active myelin injury, but the degree of complement activation is much less compared to that seen in patients with aquaporin 4 antibody associated neuromyelitis optica (nmo). in both conditions, ms as well as mogad, the basic lesion is characterized by inflammatory demyelination, partial axonal preservation and reactive astrocytic gliosis. however, the inflammatory reaction is fundamentally different. while in ms the dominant inflammatory reaction is seen around the larger drainage veins in the periventricular tissue and the meninges, in mogad the smaller veins and venules are mainly affected. finally, in mogad, infiltrating lymphocytes are mainly cd4+ t-cells with low numbers of cd8+ t-cells and b-cells; the dominant lymphocytes in active ms lesions are tissue resident cd8+ effector memory t-cells and b-cells / plasma cells. all these data indicate that mogad and ms are fundamentally different diseases. figure 1: key neuropathological features, distinguishing multiple sclerosis (ms) from the inflammatory demyelinating disease, which is associated with auto-antibodies against myelin oligodendrocyte glycoprotein (mogad) in the forebrain. a) in ms, the lesions (blue areas) are located around large cerebral veins with a predilection site around the ventricle and the subcortical white matter. they are sharply demarcated from the surrounding peri-plaque white matter and frequently display finger like perivenous extensions into the deep white matter. in the cortex, band like subpial lesions (red areas) dominate, mainly located in the deep sulci of the cortical ribbon. b) in mogad, lesions are mainly located in the optic nerves and spinal cord, but their structural details have so far not been clearly outlined. when lesions are present in the hemispheres of the forebrain, a type of tissue injury is seen, which resembles acute disseminated encephalomyelitis. it consists of perivenous sleeves of demyelination around small veins and venules (blue dotted areas), which show confluence in the lesion center (dark blue area). when the cortex is affected small perivenous intra-cortical demyelination is seen most frequently (red dotted areas). c) in ms, the lesions form around larger veins with prominent perivascular spaces. the perivascular spaces contain mainly b-cells (red dots) and cd8+ t-lymphocytes (blue dots), the latter also disperse into the plaque parenchyma. cd4+ t-cells (green dots) are very sparse. a characteristic feature of active ms lesions is the radial expansion, reflected by a rim of activated macrophages at the lesion edge (blue dotted area). d) in contrast, in mogad, numerous small perivenous sheaths of demyelination are present, which arise around small veins and venules (blue dotted areas). in the inflammatory infiltrates the cd4+ t-cells dominate, while there is a much lower contribution of cd8+ t-cells (blue dots) or b-cells and plasma cells (red dots). confluence of the perivenous lesions results in larger demyelinated plaques. interestingly, the pathology of mogad closely resembles the pathology seen in experimental models of autoimmune encephalomyelitis (eae), induced by immunization of rats, guinea pigs, or primates with cns tissue myelin or recombinant mog (höftberger et al. 2020). thus, eae appears to be a very suitable model for mogad, but much less for ms. support of this view is provided in a recent study, on archival material from a patient, who died in the 1950s with an acute ms-like inflammatory demyelinating disease after misguided repeated immunization with brain tissue (höftberger et al. 2015). new molecular biology technologies allowed resurrection of a pathogenic auto-antibody from the archival formaldehyde fixed and paraffin embedded tissue, which was found to be directed against a conformational epitope of mog and induced demyelination after transfer in vivo (beltran et al. 2020). 4) is subpial demyelination a unique feature of multiple sclerosis pathology? for a long time, ms has been regarded a demyelinating disease affecting the white matter. however, with the availability of highly sensitive immunocytochemical methods, which allow staining of the very thin myelinated fibers within the grey matter (peterson et al. 2000), it became clear that demyelination in the grey matter in the ms brain is extensive (kutzelnigg et al. 2005) and may even give rise to cortico-spinal or pure cortical variants of the disease in a subset of patients (trapp et al. 2018). three types of cortical lesions have been identified: the cortico-subcortical lesions (type 1), the intracortical lesions (type 2), and the subpial lesions (type 3) (bo et al. 2003). the third type is most abundant in ms and is related to focal inflammatory aggregates in the leptomeninges (magliozzi et al. 2010; figure 2). several previous studies have indicated that subpial demyelinated lesions in the cortex may be specific for ms (moll et al. 2008, fischer et al. 2013), but this view was based on a rather limited sample of neuropathological conditions other than ms. this has now been changed in a very comprehensive study by junker et al. (2020), in which nearly every thinkable neuropathological condition has been investigated for cortical demyelination. interestingly, subpial demyelination was exclusively present in ms and not seen in any of the other conditions. the only key diagnosis missed in this case series was mogad. however, this was also specifically addressed in the systematic studies of mogad pathology outlined above (höftberger et al. 2020, takai et al. 2020), describing intracortical and cortico-subcortical lesions but absent subpial cortical lesions. in rare instances, these intracortical lesions can fuse and give rise to large focal confluent plaques of demyelination. thus, so far it seems that subpial cortical demyelination is a unique feature of ms pathology, but the evidence for its absence in mogad is thus far based on a rather small sample of cases. nevertheless, the presence of a subpial cortical lesion strongly supports a neuropathological diagnosis of ms. figure 2: patterns of cortical demyelination in ms and mogad: a) in ms, the dominant and most specific type of cortical demyelination is the subpial lesion. it is characterized by a band of demyelination spanning over several gyri and sulci (red lesions). the lesions are more extensive in the invaginations of the cortex. active cortical demyelination is associated with meningeal inflammation by t-cells (blue dots) and b-cells (red dots) and is characterized by a band of activated macrophages / microglia at the border between the cortical lesion and the surrounding normal appearing cortex (purple bands). b) in mogad, the dominant cortical pathology is the presence of small perivenous intracortical demyelinated lesions (red circles). they arise around small cortical veins and venules with inflammatory infiltrates. on rare occasions, such intracortical perivenous lesions may give rise to a focal confluent cortical lesion, which frequently also expands into the adjacent subcortical white matter. 5) what immune cells drive inflammation in multiple sclerosis? our understanding of the immune mechanisms driving inflammation in ms is largely biased by immunological studies of experimental autoimmune encephalomyelitis, which now turns out to have more relevance for mogad than for ms. key mechanisms of inflammation, defined from such eae studies, such as the key role of mhc class ii restricted t-cells responses, the involvement of cd4+ th17 cells, or the central role of gm-csf driven myeloid cell activation (schreiner and becher 2015) are not convincingly supported by recent therapeutic trials targeting the respective immunological pathways (baker et al. 2017). in contrast, most effective therapeutic success is seen with drugs that target t-cells and b-cells together or even b-cells alone. thus, it is still an unresolved question, as to what cells and immune mechanisms trigger or drive inflammation in ms. pathology can help clarify this issue by providing a concise account of the types of inflammatory cells seen in different stages in the evolution of ms lesions. until a few years ago, information about the composition of inflammatory infiltrates in ms was restricted to small studies performed on a limited number of patients and lesions. since 2017, however, there are now three large studies available, which performed a phenotyping of inflammatory cells in an overall sample of more than 200 ms patients and which included all stages of the disease (van nierop et al. 2017, machado santos et al. 2018, fransen et al. 2020). all studies reached a similar conclusion that the dominant lymphocytes in the ms lesions are tissue resident cd8+ effector memory t-cells (ttrm), while infiltration with cd4+ t-cells is sparse. this is the case in all types of ms, including fulminant acute ms, relapsing remitting ms as well as primary or secondary progressive ms and is the same in all activity stages of the lesions. in active lesions, a subset of cd8+ cells show focally and temporally restricted activation, and b-cells are much more numerous in early stages of the disease and in active lesions than in inactive lesions at late stages (machado santos et al. 2018). in a recent study, imaging mass cytometry was applied to the biopsy tissue of a single ms patient who suffered from a rebound exacerbation after cessation of natalizumab treatment (ramaglia et al. 2019). also, in this study, the infiltrates contained t-cells and b-cells, but the relative proportion of cd4+ t-cells was higher compared to that seen in the previous studies. however, activation of t-cells, visualized by the expression of the transcription factor nfat or proliferation markers, was seen only in cd8+ t-cells. besides their absolute numbers, the activation state of leukocytes is relevant for driving the inflammatory reaction. similarly as before (machado santos et al. 2018), the most frequent b-cell phenotypes were igg+ cd38+ plasmablasts. thus, overall, the pathological data are in line with the observed effects of anti-inflammatory treatments in ms patients. 6) what do the inflammatory cells in ms lesions recognize? an important open question is what antigen is recognized by the cd8+ tissue resident memory t-cells. since such t-cells acquire their phenotype and persist after effective clearance of their cognate antigen and become reactivated when the antigen re-appears, it is unlikely that these cells recognize a classical auto-antigen, which is present in the cns in excess all the time. in line with this view, no reactivity of cd8+ t-cells from ms brain lesions has been seen against the commonly tested myelin antigens (van nierop et al. 2017). several potential candidate antigens have recently been identified. since accumulating epidemiological evidence associates epstein barr virus infection with ms (bar-or et al. 2020, levin et al. 2010), one hypothesis is that b-cells with latent ebv infection are present within the cns of ms patients (serafini et al. 2007, veroni et al. 2018) together with ebv-reactive cd8+ tissue resident effector memory t-cells (serafini et al. 2020). here, the hypothesis is that inflammatory activity is triggered and propagated when the virus is activated in latently infected cells and recognized by the specific t-cells (serafini et al. 2020). as a note of caution, several other groups have tried to confirm the specific presence of ebv infected b-cells in the ms brain and lesions and have failed (lassmann et al. 2010, van nierop et al. 2017). the reason for these discrepancies is still unresolved. in a similar approach, an interaction between brain resident cd8+ cells and b-cells has been described, where the b-cells did not express ebv (van nierop et al. 2017). this suggests that the cells recognize a b-cell autoantigen. a possible mechanism for how such autoimmunity against b-cells may be induced is provided by jelcic et al. (2018). ms b-cells, possibly in relation to their ebv infection status, expand by auto-proliferation and present a b-cell specific auto-antigen to cd4+ t-cells, which also expand and infiltrate the brain and spinal cord. within the cns, these t-cells are activated by antigen recognition on infiltrating b-cells. in addition, the respective auto-antigen (rasgrp2) is also expressed in neurons, which may further propagate inflammation and tissue injury. whether this mechanism also accounts for activation of cd8+ t-cells, the dominant inflammatory cells in ms lesions, is unknown. a different approach has been followed by konjevic sabolek et al. (2019). in this study, the interaction of cd8+ t-cells with local target cells was identified by the polarized location of cytotoxic granules at the site of contact. when this was seen, the target cell was isolated and its nature was determined in gene expression studies and by immunohistochemistry. in this study, the only cells which interacted with cytotoxic cd8+ t-cells in ms lesions were myeloid cells, expressing markers of macrophages and microglia, suggesting these may harbor the target antigen(s). finally, other studies suggest that the target antigen recognized by t-cells in ms lesions may be the stress protein alpha b crystallin, which is in ms lesions most prominently expressed in active lesions (van noort et al. 2010). in summary, the new data from systematic phenotypical and functional studies on the inflammatory response within ms lesions provide groundbreaking new insights into the pathophysiology of the disease and question the concept of ms being an autoimmune disease against myelin. however, the results are in part very divergent and, to some extent, contradicting. whether this indicates heterogeneous disease mechanisms between individual patients or patient subgroups will be seen in the future. 7) which microglia or macrophage phenotypes are associated with tissue damage in the brain? microglia and recruited macrophages play a major role in the induction of tissue injury, not only in inflammatory brain lesions but also in neurodegenerative diseases. for a long time, it was difficult to distinguish between activated microglia and recruited macrophages within brain lesions. this has changed with the introduction of markers, which are selectively expressed on microglia, such as the membrane protein tmem 119 and the marker for homeostatic microglia p2ry12. these markers have been used in a number of studies on various different inflammatory and neurodegenerative conditions and revealed a surprisingly uniform reaction pattern of myeloid cells in human diseases (zrzavy et al. 2017, 2018, hayashida et al. 2020, jäckle et al. 2020). in essence, at sites of initial tissue injury, resident microglia become activated in a pro-inflammatory pattern. this initial step is followed by recruitment of myeloid cells from the circulation, and the recruited cells also show pro-inflammatory activation or, at later stages of lesion maturation, an intermediate phenotype. the distinction of proversus anti-inflammatory phenotypes is now seen in a much more critical way, since some pro-inflammatory actions, such as the interaction of macrophages with target cells may contribute to tissue damage but may also be essential for neuroprotection and regeneration through the clearance of debris (cignarella et al. 2020). an important pro-inflammatory type of activation, which seems to play a major role in the induction of tissue injury, is the expression of proteins involved in oxidative stress, such as the expression of the nox2 complex (nadph oxidase complex 2), a prominent marker of microglia expressed in active lesions in different inflammatory conditions as well as vascular or neurodegenerative diseases in humans (zrzavy et al. 2017, fischer et al. 2013). to define the patterns of microglia activation in such diseases may finally result in more selective and efficient neuroprotective treatments. this is now possible with new technologies, such as single-cell rna sequencing or immunocytochemical methods, which allow the simultaneous detection of multiple protein antigens within the same section, such as imaging mass cytometry. in some studies of models of autoimmune encephalomyelitis and ms, these techniques have been used and confirmed in an elegant way the patterns of microglia activation and macrophage recruitment, which have been described before with more conventional techniques as summarized above (masuda et al. 2019, ramaglia et al. 2020). however, they additionally showed that microglia and macrophages with different phenotypes are present side-by-side in the same lesion. this may indicate selective activation signals in specific subpopulations of cells. not surprisingly, the data showed that different ms lesions, which were in the same activity stage but still displayed some distinct morphological features, were infiltrated by different subsets of myeloid cells (masuda et al. 2019). however, these studies did not reveal the high expression of molecules involved in oxidative injury, which had been prominent in earlier more conventional neuropathological studies. a possible explanation for these discrepancies is provided by an elegant study, which specifically focused on microglia activation and oxidative injury in autoimmune encephalomyelitis (mendiola et al. 2020). the authors first defined the molecular phenotype of microglia specifically selected from areas of oxidative injury. incorporating this profile in the analysis of single-cell rna sequencing data and immunocytochemistry, they identified specific microglia and macrophage subpopulations, which trigger oxidative stress, and showed that these are selective subtypes of cells within the population of activated myeloid cells. these particular cell types also express molecules that are important in the coagulation cascade and in glutathione metabolism. in addition, they produce molecules, which counteract oxidative stress. one of these molecules acivivin, a glutathione regulating compound, suppresses the inflammatory tissue damage in a model of autoimmune encephalomyelitis. it will be instrumental in the future to validate directly the relevance of these findings in ms lesions and also in vascular or neurodegenerative diseases of the central nervous system. the presence of a subtype of myeloid cells triggering and resolving oxidative stress could reconcile the above discrepancies and provide a mechanism for the excessive oxidative injury in the lesions and the associations of markers for oxidative injury with markers for disturbed blood coagulation in the lesions. in the long run, these data may lead to tools for the therapeutic blockade of one of the most detrimental aspects of microglia activation in human brain disease. 8) how do vascular comorbidities influence multiple sclerosis patients? clinical epidemiology of ms has shown that patients with vascular risk factors, such as diabetes, hypercholesterolaemia, hypertension, or heart disease have a more aggressive disability progression in comparison to patients lacking these co-morbidities (marrie et al. 2010). this is also reflected by lower brain volumes (pichler et al. 2019). furthermore, persistence of inflammatory demyelinating lesions and higher lesion volumes are present in brain areas with a blood perfusion that is lower than in other brain areas (haider et al. 2016). such a synergism in neurodegeneration may in part be explained by shared effector mechanisms of tissue injury between vascular and inflammatory diseases, including microglia activation, oxidative injury, and mitochondrial damage (zrzavy et al. 2017, 2018, mahad et al. 2015). a strategically important piece in the puzzle of vascular comorbidities and ms, which was missing so far, was the lack of a comprehensive neuropathological description of systemic and intracranial vascular pathology in patients versus controls. this information has now been provided by a study, which is based on a unique archival collection of ms and control autopsy cases, where detailed information regarding systemic vascular diseases was recorded, and which was collected prior to the availability of disease modifying treatments (geraldes et al. 2020). the study shows, as expected, that systemic and intracranial vascular abnormalities increase with age in both the ms and the control group. young ms patients appear to have a moderate reduction of systemic vascular co-morbidities compared to controls, but this difference disappears with aging. within the central nervous system, ms patients showed a profound increase in small arteries with increased perivascular space, perivascular hemosiderin depositions and periarteriolar accumulation of inflammatory cells, a type of pathology which correlated in its extent with the degree of ms-related pathology. these results underline the presence of age-related vascular co-morbidities in the brain of ms patients, which may be an amplification factor for neurodegeneration and disease progression in aging patients. additionally, they indicate that, in contrast to the current view, vascular pathology is not restricted to veins but also affects small arteries, and this arterial pathology develops at least in part independently from systemic vascular risk factors. in particular, the mechanisms how inflammatory infiltrates accumulate around arteries in the ms brain requires further attention. 9) what is wrong with remyelination in multiple sclerosis? primary demyelination with preservation of axons is the pathological hallmark of inflammatory demyelinating diseases, such as ms. myelin allows saltatory conduction in axons and also protects the axons from neurodegeneration. thus, major efforts in ms research have been devoted to develop treatments which prevent demyelination or stimulate myelin repair (lubetzki et al. 2020). these repair strategies, mainly designed to provide oligodendrocyte progenitor cells in the lesions and to stimulate their differentiation into myelinating oligodendrocytes, have been developed and tested in experimental models of demyelinating disease and found to be quite effective. however, none of the strategies have yet resulted in clinically meaningful myelin repair in controlled clinical trials in ms patients. in experimental models of demyelinating disease, profound spontaneous remyelination is the rule, and remyelination stimulating therapies in essence accelerate myelin repair. although spontaneous remyelination may also occur in ms patients, in particular in fresh lesions at early disease stages, remyelination is, in general, sparse or absent. several different mechanisms have been suggested to be responsible for this remyelination failure in ms lesions. it may be due to a genuine defect in oligodendrocyte (progenitor) biology. in an elegant study, starost et al. (2020) approached this question by studying oligodendrocytes, induced from pluripotent stem cells from ms patients and controls. they show that there is no difference in the proliferation, differentiation and myelin production between such cells derived from ms patients and from controls, thus strongly suggesting that there is no genuine defect of oligodendrocytes in ms patients, though with the caveat that this was only performed in cells induced to form oligodendrocytes with expression of transcriptional factors from three ms patients and three controls. in a parallel neuropathological approach, the same group analyzed the patterns or remyelination in different disease stages of ms and in different stages of lesion formation (heß et al. 2020). they found that remyelination is highly efficient in a subset of active lesions in the early stage of the disease, and their data suggest that the remyelinating cells may be derived from mature oligodendrocytes that had survived active myelin destruction. this has also been suggested in recent studies, using radiocarbon methods to determine the time of birth of new oligodendrocytes in ms lesions (yeung et al. 2019). in contrast, in chronic active lesions at later stages of ms, very little remyelination was seen and the lack of remyelination was associated with pro-inflammatory activity of the local microglia population (heß et al. 2020). these data suggest that products of activated inflammatory cells in chronic ms lesions may not only lead to their slow expansion but also inhibit the repair of myelin. this finding is also supported by the other study (starost et al. 2020), which provides convincing evidence that pro-inflammatory mediators, in particular gamma-interferon, blocks the differentiation of inducible pluripotent stem cells into myelinating oligodendrocytes. the key importance of these studies is that they document that the process of remyelination failure in ms is complex and not fully reflected in the current experimental models of inflammatory demyelinating disease. thus, for test screening of remyelination-promoting therapies different systems have to be used, which reproduce the inflammation-associated remyelination failure. 10) what can we learn from down syndrome about inflammation and neurodegeneration in alzheimer’s disease? a large spectrum of data coming from experimental studies as well as from neuropathological and genetic investigations suggest an important role of innate immune mechanisms in the pathogenesis of alzheimer’s disease (akiyama et al. 2000). in particular, microglia phenotype and function are associated with progression of cognitive decline. however, information regarding the time course and type of microglia pathology especially in the pre-symptomatic phases of the disease is limited. analyzing down syndrome brain pathology of patients may provide some answers to these questions, since a gene dosage effect on amyloid precursor protein production predictably results in early onset alzheimer’s disease (wisniewski et al. 1985, ballard et al. 2016). this approach was followed in a recent study, in which the patterns of microglia activation and the expression of proand anti-inflammatory cytokines was analyzed in a large sample of brain tissue of patients with trisomy 21 who had died at different ages (flores aguilar et al. 2020). already, in juvenile patients, a long time before the first accumulation of soluble aß or the first deposition of aß-plaques, microglia activation and the production of proinflammatory cytokines was elevated in comparison to age matched controls. in contrast, in older patients, when initial alzheimer’s disease pathology became apparent, microglia activation decreased and was replaced by senescent microglia phenotypes as well as a reduction of the production of pro-inflammatory cytokines. these results further support the view that anti-inflammatory treatment strategies in alzheimer’s disease may be most beneficial when applied in very early (pre-symptomatic) stages of disease evolution. the data further suggest that the inclusion of patients with trisomy 21 in future clinical trials could provide a valuable contribution to improve treatment options in patients with alzheimer’s disease. conclusions neuroimmunology is a highly dynamic field providing fascinating new insights into the pathogenesis of brain inflammation and neurodegeneration. the bulk of data, however, describe experimental models and, thus, an additional step is necessary, which defines the relevance of the data for human disease. for this, neuropathological studies are essential, but they have to be based on systematic analysis of a broad spectrum of cases and controls and have to be performed with the most suitable molecular or immunological technology. in this short review the prime focus was laid on such studies of human disease, which were sufficiently powered to provide definite answers to burning questions of neuroinflammation 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(https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. neuromuscular disease: 2021 update feel free to add comments by clicking these icons on the sidebar free neuropathology 2:3 (2021) review neuromuscular disease: 2021 update marta margeta department of pathology, university of california, san francisco, ca, usa corresponding author: marta margeta · ucsf pathology, box 0511 · 513 parnassus ave., hsw-514 · san francisco, ca 94143 · usa marta.margeta@ucsf.edu submitted: 5 february 2021 accepted: 22 february 2021 copyedited by: henry robbert published: 23 february 2021 https://doi.org/10.17879/freeneuropathology-2021-3236 keywords: costameres, sarcomeres, satellite cell activation, covid-19, guillain-barré syndrome, 5q-sma, smaled, lobulated fibers, supervillin, vacuolar myopathy, perilipin 4, sarcolemmal repair, trim72, vasculitis, amyloidosis, gene therapy, large-scale electron microscopy abstract this review highlights ten important advances in the neuromuscular disease field that were first reported in 2020. the overarching topics include (i) advances in understanding of fundamental neuromuscular biology; (ii) new / emerging diseases; (iii) advances in understanding of disease etiology and pathogenesis; (iv) diagnostic advances; and (v) therapeutic advances. within this broad framework, the individual disease entities that are discussed in more detail include neuromuscular complications of covid-19, supervillin-deficient myopathy, 19p13.3-linked distal myopathy, vasculitic neuropathy due to eosinophilic granulomatosis with polyangiitis, spinal muscular atrophy, idiopathic inflammatory myopathies, and transthyretin neuropathy/myopathy. in addition, the review highlights several other advances (such as the revised view of the myofibrillar architecture, new insights into molecular and cellular mechanisms of muscle regeneration, and development of new electron microscopy tools) that will likely have a significant impact on the overall neuromuscular disease field going forward. abbreviations aav, adeno-associated virus; anca, antineutrophil cytoplasmic antibody; asm, antisynthetase syndrome-associated myositis; attr, transthyretin amyloidosis; cgrp, calcitonin-gene related peptide; covid-19, coronavirus disease 2019; ck, creatine kinase; dm, dermatomyositis; fesem, field-emission scanning electron microscopy; glud1, glutamate dehydrogenase 1; egpa, eosinophilic granulomatosis with polyangiitis; gbs, guillain-barré syndrome(s); hmgcr, 3-hydroxy-3-methylglutaryl-coa reductase; iim, idiopathic inflammatory myopathy; imnm, immune-mediated necrotizing myopathy; lmn, lower motor neuron; mmsc, muscle mesenchymal stromal cells; nmd, neuromuscular disease; sars, severe acute respiratory syndrome; sibm, sporadic inclusion body myositis; sma, spinal muscular atrophy; smaled, spinal muscular atrophy lower extremity dominant; srp, signal recognition particle; stem, scanning transmission electron microscopy; svv, small vessel vasculitis; tem, transmission electron microscopy; tregs, regulatory t cells; ttr, transthyretin. in this update, i will briefly describe ten 2020 neuromuscular field advances that i consider to be most important and/or interesting; as in the prior update (margeta, 2020b), these advances will be grouped into several different “discovery clusters” and listed in no particular order. advances in fundamental neuromuscular biology with implications for neuromuscular disease 1. new understanding of the myofibrillar architecture based on numerous studies that used traditional electron microcopy methods for evaluation of skeletal muscle ultrastructure, the myofibrillar apparatus has long been understood as consisting of independent tube-like myofibrils that are arranged in parallel and span the entire length of a fiber, with each myofibril composed of sarcomeres that are serially connected through z-discs. this traditional model of the myofibrillar apparatus was effective in explaining how the contractile forces are transmitted down the length of the fiber towards the myotendinous junction, but could not readily explain how these forces were transmitted laterally, towards the sarcolemma and the extracellular matrix (into which myofibers are anchored through costameres, the sarcolemma-associated supramolecular structures that form membrane skeleton and mediate fiber adhesion). a recent groundbreaking study by willingham et al. has used a new method (focused ion beam-scanning electron microscopy) to image muscle fibers in three dimensions at high resolution, providing a new view of the myofibrillar architecture (willingham et al., 2020). to their surprise, the authors found that sarcomeres are not organized into independent parallel myofibrils as traditionally thought, but instead form the unified, non-linear myofibrillar matrix that spans both the length and the width of a fiber and is interconnected through extensive and bidirectional sarcomere branching (fig. 1). this branching occurs in what appears to be a random pattern, with branch points that are not restricted to any specific region of the sarcomere; however, the frequency of branches is fiber-type specific, with branches approximately three times more frequent in slow twitch than fast twitch fibers (and approximately two times more frequent in slow twitch fibers than cardiac myocytes). interestingly, sarcomere branching is developmentally regulated: while present at birth, it is downregulated in the period of rapid postnatal muscle growth and then upregulated again late in development. three different mechanisms of sarcomere branching were identified: sarcomere splitting (where some of the myofilaments from one sarcomere separated from the rest, forming two distinct myofibrillar segments), myofilament transfer (where some of the myofilaments were redistributed between sarcomeres, separating from one myofibrillar segment and joining the adjacent one) and myofilament trade (where myofilament transfer between two adjacent myofibrillar segments was reciprocal/bilateral). while most of the work was performed on murine samples, the few human samples that were evaluated in this study showed essentially identical findings, except for a slightly higher branching frequency in human compared to murine fast-twitch fibers (2.4 ± 0.2 vs. 1.6 ± 0.2 branches per 10 sarcomeres); however, a more thorough investigation of inter-species differences remains to be done. figure 1. 3d renderings of the interconnected sarcomeres that form the unified myofibrillar matrix. a. 3d rendering of 115 directly connected sarcomeres within the myofibrillar matrix of a fast-twitch muscle. individual colors represent different myofibrillar segments linked by branching sarcomeres. b-d. 3d renderings at different perspectives highlight the nonlinearity and connections between myofibrillar segments. representative of 7,707 sarcomeres from four volumes from four mice. scale bars, 2 µm. (this figure and its legend were adopted from figure 1 in willingham et al., 2020; this use is permitted under the creative commons attribution 4.0 international license.) what is the relevance of this new understanding of the myofibrillar architecture for neuromuscular disease (nmd)? that must be experimentally addressed and therefore remains to be seen, but it is very likely that abnormal branching of sarcomeres contributes to poor contractile force generation in various congenital / structural myopathies, which show different degrees of organelle malpositioning and myofilament disorganization. in addition, this new discovery may have important implications for pathogenesis of muscular dystrophies: given that the myofibrillar matrix is directly connected to the costameric membrane skeleton (discussed further in advance #4 below), it is possible that disruption of these connections may result in inappropriate transfer of lateral mechanical forces from the interior of the fiber to the extracellular matrix, thereby contributing to the likelihood of segmental fiber necrosis that over time leads to exhaustion of the muscle regenerative capacity and muscle wasting. 2. deepening insights into molecular and cellular mechanisms of muscle regeneration over the past decade, much has been learned about the mechanisms of skeletal muscle regeneration; the key role of satellite cells (muscle stem cells) in this process was first demonstrated in 2011, and the work that followed since then has showed that satellite cell activation requires cytokines and growth factors released by foxp3-expressing regulatory t cells (tregs) and macrophages. two studies published last year build further on these discoveries by elucidating the intercellular crosstalk and chemical signals that mediate muscle regenerative response. the first study focused on the interplay between intramuscular peripheral nerve twigs, muscle mesenchymal stromal cells (mmscs), and tregs in skeletal muscle regeneration (wang et al., 2020). following muscle injury, mmscs are the main source of interleukin (il)-33, which drives subsequent accumulation of tregs required for satellite cell activation and muscle repair. wang et al. have found that il-33+ mmscs are located in close proximity to peripheral nerve bundles and that they titrate their il-33 production in response to calcitonin-gene related peptide (cgrp), which plays a role in pain perception and is released by sensory peripheral nerve terminals in response to tissue damage. in agreement with these findings, systemic administration of cgrp led to an increase in the muscle il-33 level and recruitment of tregs into the muscle compartment. however, it remains to be shown whether cgrp treatment will enhance muscle regeneration and – conversely – whether inhibition of cgrp signaling will attenuate effective repair following muscle injury; the latter question is particularly pressing given that anti-cgrp antibodies and cgrp antagonists have recently been approved for prevention of migraine headaches. in addition, it remains to be seen whether effective muscle repair requires only signaling from sensory nerve terminals, or whether motor nerve twigs also play a role in this process. the second and more comprehensive of the two studies centered on the nature of chemical crosstalk between macrophages and satellite cells that is required for muscle repair (shang et al., 2020). the authors of that study modulated glutamine secretion from macrophages via genetic or pharmacologic manipulation of glutamine synthetase (which generates glutamine by condensation of glutamate and ammonia) or glutamate dehydrogenase 1 (glud1, which catalyzes oxidative deamination of glutamate and thereby reduces the amount of glutamate available for glutamine synthesis); in addition, they blocked satellite cell glutamine uptake by knocking down glutamine transporter slc1a5 selectively in those cells. using these experimental approaches both in vitro and in vivo, the authors demonstrated that satellite cells take up glutamine that is secreted by monocyte-derived macrophages recruited into skeletal muscle following injury; this glutamate uptake, which leads to activation of mtor signaling, is required for satellite cell proliferation and differentiation. excitingly, shang et al. showed that increased glutamine bioavailability in injured or aged muscle translates into increased muscle regenerative capacity: mice with decreased glud1 activity showed faster and more effective fiber regeneration following acute myotoxic injury when young, as well as increased muscle mass and improved baseline physical performance when aged. while these findings need to be replicated in other model systems and ultimately human clinical trials, they hold promise not only for treatment of muscular dystrophies and other skeletal myopathies characterized by recurring muscle fiber necrosis, but also for treatment of the age-associated sarcopenia that leads to significant morbidity and mortality in elderly populations. newly defined / emerging neuromuscular diseases 3. neuromuscular complications of covid-19 2020 has been dramatically shaped by the covid-19 pandemic, which has affected the nmd field in addition to all other aspects of life. covid-19 is caused by infection with sars-cov-2, an rna virus from the coronavirus family; the disease is asymptomatic in up to 50% of patients, but can also cause severe pneumonia, acute respiratory distress syndrome, and death. neurologic complications of covid-19 were noted very early in the pandemic (mao et al., 2020) and include cns, pns, and skeletal muscle manifestations (cagnazzo et al., 2020); in this update, two forms of potential neuromuscular involvement [guillain-barré syndromes (gbs) and rhabdomyolysis / myopathy] will be discussed in more detail. gbs are a group of acute immune-mediated polyneuropathies that are characterized by ascending weakness, mild-moderate sensory abnormalities, and pain; the most common subtypes include acute demyelinating inflammatory polyradiculoneuropathy, acute motor axonal neuropathy, and the miller-fischer syndrome (dalakas, 2020). in approximately 70% of gbs patients, a flu-like illness precedes neurologic symptoms by ~1-3 weeks, and all subtypes of gbs show association with different infectious agents (both viruses and bacteria). perhaps not surprisingly, gbs was also documented in patients with covid-19, first through case reports and small case series (reviewed in caress et al., 2020; dalakas, 2020; de sanctis et al., 2020) and later through small retrospective case-control studies, one from italy (filosto et al., 2020) and another from spain (fragiel et al., 2020). based on these early reports, covid-19-associated gbs was shown to have typical clinical characteristics, with most cases falling within the acute demyelinating subtype and showing onset of neurologic symptoms a median of 10-23 days after the covid-19 diagnosis. the two retrospective case-control studies found 3to 10-fold higher gbs incidence during the study period (the 2-month pandemic peak in each country) compared to the equivalent period in 2019; however, the conclusions were limited by relatively small sample sizes and a high potential for confounding due to selection and ascertainment bias. indeed, the most comprehensive investigation of the potential link between gbs and covid-19 to date (a combined retrospective epidemiologic and prospective cohort study performed in the uk and published at the end of 2020) found no association between these two conditions (keddie et al., 2020). in fact, and somewhat counterintuitively, the authors of that study found a decrease (rather than increase) in the number of gbs cases that required ivig treatment during the 2-month pandemic peak compared to prior years; while reasons for this decrease are not entirely clear, one possible explanation is a decrease in the number of infections with known gbs-triggering pathogens as a result of drastic public health actions that were undertaken to control the pandemic. in addition to a lack of increase in gbs incidence, keddie et al. found no temporal or spatial association between covid-19 and gbs cases in the uk during the study period, further weakening the likelihood of true association between these two diseases. given these contradictory findings, additional studies should be done; however, the preponderance of current evidence suggests that the sars-cov-2 infection is not a major trigger of autoimmune polyneuropathy. what about skeletal muscle involvement in covid-19? as with other flu-like illnesses, myalgias are a common symptom of this viral disease; however, actual skeletal muscle injury [as evidenced by elevated creatine kinase (ck) levels] is seen only in a subset (~10-30%) of patients (dalakas, 2020; manzano et al., 2020). in some of these “covid-19 myopathy” cases, muscle injury is very severe, with ck levels greater than 10,000 u/l (buckholz et al., 2020; dalakas, 2020; and a number of individual case reports); the most severe case was documented in a patient on rosuvastatin therapy who was infected with sars-cov-2 and developed fatal rhabdomyolysis, with the ck level of ~1,000,000 u/l (anklesaria et al., 2020). however, very few muscle biopsies or autopsy muscle samples from covid-19 patients have been comprehensively evaluated to date; as a result, very little is known about the mechanisms that underlie covid 19-associated muscle injury. skeletal muscle expresses angiotensin-converting enzyme 2, which serves as the cellular receptor for sars-cov-2 (ferrandi et al., 2020); thus, it is conceivable that this virus can invade and directly injure muscle fibers. however, limited studies published thus far have not provided evidence for viral presence in the injured muscle (manzano et al., 2020; rosato et al., 2020; zhang et al., 2020). [one case report noted the presence of coronavirus-like particles in a postmortem muscle sample from a patient who died from covid-19 but had no symptoms of skeletal muscle injury (hooper et al., 2020); however, ultrastructural detection of coronaviruses can be challenging (dittmayer et al., 2020a) and the virus-like particles shown in that paper more closely resemble clathrin-coated vesicles or parts of the rough endoplasmic reticulum than true coronaviruses.] instead, the currently available data suggest that covid 19-associated muscle injury is most likely para-infectious / immune-mediated, and can manifest as dermatomyositis-like “type i interferonopathy” (manzano et al., 2020), myositis (zhang et al., 2020), or immune-mediated necrotizing myopathy (fig. 2). this is in agreement with the work done during the sars epidemic in early 2000s, which suggested that vasculitis or immune-mediated mechanisms, rather than direct viral infection, were a cause of sars-associated myopathy (ding et al., 2003; leung et al., 2005). given that the available studies are very limited in scope, much additional work is needed to fully elucidate the mechanisms of skeletal muscle injury in covid-19. however, an intriguing possibility is that both mechanisms play a role, as recently shown for the chikungunya infection-associated myopathy: in that emerging viral disease, muscle pathology is due to activation of the host immune response but is triggered by viral replication in muscle fibers (lentscher et al., 2020). figure 2. immune-mediated necrotizing myopathy in a patient with covid-19. a representative h&e-stained cryosection (a) shows frequent, randomly distributed degenerating/regenerating muscle fibers and focal myophagocytosis in the absence of a significant lymphocytic inflammatory infiltrate; unusual dystrophic calcifications are seen in a subset of necrotic fibers (arrows), confirmed by von kossa calcium stain (not shown). deposition of the complement membrane attack complex (c5b9-immunostained cryosection; b) is seen in the sarcoplasm of necrotic fibers as well as the sarcolemma of many intact-appearing fibers. immunohistochemical stains for lc3 (c) and p62 (d; both performed on formalin-fixed, paraffin embedded tissue) show frequent fibers with densely packed fine puncta. immunohistochemical stain for sars-cov-2 was negative and no coronavirus particles were detected on ultrastructural evaluation (not shown). the patient had a history of hepatitis c, diabetes mellitus, hypertension, and hyperlipidemia treated with atorvastatin. he was diagnosed with covid-19 based on a positive sars-cov-2 pcr test performed following a close exposure; he was asymptomatic at the time, but subsequently developed progressive lower extremity weakness and dysphagia. approximately 1 month after the first weakness symptoms, he lost his sense of smell, developed shortness of breath, and was hospitalized. at admission, his ck level was 33,000 u/l and he was still positive for sars-cov-2; muscle biopsy was performed 10 days into his hospital course (~ 2 months following initial covid-19 diagnosis). the patient was ultimately treated with prednisone, which led to improvement of his neuromuscular symptoms; he was discharged from the hospital never requiring mechanical ventilation for his covid-19 pneumonia. testing for anti-hmgcr and anti-srp antibodies was not performed. scale bar, 50 µm. 4. structural myopathy caused by supervillin deficiency the presence of occasional lobulated fibers is a relatively common and therefore nonspecific finding in muscle biopsies. in some cases, however, frequent lobulated fibers dominate the overall histopathologic picture, resulting in a descriptive diagnosis of a “lobular” (or “trabecular”) myopathy. in general, this descriptive diagnosis does not correspond to a specific, well-defined clinicopathologic entity; however, a newly defined structural myopathy caused by loss-of-function mutations in supervillin (hedberg-oldfors et al., 2020) is an exciting exception to this rule and, while probably rare, should be on the radar screen of every neuromuscular pathologist going forward. supervillin is a large, differentially spliced and ubiquitously expressed actin-binding protein that is particularly abundant in striated muscle; the 250 kda muscle isoform (also called archvillin) interacts and/or co-localizes with costameric proteins such as dystrophin and dystrophin-associated proteins, integrin/vinculin/talin complex, α-actinin, desmin, and caveolin 3 (oh et al., 2003). (as mentioned in advance #1, costameres are membrane-associated supramolecular structures that mediate muscle fiber adhesion to the extracellular matrix.) during skeletal muscle development, supervillin is located at the ends of differentiating myotubes, where it is thought to contribute to the myotendinous junction formation (oh et al., 2003). in addition, supervillin plays a role in the myofibril assembly (lee et al., 2007) and helps anchor peripheral myofibrils to the sarcolemma by connecting a z-disc protein nebulin to costameric proteins dystrophin and γ-sarcoglycan (lee et al., 2008; spinazzola et al., 2015). (highlighting the critical importance of myofibril anchoring, z-discs of peripheral myofibrils are also linked to the sarcolemma through binding of a z-disc protein filamin c to costameric proteins γ-sarcoglycan and integrin; reviewed in peter et al., 2011.) mutations in genes that encode costameric proteins mostly lead to muscular dystrophies, while mutations in myofibrillar and cytoskeletal proteins generally result in congenital myopathies; loss-of-function mutations in supervillin, which have previously not been associated with any muscle disease, are now known to cause a unique structural myopathy (hedberg-oldfors et al., 2020). the authors described four patients from two unrelated consanguineous families (two affected siblings in each family) that were ultimately shown to carry homozygous truncating mutations in supervillin, resulting in a complete loss of supervillin protein. while there were subtle clinical differences between these two families, shared clinical findings included onset in childhood/adolescence, muscle pain and stiffness without significant muscle weakness, wide neck and hypertrophy of back muscles, progressive contractures, moderately elevated ck levels, and mild cardiac involvement (mainly left ventricular hypertrophy). muscle biopsies from all four patients showed frequent lobulated type 1 fibers, myofibrillar disarray (including occasional z-band streaming and nemaline rods), subsarcolemmal protein aggregates, and autophagic vacuoles (fig. 3; see also hedberg-oldfors et al., 2020). aside from frequent lobulated fibers, these histopathologic features show some overlap with myofibrillar myopathies, a group of muscle disorders characterized by z-disc instability and defects in the chaperone-assisted selective autophagy (reviewed in margeta, 2020a); this histopathologic overlap may reflect the role of supervillin in the z-disc stabilization / sarcomere anchoring. interestingly, the currently known supervillin mutations are expected to abolish expression of all five supervillin isoforms (not just the archvillin isoform expressed in striated muscle); while these four patients did not show clinical involvement of any non-muscle tissues, the disease spectrum is likely to widen as additional patients (and mutations) are identified in the future. going forward, it will be particularly interesting to elucidate how the loss of supervillin leads to fiber lobulation and to establish whether similar mechanisms operate in other myopathies with frequent lobulated fibers. figure 3. histopathology of “supervillinopathy”. biopsy from the deltoid muscle of patient iii:2 in family 2, as visualized by light (a–c) and electron microscopy (d–g). a. a considerable number of partially atrophic lobulated muscle fibers with pointed, occasionally cap-like subsarcolemmal deposits (arrows) (h&e). b. prominent oxidative enzyme activity in these fibers (arrows) (nadh). c. atrophic lobulated fibers are almost exclusively type 1 fibers (arrows); no neurogenic pattern is observed (matpase at ph 9.4). d. subsarcolemmal accumulation of degraded myofibrils, glycogen, pleomorphic material and autophagy-associated (arrows) organelles. e. heterogeneous lipoprotein deposits including maturing lipofuscin (arrows). f. sporadically, nemaline rods are seen (arrow). g. three rods in transverse section (arrows). (this figure and its legend were adopted from figure 5 in hedberg-oldfors et al., 2020; this use is permitted under the creative commons attribution 4.0 international license.) advances in understanding of etiology and pathogenesis of neuromuscular diseases 5. genetic vacuolar myopathies: expanding etiology spectrum vacuolar myopathies are pathologically defined by the presence of autophagic vacuoles that can be seen on the light microscopic level (“rimmed vacuoles”), electron microscopic level, or both. vacuolar myopathies have varied etiologies that include myotoxic drugs, chronic inflammation, and genetic mutations; genetic forms can occur either as stand-alone myopathies or together with amyotrophic lateral sclerosis, frontotemporal lobar degeneration, and/or paget’s disease of the bone as a component of multiple system proteinopathies (reviewed in margeta, 2020a). unsurprisingly, then, the differential diagnosis of a vacuolar myopathy remains broad, and the final diagnosis can be difficult to establish even in the era of widely available genetic testing; this complexity was highlighted by a 2020 study (mair et al., 2020) that performed thorough clinicopathologic and genetic evaluation of 32 adult vacuolar myopathy cases from 30 unrelated families, and which includes a very comprehensive table of non-inflammatory causes that will be a useful resource for every practicing muscle pathologist. however, and highlighting the rapid pace of advancement in the nmd field, this table is already incomplete: a very exciting work published last year (ruggieri et al., 2020) demonstrated that a repeat expansion in the lipid droplet-coating protein perilipin 4 is the cause of another rimmed vacuolar myopathy the autosomal dominant distal myopathy that was previously linked to chromosome 19p13.3 (di blasi et al., 2004). the age at diagnosis in the originally published kindred of 19 individuals varied from 26 to 73 years, but was earlier in the later generations; the symptoms were variable, but never included dysphagia or dysphonia. pathologically, the biopsies showed some unique features: vacuoles were mainly subsarcolemmal, membrane-limited, and sometimes connected to the cell surface and/or bordered by the basal lamina, but did not include filamentous inclusions and were not congo redor thioflavin s-positive (fig. 4; see also di blasi et al., 2004; ruggieri et al., 2020). in spite of early genetic linkage of this disease to chromosome 19p, the causative mutation proved challenging to elucidate; ultimately, ruggieri et al. honed in on the correct target by performing quantitative mass spectroscopy of microdissected vacuoles, which identified perilipin 4 as the most enriched among 700 identified proteins. the subsequent analysis showed that the gene encoding perilipin 4, plin4, maps to 19p13.3; in affected patients, this gene contains a 891-nucelotide expansion of the repetitive nucleotide sequence in exon 4, resulting in extra 297 amino acids in the amphipathic domain of perilipin 4 protein. figure 4. histopathology of “perilipinopathy” (19p13.3-linked distal myopathy). a-b. gomori trichrome and h&e staining, respectively, in patients iv:3 and v:3, showing vacuoles (arrows) [rimmed, empty, or containing granular or basophilic material (arrowheads), mainly located in the subsarcolemmal region of fibers], fiber size variability, central nuclei, and mildly increased endomysial spaces. note minimal changes in v:13. c. electron micrographs unveiling vacuoles located in the subsarcolemmal region or deep in the sarcoplasm, containing small vesicles, membranous bodies, and granular debris; inset shows granular debris within a small subsarcolemmal vacuole (arrow) opening to the fiber’s surface and sarcolemmal interruption (arrowheads). (this figure and its legend were adopted from figure 1 in ruggieri et al., 2020; this use is permitted under the creative commons attribution 4.0 international license.) perilipins are proteins that localize to the surface of lipid droplets, where they interact with lipid enzymes and other regulators of lipid droplet metabolism; the five known isoforms all contain an 11-mer repeat sequence that repeats 3 times to form a 33-mer amphipathic helix required for lipid droplet localization (copic et al., 2018). perilipin 4 [the isoform most highly expressed in skeletal muscle, where it mainly localizes to the subsarcolemmal regions of type 1 fibers (pourteymour et al., 2015)] has a very long amphipathic domain that normally consists of 29-31 33-mer repeats but is extended by 9 additional 33-mers in patients with 19p13.3 distal myopathy (ruggieri et al., 2020). it is currently not understood how this expansion of the perilipin 4 amphipathic domain leads to muscle disease; however, the defect almost certainly does not involve metabolism of lipid droplets, which were similar in size and number in control and patient samples (ruggieri et al., 2020). on the other hand, the patient samples showed an increase in subsarcolemmal perilipin 4 expression and co-localization of this protein with p62/sqstm1, nbr1 and wdfy3 [autophagic adapters that play a key role in aggrephagy and granulophagy (selective autophagy of protein aggregates and stress granules, respectively)]. this finding provides a potential link between 19p13.3 distal myopathy and multiple system proteinopathies, which all show a defect in granulophagy (reviewed in margeta, 2020a). interestingly, expression of perilipin 4 in the cns is regulated by a stress granule rna-binding protein tia1 (heck et al., 2014), the gain-of-function mutations in which lead to welander distal myopathy (hackman et al., 2013; klar et al., 2013). it remains to be shown whether tia1 also regulates perilipin 4 expression in skeletal muscle and whether this regulation plays a role in pathogenesis of these two (and potentially other) distal vacuolar myopathies. 6. vasculitic neuropathy secondary to eosinophilic granulomatosis with polyangiitis: two pathogenetic mechanisms vasculitic neuropathy, the most common indication for nerve biopsy in the current neuromuscular pathology practice, can be secondary to many different forms of vasculitis. based on the most recent consensus nomenclature (jennette et al., 2013), small vessel vasculitis (svv; the category of vasculitis that is most likely to involve peripheral nerves) is divided into two subcategories – immune complex svv (which shows abundant immunoglobulin deposition in the walls of affected vessels) and anca (antineutrophil cytoplasmic antibody)-associated svv (where affected vessel walls show a paucity of immunoglobulin deposits). systemic anca-associated svv includes three distinct subcategories: microscopic polyangiitis, granulomatosis with polyangiitis (previously known as wegener’s granulomatosis), and eosinophilic granulomatosis with polyangiitis (egpa; previously known as churg-strauss syndrome). interestingly, some patients with anca-associated svv do not actually have detectable anca; it is not entirely clear whether that is because they have anca that cannot be detected with current methods, because they have novel, yet-to-be discovered anca, or because pathogenesis of their vasculitis actually does not involve anca at all (jennette et al., 2013). a large clinicopathologic study of egpa-associated neuropathy published last year (nishi et al., 2020) sheds some light on this question by providing evidence for different pathogenic mechanisms in anca-positive and anca-negative forms of egpa-associated neuropathy. egpa is a form of systemic svv that is associated with asthma, eosinophilia, and peripheral neuropathy; neuropathy is common and affects 50-75% of patients. among the three subtypes of systemic anca-associated svv, egpa is least likely to show anca positivity: just 30-40% of patients have anti-myeloperoxidase anca, and almost none have anti-proteinase 3 anca (nishi et al., 2020). to explore differences between anca-positive and anca-negative forms of egpa, nishi and co-authors have retrospectively investigated 82 consecutive patients who were diagnosed with egpa-associated neuropathy and underwent sural nerve biopsy; 33% of study subjects were positive for anti-myeloperoxidase anca and none were positive for anti-proteinase 3 anca. clinically, anca-positive and anca-negative egpa patients were quite similar, although anca-positive patients were more likely to show involvement of the upper extremities than the anca-negative ones. pathologically, both anca-positive and anca-negative cases showed predominantly axonal form of nerve damage with a focal or multifocal pattern of axon loss suggestive of ischemic injury; however, the vascular involvement differed between the two groups. nerve biopsies from anca-positive patients were more likely to show frank vasculitis and/or destruction of vascular structures, with the mean diameter of affected vessels ~170 µm (fig. 5a); in contrast, nerve biopsies from anca-negative cases were more likely to show a large number of eosinophils in the epineurial vessel lumina, epineurial vessels occluded by intraluminal eosinophils (with vessels smaller than 13 µm typically showing more than 50% occlusion; fig. 5b), and extravasation of eosinophils into the endoneurium. interestingly, the pattern of axon loss in anca-negative cases showed no topographic correlation with the location of endonurial eosinophils; rather, it was patchy (focal or multifocal), suggestive of nerve ischemia. taken together, these findings suggest that there are two distinct pathogenetic mechanisms for ischemic injury in egpa-associated neuropathy: frank vessel destruction in anca-positive cases and vessel occlusion by eosinophils in anca-negative cases (the latter mechanism is conceptually similar to vessel occlusion by intravascular large b-cell lymphoma, which causes tissue ischemia without directly destroying vascular structures). while both patterns of vessel involvement can be seen in skeletal muscle (fig. 5) in addition to peripheral nerves of patients with egpa, it remains to be seen whether differences between anca-positive and anca-negative egpa cases will be conserved across all affected organ systems. in addition, it needs to be established whether similar mechanistic differences distinguish anca-positive and anca-negative cases in the other two types of anca-associated svv. finally, given the almost complete lack of vessel wall inflammation in anca-negative egpa cases, it needs to be re-evaluated whether this clinicopathologic syndrome should remain classified as a vasculitis or whether a different disease category would be more appropriate. figure 5. two patterns of vascular involvement in eosinophilic granulomatosis with polyangiitis. a. muscle biopsy from a patient with anca-positive egpa shows necrotizing vasculitis affecting small intramuscular artery; the inflammatory infiltrate includes eosinophils. b. the lumen of a very small perimysial artery (arrow) is nearly completely occluded by intraluminal eosinophils in a muscle biopsy from a patient with egpa; anca status was not reported. representative images from h&e-stained sections of formalin-fixed, paraffin-embedded tissue are shown for both cases. scale bar, 20 µm. 7. spinal muscular atrophies: skeletal muscle involvement spinal muscular atrophies (smas) are a group of genetic disorders in which the loss of lower motor neurons (lmns) in the anterior horn of the spinal cord, which typically occurs early in life, results in neurogenic muscle atrophy and weakness. the most common sma subtype, 5q-sma, accounts for ~95% of sma cases and is caused by autosomal recessive, loss-of-function mutations in both copies of smn1 (survival motor neuron 1) gene; the clinical phenotype is modified by the presence of the paralog gene, smn2, which yields only a small number of full-length transcripts and is present in a variable number of copies in different individuals, with more copies resulting in a milder clinical phenotype and better survival. a different sma subtype, known as smaled (“spinal muscular atrophy lower extremity predominant”), is caused by autosomal dominant mutations in dync1h1 and bicd2 genes, which impair centripetal microtubular transport that moves intracellular cargo from the cell periphery to the perinuclear region [dync1h1 encodes a subunit of the dynamin/dynactin retrograde transport complex, while bicd2 encodes a cargo adapter protein that directly binds to dync1h1 (koboldt et al., 2020)]. pathogenesis of both 5q-sma and smaled was thought to involve loss of lmns that occurs in a cell-autonomous manner; however, two studies published last year (kim et al., 2020; rossor et al., 2020) highlight the important and somewhat unexpected role of skeletal muscle in these disease processes. in addition to the lmn loss that is a defining feature of the sma phenotype, bicd2 deficiency causes cerebellar hypoplasia through a pathway that involves bergmann glia in a non-cell autonomous manner. to investigate whether the lmn loss in smaled also involves non-cell autonomous mechanisms, rossor et al. selectively deleted bicd2 gene from either lmns or skeletal muscle (rossor et al., 2020). while neither mouse model showed altered gait or decreased life expectancy, a subtle loss of small diameter (gamma) motor axons was observed in the mice lacking bicd2 in skeletal muscle; in contrast, there was no evident axon loss (or any other mn phenotype) in the mice lacking bicd2 expression in lmns. further experiments demonstrated that skeletal muscle from bicd2-/mice showed a reduction in the number of muscle spindles but no neurogenic changes indicative of the loss of alpha lmns; similar findings were seen in mice lacking dync1h1. finally, the authors used an in vitro assay to show a decreased secretory activity in fibroblasts from a smaled patient with a bicd2 mutation when compared to fibroblasts from a control patient. based on these findings, rossor et al. proposed that the lmn loss in smaled is non-cell autonomous and involves reduced neurotrophin secretion from skeletal muscle, which limits neurotrophin availability and leads to excess lmn apoptosis during development. while this hypothesis is intriguing, it is currently not clear whether a similar pathogenetic mechanism operates in human disease; in particular, biopsies from smaled patients show a mixture of neurogenic and myopathic changes that is not well replicated by the existing mouse models. in addition, it is not clear why diminished neurotrophin availability would preferentially affect lmns that innervate lower extremities. nonetheless, the results of this study are sufficiently compelling to warrant further investigation. kim et al. have used a similar experimental approach to explore the role of skeletal muscle in the 5q-sma pathogenesis (kim et al., 2020). intriguingly, they found that the lack of smn expression in skeletal muscle results in an age-dependent myopathy that is usually obscured by the concurrent neurogenic process driven by the lmn loss. in the presence of a single copy of smn2 gene, smn1 deficiency in skeletal muscle led to an early-onset myopathy and severely shortened life span, with most mice dying by the postnatal day 25. a milder phenotype was seen in mice deficient for smn1 that carried two copies of snm2 gene: at a young age, their skeletal muscles appeared normal but showed impaired regeneration following a myotoxic insult; by 6-7 months of age, outright myopathic changes and impaired functional muscle performance were observed, leading to early mortality with only 14% of mutants (but 95% of controls) viable at 18 months. importantly, restoration of muscle smn levels in 7-month-old mice mitigated the observed muscle pathology. conceptually, this study reinforces the paradigm observed with many genetic diseases: a treatment that is effective but targets only the most severely affected organ system typically unmasks the effects of the same mutation on other organ systems that were previously not apparent, but now need to be addressed. for patients with 5q-sma, the implications are more practical: nusinersen treatment, which is given intrathecally and therefore increases smn expression in the spinal cord but not in skeletal muscle, may need to be combined with (or replaced by) a treatment that also increases smn levels in peripheral tissues; one candidate for such therapy is aav9-smn, an aav9 (adeno-associated virus 9) vector carrying human smn transgene that was approved for 5q-sma treatment in 2019, but is currently restricted to patients younger than 2 years of age [for more information on emerging treatments for 5q-sma, see the prior update in this article series (margeta, 2020b)]. additional clinical trials that specifically focus on myopathic pathology in 5q-sma will be needed to address this clinically important issue. moreover, it will be important to determine how smn deficiency leads to the failure of muscle maintenance, which seems to underpin this late-onset muscle pathology. 8. idiopathic inflammatory myopathies: the role of autoantibodies targeting sarcolemmal repair proteins based on the current clinicoseropathologic criteria, idiopathic inflammatory myopathies (iims) are divided into four distinct subcategories [dermatomyositis (dm), anti-synthetase syndrome-associated myositis (asm), immune-mediated necrotizing myopathy (imnm), and sporadic inclusion body myositis (sibm); for a brief overview of the diagnostic criteria and other recent advances in the iim field, see last year’s update in this article series (margeta, 2020b)]. iim pathogenesis is complex and only partly understood, but an interesting study published last year uncovered a feed-forward mechanism that contributes to iim progression by linking development of autoantibodies against trim72 (also known as mitsugumin 53) with defective sarcolemmal repair and ongoing exposure of intracellular autoantigens (mcelhanon et al., 2020). trim72 is a 53 kda member of the trim family of proteins; it is highly expressed in striated muscle and has many different cellular functions, including a key role in the plasma membrane repair. following sarcolemmal injury, trim72 nucleates and then coats intracellular lipid vesicles that accumulate at the site of the membrane break; fusion of these vesicles with damaged sarcolemma creates a “patch” that seals the membrane defect in a process that requires ca2+ as well as interaction of trim72 with dysferlin and caveolin 3 (reviewed in benissan-messan et al., 2020). while mutations in dysferlin and caveolin 3 lead to limb-girdle muscular dystrophies 2b and 1c, respectively, mutations in trim72 have not yet been linked to any human disease; however, the myocardium of trim72-deficient mice is more vulnerable to ischemia (cao et al., 2010), highlighting the importance of this protein for striated muscle integrity. building on that body of work, mcelhanon et al. have now implicated trim72 in the iim pathogenesis by using an adaptive transfer animal model of iim. in this model, lymph node cells from syt7-/foxp3-/γ double mutant mice (which have defective membrane repair and are prone to autoimmunity because they lack tregs) are transferred into immunologically naïve rag1-/mice, who one week after transfer develop an inflammatory myopathy. intriguingly, endomysial inflammatory infiltrates composed of t cells and macrophages were present only in the proximal muscles of the recipient mice, while “leaky” muscle fibers with compromised sarcolemma were identified in both proximal and distal muscles. the presence of myofiber damage in non-inflamed distal muscles raised the possibility that humoral rather than cellular autoimmunity caused impaired sarcolemmal repair in this mouse model, leading the authors to investigate whether the recipient mice developed autoantibodies against one or more proteins involved in this process. indeed, anti-trim72 autoantibodies were detectable in the sera of the recipient mice as well as in 10-30% (depending on the chosen cutoff) of sera from 103 human iim patients. importantly, the authors showed that anti-trim72 antibodies were pathogenic: treatment of isolated normal muscle fibers by either purified anti-trim72 antibodies or by human iim sera positive for these antibodies caused a membrane repair defect (as measured by an in vitro membrane resealing assay), while depletion of anti-trim72 antibodies from human iim sera strongly attenuated this effect. based on these findings, the authors proposed a new model of iim pathogenesis, in which sarcolemmal disruption caused by any number of events (including a viral or bacterial infection, trauma, or overexertion) results in exposure of sarcolemmal repair proteins to the immune system; in susceptible individuals, this exposure leads to development of autoantibodies that target trim72 and/or other repair proteins, compromising membrane resealing and leading to continuous autoantigen exposure in a feed-forward loop. while this model needs to be experimentally tested, it does provide possible explanation for association of covid-19 (and other viral illnesses) with immune-mediated muscle pathology (discussed in advance #3). one limitation of this otherwise very nicely done study is the outdated classification of the patient iim sera used for experiments (they were classified into dm and polymyositis categories, presumably because they were obtained from a specimen bank that performed specimen collection before introduction of the current iim diagnostic criteria). nevertheless, the study included information about the status of a subset of myositis-specific antibodies in the same iim specimen set, providing at least partial clues to more up-to-date classification. based on this limited information, anti-trim72 antibodies were present in a significant fraction of iim patients with dm-associated antibodies (anti-mi-2, anti-tif1γ, and anti-sea), as well as one of two patients with imnm-associated anti-srp antibodies; in contrast, they were not detected in any of patients with the asm-associated anti-jo-1 antibodies. while these findings need to be replicated and extended by evaluation of patients from all currently defined iim subcategories (including sibm and other subtypes of imnm), they do raise the possibility that this new pathomechanism plays a bigger role in pathogenesis of dm and imnm than asm. in that context, it is intriguing that tif1γ / trim33 – which is targeted by one of the dm-associated myositis-specific antibodies – is another member of the trim protein family, although in contrast to trim72 it does not seem to play a significant role in skeletal muscle repair (parks et al., 2019). advances in neuromuscular disease diagnostics 9. large-scale electron microscopy transmission electron microscopy (tem) remains an important diagnostic tool in the contemporary neuromuscular pathology workflow, but some practical limitations diminish its potential usefulness. in particular, it is typically necessary to maximize the use of these expensive instruments while minimizing the number of people who must obtain specialized training required to operate them; as a result, diagnostic ultrastructural evaluation is typically performed on static images of select areas of interest that were previously captured by the trained laboratory staff. while reasonably effective, this approach makes it challenging to relate the captured nanoscale images to the microscale tissue architecture appreciated by light microscopy; in addition, depending on the staff expertise level, there is always a possibility that the captured images are not fully representative of the underlying pathology, leading to diagnostic errors. to circumvent some of these limitations, large-scale em [also known as “virtual em” or nanotomy (short for nanometer-scale anatomy)] has been developed over the last decade; while the first use of this technique in the nmd field was reported in 2003 (sullivan et al., 2003), a wider adoption did not happen until last year (dittmayer et al., 2020b; dittmayer et al., 2020c; rocha et al., 2020). in large-scale em, the entire ultrathin sections are digitized by automated acquisition of multiple overlapping image tiles that are then stitched into a single large image; the resulting high-resolution digital images can then be evaluated on a standard computer workstation, enabling easy navigation between micrometer and nanometer scales across the entire ultrathin section. initial applications of this technology were based on stitching of multiple traditionally acquired tem images (sullivan et al., 2003; faas et al., 2012; lee and mak, 2011); while providing a proof-of-principle for virtual em as a diagnostic and research tool, this approach had two major limitations (1) imperfect image quality due to interference from the specimen support grids and various preparation flaws, and (2) a long time required to capture and stich a very large number of standard tem images. in the years that followed, these limitations were addressed by replacing traditional tem by field-emission scanning em (fesem); one fesem image captures the field of view that is equivalent to ~100 tem images, thus significantly reducing the amount of required image stitching and accelerating the entire process (kuipers et al., 2016). in fesem, imaging of ultrathin sections can be performed using a conductive silicon substrate that does not cause any visual interference, producing very sharp tem-like images through detection of backscattered electrons (dittmayer et al., 2018); this solves problem 1, but not entirely problem 2 because imaging speed remains slow (kuipers et al., 2016). alternatively, fesem can be used for digitization of sections prepared with conventional tem slot grids and imaged in a transmission mode with a scanning transmission electron microscopy (stem) detector (kuipers et al., 2016); this solves problem 2 due to faster imaging, but preparation of samples with minimal flaws remains challenging. despite remaining limitations, the capabilities of this new technology are already impressive: if stem is combined with conventional modern systems, automated digitization of entire ultrathin sections at a 7 nm-pixel resolution can be performed in about 12 h (dittmayer et al., 2020c), while specialized high-throughput imaging systems based on fesem and tem technology can reduce the imaging time to about 10-20 min. when will large-scale em become available for routine diagnostic use? that is difficult to predict, given that the most sophisticated version of the technique requires purchase of new, more advanced em instruments; however, the cost could be justified by anticipated improvements in diagnostic accuracy, opportunities for digital pathology research, and reduction in the time spent on image acquisition by the laboratory staff. in the meantime, one can get a taste of this exciting new technology by viewing open-source datasets available at http://www.nanotomy.org/ advances in neuromuscular disease treatment 10. long-term efficacy and safety: gene-silencing therapies for transthyretin amyloid neuropathy transthyretin amyloidosis (attr) is caused by deposition of either wild-type or mutant transthyretin (ttr) in different tissues throughout the body; these two disease forms are referred to as attrwt and attrv (“v” for variant), respectively. ttr functions as a carrier for thyroxin and retinol/vitamin a and is primarily synthetized by the liver; clinical symptoms of attr are largely due to accumulation of ttr amyloid in the heart and peripheral nerves, but skeletal muscle can also be affected in rare cases (fig. 6 and pinto et al., 2020; see also fig. 1 in lam et al., 2015). interestingly, recent work has shown that tissue damage in attr is caused not only by the amyloid fibril deposition but also by toxicity of nonfibrillar transthyretin oligomers, which directly damage endothelial cells and nerve fibers (reviewed in koike and katsuno, 2020). figure 6. transthyretin amyloid deposition in a peripheral nerve and skeletal muscle of a patient with attrv. sural nerve biopsy (a-d) shows accumulation of congo red-positive amyloid substance in the arterial walls (a); the deposits demonstrate apple-green birefringence under the polarized light (b) and are immunoreactive with anti-transthyretin antibody (c). marked loss of myelinated axons is evident on toluidine blue stain (d). sections from a gastrocnemius muscle biopsy (e-j) show deposition of ttr amyloid in the arterial walls (e-g) and fiber sarcoplasm (h-j); well-developed neurogenic changes were also present (not shown). subsequent to the biopsy diagnosis of ttr amyloid neuropathy and myopathy, the patient was found to carry a pathogenic ttr p.phe84leu mutation and was diagnosed with attrv; he was treated by inotersen and initially stabilized, but then continued to progress and will likely be switched to the patisiran therapy going forward. panels a-c and e-j, formalin-fixed, paraffin embedded tissue; panel d, epon-embedded tissue. panels a-b, e-f, and h-i, congo red stain; panels c, g and j, transthyretin immunoperoxidase stain; panel d, toluidine blue stain. scale bars, 50 µm. fully assembled ttr is a conformationally stable homotetramer; in contrast, individual ttr monomers are prone to misfolding and aggregation. thus, the currently approved attrv therapies aim either to stabilize the tetrameric quaternary structure of ttr (tafimidis or diflunisal; approved in the early 2010s) or to inhibit ttr synthesis through gene silencing approaches (inotersen or patisiran; both approved in 2018). untreated attrv is a rapidly progressive disease with a poor prognosis, and the availability of several disease-modifying treatments was an incredible breakthrough for the nmd field. at the same time, many important treatment-related questions remained open, and had to be addressed through post-approval studies. for example, at the time of approval little was known about the long-term efficacy and safety of ttr-silencing drugs. in addition, it was not clear how different attrv treatment options compare to each other. while much work still remains to be done, at least some of these questions have been decisively answered by studies published in 2020. inotersen is an antisense oligonucleotide that inhibits ttr production and thereby slows the progression of attrv polyneuropathy; based on a 2-year update from the open-label extension of the neuro-ttr clinical trial (which led to its initial approval), no new safety concerns were identified and the drug continued to demonstrate therapeutic benefits. however, the outcomes were significantly better in study subjects who were originally placed in the experimental arm than in those who were originally in the placebo arm, indicating that early treatment is critical for inotersen efficacy (brannagan et al., 2020). similar results were seen with patisiran, which is an rna-interference therapeutic that also inhibits ttr synthesis; a 12-month update from the open-label extension of the apollo clinical trial (which led to initial approval of this drug) also demonstrated no new safety concerns and continued efficacy, with sustained benefit in patients who were originally part of the patisiran arm and a new improvement in patients who were originally part of the placebo arm. interestingly, the frequency of deaths was higher in the initial placebo group than in the initial patisiran group, again highlighting the benefits of early treatment (adams et al., 2021). finally, an indirect comparison of the data from neuro-ttr and apollo clinical trials demonstrated that patisiran was generally more effective than inotersen, with a greater treatment benefit observed on all polyneuropathy and quality of life measures evaluated by both trials (gorevic et al., 2021). taken together with a prior study that showed a greater efficacy of inotersen compared to tafimidis in patients with attrv polyneuropathy (plante-bordeneuve et al., 2019), these data suggest that patisiran is the best currently available treatment for attrv polyneuropathy. it needs to be noted that both these indirect comparison studies were funded by alnylam pharmaceuticals, the manufacturer of patisiran; however, given the magnitude and robustness of the observed therapeutic differences, it is unlikely that the results were significantly confounded by the sponsor’s financial interests. despite these advances, a lot remains to be explored. for example, it needs to be established whether ttr-silencing therapies will also be effective for treatment of attrwt polyneuropathy, and if they are whether their therapeutic benefit will be superior to that of tafimidis (which has been approved for attrwt treatment). similarly, both ttr-silencing drugs still need to be evaluated as treatments for attrv (and attrwt) cardiomyopathy: initial exploratory studies showed that inotersen led to no improvement in cardiac function while patisiran stopped (and even reversed) the progression of cardiac disease in attrv patients (reviewed in adams and slama, 2020), but randomized clinical trials addressing the cardiac aspect of attr still need to be done. disclosure statement the author receives research support from audentes therapeutics as a member of the muscle biopsy review committee for the aspiro clinical trial (nct03199469), which is evaluating the safety and efficacy of gene transfer therapy for x-linked myotubular myopathy. acknowledgements i am grateful to ms. christine lin for assistance with figure preparation. references adams, d., polydefkis, m., gonzalez-duarte, a., wixner, j., kristen, a.v., schmidt, h.h., berk, j.l., losada lopez, i.a., dispenzieri, a., quan, d., et al. 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(2020). covid-19-associated myositis with severe proximal and bulbar weakness. muscle nerve 62, e57-e60. copyright: © 2021 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. neuropathologists play a key role in establishing the extent of covid-19 in human patients feel free to add comments by clicking these icons on the sidebar free neuropathology 1:11 (2020) opinion piece neuropathologists play a key role in establishing the extent of covid-19 in human patients lokman cevik, michele joana alves, josé javier otero div. of neuropathology, dept. of pathology, the ohio state university school of medicine, columbus, oh, usa corresponding author: josé javier otero, md, phd · the ohio state university college of medicine · department of pathology · 4166 graves hall · 333 w 10th avenue · columbus, oh 43210 · usa jose.otero@osumc.edu submitted: 01 april 2020 accepted: 02 april 2020 published: 02 april 2020 https://doi.org/10.17879/freeneuropathology-2020-2736 keywords: covid-19, sars-cov2 abstract sars-cov2 infection causes covid-19, and represents the most emergent health care crisis of our generation. ample evidence in the scientific literature suggests that sars-cov, mers-cov, and endemic human coronaviruses infect brain cells. we delineate a rationale for encouraging evaluation of the brain, and in particular the brainstem, in covid-19 so that potential neuropathological mechanisms can be delineated.   sars-cov2 infection causes covid-19, and represents the most emergent health care crisis of our generation. sars-cov2 is a member of the betacoronavirus family that includes the severe acute respiratory syndrome cov (sars-cov) and middle east respiratory syndrome cov (mers-cov), both of which have caused fatal infections in the past two decades (huang et al., 2020). although most people recover from the disease, sars-cov2 can cause severe respiratory distress syndrome, particularly in older patients and patients with underlying comorbidities (mahase, 2020). most of the patients who require intensive care ultimately become unable to breathe spontaneously (wang et al., 2020). although sars-cov2 shows its effects predominantly on the respiratory system, the primary pathophysiology behind the respiratory dysfunction and mortality remains elusive. previous findings of sars-cov infection and other coronaviruses in the nervous system bring to mind the possibility that sars-cov2 infection can cause respiratory failure by disrupting the cardiorespiratory center in the brainstem and are briefly reviewed below. it is known that most coronaviruses share similar viral structures and infection pathways (baig et al., 2020). these structural and pathophysiological similarities between other coronaviruses and sars-cov2 likely indicate that pathophysiological insights from other coronavirus studies may be generalizable to sars-cov2. sars-cov and sars-cov2 have high protein homology (baig et al., 2020), and sars-cov2 uses the same receptor to enter the host cells with at least 10 times higher affinity relative to sars-cov (wrapp et al., 2020). although multiple candidate receptors have been proposed, cellular infection through interaction with the angiotensin (ang) converting enzyme (ace2), a transmembrane carboxypeptidates sharing homology to ace1’s extracellular domain but with unique transmembrane and intracellular domains (riordan, 2003), seems to be critical in the pathogenesis in sars-cov (baig et al., 2020). although ace2 is capable of modifying angiotensin i, it catalyzes angiotensin i to ang(1-9) rather than to ang ii, and plays diverse physiological roles (reviewed by (clarke and turner, 2012)). ace2 is expressed in airway epithelia, lung parenchyma, vascular endothelia, kidney cells, small intestine cells (li et al., 2020) and also in the brain, particularly in glial cells and neurons (baig et al., 2020). we also note that endemic human covs have neuroinvasive potential (desforges et al., 2014). with this in mind, several studies have explored the extent to which zoonotically transmitted covs affect human brain function. studies using different mouse lines transgenic for the expression of ace2 showed extensive virus replication in the brain, likely mediated through retrograde transport through the olfactory bulb. among the brain regions affected by the virus, thalamus, cerebrum and brainstem were severely impacted. while in the k18-hace2 transgenic line, the sars-cov infection induces neuronal death as a result directly from the neuronal and not pulmonary infection. an abundant neuronal loss was found along with increased inflammatory cytokines, and proliferation of microglia, but not of astrocytes (mccray et al., 2007; netland et al., 2008). additional studies using transgenic mice demonstrated enhanced levels of il-6, il-12p40, g-csf, cxcl1, mip-a and mcp-1 in brain homogenates 3 days after the infection resulting in an inflammatory cytokine reaction (tseng et al., 2007). such cytokine elevations and extensive neuronal pathologies in the brainstem were also noted in mers-cov infections (li et al., 2016). in this same study, the authors showed evidence of viral replication in primary and porcine astrocytes and human glioblastoma and neuroblastoma cell lines (li et al., 2016). it is not well established how sars-cov infection affects astrocytes in vivo and how it mediates the neuronal damage of this syndrome, despite the fact that ace2 is expressed in isolated astrocytes from brainstem, cerebellum and medulla (gallagher et al., 2006; gowrisankar and clark, 2016). on the other hand, expression of ace2 at mrna and protein levels in neurons is also documented. in vivo findings have shown ace2 expression in neurons of the paraventricular nucleus (pvn), area postrema (ap), dorsal motor nucleus of the vagus (dmnv), nucleus of tractus solitarii (nts), the rostroventrolateral medulla (rvlm), and the nucleus ambiguous (na), all brain structures related to cardiovascular and respiratory function (doobay et al., 2007). furthermore, we do not know the extent to which sars-cov affects brains of newborn babies. for instance, a timely study emerging from china performed on covid-19 outcomes on pediatric patients demonstrated that the severity of covid-19 in children less than 1-year-old was very high (53.8% showing critical course) relative to other age groups (dong et al., 2020). neurological manifestations of covid-19 have also been documented. in a preprint study from china, one of 3 patients suffering from sars-cov2 had neurological manifestations, including dizziness, headache, impaired consciousness, hypogeusia (reduced ability to taste) and hyposmia (reduced ability to smell) (mao et al., 2020), the latter symptoms suggesting neuronal involvement of areas in proximity to the olfactory bulb. another possibility of trans-synaptic transfer of sars-cov2 is the usage of neuroanatomic interconnections of the respiratory and gastrointestinal system to nuclei of the brainstem as has been noted by the avian influenza virus (li et al., 2020). finally, sars-cov2 could disseminate through the blood to the brain via crossing endothelial cells that express the ace2 receptor as another way of transfer (baig et al., 2020). in conclusion, there is growing evidence that the brain could be the main trigger in the severity of covid-19, but there is a paucity of evidence derived from human patients. according to the interim guidance of the american centers for disease control (cdc) in march 2020, cdc recommends collecting swab specimens, samples for postmortem microbiologic and infectious disease testing, formalin-fixed autopsy tissues from lung, upper airway, and other major organs, if an autopsy is performed for a confirmed covid-19 case (center of disease control and prevention, 2020, march 25). however, additional concerns have also been raised regarding the use of oscillating saws, a tool commonly used during brain procurement, as these saws have been shown to promote aerosolization. nevertheless, we believe that documenting the extent of cns involvement in severe manifestations, including documenting which brainstem nuclei may be affected in the autopsies is crucial to clarify the pathophysiology of covid-19. we also recognize that in areas ravaged by the scale of infection and covid-19 disease, investment in personal protective equipment for autopsy would represent an unwise decision when front-line workers remain unprotected. we therefore suggest that a coordinated effort between health systems work together to meet this goal. drawing inspiration from the neuropathological evaluations of brains of hiv infected patients (petito et al., 2003), we suggest that a study design composed of 20 brains procured from covid-19 infected decedents be performed, with extensive sampling of brainstem nuclei to include structures implicated in human control of respiration, including the locus coeruleus (nobuta et al., 2015), ventral medulla (rudzinski and kapur, 2010), and prebotzinger complex (schwarzacher et al., 2011). it would also be important to evaluate covid19 infected decedents without significant neurological manifestations as potential controls. in this way, highly impactful descriptive studies of covid-19 disease can be achieved and the burden of investing in personal protective equipment can be shared by various centers. references baig, a.m., khaleeq, a., ali, u., syeda, h., 2020. evidence of the covid-19 virus 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with overexpression of the brain renin-angiotensin system. am j physiol regul integr comp physiol 292, r373-381. gallagher, p.e., chappell, m.c., ferrario, c.m., tallant, e.a., 2006. distinct roles for ang ii and ang-(1-7) in the regulation of angiotensin-converting enzyme 2 in rat astrocytes. am j physiol cell physiol 290, c420-426. gowrisankar, y.v., clark, m.a., 2016. angiotensin ii regulation of angiotensin-converting enzymes in spontaneously hypertensive rat primary astrocyte cultures. j neurochem 138, 74-85. huang, c., wang, y., li, x., ren, l., zhao, j., hu, y., zhang, l., fan, g., xu, j., gu, x., cheng, z., yu, t., xia, j., wei, y., wu, w., xie, x., yin, w., li, h., liu, m., xiao, y., gao, h., guo, l., xie, j., wang, g., jiang, r., gao, z., jin, q., wang, j., cao, b., 2020. clinical features of patients infected with 2019 novel coronavirus in wuhan, china. lancet 395, 497-506. li, k., wohlford-lenane, c., perlman, s., zhao, j., jewell, a.k., reznikov, l.r., gibson-corley, k.n., meyerholz, d.k., mccray, p.b., jr., 2016. middle east respiratory syndrome coronavirus causes multiple organ damage and lethal disease in mice transgenic for human dipeptidyl peptidase 4. j infect dis 213, 712-722. li, y.c., bai, w.z., hashikawa, t., 2020. the neuroinvasive potential of sars-cov2 may play a role in the respiratory failure of covid-19 patients. j med virol. mahase, e., 2020. coronavirus covid-19 has killed more people than sars and mers combined, despite lower case fatality rate. bmj 368, m641. mao, l., wang, m., chen, s., he, q., chang, j., hong, c., zhou, y., wang, d., li, y., jin, h., hu, b., 2020. neurological manifestations of hospitalized patients with covid-19 in wuhan, china: a retrospective case series study. medrxiv, 2020.2002.2022.20026500. mccray, p.b., jr., pewe, l., wohlford-lenane, c., hickey, m., manzel, l., shi, l., netland, j., jia, h.p., halabi, c., sigmund, c.d., meyerholz, d.k., kirby, p., look, d.c., perlman, s., 2007. lethal infection of k18-hace2 mice infected with severe acute respiratory syndrome coronavirus. j virol 81, 813-821. netland, j., meyerholz, d.k., moore, s., cassell, m., perlman, s., 2008. severe acute respiratory syndrome coronavirus infection causes neuronal death in the absence of encephalitis in mice transgenic for human ace2. j virol 82, 7264-7275. nobuta, h., cilio, m.r., danhaive, o., tsai, h.h., tupal, s., chang, s.m., murnen, a., kreitzer, f., bravo, v., czeisler, c., gokozan, h.n., gygli, p., bush, s., weese-mayer, d.e., conklin, b., yee, s.p., huang, e.j., gray, p.a., rowitch, d., otero, j.j., 2015. dysregulation of locus coeruleus development in congenital central hypoventilation syndrome. acta neuropathologica. petito, c.k., adkins, b., mccarthy, m., roberts, b., khamis, i., 2003. cd4+ and cd8+ cells accumulate in the brains of acquired immunodeficiency syndrome patients with human immunodeficiency virus encephalitis. j neurovirol 9, 36-44. riordan, j.f., 2003. angiotensin-i-converting enzyme and its relatives. genome biol 4, 225. rudzinski, e., kapur, r.p., 2010. phox2b immunolocalization of the candidate human retrotrapezoid nucleus. pediatr dev pathol 13, 291-299. schwarzacher, s.w., rub, u., deller, t., 2011. neuroanatomical characteristics of the human pre-botzinger complex and its involvement in neurodegenerative brainstem diseases. brain 134, 24-35. tseng, c.t., huang, c., newman, p., wang, n., narayanan, k., watts, d.m., makino, s., packard, m.m., zaki, s.r., chan, t.s., peters, c.j., 2007. severe acute respiratory syndrome coronavirus infection of mice transgenic for the human angiotensin-converting enzyme 2 virus receptor. j virol 81, 1162-1173. wang, d., hu, b., hu, c., zhu, f., liu, x., zhang, j., wang, b., xiang, h., cheng, z., xiong, y., zhao, y., li, y., wang, x., peng, z., 2020. clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus-infected pneumonia in wuhan, china. jama. wrapp, d., wang, n., corbett, k.s., goldsmith, j.a., hsieh, c.l., abiona, o., graham, b.s., mclellan, j.s., 2020. cryo-em structure of the 2019-ncov spike in the prefusion conformation. science 367, 1260-1263. copyright: © 2020 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. neurotrauma: 2021 update feel free to add comments by clicking these icons on the sidebar free neuropathology 2:4 (2021) review neurotrauma: 2021 update daniel p. perl j. edward hebert school of medicine, uniformed services university, bethesda, md, usa corresponding author: daniel p. perl, md · j. edward hebert school of medicine · uniformed services university · bethesda, md 20814 · usa daniel.perl@usuhs.edu submitted: 12 february 2021 accepted: 09 march 2021 copyedited by: shino magaki published: 15 march 2021 https://doi.org/10.17879/freeneuropathology-2021-3264 keywords: traumatic brain injury, chronic traumatic encephalopathy, biomarkers, neuroinflammation, microglia, tau abstract despite the interruptions and restrictions to the progress of science that the covid-19 pandemic has introduced, 2020 was marked by a number of important advances in the field of neurotrauma. here, i will highlight what i believe are among the most important contributions. this year there were notable advances towards providing clinically useful information on neurotrauma outcome through the use of fluid biomarkers. i also introduce fascinating approaches to studying the role of microglia in nervous system repair and neuroinflammatory mechanisms leading to dysfunction through the use of colony-stimulating factor 1 receptor inhibitors, especially plexxikon 5622 (plx5622). oral administration of this compound is able to deplete microglial elements and then, following withdrawal from the drug, a new population of microglia then repopulates the brain. use of this approach in traumatic brain injury experimental models has produced important insights into the pathogenetic role of microglia in responding to this process. important new data on the nature and distribution of tau involvement of neurons and astrocytes in cases of chronic traumatic encephalopathy (cte) also appeared suggesting differences and similarities to alzheimer’s disease. additionally, the use of tau-specific pet scan ligands in at-risk populations has suggested that this approach may be able to identify cases with cte. lastly, we note the death in the past year of a major contributor to the field of neurotrauma neuropathology, professor j. hume adams. introduction for virtually all of us, 2020 will mostly be remembered as the year of covid-19. with numerous calls for mask wearing, social distancing, partial-to-complete shutdowns of offices and laboratories (including our own) and other similar restrictions, this pandemic has definitely had a dampening effect on everyone’s scientific productivity. nevertheless, by viewing the traumatic brain injury (tbi) literature that appeared during 2020, it is hard to see any significant slowing of progress. i have the impression that with most scientists working within a telecommuting mode, rather than being active in the lab, many took the time to get to accumulated data waiting to be analyzed, write papers waiting to be written, etc., and thus scientific output did not appear to slow, and possibly even increased. it might be predicted that following the achievement of widespread vaccination allowing for a final full return to our lab benches, there will likely be a lag in output as new experiments need to be put in place and additional data collected. time will tell. nevertheless, in surveying the field of neurotrauma, many important contributions were reported in 2020 and here i would like to pick some of what i believe were among the more important advances that were published. this is an individual viewpoint, reflecting my own biases, and i will apologize, in advance, if i have omitted any of your favorites on this year’s list. so, among the more than 2,000 publications appearing in the literature on neurotrauma in 2020, here are those that i chose to highlight for you. contributions to the development of fluid biomarkers for tbi: finally some approaches that appear to be useful over the past few years, a large number of groups have engaged in efforts to identify useful blood-based biomarkers in the field of tbi that hopefully would be able to reflect outcome, especially with respect to predicting a transition to clinically persistent sequelae. by and large, the results of these efforts have been relatively inconsistent and have not achieved robust clinical validity. however, in 2020 a group reported [1] that increases in exosome and plasma levels of neurofilament light (nfl) chain protein in patients with repeated mild traumatic brain injuries (mtbis) were predictive of subsequent development of chronic post-concussion syndrome, post-traumatic stress disorder (ptsd) and depression. the study involved a cohort of 195 retired service members enrolled in the chronic effects of neurotrauma consortium longitudinal study. the cohort received detailed assessments to document and quantify episodes of prior mtbi and were grouped into repetitive mtbi (3 or more episodes), 1-2 mtbis and controls with no tbi history. these assessments were obtained through interviews with the subjects. these data have particular importance, because they were obtained from retired service members, and thus their tbi exposures mostly occurred years prior to obtaining the biomarker assay. the results reported suggest that their assays are reflecting a chronic, ongoing pathologic process that extended from the mtbi exposures to the time when the blood was drawn. the nature of that process remains unclear, although in their discussion the authors noted literature pointing towards the elevation of nfl levels in association with axonal injury. the use of isolated exosomes, brain-derived membrane-bound components that cross the blood-brain-barrier to enter the blood stream, is particularly noteworthy. since these exosomes can be linked back to a central nervous system origin, it suggests that this approach is capable of providing indicators of more specific brain-related pathology and thus more closely reflect relevant biologic processes. since the results were predictive for the development of depression and symptoms indicative of ptsd, this result represents another piece of evidence for a biologic substrate of the long-term persistent behavioral consequences of tbi. further advances in the blood biomarker field were reported by okonkwo et al. [2] in a study where the predictive accuracy of determining glial fibrillary acidic protein (gfap) levels was compared with s100 calcium-binding protein b (s100b) assays in the blood of tbi patients. the u.s. centers for disease control and prevention reports that each year about 4,800,000 evaluations for tbi occur in emergency departments in the united states. about 80-90% of these patients have mild forms of tbi (mtbi, as defined by a glasgow coma scale score of 13-15) and only 10% of the mtbi patients will subsequently demonstrate abnormalities on computed tomography (ct) studies of the head and thus need further evaluation and monitoring. accordingly, rapid reliably predictive studies are needed to lighten this huge clinical diagnostic load and better identify which patients need further ct scrutiny. in the united states, assays of serum gfap have been approved by the u.s. food and drug administration (fda) so that they can be used in a clinical setting to determine the need for a head ct within 12 hours of an episode of mtbi. alternatively, in europe, approval has been granted for s100b assays that are currently used to serve this clinical assessment function. importantly, in the study now reported, plasma gfap was determined using a newly developed point-of-care prototype assay that can be completed within 15 minutes. serum s100b was determined by a more standard laboratory-based analytic method that requires transfer of the specimen to the laboratory, adding considerably greater time to provide a report. the endpoint used for the 1,359 tbi patients in the study, who ranged from mild to more severe degrees of injury, was the ability to predict the presence of structural abnormalities on a head ct scan. using a predetermined cut-off value for serum gfap of 22 pg/ml, this study found that gfap levels were an excellent predictor of ct abnormalities and could do so even on samples taken up to 24 hours from the time of trauma. gfap assays substantially outperformed that of s100b as a ct abnormality predictor. further, the mean level of gfap correlated with increasing numbers of distinct lesions identified on head ct, suggesting a dose response. indeed, patients with severe to moderate tbi (gcs 3-12) had 10-fold greater levels than the levels assayed on patients with mtbi (gcs 13-15). overall, the gfap assay had a predictive sensitivity of 0.987. it should be pointed out that the point-of-care assay, with its rapid reporting time (15 minutes) is much faster than the currently fda approved lab-based methodology and, by its nature, can be performed in obscure locations without access to laboratory facilities. it is my understanding that this new and faster technology is in the process of being evaluated by the fda for approval as a clinical diagnostic tool. this promises to become an extremely valuable approach for clinicians faced with a staggering number of tbi patients to evaluate. a rapid point-of-care assay method will also be of great value for military medicine where many tbis are clinically evaluated downrange in isolated battlefield locations. assays on the playing field of contact sport events might also find this approach quite attractive for clinicians involved in such evaluations. the role of microglia in repair of tbi microglia are abundant cellular constituents of the brain that play important roles in brain development, maintenance and the pathogenesis of most disease states. we are taught to consider microglia as playing a central role in virtually all nervous system repair processes, including that of tbi. indeed, although the precise mechanism for their action remains unknown, microglia have been thought to mediate ongoing neuroinflammatory damage to the brain that is responsible for at least some of the long-term functional sequelae of tbi and even aspects of potential subsequent neurodegenerative phenomena. recently, a series of drugs have been identified that inhibit colony-stimulating factor 1 receptor (csf-1r), which microglia depend upon for their survival. these drugs penetrate the brain after oral administration and within two to three weeks are able to almost completely deplete the entire microglial cellular compartment of the brain in an exposed animal. following withdrawal of the drug, there will be a steady repopulation of the brain by microglia (figure 1). importantly, the repopulated microglia appear to be a new rejuvenated population of cells without a prior history of antigen exposure. the primary agent used in such microglial repopulation experiments is referred to as plexxikon (plx) 5622. figure 1. microglia are depicted in 8 weeks old cx3cr1-gfp mice (microglia – green; dapi – blue) a) control mouse, normal diet, b) 3 weeks following a diet containing 1200 mg/kg of plx5622 (note virtual complete absence of microglia) and c) after 3 weeks on the plx5622 diet then 2 weeks on the control diet (note the return of prominent green staining from the repopulated microglia). from ongoing studies of k. whiting and z. galdzicki of uniformed service university neurosciences program. the use of drugs such as plx5622 have begun to be used to explore the specific roles that microglia play in the repair processes and sequelae of experimental tbi, such as occurs with controlled cortical impact. henry and colleagues [3] administered plx5622 one month after controlled cortical impact in mice and then withdrew the drug to allow for microglial repopulation. by 3 months post-injury, they noticed that the plx5622 treated animals possessed a smaller cortical lesion, reduced hippocampal neuron cell death and decreased expression of nox2 and nlrp3 inflammasome-associated neuroinflammatory modulators, when compared to non-treated animals receiving equivalent controlled cortical impact injury. the plx5622 treatment animals also showed improved long-term motor and cognitive function. these intriguing experiments demonstrated that removal of microglia in the chronic phase of tbi repair reduced subsequent neuroinflammation as well as lessening subsequent motor and cognitive functional deficits. further, it showed that such inflammatory effects extended far longer than has been traditionally believed. willis and coworkers [4] also used plx5622 for microglial depletion and repopulation in a mouse model of tbi (they too used closed cortical impact), however these workers showed that removal of microglia had little effect on the clinical outcome of their tbi model. nevertheless, examination of the treated animals showed neuroprotective effects that appeared to aid in recovery. these beneficial effects were mostly modulated through interleukin-6 (il-6) signaling via the soluble il-6 receptor and its support of neurogenesis. these authors suggested that the presence of activated microglia associated with neurotrauma may not have a negative effect on outcomes and that it would appear that, as they state, “these cells lack an ability to support endogenous repair processes.” obviously, more needs to be done to further dissect out the role that microglia play in the brain’s response to tbi. the timing of when the microglial removal and repopulation takes place in these models appears to be critical to the results obtained. it may be predicted that the use of tools such as plx5622 will greatly help in unraveling such issues. in the discussion, the authors wonder if plx5622 might even represent a clinically feasible therapeutic approach to reducing some of the long-term complications of tbi. plx5622 has received limited fda approval for clinical use in patients with other non-tbi indications. for those who are interested in pursuing microglial depletion and repopulation, in a companion publication, this group has provided detailed protocols for such experiments in mice [5]. this would appear to be a valuable new tool for the investigation of the role of microglia, both positive and negative, in the pathogenesis of not only neurotrauma but many other disease processes [6, 7]. studies of the long-term effects of subconcussive blast exposure in breachers the effects of blast tbi on the brain have mostly been studied either experimentally in small animal models or in humans following a single significant blast event, typically related to exposure to an improvised explosive device (ied). it should be kept in mind that, in the combat setting, blunt impact tbi also commonly occurs in conjunction with these blast injuries. breaching represents a process where explosions are used to blast open doors and thus gain entry to buildings (figure 2). as such, breaching is associated with blast exposure in the absence of impact trauma. stone and colleagues [8] reported studies comparing a cohort of career breachers (typically breaching instructors) to a matched but minimally exposed control group. of the 20 experienced breachers studied, they reported having experienced an average of 4,628 breaching blast exposures over their careers, as opposed to the control group (n=14) who experienced an average of 3 exposures. keep in mind that these exposures are all considered to be sub-concussive in nature. figure 2. breaching is a procedure whereby an explosive charge, typically placed on a door, is used by combatants to enter a building and engage the enemy. service members participate in breaching in both training exercises and on the field of battle. repeated training exercises, such as portrayed here, expose participants to numerous sub-concussive blast wave exposures, in the absence of impact tbi. courtesy of sofia echelmeyer. using detailed neuroimaging approaches, they found, somewhat surprisingly, evidence of a significant degree of cerebral cortical thickening in the breacher group. this change was widespread, throughout the cerebral cortex. the nature of this cerebral cortical enlargement remains unclear, as no neuropathologic studies of deceased career breachers have yet to be reported. the authors also noted differences between the heavily exposed and control groups related to regional blood flow, various neuropsychological assessment results and serum biomarkers, however none of these differences survived bonferroni correction for multiple comparisons. this suggested that further studies using a larger cohort would be instructive. service members carrying out other duties, such as those who spend a career firing high caliber artillery ordinances or explosive ordinance disposal personnel, may also have an equivalent degree of blast exposure, and it would appear that similar studies of these groups are warranted. tbl-related neuroinflammatory markers neuroinflammatory response to tbi is a subject that has received considerable attention in both experimental models and clinical settings. clinically, studies of cytokine expression have mostly focused on moderate to severe tbi patients which have demonstrated slightly elevated plasma cytokine levels in the acute phase post-injury. post-mortem studies by johnson and colleagues [9] have demonstrated that activated microglia can persist for decades after the initial traumatic incident. a recent study by chaban and colleagues [10] published in 2020 looked at plasma levels of 12 different cytokines in a cohort including 207 patients with mtbi and 82 matched uninjured community controls. plasma samples were drawn at admission for evaluation of the tbi, as well as 2 weeks, 3 months and 12 months following the injury. brain magnetic resonance imaging (mri) was also performed on all the participants. comparing the mtbi group with the controls, they found significant elevations in plasma interferon gamma, il-8, macrophage inflammatory protein-1 beta, monocyte chemoattractant protein-1, il-17a, il-9, tumor necrosis factor, and basic fibroblast growth factor at all time points, whereas a number of the other cytokines remained unchanged. the presence of persistent cytokine levels did not correlate with the mri findings, suggesting that the cytokine changes were not related to the extent of tissue damage. this is one of the few longitudinal studies of plasma neuroinflammatory markers of patients with mtbi that showed evidence of persistent systemic inflammation lasting up to a year. the nature of intracellular tau accumulations in cases of chronic traumatic encephalopathy (cte) tbi, especially repeated impact tbi, constitutes a risk factor for the development of chronic traumatic encephalopathy (cte), a tauopathy associated with neurofibrillary tangles (nfts) and tau accumulation in astrocytes, primarily in the form of what are referred to as thorn-shaped astrocytes (tsas). arena and colleagues [11] reported a detailed immunohistochemical study on the nature of tau accumulations in nfts and tsas in cases of cte and compared them to staining results obtained in several other forms of tau-related neurodegenerative disorders (alzheimer’s disease, frontotemporal dementia, pick’s disease) and other tau-related conditions seen in the elderly (age-related tau astrogliopathy, or artag, and primary age-related tauopathy, or part). this study showed that the nfts of the cte cases contained both 3r and 4r isoforms that are also classically seen in association with the tangles of alzheimer’s disease and part. the tsas of cte stained virtually entirely for 4r tau, similar to what is seen in cases of artag but distinct from what has been observed in alzheimer’s disease. the use of antibodies directed towards various post-translational modifications of tau (primarily related to the presence of specific phosphorylation residues) also showed consistent similarities between the tsas of cte and those of alzheimer’s disease and artag. further, the authors employed recently developed anti-tau antibodies that are configuration-dependent [12] (gt-7 and gt-38) which showed that the nfts in the depths of sulci in the cases of cte were strongly immunoreactive, which is consistent with the staining reaction that is seen in the nfts of alzheimer’s disease. however, in all but a few of the cte cases, the astrocytes failed to stain with these antibodies. in their discussion, the authors noted the similarity between the tau immunophenotype seen in the nfts of cte and that of alzheimer’s disease and part. in contrast, the tau-positive astrocytes of cte were indistinguishable from those of artag but distinctly different from the alzheimer’s disease results. this led to the suggestion that cte and artag may share pathogenetic mechanisms that separate these two diseases from that of alzheimer’s disease. clearly, these are concepts that need to be explored further and may be of importance in sorting out the complex and confusing nosology and diagnostic criteria for these various neuropathologic entities. obviously, additional work needs to be done and neuropathologists involved in this area will follow it with interest. animal models of cte and the use of pet ligand for tau for clinical diagnosis of cte further on tau and its association with repeated tbi, dickstein and colleagues [13] investigated various biomarkers in rats that were experimentally exposed to repeated low-level blast over-pressures as well as military service members who had been exposed to ieds on the battlefield and subsequently suffered persistent behavioral, cognitive and/or memory complaints. in the rats, six weeks following blast exposure, by western blot, abnormally phosphorylated tau (thr181, p-tau) was shown to be increased in the right anterior cortex and right hippocampus but not in contralateral locations. by ten months post-exposure, p-tau levels had further increased and were now more likely to be encountered bilaterally. using immunohistochemistry, involved animals primarily showed fine dendritic staining although some did show perikaryal accumulations, a most unique observation among repeated tbi experimental rodent models. the human military cases were studied by pet scanning using [18f]av45 (flortaucipir), a pet ligand that is reported to be selective for tau. half of the ten blast-exposed service members receiving pet scans showed excessive accumulation of the ligand at gray-white matter junctions in frontal, parietal and temporal regions, a pattern the authors interpreted to be “a typical localization of cte tauopathy.” in addition, levels of plasma nfl were elevated in the blast-exposed subjects who showed excess [18f]av45 retention on pet analysis. this study suggests the potential value of the rat as an experimental model of tau accumulation following repeated blast exposure. this stands in direct contrast to the mostly negative findings in mice exposed to repetitive impact tbi. the reported results also point to the potential for further use of tau pet ligands in the study of human subjects at risk for cte. of note, since in the human studies they engaged living patients, no opportunity for neuropathologic confirmation of the proposed diagnosis of cte could be made. follow-up from last year’s neurotrauma 2019 report in my contribution last year, i highlighted a paper suggesting that the use of tranexamic acid treatment showed a significant lowering of mortality in a very large randomized placebo-controlled clinical trial in patients with more severe forms of acute tbi [14]. i felt this was important in view of the dismal record, despite numerous attempts, for introducing therapeutic agents with evidence-based positive results in the treatment of tbi patients. at the time, i was encouraged by the size of the study and their positive results. since tranexamic acid appears to be quite safe and is a very inexpensive drug, i was further encouraged by this approach. i also used this publication to highlight the importance of vascular pathology and bleeding in tbi pathophysiology, morbidity and mortality. despite my optimism over the positive results of prehospital administration of tranexamic acid in tbi patients, in 2020, bossers et al. [15] reported the results of a multicenter cohort study of using the drug to treat 1,827 severe tbi patients. this new study showed that prehospital administration of tranexamic acid produced increased mortality in those patients receiving the drug. of course, one negative study does not settle matters here, but these results suggest that care must be taken before proceeding in this direction. it would appear that, once again, those who seek an effective therapy for tbi patients are left with few, if any, therapeutic approaches showing efficacy. the toolbox for clinicians dealing with the effects of tbi continues to remain virtually empty of drugs that carry evidence of improved clinical outcome. finally for those of us with an interest in the neuropathology of neurotrauma, there is one additional event that occurred in 2020 that i felt was important to note. on april 12, 2020, professor james hume adams died [16]. professor adams was a pioneering, major contributor to our understanding of the neuropathology of neurotrauma, spending virtually all of his career studying the disorder. he trained in neuropathology at the institute of psychiatry at the maudsley hospital in london, where he initially came in contact with several people with an interest in neurotrauma, namely sabina stritch, peter daniel and j.a.n. corsellis. it is clear that the seeds for his life-long interest in neurotrauma were planted at this time. he subsequently took a position in glasgow at the department of pathology, western infirmary, eventually moving to the new facility at the southern general hospital. while in glasgow, he developed and ran the glasgow database of human head injury, and its brain bank repository has represented a major resource for the study of the effects of impact tbi on the human brain. importantly, this unique facility represents one of the only available collections in which one would be able to characterize the long-term effects of impact tbi. over the years, this facility has been extensively used by the glasgow group, now under the direction of dr. willie stewart. professor adams was best known for his seminal contributions in describing and then further characterizing diffuse axonal injury (dai), both clinically and experimentally. much of this work was carried out in conjunction with colleagues in glasgow and at the university of pennsylvania. the critical concepts related to the effects of trauma on the brain that he first proposed continue to be understood and used to this day. although in more recent years he had been in retirement as an emeritus professor, the influence of this work continues and according to his obituary, the 60 papers he published with the university of pennsylvania group on neurotrauma have been cited more than 9,000 times. in 2020, we lost a towering figure who contributed greatly to the field of neurotrauma. acknowledgement the opinions expressed herein are those of the author and are not necessarily representative of those of the uniformed services university of the health sciences, the united states department of defense, the united states army, navy or air force. references 1. guedes, v. a. et al. exosomal neurofilament light: a prognostic biomarker for remote symptoms after mild traumatic brain injury? neurology, 2020. 94(23): p. e2412-e2423. 2. okonkwo, d. o. et al. point-of-care platform blood biomarker testing of glial fibrillary acidic protein versus s100 calcium-binding protein b for prediction of traumatic brain injuries: a transforming research and clinical knowledge in traumatic brain injury study. j neurotrauma, 2020. 37(23): p. 2460-2467. 3. henry, r. j. et al. microglial depletion with csf1r inhibitor during chronic phase of experimental traumatic brain injury reduces neurodegeneration and neurological deficits. j neurosci, 2020. 40(14): p. 2960-2974. 4. willis, e. f. et al. repopulating microglia promote brain repair in an il-6 dependent manner. cell, 2020. 180(5): p. 833-846. 5. willis, e. f. and vukovic, j. protocol for brain-wide or region-specific microglia depletion and repopulation in adult mice. star protoc, 2020. 1(3): p. 100211. 6. qu, w. et al. inhibition of colony-stimulating factor 1 receptor early in disease ameliorates motor deficits in sca1 mice. j neuroinflammation, 2017. 14(1): p. 107. 7. tahmasebi, f. et al. the effect of microglial ablation and mesenchymal stem cell transplantation on a cuprizone-induced demyelination model. j cell physiol, 2021. 236(5): p.3552-3564. 8. stone, j. r. et al. functional and structural neuroimaging correlates of repetitive low-level blast exposure in career breachers. j neurotrauma, 2020. 37(23): p. 2468-2481. 9. johnson, v. e. et al. inflammation and white matter degeneration persist for years after a single traumatic brain injury. brain, 2013. 136(pt 1): p. 28-42. 10. chaban, v. et al. systemic inflammation persists the first year after mild traumatic brain injury: results from the prospective trondheim mild traumatic brain injury study. j neurotrauma, 2020. 37(19): p. 2120-2130. 11. arena, j. d. et al. tau immunophenotypes in chronic traumatic encephalopathy recapitulate those of ageing and alzheimer's disease. brain, 2020. 143(5): p. 1572-1587. 12. gibbons, g. s. et al. detection of alzheimer disease (ad)-specific tau pathology in ad and nonad tauopathies by immunohistochemistry with novel conformation-selective tau antibodies. j neuropathol exp neurol, 2018. 77(3): p. 216-228. 13. dickstein, d. l. et al. brain and blood biomarkers of tauopathy and neuronal injury in humans and rats with neurobehavioral syndromes following blast exposure. mol psychiatry, 2020. 14. crash-3 trial collaborators. effects of tranexamic acid on death, disability, vascular occlusive events and other morbidities in patients with acute traumatic brain injury (crash-3): a randomised, placebo-controlled trial. lancet, 2019. 394(10210): p. 1713-1723. 15. bossers, s. m. et al. association between prehospital tranexamic acid administration and outcomes of severe traumatic brain injury. jama neurol, 2020. 16. graham, d. and smith, c. obituary of emeritus professor j hume adams. neuropathol appl neurobiol, 2020. 46(6): p. 618-620. copyright: © 2021 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. from neurology to neuropathology and back feel free to add comments by clicking these icons on the sidebar free neuropathology 2:10 (2021) reflections from neurology to neuropathology and back arnulf h. koeppen research service (151), va medical center, albany, ny, usa corresponding author: arnulf h. koeppen · research service (151) · va medical center · 113 holland avenue · albany, ny 12208 · usa arnulf.koeppen@va.gov submitted: 13 april 2021 accepted: 17 april 2021 copyedited by: deanna c. fang published: 21 april 2021 https://doi.org/10.17879/freeneuropathology-2021-3333 keywords: neuropathology, neurology, reflections, autobiography chemistry or medicine 1958-1963? in the fifties, high school students in germany had to select their career goals without much guidance or college education. i was a student at a gymnasium of mathematics and natural sciences in osterode, a provincial town near the harz mountains in lower saxony of germany. my father, a general practitioner in a small town in the harz, had different ideas. he wanted me to be a medical student and ultimately take over his practice. the university town, göttingen, was located 50 km from our home. göttingen was then a small town, but medical education was excellent and diverse. i stayed in a room near the max planck institute where otto hahn, the nobel laureate in nuclear fission, was still active. my inaugural dissertation involved measurements of nuclear sizes in rat liver, adrenals, and endometrium after the animals were treated with drugs affecting the autonomic nervous system (koeppen, 1963). my interest in the neurosciences began during my last year of medical school. the inaugural dissertation received the annual faculty award in 1963; and the sum of 800 deutschmarks was sufficient to defray my cost of a trip by ocean liner to the united states where a non-profit organization, the ventnor foundation, had offered me a position as a medical assistant (intern) at a new jersey hospital. after an internship of 18 months, i joined the neurology training program at montefiore hospital and medical center in new york city in 1965. neurology education was disappointing, but i met the late drs. harry zimmerman, robert terry, and asao hirano who stimulated my interest in neuropathology and, above all, clinicoanatomic correlation in neurology and neurosurgery. after one year at montefiore, i was recruited to the department of neurology and psychiatry of northwestern university school of medicine in chicago. my first assignment was to the veterans affairs (va) medical center in hines, illinois, where the late dr. kevin d. barron was the chief neurologist. dr. barron had been trained by drs. zimmerman and terry and continued to practice neuropathology at northwestern and the va medical center. dr. barron was a master of clinicoanatomic correlation and stressed that neurologists should have training in neuropathology. his training had a major impact on my further career as a neurologist and neuropathologist. while opposition to neurologists and psychiatrists practicing neuropathology was evolving, we were reminded of non-pathologists who had made major contributions to the practice of neuropathology. we thought of alois alzheimer and his 1906 illustrations of neurofibrillary tangles, and of king engel who gave us modified trichrome in the diagnosis of mitochondrial myopathy. my training in neuropathology from 1969 to 1971 was enough to gain acceptance to the neuropathology board examination in 1980, and, since that time, i was convinced that i was a better neurologist because i was also a neuropathologist. during my time in chicago, dr. barron insisted that i should learn neurochemistry as well, and i was assigned to the research laboratory of the late dr. joseph bernsohn. over a one-year period, i learnt standard and advanced laboratory techniques, and my initial interest in chemistry came to belated fruition. since joining the department of neurology at albany medical college in 1969, i have maintained a neurochemical and neuropathological laboratory at the albany va medical center (figure 1) while also running the clinical neurology service. among my valued neurochemical discoveries was a sensitive assay of brain malonyl-coenzyme a, a critical molecule in the biosynthesis of fatty acids (mitzen and koeppen, 1984). the autopsy practice generated access to specimens from unusual cases, such as pelizaeus-merzbacher disease (pmd). the neurochemical laboratory helped me identify the cause of the disease, namely, a deficiency of proteolipid protein (plp) (koeppen, et al., 1987). the mutation in this patient with pmd was a point mutation (hudson, et al., 1989) though later, the mutation in most cases was identified as a duplication of the plp gene. the plp protein is unusual because it is soluble in a mixture of chloroform and methanol (folch and lees, 1951). through the work on pmd, i met the co-discoverers of plp, the late dr. marjorie b. lees (figure 2), and the late prof. franz seitelberger who had advanced the existence of a “connatal” form of pmd. prof. seitelberger invited me to the neurological institute in vienna where i gave a lecture on plp and pmd. profs. kurt jellinger and herbert budka were in audience. i also discovered that the late neuropathologist, dr. wolfgang zeman (figure 3), had written to dr. lees in 1963, proposing that plp was a likely candidate in the genetic mutation underlying the disease. pelizaeus-merzbacher disease is an x-linked disorder, and pelizaeus, a german balneologist, understood the mechanism of x-linked transmission before the existence of an x-chromosome was known. pelizaeus (1885) stated succinctly that the “disease goes through the mother but does not do anything to her”. it is peculiar that at the time of my work on pmd, a spontaneous x-linked central nervous system myelin deficiency in rats was discovered in albany, ny. the animal model was called "myelin-deficient (md) rat", and the neuropathologic and genetic similarity to pelizaeus-merzbacher disease was remarkable (koeppen et al, 1988). pmd patients also show a lack of other myelin proteins, and the injection of a white matter homogenate into guinea pigs (type 13) did not cause experimental allergic encephalomyelitis (eae). antibodies to plp were not commercially available, and i isolated the protein from frozen normal human white matter. injection into rabbits caused encephalitis that closely resembled eae. one rabbit with advanced encephalitis generated a high-titer anti-plp antibody that worked very well on paraffin sections of human central nervous tissue. the antibody became very popular, and i forwarded samples to several foreign and domestic myelin investigators. figure 1. laboratory staff in albany, ny. from left to right: alyssa sossei, research assistant; sarah bjork, medical student; r. liane ramirez, research assistant; the author; pamela kruger, post-doctoral scientist. a new goal: the hereditary ataxias in 1972, a patient with autosomal dominant ataxia presented with an unusual disturbance of ocular motility that became known as supranuclear pseudoophthalmoplegia. many years later, the mutation was identified as a cytosine-adenine-guanine (cag) trinucleotide repeat expansion on chromosome 12, and the disease was named spinocerebellar ataxia type 2 (sca-2). the mutation was like the cag trinucleotide expansions in sca-1 and sca-3 (machado-joseph disease). sca-6, sca-7, and sca-17 were later also identified as the consequence of cag trinucleotide repeat expansions. the expansions are expressed by changing the length of polyglutamine in the cognate proteins, hence, became known as polyglutamine (or polyq) diseases. antibodies raised against the polyq stretch of the normal tata-binding protein, designated “1c2”, became useful in the visualization of intranuclear inclusion bodies in the ataxic polyq diseases and huntington's chorea. stimulated by the experience with a single patient with supranuclear pseudoophthalmoplegia, i traveled to scotland, his ancestral home, to search for other members of this family. over a period of 40 days, i could not find any but discovered unrelated patients with an identical disturbance of eye movements. at that time, commercial genetic testing for sca was not yet fully available, and the lack of saccades in some patients with ataxia became a "genetic marker" of the disease. the discovery of supranuclear pseudoophthalmoplegia remains important but due to its variability can no longer be called a genetic marker. the scottish experience (koeppen, et al, 1977) prompted me to apply to the alexander-von-humboldt foundation for a research fellowship. the foundation agreed, and i moved to the university of hamburg, germany, where i found a warm welcome in the department of human genetics and the neuropathological institute of the eppendorf university hospital. the plan was to backtrack from autopsy records to surviving family members. the director of eppendorf neuropathology was prof. hans-joachim colmant, a psychiatrist. he made available a huge archive of neuropathological specimens in the basement of the hospital that had survived the bombings of world war ii. the chief psychiatrist during the war, prof. hans bürger-prinz, decided that the collection of slides, reaching back to 1891, should be protected against the allied bombings and moved the collection to a secure location. beyond specimens of ataxia, the collection also contained invaluable other cases, including jakob's original 1920 specimens of prion disease. it was an exciting experience to study these slides and be allowed to make photographs. german neuropathologists favored cresyl violet as their routine stain, and it was apparent that this stain does not properly show the sponginess that is readily apparent on hematoxylin-and-eosin. my stay in hamburg was intermittent, but i benefitted from the collection at the eppendorf hospital over an aggregate period of 2 years. on return to albany, ny, i began to collect autopsy specimens of hereditary ataxia in a tissue repository and, to this day, make available fixed and frozen samples to other ataxia investigators. figure 2. marjorie b. lees another change in direction: from dominant to recessive ataxia, 1996-2021 after dr. massimo pandolfo and his group discovered the mutation in friedreich ataxia (fa) in 1996, my interest changed, and my research now focuses on this autosomal recessive disease. though fa is a monogenic disease, the pathology offers a fascinating array of lesions in multiple organs: brain, spinal cord, dorsal root ganglia, sensory peripheral nerves, eyes, heart, and the insulin-producing beta cells of the pancreas. many clinicians and fa researchers consider fa a "neurodegenerative" disease, implying the existence of onset, progression, and death. the term neurodegenerative also means atrophy, and clinical onset is thought to be related to a degree of atrophy that translates to nervous system dysfunction. neuropathologists, however, will agree that fa is also a hypoplastic disease as far as the spinal cord and dorsal root ganglia are concerned. sectioning of the spinal cord longitudinally to represent dorsal root entry zones as obersteiner and redlich did in 1894 in vienna invariably showed loss of demarcation between the peripheral nervous tissue of dorsal roots and the central nervous tissue of the spinal cord (koeppen, et al, 2017). a surprising observation in fa was the protrusion of glial tissue from the spinal cord into dorsal roots, in support of dysfunction of a local barrier called the boundary cap. this work received the 2018 stout award from the united states and canadian academy of pathology for “resolving a scientific medical problem by studying its anatomic features”. this award is my most prized recognition. figure 3. wolfgang zeman the mystery of brain iron in 1967, i participated in the examination of a brain that was incrusted by hemosiderin. the diagnosis, superficial siderosis, was not difficult, but the disorder was thought to be rare, and magnetic resonance imaging with its extraordinary ability to detect brain iron, was still to come. dr. kevin d. barron and i studied the specimen in detail, and i entered a 10-year period of research in normal and pathological brain iron (koeppen et al, 1992-2002). this iron work correlated well with my later studies in fa because other researchers thought that this disease was due to iron accumulation in mitochondria of all affected organs. america, 1964-2021 my fifty-seven years in the united states constitute two thirds of my life. my early youth was troubled by the experience of world war ii, america has been good to me. i waved goodbye to europe when the maasdam, a passenger liner of the holland-america line put out to sea in late january 1964, and i greeted the statue of liberty 10 days later. i was never homesick, but occasionally think of my time as a refugee at the end of world war ii. in january 1945, my mother packed some essentials and took me, two brothers, and a sister by bus to the west, leaving the then german province of silesia. at that time, our father was a prisoner of war in an american camp near naples, italy, and did not return to germany until 1947. things did not stabilize until 1949 when the family walked across the border between the newly established communist german democratic republic and the american zone of west germany. food, drink, and clothing were scarce, and we spent one year in a displaced-persons camp. the privations ended in 1950 when the german economy improved with the outstanding help of the marshall plan. after i became more established in the united states, i raised a family and met many good people. i want to thank my teachers of neurology and neuropathology, among whom my friend and colleague for 47 years, the late dr. kevin d. barron, was the most important (figure 4). the united states continues to fascinate me, and, during my chicago years, the sky of the midwest seemed higher and bluer. i did not forget my european origin or my years of medical education in germany. i am still trilingual (english, german, and spanish) and enjoy the occasional opportunity to practice three languages. i also became an amateur radio operator (a “ham” in american lingo), and my last conversation with my late father was by ham radio. while on sabbatical in hamburg, i received the call sign of dj0rf wherein the “dj0” was reserved for foreign nationals who had an amateur radio license in their home countries. ham radio will be my main pursuit after i finally retire from research or the practice of neurology and neuropathology. figure 4. kevin d. barron and former trainees. from left to right; suhandsu chokroverty, mbbs; the late kevin d. barron, md, the late cesar mayo, md; ahk; and the late artemio ordinario, md. the group photo was taken during the combined 1967 meeting of the american association of neuropathologist and the american neurological association in atlantic city, new jersey. a final word: do we teach enough clinicoanatomic correlation? we hear a lot from proponents of “evidence-based” medicine but rarely about the art of medicine. clinicoanatomic correlation benefits from both, and thinking of brain structure while considering a differential diagnosis is a rewarding exercise. senior physicians will agree that anatomy and pathology are the basis of medicine. telehealth has arrived and is perhaps not in line with the art of medicine. its supporters may argue that it is more artful than face-to-face and hands-on medicine. whatever we prefer as clinicians, we should strive to emphasize clinicoanatomic correlation in our teaching and convey the joy of a successful diagnosis based on traditional knowledge of anatomy. such thought should, of course, precede the ordering of advanced imaging. in a recent survey of neurology training programs in the united states, 86% of program directors considered a rotation through neuropathology essential (rayi, et al, 2020). let me add my voice to this goal. references folch j, lees m. proteolipides, a new type of tissue lipoproteins. their isolation from brain. j biol chem 1951; 191 807-17 hudson l.d., puckett c., berndt .j, chan j., gencic s. mutation of the proteolipid protein (plp) gene in human x-linked myelin disorder. proc natl acad sci 1989; 86: 8128-31 koeppen a.h.: (transl.) the influence of pharmacological agents of the autonomic nervous system on the cellular nuclei of the liver, uterus, and adrenal cortex measured by karyometric methods). inaugural dissertation of university of göttingen medical school, 1963 koeppen a.h., hans m.b., shepherd d.i., best p.v.: adult-onset hereditary ataxia in scotland. arch neurol 1977; 34: 611-18 koeppen a.h., ronca n.a., greenfield e.a., hans m.b.: defective biosynthesis of proteolipid protein in pelizaeus-merzbacher disease. ann neurol 1987; 21: 159-70 koeppen a.h., barron k.d., csiza c.k., greenfield e.a.: comparative immunocytochemistry of pelizaeus-merzbacher disease, the jimpy mouse, and the myelin-deficient rat. j neurol sci 1988; 84: 315-27 koeppen a.h., borke r.c.: experimental superficial siderosis of the central nervous system. i. morphological observations, j neuropathol exp neurol.1991; 50: 579-9 koeppen a.h., hurwitz c.g., dearborn r.e., dickson a.c., borke r.c., chu r.c.: experimental superficial siderosis of the central nervous system: biochemical correlates. j neurol sci 1992; 112: 38-45 koeppen a.h., dickson a.c., chu r.c., thach r.e.: the pathogenesis of superficial siderosis of the central nervous system. ann neurol1993; 34: 646-53 koeppen a.h.: the history of iron in the brain. j neurol sci 1995; 134 (suppl.): 1-9 koeppen a.h.: a brief history of brain iron research. j neurol sci 2002; 207: 95-7 koeppen a.h., becker a.b., qian j., gelman b.b., mazurkiewicz j.e.: friedreich ataxia: developmental failure of the dorsal root entry zone. j neuropathol exp neurol 2017; 76 (11): 969-77 mitzen e.j., koeppen a.h.: malonate, malonyl-coenzyme a and acetyl-coenzyme a in developing rat brain. j neurochem 1984; 43: 499-506 obersteiner h., redlich e. (transl): on the nature and pathogenesis of the tabetic dorsal column degeneration. arbeit neurol inst wien univ 1894; 1-3: 158-72 pelizaeus f.: (transl) on a peculiar form of spastic paralysis with brain symptoms on hereditary basis (multiple sclerosis). arch psychiat nervenkrankh 1885; 16: 698-710 rayi a. et al.: how are residents trained in neuropathology? a survey of neurology program directors in the united states. j neuropathol exp neureol 2020; 79:1218-22 copyright: © 2021 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. 65th annual meeting of the german society of neuropathology and neuroanatomy (dgnn) meeting abstracts feel free to add comments by clicking these icons on the sidebar free neuropathology 2:22 (2021) meeting abstracts 65th annual meeting of the german society of neuropathology and neuroanatomy (dgnn) meeting abstracts september 1–3, 2021 submitted: 23 august 2021 accepted: 23 august 2021 published: 25 august 2021   dear participants, dear colleagues, it is our great pleasure to welcome you to the 65th annual meeting of the german society of neuropathology and neuroanatomy the brain and nerve microenvironment in health and disease which will be held as a virtual meeting from september 1–3, 2021. the meeting will bring together basic and clinical researchers, physicians as well as junior scientists and phd students from different disciplines of basic and clinical neuroscience. we will have outstanding lectures by and with some of the most renowned international experts in the field of neuro-oncology, neuroinflammation, neurodegeneration and muscle and nerve diseases and look forward to exciting scientific discussions. there will also be a special and timely section on the effects of covid-19 on the central and peripheral nervous system. the three days will offer exciting insights into different areas of basic and clinical neuroscience. we have also encouraged early career scientists to present their scientific findings in short talks and poster presentations. we are therefore particularly thankful that the abstracts of the meeting, which in their sum provide the best overview of the high scientific standing of the field, will be published in free neuropathology. finally, we would like to thank you all for your active contribution to this conference in these difficult times. we also thank all supporters for their financial help. we wish you a stimulating and exciting conference. yours sincerely, prof. dr. med. till acker conference chair (justus liebig university giessen) pd. dr. med. anne schänzer dr. med. hildegard dohmen https://doi.org/10.17879/freeneuropathology-2021-3500 keywords: german society for neuropathology and neuroanatomy, dgnn, meeting abstracts, 65th meeting sep. 2021 contents neuroinflammation ni-01 ni-02 ni-03 neurooncology adult i (from mechanisms to translation) noa-01 noa-02 noa-03 noa-04 noa-05 noa-06 noa-07 muscle & nerve diseases mnd-01 mnd-02 neurodegeneration / covid-19 and neuropathology nd-01 nd-02 nd-03 neurooncology pediatrics nop-01 nop-02 postersession i – neuroinflammation pi-01 pi-02 pi-03 postersession ii – neurooncology pii-01 pii-02 pii-03 poster session iii – muscle & nerve piii-01 piii-02 piii-03 piii-04 poster session iv – neurodegeneration piv-01 piv-02 piv-03 piv-04 piv-05 piv-06     neuroinflammation ni-01 free neuropathol 2:22:4 microbiota-derived acetate enables the metabolic fitness of the cns innate immune system during health and disease d. erny1 1 institut für neuropathologie, neurozentrum, freiburg i. br., germany introduction: as tissue macrophages of the central nervous system (cns) microglia constitute the pivotal immune cells of this organ. microglial features are strongly dependent on environmental cues such as commensal microbiota. gut bacteria are known to continuously modulate microglia maturation and function by the production of short-chain fatty acids (scfa). however, the precise mechanism of this crosstalk is unknown. objectives & results: here we determined that the immature phenotype of microglia from germ-free (gf) mice is epigenetically imprinted by h3k4me3 and h3k9ac on metabolic genes associated with substantial functional alterations including increased mitochondrial mass and specific respiratory chain dysfunctions. we identified acetate as the essential microbiome-derived scfa driving microglia maturation and regulating the homeostatic metabolic state, and further showed that it is able to modulate microglial phagocytosis and disease progression during neurodegeneration. conclusion: these findings indicate that acetate is an essential bacteria-derived molecule driving metabolic pathways and functions of microglia during health and perturbation.   ni-02 free neuropathol 2:22:5 multiple sclerosis immunopathological patterns and their mri correlates i. metz1, r. gavrilova2, s. weigand3, y. guo2, n. zalewski2, m. gloth1, o. tobin2, h. lassmann4, j. tillema2, b. erickson2, j. parisi5, b. popescu6, s. becker7, f. könig8, j. frischer9, w. brück1, c. lucchinetti2 1 university medical center göttingen, georg-august-university, institute of neuropathology, göttingen, germany 2 mayo clinic, department of neurology, rochester, ny, united states 3 mayo clinic, department of health sciences research, rochester, ny, united states 4 medical university vienna, department of neuroimmunology, center for brain research, vienna, austria 5 mayo clinic, department of laboratory medicine and pathology, rochester, ny, united states 6 university of saskatchewan, department of anatomy, physiology and pharmacology, and cameco ms neuroscience research center, saskatchewan, canada 7 university medical center göttingen, department of palliative medicine, göttingen, germany 8 klinikum kassel, institute of pathology, kassel, germany 9 medical university vienna, department of neurosurgery, vienna, austria introduction: early active multiple sclerosis lesions can be classified into three main immunopathological patterns of active demyelination (patterns i-iii) by histology. these patterns are inter-individually heterogeneous but intra-individually stable. in pattern i and ii, a t-celland macrophage-associated demyelination is suggested, with pattern ii only showing signs of a humoral immune response. pattern iii is characterized by inflammatory lesions with an oligodendrocyte degeneration. patterns suggest pathogenic heterogeneity, and we postulated that they have distinct mri correlates that may serve as biomarkers. objectives: to analyze the mri correlates of multiple sclerosis immunopathological patterns. patients and methods: we evaluated in an international collaborative retrospective cohort study the mri lesion characteristics of 789 pre-biopsy and follow-up mris in relation to their histopathologically classified immunpathological patterns (n=161 subjects) and lesion edge features (n=112). results: a strong association of a ring-like enhancement and a hypointense t2-weighted rim (t2w rim) with pattern i and ii, but not pattern iii, was observed. only a fraction of pattern iii patients showed a ring-like enhancement, and this was always atypical. ring-like enhancement and t2w rims colocalized, and ring-like enhancement showed a strong association with macrophage rims as shown by histology. a strong concordance of mri lesion characteristics, meaning that different lesions showed the same features, was found, indicating lesion homogeneity within individual patients. conclusion: we provide robust evidence that mri characteristics reflect specific histological features of ms immunopatterns and that ring-like enhancement and t2w hypointense rims might serve as a valuable non-invasive biomarker to differentiate pathological patterns of demyelination.   ni-03 free neuropathol 2:22:7 microvascular vulnerability to cytokine toxicity is a critical initiating factor in cerebral interferonopathies b. viengkhou1, e. hayashida1, s. mcglasson2, k. emelianova2, y. crow2, a. pagenstecher3, d. hunt2, m. hofer1 1 the university of sydney, charles perkins centre, sydney, australia 2 the university of edinburgh, edinburgh, united kingdom 3 university of marburg, dept. neuropathology, marburg, germany cerebral interferonopathies are a group of devastating diseases characterised by overproduction of interferon-alpha (ifn-α), leading to diffuse brain damage. importantly, the critical cellular mediators of ifn-α are unknown. here we use a mouse model of brain-targeted ifn-α production to identify endothelial cells as the major mediator of ifn-α neurotoxicity. specifically, we have created a single cell atlas of the "cerebral interferome" using single cell rna sequencing of brain cells of wild type and ifn-α transgenic mice. by linking this dataset with neuropathological studies of cerebral interferon overproduction, we identified a role for cerebral endothelial cells in the initiation of brain disease. we then confirmed a critical role for endothelial interferon signalling through targeted deletion of the type i interferon receptor ifnar on endothelial cells of the transgenic mice. remarkably, this intervention led to near-complete rescue of the complex multicellular disease phenotype. importantly, we show that diffuse aspects of interferonopathic brain disease such as calcification and neuronal loss arise as a downstream consequence of cytokine-induced microvascular disease. in conclusion, our data identify damage to the cerebral microvasculature as the critical first step in interferon neurotoxicity, and pinpoint ifnar signalling within endothelial cells as a key site for potential therapeutic intervention.   neurooncology adult i (from mechanisms to translation) noa-01 free neuropathol 2:22:8 molecular mechanism of therapy resistance in malignant melanoma brain metastasis e. schumann1, r. koll1, j. onken2, k. jürchott3, t. redmer4, j. radke1 1 charité – universitätsmedizin berlin, department of neuropathology, berlin, germany 2 charité – universitätsmedizin berlin, department of neurosurgery, berlin, germany 3 charité – universitätsmedizin berlin, institute of medical immunology, berlin, germany 4 university of veterinary medicine vienna, department of medical biochemistry, vienna, austria introduction: malignant melanoma (mm) has the highest potential to disseminate to the cns. about 45% of patients suffer from brain metastasis, which likely proceed continuously during the course of disease. genetically and molecularly distinct subclones lead to tumor heterogeneity, therapy resistance and poor prognosis. previous studies suggested increased metastatic spread to and within the brain during braf inhibitor (brafi) therapy, which is caused by upregulation of a subset of molecular drivers that control migratory and invasion such as the nerve growth factor receptor cd271/ngfr. objectives: to investigate the molecular features of migration and invasion of patient derived cell lines from mm brain metastases (bm) that were therapy-responsive or therapy-resistant to brafi, radiotherapy and immune checkpoint inhibitors. patients & methods: we isolated and cultured patient derived cell lines from mm bm (n = 9) and dna-sequencing (n = 5) and transcriptome analyses (n = 2) of cell lines and concordant tumor (n = 2). the incucyte® live-cell analysis was used to perform high throughput scratch wound assays with patient derived cell lines, which were genetically modified to overexpress or downregulate the expression of ngfr. results: transcriptome profiling of brafi resistant mm bm revealed that the invasive potential increased during disease progression, which was accompanied by upregulation of ngfr expression. this phenotype was preserved in patient derived cells lines, which demonstrated significantly higher potential of two-dimensional in vitro migration (90% vs. 76% after 100 hours). furthermore, cd271 knockdown was associated with loss-of-expression of several genes involved in migration and invasion such as erbb3, tcf19 and baalc. conclusion: brain metastases are the major cause of death in metastasized mm. our study provides a longitudinal perspective on the progression of brain metastasis and their mechanisms leading to therapy resistance.   noa-02 free neuropathol 2:22:9 molecular characterization of sporadic endolymphatic sac tumours and comparison to von hippel-lindau disease-related tumours l. schweizer1, f. thierfelder1, c. thomas2, p. soschinski2, h. y. kim1, r. jödicke1, n. woltering2, a. förster1, d. teichmann1, c. siewert1, k. klein1, s. schmid1, m. nunninger3, u. w. thomale4, j. onken5, h. mühleisen6, j. schittenhelm7, m. tatagiba8, a. von deimling9, d. e. reuss9, d. a. solomon10, f. l. heppner1, a. koch1, c. hartmann11, o. staszewski12, d. capper1 1 charité – universitätsmedizin berlin, department of neuropathology, berlin, germany 2 university hospital münster, department of neuropathology, münster, germany 3 charité – universitätsmedizin berlin, department of radiology, berlin, germany 4 charité – universitätsmedizin berlin, department of neurosurgery, division pediatric neurosurgery, berlin, germany 5 charité – universitätsmedizin berlin, department of neurosurgery, berlin, germany 6 synlab labor für pathologie mutlangen, mutlangen, germany 7 institute of pathology and neuropathology, university of tübingen, department of neuropathology, tübingen, germany 8 university hospital tübingen, department of neurosurgery, tübingen, germany 9 heidelberg university hospital, department of neuropathology, institute of pathology, heidelberg, germany 10 university of california, division of neuropathology, department of pathology, san francisco, ca, united states 11 hannover medical school (mhh), department of neuropathology, hannover, germany 12 faculty of medicine, university of freiburg, institute of neuropathology, freiburg i. br., germany introduction. endolymphatic sac tumours (elsts) are low-grade tumours arising from the endolymphatic sac in the temporal bone. elsts can be locally aggressive and may even expand into the posterior fossa representing a rare differential diagnosis for tumours of the cerebellopontine angle. although inactivation of the von hippel-lindau gene (vhl) on chromosome 3p25 is considered to be the major cause of hereditary endolymphatic sac tumours (elsts), the genetic background of sporadic elst is largely unknown. objectives. the aim of this study was to determine the prevalence of vhl alterations in sporadic elsts and compare their characteristics to vhl-disease-related tumours. materials and methods. we analyzed 11 sporadic and 11 vhl-disease-related elsts by targeted sequencing and dna methylation analysis. results. vhl mutations and small deletions detected by targeted deep sequencing were identified in 9/11 sporadic elsts (82%). no other cancer-related genetic pathway was altered except for tert promoter mutations in two sporadic and one vhl-disease-related elsts (15%). loss of heterozygosity of chromosome 3 was found in 6/10 (60%) vhl-disease-related and 10/11 (91%) sporadic elsts resulting in biallelic vhl inactivation in 8/10 (73%) sporadic elsts. dna methylation profiling did not reveal differences between sporadic and vhl-disease-related elsts, but reliably distinguished elst from morphological mimics of the cerebellopontine angle. vhl patients were significantly younger at disease onset compared to sporadic elsts (29 vs. 52 years, p < 0.0001, fisher's exact test). vhl-disease status was not associated with an increased risk of recurrence, but the presence of clear cells was found to be associated with shorter progression-free survival (p = 0.0002, log-rank test). conclusion. biallelic inactivation of vhl is the main mechanism underlying elsts, but unknown mechanisms beyond vhl may rarely be involved in the pathogenesis of sporadic elsts. fig. 1 fig. 2   noa-03 free neuropathol 2:22:12 establishment and characterization of a meningioma cell model with ragc knockout r. diaz peregrino1, j. kahr1, n. waldt1, e. kirches1, c. mawrin1 1 otto-von-guericke universität magdeburg, institut für neuropathologie, magdeburg, germany amino acid sensors are essential for cell growth and carcinogenesis via the mtorc1 pathway. iomm lee cells are suitable to study the meningioma pathogenesis, and amino acid sensors like ragc can be investigated in these cells. the aims of this work are (i) generate a meningioma cell line with ragc deficiency; (ii) determine its morphology; (iii) characterize it in vitro using cell viability assay, trypan blue cell counting assay, colony formation assay, and brdu assay; (iv) and measure its motion. ragc knockout was achieved through crispr-cas9 system and corroborated by western blot. the area and circularity of iomm ragcko and iomm scr (scramble control) cells were determined by phalloidin staining. in vitro assays at 24 and 48 hours were performed to scr and ragcko cells cultured in normal and leucine-depleted medium. wound healing assay, velocity, displacement, acceleration, and percentage of moving cells were measured in both cell lines seeded in the standard and leucine-free medium. cell area was similar between cell genotypes, but ragcko cells were more elongated than scr cells. based on cell genotype comparison, ragcko cells were more metabolically active and displayed more cell proliferation than scr cells. centered on media comparison, cell numbers and cell proliferation decayed in both cell lines when cultivating them in the leucine-free medium. only ragcko cells had a reduction in atp production under leucine withdrawal. finally, ragcko cells exhibited higher motility than scr cells judged by the motion parameters. moreover, the motility of ragcko cells was resistant towards leucine depletion and even increased in comparison to scr cells. ragcko cells displayed increased proliferation and atp production in leucine-supplied scenarios and accelerated migration under leucine-deprived conditions. changes in akt and pp70s6 signaling may be involved in both phenomena. furthermore, the morphological modifications of ragcko cells might enhance their motion.   noa-04 free neuropathol 2:22:13 the role of 2-oxoglutarate homeostasis in tumour invasion and metastasis in glioblastoma a. alserw1, n. bögürcü-seidel1, s. seidel2, s. gräf1, s. zukunft3, i. fleming3, s. günther4, m. looso4, a. németh1, b. garvalov1,5, t. acker1 1 justus-liebig-university gießen, institute for neuropathology, gießen, germany 2 institute of cell biology and neuroscience and buchmann institute for molecular life sciences (bmls), goethe university, frankfurt a. m., germany 3 goethe university frankfurt, institute for vascular signalling, frankfurt a. m., germany 4 max planck institute for heart and lung research, bioinformatics core unit (m.l., p.g.), bad nauheim, germany 5 european center for angioscience (ecas), department of microvascular biology and pathobiology, mannheim, germany introduction: metabolic reprogramming has been associated with tumour invasion and metastasis. epithelial/proneural-to-mesenchymal transition (emt/prmt) is a key process behind cancer cell dissemination, which is regulated by several transcription factors, including snail. 2-oxoglutarate (2-og) is a metabolite whose intracellular level depends on the activity of isocitrate dehydrogenase 1 (idh1). the metabolite acts as a co-substrate for 2-og-dependent dioxygenases (2-ogdds) which regulate different cellular activities. while mutant-idh-induced, 2-ogdd-dependent tumorigenic mechanisms have been described in detail, it is less well understood how wildtype idh and 2-og levels control emt and invasion. objectives: we aim to define the homeostatic function of idh1 and 2-og in the regulation of emt and tumour progression primarily in glioblastoma, as well as in breast and lung cancer models. materials and methods: boyden chamber, western blot, if, rt-qpcr, rnaseq and metabolite ms assays are employed to determine the role of idh1 and 2-og levels on emt regulators and cell invasion. idh1 knockdown and several functional assays are used to further explore the underlying mechanisms. results: we observed that tgfβ; an established emt inducer results in idh1 downregulation, with a concomitant increase in snail expression and invasion. strikingly, 2-og supplementation reverts tgfβ induced snail at the mrna and protein level as well as cellular invasion in vitro. in line with a functional role of idh1 in emt, idh1deficiency promotes invasion and metastasis and enhances a cancer stem cell phenotype. importantly, we identify hif and mtor signalling as two crucial mechanistic pathways involved in snail regulation by 2-og. conclusion: collectively, our findings emphasize the interplay between metabolism and metastasis and reveal novel mechanistic insights into the role of wildtype idh and intracellular 2-og levels in the metabolic control of tumor invasion and emt/prmt.   noa-05 free neuropathol 2:22:14 infratentorial idh mutant astrocytoma is a distinct subtype r. banan1, d. stichel1, a. bleck2, b. hong3, u. lehmann4, a. suwala1, a. reinhardt1, d. schrimpf1, r. buslei5, c. stadelmann-nessler6, k. ehlert7, m. prinz8, t. acker9, j. schittenhelm10, d. kaul11, l. schweizer12, d. capper12, p. n. harter13, n. etminan14, d. t. w. jones15, s. m. pfister16, c. herold mende17, w. wick18, f. sahm1, a. von deimling1, c. hartmann2, d. e. reuss1 1 heidelberg university hospital, department of neuropathology, institute of pathology, heidelberg, germany 2 hannover medical school (mhh), department of neuropathology, institute of pathology, hannover, germany 3 hannover medical school (mhh), department of neurosurgery, hannover, germany 4 hannover medical school (mhh), institute of pathology, hannover, germany 5 klinikum bamberg, institute of pathology, bamberg, germany 6 university medical center göttingen, institute of neuropathology, göttingen, germany 7 university of greifswald, department of pediatric oncology and hematology, greifswald, germany 8 university of freiburg, institute of neuropathology, freiburg i. br., germany 9 institute of neuropathology, university of gießen, gießen, germany 10 university hospital tübingen, department of neuropathology, tübingen, germany 11 charité – universitätsmedizin berlin, department of radiation oncology and radiotherapy, berlin, germany 12 charité – universitätsmedizin berlin, department of neuropathology, berlin, germany 13 goethe university frankfurt, institute of neurology (edinger institute), frankfurt a. m., germany 14 university hospital mannheim, university of heidelberg, department of neurosurgery, mannheim, germany 15 german cancer research center (dkfz), pediatric glioma research group, heidelberg, germany 16 university hospital heidelberg, department of pediatric oncology and hematology, heidelberg, germany 17 university hospital heidelberg, department of neurosurgery, division of experimental neurosurgery, heidelberg, germany 18 university of heidelberg medical center, neurology clinic, heidelberg, germany introduction: diffuse idh-mutant astrocytic tumors are rarely diagnosed in infratentorial structures. only few studies have reported single cases of infratentorial diffuse gliomas harboring idh mutations, while suffering small case numbers. objectives: in this multi-institutional study, we aimed to analyze infratentorial idh-mutant astrocytomas in a larger series with respect to clinical and molecular parameters. patients and methods: tumor samples of patients with diffuse infratentorial astrocytomas from different german neuropathology departments were investigated using immunohistochemistry, pyrosequencing, genome-wide dna methylation analysis and ngs panel sequencing. overall survival data were assessed using kaplan–meier method. results: we identified a series of 43 infratentorial idh-mutant astrocytomas from 42 patients. about 80% of idh mutations were of non-idh1-r132h variants which are rare in supratentorial astrocytomas. most frequently, idh1-r132c/g and idh2-r172s/g mutations were present. moreover, atrx-loss and mgmt promoter methylation typically associated with idh mutations in supratentorial astrocytomas, were significantly less frequent in the infratentorial compartment. panel sequencing revealed two samples with idh1-r132c/h3f3a-k27m co-mutations. methylation analysis and copy number profiling provided further evidence for a molecular distinctiveness of these tumors. clinical outcome of patients with infratentorial idh-mutant astrocytomas was significantly better than in patients with diffuse midline gliomas, h3k27m-mutant (p < 0.005) and poorer than in those with supratentorial idh-mutant astrocytomas (p = 0.028). conclusion: the presented data highlight the very existence and distinctiveness of infratentorial idh-mutant astrocytomas implying that molecular testing is critical for detection of these tumors, since many of these tumors cannot be identified by immunohistochemistry applied for the mutated idh1-r132h protein or loss of atrx.   noa-06 free neuropathol 2:22:16 dna methylation profiling and molecular grading of stereotaxic brain tumor biopsies k. filipski1,2,3, j. hench4, m. armbrust1, p. s. zeiner2,3,5, t. i. hartung1, e. steidl6, j. quick-weller7, t. fenton8, j. p. steinbach2,3,5, m. czabanka7, d. capper9,10, s. frank4, k. h. plate1,2,3, m. t. forster7, p. n. harter1,2,3 1 university hospital frankfurt, neurological institute (edinger institute), frankfurt a. m., germany 2 german cancer consortium (dktk), german cancer research center (dkfz), heidelberg, germany 3 frankfurt cancer institute (fci), frankfurt a. m., germany 4 basel university hospital, department of neuropathology, institute of pathology, basel, switzerland 5 university hospital frankfurt, dr. senckenberg institute of neurooncology, frankfurt a. m., germany 6 university hospital frankfurt, institute of neuroradiology, frankfurt a. m., germany 7 university hospital frankfurt, department of neurosurgery, frankfurt a. m., germany 8 university of kent, school of bioscience, kent, united kingdom 9 charité – universitätsmedizin berlin, department of neuropathology, berlin, germany 10 german cancer consortium (dktk), partner site berlin, german cancer research center (dkfz), heidelberg, germany introduction: dna methylation profiling of cns tumors emerges as a powerful tool to support and refine histological diagnoses. tumor entity-specific dna methylation signatures allow for alignment with methylation classes and copy number profiling (cnp), the latter being of particular interest in terms of molecular grading regarding the upcoming who classification. when gross tumor resection is precluded, stereotaxic biopsies might not be representative of the bulk tumor, underlining the need for objective molecular diagnostic parameters. objective: here we investigated the diagnostic potential and accuracy of dna methylation profiling and molecular grading on small stereotaxic brain tumor biopsies. materials & methods: ffpe samples of 237 patients were subjected to dna methylation analysis including the heidelberg brain tumor classifier, cnp and tumor deconvolution using methylcibersort. results: median dna concentration was 6.26 ng/µl. 90.5% of stx samples (n=232) allocated to previously defined dna methylation classes, with 61.6% reaching classifier scores of > 0.84. combined gain of chromosome 7 and loss of chromosome 10 or egfr amplification were detectable in 61% and 38% of idh wildtype glioma (n=156), respectively. 64.5% of histological lower grade, idh wildtype gliomas (n=31) were eligible for upgrading to molecular glioblastoma. 58% of idh mutant gliomas (n=31) presented with 1p/19q codeletion and 23% with homozygous deletion of cdkn2a/b. recursive partitioning pointed at a minimum of 29.42% cancer cells for a 0.83 probability to reach a matching calibrated score in glioblastoma, idh wildtype. a dna input threshold of 3.53 ng/µl yielded matching dna methylation classes with a probability of 0.69. conclusion: our findings demonstrate broad applicability of dna methylation profiling and molecular grading to challengingly small, ill-defined brain tumor specimens with a special impact on grading of histologically lower grade, non-idh mutant diffuse gliomas.   noa-07 free neuropathol 2:22:18 prediction of meningioma methylation classes using ai-assisted digital histopathology j. sehring1, h. dohmen1, m. manke1, d. amsel1, a. németh1, a. mukhopadhyay2, t. acker1 1 justus-liebig-university gießen, institute of neuropathology, gießen, germany 2 technical university darmstadt, medical and environmental computing, darmstadt, germany introduction: current studies suggest that dna methylation-based molecular classification of meningioma has a higher predicting power for tumor recurrence than the current histology-based who classification. however dna methylation profiling is costly, time-consuming and not widely available. therefore, an ai-histology-based prediction of the six dna methylation classes would be very beneficial and could additionally complement the molecular classification. deep neural networks and their visual decoding power offer a promising tool to address this problem. objectives: our goal is to predict meningioma methylation classes using ai-assisted digital histopathology. materials and methods: h&e stained histological slides were digitized with a hamamatsu nanozoomer s360 to obtain whole slide images(wsi). multiple image patches per patient were extracted and encoded into feature space using a pre-trained resnet50. those features were aggregated in a multiple instance learning setting using an attention network to predict the methylation class. results: we have collected wsi data of 167 patients so far. most specimens belong to the most frequently diagnosed methylation subgroups, namely benign-1 (n=36), benign-2 (n=55) and intermediate-a (n=60). three separate two-class classifications (benign-1 vs. benign-2, benign-1 vs. intermediate-a and benign-2 vs. intermediate-a) are currently under development. patients were allocated into training, validation and test sets (0.7/0.1/0.2) and we evaluated the patient-wise balanced accuracy on the test set in a 5-fold cross-validation. in addition, the attention weights for the image patches of a wsi were visualized for interpretability. conclusion: we employ a framework for the prediction of methylation classes based on histological features. visualization is used for an enhanced interpretability of the black-box network. this ai-framework may support, complement, and accelerate meningioma classification in the future.   muscle & nerve diseases mnd-01 free neuropathol 2:22:19 in the spotlight: nk cell receptors in inclusion body myositis c. nelke1, m. pawlitzki2, t. ruck1 1 university hospital duesseldorf, deparment for neurology, neurology, düsseldorf, germany 2 university hospital münster, department of neurology with intitute of translational neurology, münster, germany introduction: inclusion body myositis (ibm) is the most common inflammatory idiopathic myopathy (iim) of old age. as of yet, effective treatment approaches are lacking. a deeper immunological understanding is necessary to overcome this roadblock. as natural killer (nk) cells orchestrate the extent of inflammatory responses by cytolytic properties in iim capable of inducing bystander self-tissue damage, we aimed to characterize nk cell patterns in ibm. objectives: to determine nk cell patterns in ibm and identify drivers of nk cell activation. patients & methods: we included 20 ibm patients and 20 age and sex matched non diseased controls (ndc). peripheral blood was collected and peripheral blood mononuclear cells (pbmc) were isolated by density gradient centrifugation. differences between groups were analyzed using unpaired student"s t test or mann–whitney-u test as appropriate. anti-cn-1a-antibodies were detected in patient serum using the euroimmun immunoassay. results: the activated cd56dim population was reduced in peripheral blood of ibm patients as compared to controls. comparing surface expression as measured by mean fluorescence intensity, we observed a significant increase of nkg2a and nkg2d levels on the cd56dim nk cell subset in ibm patients as compared to ndc. other surface receptors remained unchanged. next, we were interested whether the antibody status of ibm patients affects expression of surface receptors. consequently, comparison of anti-cn-1a positive and anti-cn-1a negative patients revealed that nkg2a level do not differ, while increased nkg2d levels were associated with anti-cn-1a-ab positivity. conclusion: here, we report distinct nk cell patterns in ibm patients. activated cd56dim nk cells are reduced in peripheral blood as compared to ndc. nkg2a and nkg2d levels are increased on cd56dim nk cells. lastly, increased nkg2d levels were associated with anti-cn-1-ab serostatus. fig. 1 fig. 2   mnd-02 free neuropathol 2:22:21 mutations in bves can lead to early onset of cardiac symptoms without clinical signs of muscle dystrophy in children and adolescents a. gangfuß1, a. hentschel2, m. gonzales3, a. schönecker4, a. töpf5, a. sickmann2, a. nishimura6, u. schara-schmidt1, a. hahn7, a. roos1, a. schänzer6 1 university duisburg-essen, department of pediatric neurology, centre for neuromuscular disorders, centre for translational neuroand behavioral sciences, essen, germany 2 leibniz-institut für analytische wissenschaften isas e.v., dortmund, germany 3 justus-liebig-university gießen, pediatric heart center, gießen, germany 4 university duisburg-essen, department of pediatric cardiology, essen, germany 5 newcastle university and newcastle hospitals nhs foundation trust, the john walton muscular dystrophy research centre, translational and clinical research institute, newcastle upon tyne, united kingdom 6 newcastle university and newcastle hospitals nhs foundation trust, the john walton muscular dystrophy research centre, translational and clinical research institute, newcastle upon tyne, united kingdom 7 justus-liebig-university gießen, department of child neurology, gießen, germany introduction: limb-girdle muscular dystrophies occur in children and adults and may be associated with a high variability of the clinical phenotype. in some subtypes, cardiac involvement is the first and thus key symptom. recently, blood vessel endothelial substance (bves), a gene encoding popeye domain containing protein 1 (popdc1), has been implicated as a causative for limb-girdle dystrophy type r25 (lgmdr25), associated with cardiac arrhythmia and variable skeletal muscle involvement. until today, only 10 adult patients with this rare autosomal recessive lgmd have been described. objectives: with this study, we aim to broaden the phenotypic spectrum and improve the pathophysiological understanding of lgmdr25. materials & methods: hence, we describe the clinical phenotype of four affected children of two families with homozygous variants (c.457>t; p.gln153ter and c.578t>g, p.ile193ser) in blood vessel endothelial substance (bves). additionally, we performed detailed muscle biopsy analysis from two affected patients including immunofluorescence, electron microscopic and proteomic studies. results: highly elevated creatine kinase and cardiac involvement are present in all patients. muscle weakness was not present in any of the patients, considering the oldest being 19 years old. however, histological and ultrastructural analysis revealed a myopathy. immunohistochemistry showed an absence of sarcolemnal expression of bves confirming the diagnosis. additional proteomic analyses were indicative for an impairment of the calcium balance and thus a potential influence on the contractile apparatus. conclusion: to conclude, lgmdr25 should be considered in children with cardiac symptoms and highly elevated creatine kinase, even in the absence of muscle weakness. a muscle biopsy is helpful to confirm the diagnosis and to increase the understanding of the disease pathology.   neurodegeneration / covid-19 and neuropathology nd-01 free neuropathol 2:22:23 combining postmortem single cell analysis with an induced pluripotent stem cell model to study dysregulated pathways in frontotemporal dementia o. al dalahmah1, r. kühn1, t. nguyen1, m. deture2, m. siegelin1, d. dickson2, j. p. vonsattel1, j. goldman1, p. canoll1, g. hargus1 1 columbia university, department of pathology and cell biology, new york, ny, united states 2 mayo clinic, jacksonville, fl, united states introduction: frontotemporal dementia (ftd) is a group of early-onset dementias leading to an impairment of behavior, language and cognition. ftd can be caused by mutations in the mapt gene encoding the microtubule-associated protein tau resulting in pronounced atrophy of the frontal and temporal lobes, basal ganglia and brain stem areas. objectives: currently, the underlying mechanisms of neurodegeneration are not clearly understood and curative options do not exit. materials & methods: here, we performed single nucleus rna sequencing (snrna-seq) on postmortem brain tissue from ftd patients carrying the maptn279k mutation and from healthy control individuals to identify dysregulated pathways in patient cells at single cell resolution. results: we found significant changes in pathways related to cell metabolism and neuroinflammation in patient neurons that we tested further in a stem cell model of ftd using patientand control individual-derived induced pluripotent stem cells (ipscs). patient ipsc-derived neurons with the maptn279k mutation demonstrated tau pathology, an impairment of neurite outgrowth and an increased but reversible oxidative stress response. ftd neurons also showed altered metabolic profiles with an increased basal mitochondrial respiration and increased atp production indicating an increased energy demand in these cells. interestingly, ftd neurons also had a significant effect on the survival of host neurons and on glial cell responses when transplanted into the brains of immunocompromised mice indicating a potential immunomodulatory role of patient neurons in vivo. conclusion: these findings demonstrate that a combinatorial approach applying snrna-seq on patient brain tissue and a dynamic ipsc model comprises a powerful tool to identify disease phenotypes in neural cells at risk in ftd. such stem cell model could also be used as a cellular platform for high-throughput drug screening assays to identify potential therapeutic targets in ftd.   nd-02 free neuropathol 2:22:24 deep learning assisted quantitative assessment of histopathological markers of alzheimer"s disease and cerebral amyloid angiopathy v. perosa1,2, a. scherleck3, m. kozberg1, l. smith4, t. westerling-bui4, c. a. auger5, s. vasylechko6, s. m. greenberg1, s. j. van veluw1,5 1 massachusetts general hospital, 1j. philip kistler stroke research center, boston, ma, united states 2 otto-von-guericke university, department of neurology, magdeburg, germany 3 rush university medical center, 3rush alzheimer disease center, chicago, il, united states 4 aiforia inc, cambridge, ma, united states 5 massachusetts general hospital, massgeneral institute for neurodegenerative disease, charlestown, ma, united states 6 boston children's hospital, computational radiology laboratory, boston, ma, united states introduction: traditionally, analysis of neuropathological markers in neurodegenerative diseases has relied on visual assessments of stained sections. resulting semiquantitative scores often vary between individual raters and research centers, limiting statistical approaches. to overcome these issues, we have developed six deep learning-based models, that identify some of the most characteristic markers of alzheimer"s disease (ad) and cerebral amyloid angiopathy (caa). methods: deep learning-based models were trained to differentially detect parenchymal amyloid β (aβ)-plaques, vascular aβ-deposition, iron and calcium deposition, reactive astrocytes, microglia, as well as fibrin extravasation. the models were trained on digitized histopathological slides from brains of patients with ad and caa, using a workflow that allows neuropathology experts to train convolutional neural networks (cnns) on a cloud-based graphical interface. results: validation of all models indicated a very good to excellent performance compared to three independent expert human raters. furthermore, the aβ and iron models were consistent with previously acquired semiquantitative scores in the same dataset and allowed the use of more complex statistical approaches. discussion: the presented workflow is easy for researchers with pathological expertise to implement and is customizable for additional histopathological markers. the implementation of deep learning-assisted analyses of histopathological slides is likely to promote standardization of the assessment of neuropathological markers across research centers, which will allow specific pathophysiological questions in neurodegenerative disease to be addressed in a harmonized way and on a larger scale.   nd-03 free neuropathol 2:22:25 exercise ameliorates alzheimers pathology across generations a. herring1, y. münster1, n. kurapati1, r. khadzhiev1, k. keyvani1 1 university duisburg-essen, institute of neuropathology, essen, germany introduction: an active lifestyle reduces the risk of dementia and cognitive decline in alzheimer"s diseases (ad) patients. animal studies have shown that environmental enrichment increases the cognitive reserve and interferes with the pathomechanisms of ad over a very extended period of ontogenesis, from in utero stimulation via maternal running during pregnancy until late exercise in a full-blown stage disease. objectives: here we asked whether protective effects of exercise against ad can be inherited across several generations and if so, what epigenetic modifications enable transgenerational transmission. materials & methods: in the founder generation (f0), tgcrnd8 mice (disease onset around postnatal day 90, p90) and wildtype littermates had either access to voluntary wheel running for six months, starting at p30, or were housed without exercise (control). from p90, either exercised mice were mated or mice that experienced a sedentary life style. the offspring was solely housed under control conditions and crossed until the third progenitor generation. the progeny (f1-f3) was phenotyped for memory and explorative behaviour as well as amyloid beta (aβ) pathology in an advanced stage disease (p210). to detect epigenetic transmitters, we determined the cerebral expression profile of micrornas involved in aβ metabolism. results: we here show that great-grandparental running reduced aβ plaque burden, enhanced cerebral levels of the aβ efflux transporter mdr1 and further increased recognition memory and explorative behaviour in transgenic mice until the f3 generation. further, the cerebral expression of micrornas that promote aβ pathology (mirnas 342-3p, 16-5p, 9-5p) was strongly reduced following ancestral exercise. conclusion: we conclude that protective effects of exercise can propagate across generations. ongoing experiments aim to decode further epigenetic modifications that enable transgenerational inheritance.   neurooncology pediatrics nop-01 free neuropathol 2:22:26 co-activation of sonic hedgehog and wnt signaling in murine retinal precursor cells drives ocular lesions resembling intraocular medulloepithelioma m. dottermusch1,2, p. sumislawski1, j. krevet3,4, h. voß5, m. middelkamp1, h. bartsch6, k. sotlar6,7, a. korshunov8, m. glatzel1, u. schüller1,4,9, j. neumann1,2 1 university medical center hamburg-eppendorf, institute of neuropathology, hamburg, germany 2 university medical center hamburg-eppendorf, center for molecular neurobiology (zmnh), hamburg, germany 3 ludwig-maximilians-university munich, center for neuropathology, münchen, germany 4 research institute children’s cancer center hamburg, hamburg, germany 5 university medical center hamburg-eppendorf, institute of clinical chemistry and laboratory medicine, hamburg, germany 6 ludwig-maximilians-university munich, department of pathology, münchen, germany 7 paracelsus medical university, department of pathology, salzburg, austria 8 german cancer consortium (dktk), german cancer research center (dkfz), clinical cooperation unit neuropathology (g380), heidelberg, germany 9 university medical center hamburg-eppendorf, department of pediatric hematology and oncology, hamburg, germany introduction: intraocular medulloepithelioma (io-mepl) is a rare embryonal ocular neoplasm, which prevalently occurs in young children and is commonly treated by enucleation of the eye. io-mepls share common histomorphological features with variants of cns embryonal tumors with multilayered rosettes (etmr), referred to as intracranial medulloepitheliomas. while sonic hedgehog (shh) and wnt signaling pathways are crucial for etmr pathogenesis, the impact of these pathways on human io-mepl development is unclear. objectives: our objective in this study was to explore the relevance of shh and wnt signaling in human io-mepls and demonstrate the effect of simultaneous shh and wnt activation in mouse retinal precursor cells. materials & methods: using nanostring technology, gene expression data was obtained from ffpe tumor samples of 8 human io-mepls, as well as 16 intracranial embryonal tumors comprising etmrs, shh-medulloblastomas (mbs), wnt-mbs and group 4-mbs. in order to unravel the function of shh and wnt signaling for io-mepl pathogenesis in vivo in a time point and cell specific manner, the tamoxifen inducible creert2-lox system was utilized to activate shh and wnt signaling in sox2or raxexpressing retinal precursor cells. results: io-mepls and etmrs displayed similar gene expression patterns and significant simultaneous overrepresentation of both shh and wnt target genes. co-activation of both pathways in early sox2or raxexpressing precursor cells resulted in infiltrative ocular lesions that displayed extraocular expansion. histomorphological, immunohistochemical and molecular features of these lesions revealed strong concordance with those of human io-mepls. conclusion: we report a relevant role of wnt and shh signaling in io-mepl and report a mouse model for this rare disease, setting the foundation for a targeted therapeutic approach with the aim of more commonly feasible eye salvage in affected patients.   nop-02 free neuropathol 2:22:28 characterisation of spinal diffuse midline gliomas l. stegat1, u. schüller1,2,3, a. wefers1 1 university medical center hamburg-eppendorf, institute of neuropathology, hamburg, germany 2 university medical center hamburg-eppendorf, department of pediatric hematology and oncology, hamburg, germany 3 research institute children’s cancer center hamburg, hamburg, germany introduction: diffuse midline gliomas (dmgs) are malignant gliomas that grow in midline structures of the central nervous system. due to their aggressive and diffuse growth and a two-year survival rate of less than 10%, dmgs are graded as who grade iv. depending on the localization, median age of patients is about 11-20 years. genetically, tumors are defined by a k27m-mutation in one of the highly homologous genes encoding histone protein h3. while some studies state that the global dna methylation pattern is correlated with the mutated histone gene, others suggest that it differs between dmgs of different localisations. objectives: since dmgs most frequently occur in pons and thalamus, comparatively little is known about spinal dmgs. therefore, we histologically, molecularly and clinically characterised spinal dmgs and analysed in which aspects they differ from dmgs of other localizations as this may have diagnostical and/or clinical implications. materials & methods: our cohort currently consists of 17 spinal dmgs plus dmgs of different localisations. histological and immunohistochemical analysis as well as molecular analyses (dna methylation, dna panel sequencing) were done from ffpe tissue. clinical data were analysed with prism. results: spinal dmgs were histologically very heterogeneous, both with regard to different areas of single tumors as well as when comparing different tumors. preliminary data suggest that there may be some differences to dmgs of different localisations, e.g. with regard to cell differentiation. first t-sne analyses of dna methylation data did not indicate a separation of spinal dmgs from those of different localisations. 10/11 spinal dmgs sequenced so far were h3f3a k27m-mutant, while one tumor had a hist1h3b k27m mutation. mean age was 22 years. conclusion: first analyses suggest slight histological differences of spinal dmgs compared to those of other localisations. further histological, molecular and clinical analyses are ongoing.   postersession i – neuroinflammation pi-01 free neuropathol 2:22:29 neurofilament light chains in serum are a biomarker for the acute axonal damage in ms lesions: a histological-serological correlative study a. s. beutler1, n. kruse1, l. stork1, m. gloth1, c. stadelmann-nessler1, w. brück1, i. metz1 1 university medical center göttingen, georg-august-university, institute of neuropathology, göttingen, germany introduction: multiple sclerosis (ms) is an inflammatory demyelinating disease of the cns, associated with a variable axonal damage. the axonal damage is responsible for the irreversible clinical disability, and within the cns no relevant neuroaxonal regeneration occurs. thus, in ms patients it is of utmost importance to monitor and hinder the axonal damage from the earliest stages on. a promising biomarker are neurofilament light chains measured in serum (snfl). however, snfl do not provide any information regarding the localization of the neuroaxonal damage, and multiple factors influence snfl levels. objectives: to perform a histopathological-serological correlative study to show the pathological correlate of elevated snfl levels in ms patients. patients and methods: 106 ms subjects with biopsy tissue and blood sample available were characterized for their acute axonal damage with the anti-app staining and the chronic axonal damage with the bielschowsky silver staining. results were correlated with the neurofilament light chain levels in serum measured with the single molecular array (simoa) technique. results: high snfl levels were present in biopsied ms patients (median 59 pg/ml) compared to healthy controls (19 pg/ml), and levels were influenced by the age of patients, relapses prior blood sampling as well as the time interval after biopsy. correlating histological parameters with snfl, multiple regression analyses showed that the acute axonal damage within early active ms lesions significantly contributed to the snfl (p<0.01), but not the axonal density. importantly, snfl correlated with the clinical disability at biopsy as well as at last follow-up. conclusion: snfl mirror the acute axonal damage in early ms lesions as shown by histology, and correlate with the clinical disability of patients. they are thus a promising biomarker to monitor the axonal damage ex vivo in ms patients. this study was sponsored by novartis pharma gmbh.   pi-02 free neuropathol 2:22:30 mgmt-promoter methylation in non-neoplastic diseases of the central nervous system s. teuber-hanselmann1, k. worm2, a. junker1 1 universitätsklinikum essen, institut für neuropathologie, essen, germany 2 universitätsklinikum essen, institut für pathologie, essen, germany introduction: o6-methylguanine-dna-methyltransferase (mgmt) plays a pivotal role in cellular defense by repairing alkylated dna. hypermethylation of the mgmt promoter region results in gene silencing, an effect seen in various tumors, like gliomas. mgmt hypermethylation was postulated to be attributed to cancers or to malignant transformation of precancerous lesions and that consequently it could be used as a tumor biomarker. but while mgmt hypermethylation was also detected in various peripheral inflammatory diseases, it has not yet been investigated in non-neoplastic cns diseases. objectives: to investigate mgmt promoter methylation levels in various non-neoplastic cns diseases. methods: mgmt promoter methylation and expression levels of dna demethylases tet1 and tet2 were investigated via pyrosequencing and immunohistochemistry on human autopsy or biopsy ffpe tissue samples with pml (10), ms (28), toxoplasmosis (6), cmv (1), hsv1 (1) or hiv (2) infection, mycotic encephalitis (4), brain abscesses (3), central pontine / extrapontine myelinolysis (cpm/epm) (8), wallerian degeneration (3) and controls without cns pathologies (8). results: slight mgmt hypermethylation was seen in non-neoplastic cns diseases, all of which were associated with damaged myelin sheaths, i.e. inflammatory and demyelinating (ms and pml), as well as non-inflammatory metabolic or degenerative cns diseases (cpm/epm, wallerian degeneration), while there were no associations with infectious non-demyelinating diseases or distinct pathogens. reduced expression levels of tet1 could possibly be the cause for mgmt hypermethylation. conclusion: mgmt hypermethylation is not restricted to neoplastic or infectious diseases but occurs in chronic cns diseases that lead to damage of the myelin sheath in various ways. while this gives new insights into epigenetic and pathophysiological processes involved in de-/remyelination, it also reduces the specificity of mgmt hypermethylation as a tumor biomarker.   pi-03 free neuropathol 2:22:31 chronic retrotransposon expression in mouse glial cells is associated with spatial learning deficits without an overt inflammatory response r. sankowski1, g. monaco1, m. prinz1 1 universitätsklinikum freiburg, institut für neuropathologie, freiburg i. br., germany introduction: retrotransposons are mobile genomic elements comprising approximately 40 percent of the human and mouse genomes. activation of retrotransposons has been associated with an inflammatory response in the brain and peripheral tissues. we have previously shown that chronic retrotransposon activation in b cell deficient mice was associated with astrogliosis and spatial learning deficits. objective: we were wondering about cell-type dependent retrotransposon expression in the b cell deficient mouse model of constitutive retrotransposon activation. materials & methods: here, we have utilised single-nucleus and single-cell rna-sequencing to profile the expression of retrotransposons in hippocampi and cortices of b cell deficient mice. results: we could identify increased expression of long-terminal repeat and non-long terminal repeat retrotransposons in microglia and astrocytes compared to other cell types. surprisingly, these expression changes were not associated with an increased expression of genes encoding inflammatory mediators. we confirmed these results using bulk rna sequencing. conclusion: our findings underscore the ability of brain tissues to attenuate an inflammatory response despite continuing presence of an immunogenic agent with direct therapeutic implications for unresolving chronic inflammatory conditions.   postersession ii – neurooncology pii-01 free neuropathol 2:22:32 dna methylation-based classification of sinonasal tumors p. jurmeister1, s. glöß2, r. roller3, s. schmid2, r. fritz3, a. thieme2, c. friedrich3, i. hoffmann3, m. leitheiser3, p. mertins4, f. klauschen1, d. capper2 1 ludwig-maximilians-university munich, institute of pathology, münchen, germany 2 charité – universitätsmedizin berlin, department of neuropathology, berlin, germany 3 charité – universitätsmedizin berlin, institute of pathology, berlin, germany 4 max-delbrück-centrum für molekulare medizin, berlin, germany introduction: the histopathological diagnosis of sinonasal tumors is challenging as it encompasses a heterogenous spectrum of diverse differential diagnoses as well as poorly defined, disputed entities such as sinonasal undifferentiated carcinomas (snucs). objectives: in this study, we aimed to develop a dna methylation-based classification algorithm for sinonasal tumors and to elucidate the molecular background of snucs. materials & methods: we established a cohort of dna methylation profiles from 374 sinonasal tumor specimens, covering 17 tumor entities and normal control tissue. furthermore, we performed dna sequencing and mass spectrometry-based proteomics of selected cases. results: a machine learning algorithm was able to reliably classifiy sinonasal tumors based on their dna methylation profiles with clinical-grade accuracy. we further showed that tumors with snuc morphology can be assigned to four molecular classes defined by distinct epigenetic, mutational and proteomic profiles. this included two classes with clear neuroendocrine differentiation in proteomics analysis which were characterized by either idh2 mutations or swi/snf chromatin remodeling complex (smarca4/arid1a) mutations and overall favorable clinical course. the third group comprised highly aggressive tumors, driven by smarcb1-deficiency. the fourth snuc class contained tumors that actually represented previously misclassified adenoid-cystic carcinomas, which was supported by recurrent myb translocations and the resemeblance of these specimens with serous cell of submucosal glands in proteomics analysis. conclusions: our dna methylation-based machine learning algorithm could greatly improve the diagnostics of challenging sinonasal tumors. furthermore, we provide solid proof that sinonasal tumors are not as undifferentiated as their current terminology implies, but rather represent distinct molecular groups with at least two classes showing clear neuroendocrine differentiation. fig. 1   pii-02 free neuropathol 2:22:34 deep learning aided mitotic figure count in human meningiomas based on the open-source software sliderunner s. jabari1, c. bertram2, r. coras1, a. maier3, i. blümcke1, m. aubreville4 1 university hospitals erlangen, institute for neuropathology, erlangen, germany 2 veterinärmedizinische universität wien, institut für pathologie, vienna, austria 3 lehrstuhl für mustererkennung, fau, erlangen, germany 4 image understanding and medical application of artificial intelligence, ingolstadt, germany introduction: the world health organization (who) classification of brain tumors describes three grades of meningioma: who i-iii. a major factor that distinguishes the classes is the number of mitotic figures. detecting and counting mitotic figures reliably in meningioma is, therefore, an essential part of the diagnostic workup. objectives: however, detecting mitotic figures quickly, reliably, and comprehensively in a tissue section is a major challenge. for this reason, we developed a technical assistance system that detects mitotic figures on whole-slide-images and visualizes these as a heat map showing the mitotic density in almost real-time. patients & methods: meningioma samples were retrospectively collected from the dept. of neuropathology erlangen, germany. all samples were rereviewed by an expert neuropathologist and subsequently digitized using the hamamatsu s60 digital slide scanner. the slides have been completely annotated for mitotic figures and we provide secondary annotations for mitotic figure look-alikes. additionally to a blinded two-expert manual annotation, we developed an algorithm-aided dataset, where potentially missed mitotic figures were detected by a deep neural network and subsequently assessed by a human expert. we used a pretrained deep-neuronal network approach (retinanet) to validate our data set. finally, a plugin for sliderunner, an open-source software, was built for convenience enabling real-time identification of mitotic figures in virtual slides. results: out of 69 whole slide images of the complete data set, 46 were randomly assigned to the training set and 23 to be the test set. a preliminary evaluation yielded an f1 score of 0.659. conclusion: error-prone and tedious mitotic figure counts and region of interest identification may be assisted in the future with the help of modern deep learning algorithms integrated into a user-friendly software suite.   pii-03 free neuropathol 2:22:35 intracranial ependymoma in adults, a methylome-based analysis m. träger1, l. schweizer2,3, p. vajkoczy4, k. fukuoka5, k. ichimura5, u. schüller6, l. dührsen7, m. müther8, w. paulus9, c. thomas9, j. schittenhelm10, f. eckert11, k. m. niyazi12, d. f. fleischmann12, m. dorostkar13, p. feyer14, s. a. may15, d. moskopp16, h. badakhshi17, s. wecker17, c. radke18, j. walter19, d. capper2,3, d. kaul1,3 1 charité – universitätsmedizin berlin, department of radiation oncology, berlin, germany 2 charité – universitätsmedizin berlin, department of neuropathology, berlin, germany 3 german cancer consortium (dktk), partner site berlin, german cancer research center (dkfz), heidelberg, germany 4 charité – universitätsmedizin berlin, department of neurosurgery, berlin, germany 5 national cancer center research institute, division of brain tumor translational research, tokyo, japan 6 university medical center hamburg-eppendorf, institute of neuropathology, hamburg, germany 7 university medical center hamburg-eppendorf, department of neurosurgery, hamburg, germany 8 university hospital münster, department of neurosurgery, münster, germany 9 university hospital münster, institute of neuropathology, münster, germany 10 university hospital tübingen, department of neuropathology, tübingen, germany 11 university hospital tübingen, department of radiation oncology, tübingen, germany 12 university hospital, lmu munich, department of radiation oncology, münchen, germany 13 ludwig-maximilians-university munich, center for neuropathology, münchen, germany 14 vivantes hospital neukölln, berlin, germany 15 klinikum chemnitz, department of neurosurgery, chemnitz, germany 16 vivantes klinikum im friedrichshain, department of neurosurgery, berlin, germany 17 ernst von bergmann medical center, academic teaching hospital of humboldt university berlin (charité), department of clinical radiation oncology, potsdam, germany 18 ernst von bergmann medical center, academic teaching hospital of humboldt university berlin (charité), department of pathology, potsdam, germany 19 medical center saarbruecken, department of neurosurgery, saarbrücken, germany introduction: recently dna methylation profiling became widely accepted as a method to identify biologically distinct subgroups of ependymoma. however, due to the rarity of these tumors in adults, previous studies have failed to achieve a sufficient number of patients to characterize the landscape of dna methylation profiles, their clinical characteristics and prognosis in this age group. objectives: we investigated dna methylation profiles of adult intracranial ependymoma collected from 12 different centers and sought to correlate molecular subgroups to clinical characteristics and outcome. materials and methods: tumors from 157 adult patients histologically classified as who grade ii or iii intracranial ependymoma between 1990 and 2020 were subjected to dna methylation profiling using the illumina human methylation 450k or epic bead chip platform. profiles were matched with the dkfz brain tumor classifier and detected molecular subgroups were correlated with overall survival (os) and progression free-survival (pfs). results: in 68.8% (108/157) of cases, the suggested methylation class confirmed diagnosis of an ependymal tumor with the additional assignment of a molecular subgroup (49 pfb, 36 se, 17 rela, 3 pfa, 2 spinal epn and1 myxopapillary epn) whereas 5.7% (9/157) were not compatible with the initial diagnosis of ependymoma. the remaining 25.5% (40/157) did not reach a calibrated score ≥0.9 and were considered not classifiable. median follow-up was 54 months. five and 10-year pfs rates were 54.1% and 40.6% for se, 65.2% and 37.1% for pf-b, 49% and 49% for rela tumors. the 5and 10-year os rates were 70.1% and 49.1%for se, 95.4% and 85.5% for pf-b and 100% and 75% for rela tumors. conclusion: this cohort of adult intracranial ependymoma comprises various molecular subgroups and even different tumor entities. most patients with ependymal tumors confirmed by dna methylation profiling show a favorable prognosis. however, factors that affect survival need to be determined.   poster session iii – muscle & nerve piii-01 free neuropathol 2:22:37 imaging of extended tissue areas by automated large-scale electron microscopy c. dittmayer1, h. h. goebel1,2, f. l. heppner1, w. stenzel1 1 charité – universitätsmedizin berlin, department of neuropathology, berlin, germany 2 johannes-guttenberg universität, department of neuropathology, mainz, germany introduction: traditional imaging of sections in diagnostic or research settings using transmission electron microscopy (tem) is limited by manual acquisition procedures. thus, analysis of samples is restricted to images of few preselected regions that may be non-representative and lack correlation to their microanatomical context. modern scanning electron microscopes allow unbiased digitization of entire large sections and improved analysis, but performance is limited by preparation artifacts. objectives: we aimed at an improved preparation of sections for large-scale electron microscopy (em) and at a pipeline for data processing and analysis. material & methods: we achieved wrinkle-free collection of sections by a combined glow-discharge and ethanol-based smoothening procedure and reduced filming and staining artifacts. a scanning electron microscope with a scanning transmission electron microscopy detector was used for pre-irradiation and imaging. imaging parameters were varied to achieve fast imaging and adequate image quality. we used trakem2 and nip2 for stitching of image tiles to generate coherent bigtif datasets, thus allowing in-depth analysis via qupath. results: our preparation workflow allowed to reliably prepare samples with virtual absence of limiting artifacts, such wrinkles and stain precipitates. up to 12 entire sections were automatically digitized in about 6 days using 7.3 nm pixel size and 1.0 µs dwell time. our data processing pipeline allowed batch generation of bigtif files with minimal operator involvement. conclusion: we propose a highly reliable workflow for preparation, imaging and data processing of samples with superior quality to enable routine large-scale em in diagnostic and research settings. we demonstrate the high pragmatic value of our workflow with data of different published and unpublished projects in the field of muscle pathology and ultrastructural characterization of sars-cov-2 in autopsy tissues.   piii-02 free neuropathol 2:22:38 nanostring technology distinguishes anti-tif-1γ+ from anti-mi-2+ dermatomyositis patients c. preuße1,2, p. eede1, l. heinzeling3,4, k. freitag1,5, r. koll1,6, w. froehlich3, u. schneider7, y. allenbach8, o. benveniste8, a. schänzer9, h. h. goebel1, w. stenzel1, j. radke1,6 1 charité – universitätsmedizin berlin, neuropathology, berlin, germany 2 münster university hospital, neurology with institute for translational neurology, münster, germany 3 university hospital of erlangen, dermatology, erlangen, germany 4 lmu klinikum, dermatology, münchen, germany 5 german center for neurodegenerative diseases (dzne) within the helmholtz association, berlin, germany 6 german cancer consortium (dktk), german cancer research center (dkfz), berlin, germany 7 charité – universitätsmedizin berlin, rheumatology, berlin, germany 8 pitié-salpêtrière university hospital, internal medicine and clinical immunology, paris, france 9 justus-liebig-university gießen, neuropathology, gießen, germany introduction: dermatomyositis (dm) is a systemic idiopathic inflammatory disease affecting skeletal muscle and skin, clinically characterized by symmetrical proximal muscle weakness and typical skin lesions. recently, myositis-specific autoantibodies (msa) became of utmost importance because they strongly correlate with distinct clinical manifestations and prognosis. antibodies against transcription intermediary factor 1γ (tif-1γ) are frequently associated with increased risk of malignancy, a specific cutaneous phenotype and limited response to therapy in adult dm patients. anti-mi-2 autoantibodies, in contrast, are typically associated with classic dm rashes, prominent skeletal muscle weakness, better therapeutic response and prognosis, and less frequently with cancer. the correlation of cancer and mi-2 autoantibodies, however, remains debated. objectives: to further investigate the both clinically distinct dm subgroups and visualize differences or similarities on gene and protein level. patients & methods: we analyzed 30 dm patients (n=15 mi2+ and n=15 tif-1γ+) and 8 non-disease controls (ndc) by nanostring, qpcr, and immunohistochmistry. results: both anti-tif-1γ+ and anti-mi-2+ patients" skeletal muscle biopsies revealed strong dysregulation of immune response-related genes compared to ndcs. in line with previous reports, these included well-known type 1 ifn-inducible genes; however, we also identified subgroup-specific differences. conclusion: our results help to stratify both subgroups and predict, which dm patients require an intensified diagnostic procedure and might have a poorer outcome. additionally, we demonstrate that the nanostring technology can be used as a very sensitive method to clearly differentiate two clinically distinct dm subgroups, namely mi2+ and tif-1γ+ dermatomyositis. potentially, this could also have implications for the therapeutic approach.   piii-03 free neuropathol 2:22:40 immunomodulatory function of gm-csf in idiopathic inflammatory myopathies d. cengiz1,2, t. müntefering2, j. schubert1, c. preuße3, w. stenzel3, s. meuth2, t. ruck2 1 clinic for neurology with institute for translational neurology, university hospital, münster, germany 2 heinrich-heine university düsseldorf, department of neurology, medical faculty, düsseldorf, germany 3 charité universitätsmedizin, department of neuropathology, berlin, germany introduction: idiopathic inflammatory myopathies (iims) are characterized by chronic inflammation of the muscle, resulting in muscle weakness and pain. pathogenesis is driven by the interaction of skeletal muscle, muscle endothelial and immune cells. cytokines play a central role in controlling these interactions. gm-csf is a cytokine, which is significantly involved in the development of autoimmune diseases and is currently being investigated in clinical trials for therapy. objectives: the role of gm-csf in the inflammatory processes of iims is largely unknown. therefore, the aim of this work is to investigate its immunomodulatory function. materials & methods: murine skeletal muscle and endothelial cells were stimulated with gm-csf and other proand anti-inflammatory cytokines. to analyze the impact of these conditions, flow cytometric analysis was performed. furthermore, the immunological relevance of gm-csf on immune cell migration and adhesion were investigated. the expression of gm-csf was also evaluated in human muscle biopsies of iim patients. results: stimulation with proand anti-inflammatory cytokines regulate the expression of gm-csf and the gm-csf receptor in muscle and endothelial cells. gm-csf led to increased expression of costimulatory molecules on skeletal muscle cells, as well as decreased expression of adhesion molecules on endothelial cells. stimulation also resulted in increased immune cell migration across muscle endothelial barriers. in human muscle biopsies of iim patients, histological analysis revealed high expression of gm-csf. conclusion: the gm-csf signaling pathway affects the cellular interactions of muscle, endothelial, and immune cells in the skeletal muscle. these findings could contribute to elucidate autoimmune processes in iims and identify new therapeutic targets.   piii-04 free neuropathol 2:22:41 effect of vasoactive therapy in patients with systemic sclerosis on dermal small nerve fibers, langerhans-cells and vessel density. l. bajors1, f. höcketstaller2, m. köhm2,3, s. seidel4, r. wolf5, a. roth1, u. drott2,6, a. schänzer1 1 justus-liebig-university gießen, institute of neuropathology, gießen, germany 2 goethe university frankfurt, department of rheumatology, frankfurt a. m., germany 3 goethe university frankfurt, fraunhofer project group translational medicine and pharmacology, ime, frankfurt a. m., germany 4 goethe university frankfurt, institute of cell biology and neuroscience and buchmann institute for molecular life sciences (bmls), frankfurt a. m., germany 5 philipps university marburg, department of dermatology and allergology, marburg, germany 6 median klinik schlangenbad, department of rheumatology, wiesbaden, germany introduction: systemic sclerosis (ssc) is an orphan immune-mediated disease affecting multiple organ systems. most commonly, clinical symptoms such as skin fibrosis and digital ulceration occur early in disease course. here, vasculopathy is an underlying aetiology. additional, small nerve fibers (snf) might be affected in in ssc patients and contribute to disease progression. material and methods: 21 ssc patients received intravenous medication iloprost for 10 days (95% females; 56,1±11,9 years). 7 healthy subjects served as controls (71% f; 51,6±18,3 y). skin biopsies were taken from the volar forearm before and 3 months after treatment. epidermal nerve density (end), langerhans cells (lc) and vessel density (vd) were analysed at immunofluorescence-stained sections using a zeiss axioscanner and imagej software. the skin pathology was categorized with a histological skin score. results: the ssc group showed no significant difference of end and lc density (13,09±4,26 snf/mm; 259,8±121,5 lc/mm²) compared to controls (10,8±3,11 sfn/mm, p=0,198; 335,4±124,86 lc/mm², p=0,091) or after treatment (12,71±5,82 snf/mm, p=0,49; 305,4±98,5 lc/mm², p=0,098). the vd was significantly increased in the ssc group compared to controls (3,01±1,10µm²/100µm²; 2,18±1,12µm²/100µm²; p=0,046). in the ssc group, the vd was significantly decreased after treatment (2,51±0,78µm²/100µm²; p=0,044). the histological skin score showed overall low fibrotic alterations in both groups. conclusion: in our study, ssc patients showed a higher vd compared to controls and vd was affected during short-term iloprost-therapy. megacapillaries is a key feature of ssc and the results should be correlated with results of capillary microscopy. end and lc were not altered during treatment in ssc patients. maybe these findings depend on the low skin score showed in the patients. to investigate further correlations, the results will be correlated with clinical parameters.   poster session iv – neurodegeneration piv-01 free neuropathol 2:22:42 the interplay between neuroimmune activation and blood brain barrier dysfunction in alzheimer's disease m. armbrust1, a. dominguez-belloso1, r. figueiredo2, k. gneiding1, s. guérit1, s. thom1, p. winter2, k. h. plate1, p. n. harter1, k. devraj1, s. liebner1 1 goethe university hospital, neurological institute, edinger institute, frankfurt a. m., germany 2 genxpro gmbh, frankfurt a. m., germany introduction: alzheimer's disease (ad) is characterized by amyloid-β aggregation, hyperphosphorylated tau protein, neuroimmune activation, neuronal loss and blood brain barrier (bbb) dysfunction. the contribution of the latter to disease genesis and progression is poorly understood. objectives: to investigate wnt/β-catenin and bbb-specific alterations in ad. materials and methods: transgenic mice harbouring triple-mutated (swdi) human app were used for 3'-rna-sequencing (massive analysis of cdna ends: mace) of isolated mouse brain microvessels (mbmvs) and of facs-sorted cells of the neurovascular unit (endothelial cells (ecs), mural cells, astrocytes and microglia (mg)). an activation of the wnt/β-catenin pathway in ecs was conducted in a cre-loxp-based β-catenin gain-of-function (gof)-ad mouse model. tracer permeability assays, immunohistochemistry and immunofluorescence analyses as well as behavourial tests were performed in ad and ad-gof mice. results: gene ontology analysis revealed a downregulation of wnt/β-catenin signalling in mbmvs and facs-sorted ecs of ad mice. dkk2, encoding dickkopf wnt pathway inhibitor 2, was upregulated in mbmvs and mg. in human ad brain tissue, dkk2 was detected in mg. additionally, an increase in bbb permeability correlating with cognitive decline and a decrease of vascular lymphoid enhancer-binding factor 1 expression, a target and key player in wnt/β-catenin signalling, was observed in ad mice. a presymptomatic activation of the wnt/β-catenin pathway in ecs of gof-ad mice partially prevented cognitive decline. conclusion: dkk2 upregulation in microvessel-associated mg could contribute to the wnt/β-catenin pathway repression observed at the bbb in ad. together with the prevention of cognitive decline after presymptomatic activation of the wnt/β-catenin pathway, our findings indicate a potential link between neuroimmune activation and bbb dysfunction and propose the wnt/β-catenin pathway as a potential therapeutic target.   piv-02 a deep learning approach for monitoring parietal-dominant alzheimer's disease in world trade center responders at midlife not available for publication   piv-03 free neuropathol 2:22:44 amyloid-β dimers (aβ-s8c) are antiprions inhibiting seeded nucleation in vivo e. van gerresheim1, a. müller-schiffmann1, s. schäble2, c. korth1 1 heinrich-heine university düsseldorf, neuropathology, düsseldorf, germany 2 heinrich-heine university düsseldorf, experimental psychology, düsseldorf, germany introduction: alzheimer"s disease (ad) is a neurologic disorder, were amyloid-beta (aβ) aggregation causes massive neuronal death. aβ expansion can follow prion-like replication from aβ seeds. with the current animal models, based on the overexpression of aβ, used to study the aβ cascade and prion-like expansion, it is impossible to distinguish between exogenous de novo seeding and accelerated spontaneous plaque development. so far, seeding of aβ in wild type animals has not been successful. objectives: here we studied the effects of aβ-s8c dimers on aβ prion spreading and induced behavioral changes. materials and methods: tgdimer mice, expressing human app751 (swe, k670 n/ m671l, s679c), only develop aβ-s8c dimers and do not develop aβ plaques, but do associate to existing plaques, were crossed with gfap-luc mice, enabling bioluminescence (bli) monitoring of aβ aggregation-associated astrogliosis. mice were intra-hippocampal inoculated with aβ aggregates. wild type and app23 mice, aβ aggregate inoculated, were used as controls. cognition was probed in the observer-free paradigm, the cognitionwall, for reward-dependent learning and burrowing as species-specific behavior. results: we observed no aβ aggregation-associated astrogliosis or impaired cognition in the tgdimer mice inoculated with aβ aggregate. for the positive control, the app23 mice, an increase in bli signal started at 11 months of age and at 18 months of age, app23 mice showed impaired discrimination learning in the cognitionwall. conclusions: aβ-s8c dimers inhibit aβ aggregate spreading and are thus anti-prions. they may have a regulatory role for aggregate spreading suggesting that aβ prion effects are actively regulated rather than a passive process. the difference in time of start of aβ aggregate spreading and impaired discrimination learning in app23 mice suggests that behavioral consequences are disparate from initial neuropathological signs.   piv-04 free neuropathol 2:22:45 a decline in peroxisomal numerical abundance occurs late during alzheimer´s disease progression and is particulary found in the hippocampus e. semikasev1, b. ahlemeyer1, t. acker2, a. schänzer2, e. baumgart-vogt1 1 institute of anatomy and cell biology, medical cell biology, jl university, gießen, germany 2 institute of neuropathology, jl university, gießen, germany peroxisomal biogenesis disorders are inherited metabolic diseases caused by mutations in the pex genes. in severe forms, patients have profound defects in the brain demonstrating the importance of this organelle for neuronal survival and function. additionally, dysfunction of the peroxisomal metabolism, e.g. plasmalogen synthesis or the degradation of very long-chained fatty acids, has to been linked to neurodegenerative diseases such as alzheimer´s disease (ad). to study the role of peroxisomes in ad, we analyzed the density of this organelle in the perikarya of pyramidal cells in the hippocampus (cornu ammonis, subiculum and the entorhinal cortex layer iii) as well as neocortical regions (frontal, temporal, parietal und occipital) of brain from patients with different neuropathological stages. we used double immunofluorescence staining techniques to detect pex14 (as marker for the total peroxisome numerical abundance) and β-amlyoid plaques or neurofibrillary tangles (nfts)s in different brain regions. human brains were genderand age-matched and classified into 5 patient groups based to the abc-score. all 42 brain samples were from the institute of neuropathology in gießen. quantification of peroxisome number per area (density) was performed by imagej free software. with ongoing stages of ad, we measured a progressive decline in the density of peroxisomes in pyramidal cells of all hippocampal and neocortical areas. the effect was even more pronounced when we included known ad risk factors such as vascular angiopathies. interestingly, the decrease in the density of peroxisomes at late stages in ad was more evident in patients ≤80 years old than in patients >81 years old. however, we did not find a correlation between the intraneuronal peroxisome density and the amount of β-amyloid and nfts. our data suggest that peroxisomes may play a protective role in ad and their decline in numerical density – especially in the hippocampus – occurs late during ad progression.   piv-05 free neuropathol 2:22:46 its not all about covid19 incidental finding of hereditary neuroferritinopathy in a 78-year-old woman diagnosed with covid-19 associated pneumonia v. umathum1,2, d. amsel1, a. weber3, c. becker4, c. kasan4, a. may5, t. acker1, a. schänzer1 1 justus-liebig-university gießen, institute of neuropathology, gießen, germany 2 bundeswehrkrankenhaus ulm, institute of pathology and molecular pathology, ulm, germany 3 justus-liebig-university gießen, institute of human genetics, gießen, germany 4 institute of pathology, cytology and molecular pathology, wetzlar, germany 5 agaplesion evangelisches krankenhaus, department of pulmonology, gießen, germany introduction: neuroferritinopathy (nf) is an autosomal-dominant movement disorder due to mutations in the l-ferritin gene (ftl) on chromosome 19q13.33. nf belongs to neurodegeneration with brain iron accumulation (nbia) disorders, a group of hereditary neurodegenerative diseases with a prevalence of 1/1.000.000. patients with nf present clinically with progressive extrapyramidal movement symptoms with variable phenotype. histopathologically, nf is characterized by iron deposits and formation of ferritin inclusion bodies (ibs). clinical history: a 78-year-old woman was hospitalized with a severe covid-19 pneumonia and after three months died from covid19-associated pneumonia. during her 3-month clinical course she showed neurological symptoms of delirium and restlessness. cranial mri was not performed. neuropathological findings: brain weight was 1172 g. no abnormalities were seen in cross sections. histological examination revealed frequent atypical iron accumulation throughout the brain particularly in the basal ganglia. the nuclear spherical iron inclusions were localized in neurons, microglia and oligodendrocytes stained for prussian blue and immunohistochemically with antibodies against anti-ferritin light chain. electron microscopy showed fragmented nuclei with inclusions and chromatin accumulation toward the membrane consistent with ibs. moderate microcystic vacuolization and partial loss of myelination of nerve fibres were evident. discussion and conclusion:131 cases of nf have been described so far in the literature with 80% in france and the uk and 20% throughout the world. movement disorders or cognitive impairments were not reported in our patient, maybe due to a lack of full penetrance that occurs at the age of 60, in general. in our patient, the brain pathology is suspicious for a nf. most of the patients harbour an adenine insertion at position 460-461 of the ftl-gene. whole exome sequencing from brain tissue is in progress to confirm the diagnosis.   piv-06 free neuropathol 2:22:47 role of purinergic receptor p2y12 in alzheimer's disease r. maruccia1,2, a. a. gabr1, m. j. pietrowski1, b. a. tambe1, a. halle1,2 1 deutsches zentrum für neurodegenerative erkrankungen, bonn, germany 2 universitätsklinikum bonn, institut für neuropathologie, bonn, germany microglia have recently moved into prime focus of ad research due to the discovery of risk loci and genes that are linked to immune mechanisms, some of them highly expressed in microglia. however, immune signaling pathways in microglia that modulate ad progression, for example by removing aβ plaques, have remained poorly understood. the purinergic receptor p2y12 is activated by extracellular atp and adp and is highly expressed in platelets and microglia. p2y12 is crucial for several microglial functions such as chemotaxis, blood-brain barrier closure, synaptic plasticity, interaction with neuronal somata and phagocytosis. thus, a compromised microglial phagocytic removal of aβ and impaired plaque barrier formation upon p2y12 deletion may affect ad progression. here we analyze 3d-reconstructed confocal images of brain tissue from ad models to assess the role of p2y12 in the progression of aβ pathology, microglia-plaque interaction and inter-cellular interaction between microglial cells under neurodegenerative conditions. our preliminary findings suggest a region-dependent modulation of microglial coverage and co-localization with plaques in models with p2y12 deletion and therefore that this receptor may affect microglial interaction with aβ plaques. future studies investigating plaque load changes and the interplay between microglia, astrocytes and neurons will be needed to better understand inter-cellular pathways that contribute to p2y12-related effects in ad pathology. copyright: © 2021 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. detailed neuropathologic report of covid-19 complicated by large intracerebral hemorrhage and periventricular lesions with macrophagic infiltrates feel free to add comments by clicking these icons on the sidebar free neuropathology 2:7 (2021) case report detailed neuropathologic report of covid-19 complicated by large intracerebral hemorrhage and periventricular lesions with macrophagic infiltrates adrian levine1, carol lee2, craig fava3, frankie tsang4, kelly macneil5, stephen t. yip1,5, veronica hirsch-reinshagen1 1 department of pathology and laboratory medicine, university of british columbia, vancouver, bc, canada 2 division of forensic pathology, royal columbian hospital, vancouver, bc, canada 3 division of critical care, royal columbian hospital, bc, canada 4 british columbia center for disease control, vancouver, bc, canada 5 cancer genetics & genomics laboratory, bc cancer, vancouver, bc, canada corresponding author: veronica hirsch-reinshagen · anatomical pathology · vancouver general hospital · 899 west 12th av · jpn rm 1401 · vancouver, bc · canada · v5z 1m9 veronica.hirsch@vch.ca submitted: 15 february 2021 accepted: 16 march 2021 copyedited by: deanna fang published: 25 march 2021 https://doi.org/10.17879/freeneuropathology-2021-3266 keywords: covid-19, cerebral hemorrhage, brain, postmortem, histology, neuropathology abstract infection with the sars-cov-2 virus affects a wide range of systems. significant involvement of the central nervous system has been described, including ischemic and hemorrhagic strokes. thus far, neuropathologic reports of patients who passed away from covid-19 have generally described non-specific findings, such as variable reactive gliosis and meningeal chronic inflammatory infiltrates, as well as the consequences of the infection’s systemic complications on the brain, including ischemic infarcts and hypoxic/ischemic encephalopathy. the neuropathological changes in patients with covid-19 and large hemorrhagic strokes have not been described in detail. we report the case of an elderly male who had a long course of covid-19 and ultimately passed away from a large intracerebral hemorrhage. in addition to acute hemorrhage, neuropathologic examination demonstrated non-specific reactive changes and chronic periventricular lesions with macrophagic and perivascular lymphocytic infiltrates without evidence of demyelination or presence of sars-cov-2 by pcr test. this manuscript expands the spectrum of reported neuropathological changes in patients with covid-19. introduction intracerebral hemorrhage (ich) is an uncommon complication of infection with severe acute respiratory syndrome coronavirus 2 (sars-cov-2) virus, affecting 0.5-7.9% of patients hospitalized with covid-191–3. therapeutic anticoagulation has been identified as the most common etiology1–3 and an important risk factor for ich in covid-194. in addition, other factors such as microand macrovascular thrombosis, endothelial dysfunction and endotheliitis may play important pathogenic roles in this setting. despite this plethora of hypothesized factors, only a few neuropathological reports have included detailed histopathological brain evaluations of covid-19 patients with large fatal ich. post-mortem neuropathological evaluation of patients with covid-19 have shown a range of histological changes and have been reviewed elsewhere5,6. the vast majority of cases show relatively mild and non-specific findings including variable and diffuse microand astrogliosis, and mild parenchymal and leptomeningeal infiltration by chronic inflammatory infiltrates, which could represent the histological features of an encephalopathy associated with severe systemic inflammation rather than specific covid-19-related changes7. in addition, many cases show variable hypoxic-ischemic encephalopathy, infarcts associated with large vessel thromboembolism and variable hemorrhagic lesions from microhemorrhages to fatal ich5,6. only three detailed neuropathological reports of patients with large ich have been published to date and include a case of cerebellar hemorrhage thought to be most likely secondary to hypertensive vasculopathy8, one of hemorrhagic transformation of a large middle cerebral artery stroke9 and two cases reported by the authors as ich in the context of lymphocytic panencephalitis, meningitis and diffuse petechial hemorrhages10. in the latter, no details are provided regarding the histological changes associated with, or the etiopathogenesis of, the ich or petechial hemorrhages. in this report, we expand on the neuropathological literature of covid-19 cases by detailing the neuropathological findings in an elderly male with covid-19 and fatal ich. some of the findings reported in this case were also seen in a younger individual with covid-19 and fatal ich for which we were unable to acquire consent for publication of clinical details. the histological similarities and differences between these two cases will be highlighted in the text. case presentation this male patient, in his early 70s, presented to hospital with bilateral pneumonia secondary to covid-19 infection that was confirmed by nasopharyngeal swab testing. parallel testing for influenza a, b and respiratory syncytial virus were negative. his prior medical history included hypertension and significant hip osteoarthritis. he was diagnosed with septic shock and transferred to the intensive care unit upon admission as he required vasopressors and mechanical ventilation. his mean arterial pressure was targeted to more than 65 mmhg while his systolic blood pressure (sbp) was kept below a ceiling of 180 to 200 mmhg to avoid ischemic brain injury. this was especially relevant in the context of his shock syndrome and prior history of hypertension. only two sbp episodes over 180 mmhg, both of less than 30 min in duration, were recorded 4 and 19 days prior to his terminal ich. during his first week of admission, he developed renal failure, which was thought clinically secondary to acute tubular necrosis in the context of multisystem organ failure. he was placed on continuous renal replacement therapy (crrt) one week after admission. this required local anticoagulation with unfractionated heparin titrated to obtain an activated partial thromboplastin time (aptt) post-crrt filter of 60 to 90 seconds. his international normalized ratio (inr) hovered around the upper normal limit of 1.2 during his entire hospitalization. other relevant laboratory findings included an elevated d-dimer, c reactive protein and procalcitonin. he did not have evidence of pulmonary embolism during his hospitalization. figure 1. (a) ct scan showed a large left frontal ich with intraventricular extension. (b) coronal sections confirmed this ich and left-to-right subfalcine herniation. (c) leptomeningeal chronic inflammatory infiltrates were most prominent in the brainstem. (d) immunohistochemistry for hla-dr, a major histocompatibility class ii cell surface receptor, highlighted the leptomeningeal chronic inflammatory infiltrates (arrow) and the mild-to-moderately increased brainstem microglial activation. nearly three weeks into his admission and two weeks after initiation of crrt, the patient showed evidence of decreased level of consciousness. a computerized tomography scan demonstrated a left frontal intracerebral hemorrhage with intraventricular extension and subfalcine herniation (fig. 1a). he died the next day after withdrawal of life-sustaining therapies. a brain-restricted autopsy was performed 7 days after death to aid in the determination of the etiology of the brain hemorrhage. gross examination of the brain revealed a weight of 1280 grams, an enlarged left hemisphere, subfalcine left-to-right herniation of the left anterior frontal lobe and bilateral hippocampal uncal herniations. coronal sections confirmed a large ich centered in the left frontal lobe in close proximity to the lateral ventricle with extension throughout the ventricular system (fig. 1b). away from the hemorrhage, no additional gross pathological changes were identified. microscopic examination revealed mild diffuse reactive changes (fig. 1c and d) and focal periventricular lesions at the angles of the ventricles (fig. 2). the diffuse reactive changes included rather mild leptomeningeal chronic inflammatory infiltrates, parenchymal reactive gliosis and very mildly increased perivascular cd3 t-lymphocytes. the leptomeningeal inflammatory infiltrates were composed predominantly of macrophages and cd3 t-cell lymphocytes found throughout the meninges, but were accentuated in the brainstem (fig. 1c). reactive gliosis, highlighted by mild-to-moderately increased immunoreactivity for human leukocyte antigen – dr isotype (hla-dr) and glial fibrillary acidic protein (gfap), was present predominantly in the brainstem and olfactory bulb. similar changes were also seen in the younger individual. no microglial nodules were identified on hematoxylin and eosin (he) stains. two possible microglial nodules were identified on hla-dr immunohistochemistry (ihc) in the medulla only. figure 2. (a) multifocal periventricular lesions showed loss of ependyma and significant infiltration by macrophages with scant accompanying lymphocytes. (b) cd68 highlighted the significant macrophagic infiltrates. (c) luxol-fast blue showed mild loss of myelin, while phosphorylated neurofilament (pnf) ihc (d) showed associated axonal pathology with significant numbers of axonal spheroids. atypical periventricular lesions (fig. 2) were seen at multiple locations including both temporal horns, left occipital horn, fourth ventricle and in close proximity to the anterior left frontal lateral ventricle at the edge of acutely hemorrhagic brain parenchyma. these lesions consisted of loss of ependymal lining, relatively well delimited macrophagic and lymphocytic infiltrates (fig. 2a and b) and reactive vessels with plump endothelial cells. axonal pathology, in the form of relatively frequent axonal spheroids without appreciable demyelination, was also present (fig. 2c and d). iron stains performed in several of these lesions were negative. the abundance of phagocytic macrophages and the absence of hemosiderin suggest that these lesions were older than the intracerebral hemorrhage and therefore not likely to be a reactive change to intraventricular blood. this type of lesion was not seen in the younger individual, who instead exhibited multifocal petechial hemorrhages and microscopic ischemic infarcts thought to be most compatible with embolic lesions. no definite evidence of thrombi or megakaryocytes was identified in either of the cases. the neocortex, hippocampi, deep grey nuclei and cerebellum were unremarkable aside from some mild age-related neurodegenerative pathology. pontine white matter tracts only showed several microscopic foci of amyloid precursor protein (app) immunoreactive axonal pathology associated with microand astrogliosis. cd68 revealed absence of mature phagocytic macrophages in these lesions. these were interpreted as foci of axonal damage secondary to traction due to the left frontal and intraventricular ich. no cerebral amyloid angiopathy, arteriolosclerosis, lacunar infarcts or other vascular changes were identified. polymerase chain reaction (pcr) test for the presence of sars-cov-2 rna in multiple sections (medulla, hippocampus and olfactory bulbs) of formalin-fixed paraffin-embedded (ffpe) brain tissue was negative, including representation of the periventricular lesions. similar results were obtained for the younger patient. discussion our cases showed three main pathological changes: (1) acute and fatal ich, (2) diffuse reactive changes and (3) additional parenchymal lesions, either as multifocal chronic periventricular lesions in the elderly individual or as multifocal microhemorrhages and infarcts in the younger patient. the histopathological features of the periventricular lesions make them most compatible with localized tissue necrosis of unclear etiology. they were negative for sars-cov-2 rna. to our knowledge, this type of lesion has not been studied extensively. a single report comparing the neuropathological substrate of periventricular white matter abnormalities in patients with major depression and in controls describes lesions with similar characteristics to those found in this case. these lesions were interpreted as corresponding most likely to ischemic insults and were found both in a patient with major depression and a control11. the pathogenesis of these lesions awaits further evaluation, but the fact that lesions similar to those seen in our older individual have been described in non-covid cases raises the possibility that they are either nonspecific in etiology (e.g. associated with an episode of severe systemic illness) and/or can be brought about by different injury mechanisms. furthermore, in our case, it is unclear whether these lesions predated the sars-cov-2 infection or developed as a consequence of it. the close relationship of the acute left frontal ich to changes suggestive of an underlying periventricular lesion raise the possibility that local loss of tissue integrity may play a predisposing role to ich in covid-19. a similar situation was observed in the younger individual, in whom multifocal petechial hemorrhages and microscopic ischemic infarcts were identified. these were interpreted as most likely due to microemboli in the context of severe covid-19 and therapeutic anticoagulation for extracorporeal membrane oxygenation (ecmo). both patients were locally or systemically anticoagulated for therapeutic reasons and the risk of ich in the setting of anticoagulation appears to be increased in patients with covid-19. a study of 10 patients with covid-19 supported on ecmo for acute respiratory distress syndrome (ards) showed a markedly increased incidence of hemorrhagic strokes compared to non-covid patients on ecmo12. the diffuse reactive changes identified in both covid cases are similar to those reported previously in the literature and are at least partially explained as a manifestation of severe illness-related encephalopathy5–7. several tissue blocks of both cases were tested for sars-cov-2 by pcr and were negative. neither patient received antiviral therapies that could have decreased sars-cov-2 tissue levels. formalin-fixed control lung tissue from an unrelated covid-19 autopsy case with a shorter postmortem interval served as a positive control. although the long post-mortem interval and formalin-fixation may have interfered with the detection of sars-cov-2 in our samples, these results are in line with previously published reports that have shown inconsistent and variable detection of sars-cov-2 in brain parenchyma by pcr and immunohistochemical methods7,8. overall, this would suggest that the brain is not a site consistently affected by high viral loads of sars-cov-2 and raises the possibility that direct infection of the cns tissue may not be the main pathogenic mechanism of covid-19 neurologic manifestations. in summary, the fatal ich in two cases of covid-19 were most likely due to a combination of anticoagulation and additional factors affecting the integrity of the cns parenchyma. additional chronic inflammatory infiltrates and glial reactive changes are at least partially explained by severe illness-related encephalopathy. references 1. kvernland, a. et al. anticoagulation use and hemorrhagic stroke in sars-cov-2 patients treated at a new york healthcare system [published online ahead of print, 2020 aug 24]. neurocrit care. (2020) 1-12. https://doi.org/10.1007/s12028-020-01077-0 2. dogra, s. et al. hemorrhagic stroke and anticoagulation in covid-19. j stroke cerebrovasc dis. (2020) 29(8):104984. https://doi.org/10.1016/j.jstrokecerebrovasdis.2020.104984 3. benger, m. v intracerebral haemorrhage and covid-19: clinical characteristics from a case series. brain behav immun. (2020) 88:940-944. https://doi.org/10.1016/j.bbi.2020.06.005 4. melmed, kr. et al. risk factors for intracerebral hemorrhage in patients with covid-19 [published online ahead of print, 2020 sep 24]. j thromb thrombolysis. (2020) 1-8. https://doi.org/10.1007/s11239-020-02288-0 5. lou, jj. et al. neuropathology of covid-19 (neuro-covid): clinicopathological update. free neuropathol. (2021) 2:2. https://doi.org/10.17879/freeneuropathology-2021-2993 6. mukerji, ss & solomon, ih. what can we learn from brain autopsies in covid-19?. neurosci lett. (2021) 742:135528. https://doi.org/10.1016/j.neulet.2020.135528 7. deigendesch, n. et al. correlates of critical illness-related encephalopathy predominate postmortem covid-19 neuropathology. acta neuropathol. (2020) 140(4):583-586. https://doi.org/10.1007/s00401-020-02213-y 8. al-dalahmah, o. et al. neuronophagia and microglial nodules in a sars-cov-2 patient with cerebellar hemorrhage. acta neuropathol commun. (2020) 8(1):147. published 2020 aug 26. https://doi.org/10.1186/s40478-020-01024-2 9. hanley, b. et al. histopathological findings and viral tropism in uk patients with severe fatal covid-19: a post-mortem study. lancet microbe. (2020) 1(6):e245-e253. https://doi.org/10.1016/s2666-5247(20)30115-4 10. von weyhern, ch. et al. early evidence of pronounced brain involvement in fatal covid-19 outcomes. lancet. (2020) 395(10241):e109. https://doi.org/10.1016/s0140-6736(20)31282-4 11. thomas, aj. et al. a neuropathological study of periventricular white matter hyperintensities in major depression. j affect disord. (2003) 76(1-3):49-54. https://doi.org/10.1016/s0165-0327(02)00064-2 12. usman, aa. et al. a case series of devastating intracranial hemorrhage during venovenous extracorporeal membrane oxygenation for covid-19. j cardiothorac vasc anesth. (2020) 34(11):3006-3012. https://doi.org/10.1053/j.jvca.2020.07.063 copyright: © 2021 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. neurodegeneration: 2021 update feel free to add comments by clicking these icons on the sidebar free neuropathology 2:9 (2021) review neurodegeneration: 2021 update john f. crary neuropathology brain bank & research core, department of pathology, nash family department of neuroscience, ronald m. loeb center for alzheimer's disease, friedman brain institute, icahn school of medicine at mount sinai, new york, ny, usa corresponding author: john f. crary , md-phd · friedman brain institute · ronald m. loeb center for alzheimer’s disease · icahn school of medicine at mount sinai · 1 gustave l. levy place box 1194 · new york, ny 10029 · usa john.crary@mountsinai.org submitted: 28 march 2021 accepted: 15 april 2021 copyedited by: jeffrey nirschl published: 21 april 2021 https://doi.org/10.17879/freeneuropathology-2021-3317 keywords: neurodegeneration, neuropathology, alzheimer disease, tauopathy, α-synucleinopathy, chronic traumatic encephalopathy, cryogenic electron microscopy, proteomics abstract this article reviews a collection of manuscripts in the field of neurodegenerative disease chosen from what are considered by the author to be among the 10 most important and potentially impactful topics or research trends of 2020 relevant to the field of experimental and diagnostic neuropathology. a deliberate effort was made to provide balance among disease categories covered. the result is a varied selection that includes not just individual papers but also research topics and trends. the association of covid-19 with longer-term neurological symptoms has launched a research trend fueled by speculation that the sars-cov-2 might trigger neurodegenerative changes. the onslaught of transcriptomic studies has begun to give way to proteomics, with three transformative studies published examining glial contributions to alzheimer disease, cerebral atherosclerosis in cognitive decline, and the complex sequence of post-translational modifications of the tau protein. plasma biomarkers for alzheimer disease have continued to make rapid advances, especially around highly sensitive assays capable of detecting different forms of abnormal hyperphosphorylated tau in peripheral blood. two studies using cryo-electron microscopy showed the power of the approach by continuing to elucidate the diversity of filamentous tau inclusions, and a third study gave the first glimpse of α-synuclein aggregates at near atomic resolution. another study continued to delineate how different α-synuclein conformers (“strains”) target specific brain regions and lead to neurodegeneration. in huntington’s disease, we saw compelling molecular data showing how cells adapt to endoplasmic reticulum stress through the unfolded protein response. finally, the role of astrocytes in chronic traumatic encephalopathy has emerged as a critical area of interest. introduction in a year like no other, experimental neuropathology research in neurodegeneration persevered. confronted with a devastating pandemic, we endured despite shuttered laboratories, working from home, supply shortages, the loss of loved ones and a myriad of other challenges. our laboratories demonstrated inspiring resilience, adapting to the new landscape and soldiering on. many rose to the challenge, finding purpose and inspiration in adversity. dr. rita levy montalcini once said, “i should thank mussolini for having declared me to be of an inferior race. this led me to the joy of working, not any more unfortunately in university institutes, but in a bedroom.” upon completing this review of just a few of the accomplishments of the past year, it was clear that this spirit is alive, the same that compelled levy-montalcini to work tirelessly at home to study chick embryos using scalpels fashioned from sewing needles. the spirit could be felt emanating from our colleagues sheltering at home, many also literally working in their bedrooms! so, it is with great pride and appreciation that we take this look back and celebrate the accomplishments of our colleagues and recognize just a few of the many transformative advances in 2020. the pandemic is a reminder that substantive scientific progress comes with great human effort and rarely a “eureka moment.” here, we also highlight collaborative efforts, research trends, groundbreaking methodologies, and tools. a concerted effort was made to seek out innovations across all neurodegenerative disease categories. given the readership and mission of this journal, we emphasized pathoanatomical studies most relevant to neuropathologists and clinical neuroscientists. 1. the long-term neuropathological sequelae of covid-19 over the past year, the sars-cov-2/covid-19 pandemic has permeated essentially every aspect of our lives, and research in neurodegenerative disease is no exception, launching a new, albeit speculative, research trend. early in the pandemic, neuropathologists began to wonder about the extent to which covid-19 might impact brain health. theoretically, this could be through direct neurotropism as some coronaviruses are known to cause encephalitis, or perhaps indirectly through complications related to critical illness and systemic disease (morgello, 2020). early reports documented encephalitis in the setting of covid-19 (reichard et al., 2020), but sars-cov-2 encephalitis was considered a rare event predominantly restricted to susceptible populations and of modest public health concern. larger autopsy series emerged, highlighting infarction and microthrombi in the brain in the acute setting (figure 1), more commonly in hospitalized patients, but this too was thought to be uncommon (bryce et al., 2020). nevertheless, researchers began to speculate that there may be long-term neurological sequelae that follow covid-19 as examples of post-viral tauopathy and neurodegeneration are well documented and now there is increasing alarm that there might be an incipient rise in neurological complications, including dementia and neurodegeneration, that might follow the pandemic (badrfam & zandifar, 2020). figure 1. post-mortem findings in a patient with sars-cov-2/covid-19. a. fresh gross image of a right hemibrain from a 68 male with covid-19 showing diffuse swelling and leptomeningeal hemorrhage. b. fixed right hemibrain from the same patient showing acute and subacute infarction in the distribution of the middle cerebral artery territory. c. luxol fast blue counterstained hematoxylin and eosin (lh& e) section showing subacute hemorrhagic infarction. d. lh& e-stained section from the neocortical white matter in a 29-year-old male covid-19 patient with acute disseminated encephalomyelitis (adem). neurologists and other clinicians have been describing persistent neurological symptomatology in covid-19 survivors, including inattentiveness, memory impairment and a constellation of other cognitive symptoms (i.e., “brain fog”). these are part of an entity now termed the post-acute covid-19 syndrome (pacs). the underlying neuropathological substrate for these findings remains unclear, but the effects of hypoxic-ischemic injury, chronic neuroinflammation, toxic encephalopathy from breakdown of the blood brain barrier are candidates. direct effects of the virus are not completely ruled out, and the potential for neurotropic strains to emerge remains a possibility. given the existence of encephalitis lethargica and measles related subacute sclerosing panencephalitis, both virus related and associated with neurofibrillary tangles, it is not unreasonable to speculate that a degenerative component of pacs might exist. critically, it is imperative to understand whether these pacs neurological symptoms are progressive and we expect a surge in translational studies in the coming years focused on the long-term changes that follow covid-19.  2. proteomic analysis: spotlight on microglial and astrocyte inflammation in alzheimer disease in the previous decades, transformative technologies have enabled large-scale genomic and transcriptomic studies of post-mortem brain tissues that have been directed towards neurodegenerative diseases, yielding a flood of data out of which has emerged a rich and complex picture of alzheimer disease (ad). but what about the proteins? owning to their relatively more complex chemical structures, measuring structural changes on the protein level in a high throughput manner is much more challenging. but now, proteomic technologies have advanced sufficiently, and we are beginning to see them take center stage. here and in the next two sections we highlight three such studies. the first study, published in nature medicine, led by nicholas seyfried and allan levey at emory university, profiled 2,000 brain samples, comparing more than 3,000 proteins from multiple cohorts, including controls, people with asymptomatic ad neuropathology and those with dementia due to ad (johnson et al., 2020). cases were derived from a total of eight collections, including the religious orders study and memory and aging project (ros-map), mayo clinic brain bank, baltimore longitudinal study of aging (blsa), baltimore coroner’s office, banner sun health research institute, mount sinai school of medicine brain bank, adult changes in thought study (act), and university of pennsylvania school of medicine brain bank. researchers identified 13 different gene sets (“modules”) based on co-expression patterns (figure 2). of these, six were correlated with ad neuropathological features (i.e., amyloid plaques, neurofibrillary tangles) or cognitive impairment. of the three that were most closely correlated with ad features, two were downregulated, including the m1 that was composed of proteins involved in synaptic function and the m3 that tracked with mitochondrial proteins. m4 had the strongest correlation with disease traits and contained astrocyte and microglial proteins involved in sugar metabolism. intriguingly, many of the proteins in the m4 module were encoded by genes that are ad risk loci. importantly, regardless of their precise function and role in disease, 27 of them were detected in csf, underscoring their potential utility as biomarkers. because this was funded by the accelerating medicine partnership ad (amp-ad) project, all of these data are publicly available for the research community and will surely catalyze continued proteomic studies. figure 2. protein network analysis in post-mortem alzheimer disease (ad) brain. a. brain tissue from the dorsolateral prefrontal cortex from ad patients and compared to cognitively normal subjects with age-related pathology and pathologically negative controls from four cohorts. b. a correlation network consisting of 13 modules using 3334 proteins was created and correlated with neuropathological assessments, cell type markers, and the associated biological processes identified using gene ontology (go) analysis. images courtesy of dr. erik c. johnson, allan i. levy, nicholas t. seyfried, reproduced with permission under the creative commons license. 3. proteomic analysis: the contribution of cerebral atherosclerosis to cognitive decline another proteomics paper published in nature neuroscience, also from emory university led by nicholas seyfried, allan levey, and thomas s. wingo, examined cerebral atherosclerosis as it relates to cognitive decline and ad (wingo et al., 2020). here, the investigators performed a proteome-wide association study (pwas), which is similar to a genome-wide association study (gwas), to look more broadly at relevant neuropathological changes, including amyloid-beta deposition, neurofibrillary tangles, infarcts (both macro and microinfarcts), cerebral amyloid angiopathy, tar dna-binding protein 43 (tdp-43, transactive response dna binding protein 43 kda), lewy body pathology, hippocampal sclerosis, and atherosclerosis. out of this analysis, 114 proteins emerged, independently of cerebrovascular risk factors (e.g., hypertension, diabetes, smoking; figure 3). strikingly, many were from oligodendrocytes involved in myelination. other genes were involved in mrna processing and splicing. they performed co-expression network analysis and 31 modules were identified, with five being linked independently to cerebral atherosclerosis. this analysis pointed to oligodendrocyte function alongside downregulation of mrna processing in neurons and astrocytes with impaired synaptic functioning in brains of individuals with cerebral atherosclerosis. this may be related to another finding of higher levels of neurofilament light (nfl) and medium (nfm) which were higher in cases with atherosclerosis, perhaps reflecting axonal injury. this study will drive continued research into the effects of cerebral atherosclerosis on the brain proteome and their contribution to ad. figure 3. co-expression network analysis of protein modules in post-mortem human brain. a, b. a total of 31 modules were identified and protein signatures mapped to cell type. c. these modules were then associated with neuropathological outcomes. d. differences in module eigen proteins were seen modules 3 and 9 for atherosclerosis and alzheimer disease-related dementia. images curtesy of drs. thomas wingo, aliza wingo, nicholas seyfried, allan i. levey (emory university school of medicine). 4. proteomic analysis: a high-resolution quantitative map of post-translational modifications of tau proteoforms neurofibrillary tangles exist in a spectrum of morphological forms that are immediately recognizable using immunohistochemistry with antisera targeting hyperphosphorylated tau (p-tau). the earliest detectable neurofibrillary change is the presence faint granular p-tau labeling in pre-tangles; this staining accumulates and coalesces into intracellular tangles that eventually become extracellular (“tombstone” or “ghost”) tangles following neuronal death. understanding the molecular events that correspond to this sequence would be extremely helpful for developing the next generation of diagnostics (see below) and therapeutics. the pathobiology of tau pathology is complex, with multiple tau isoforms undergoing innumerable secondary structural modifications in disease states beyond just hyperphosphorylation, including truncation, acetylation, ubiquitination, glycosylation and methylation. it has been challenging to temporally map the order of these events using traditional approaches (e.g., phospho-specific tau antisera-based methods) which lack the ability to synchronously and comprehensively detect these critical changes. in the last proteomics paper, published in cell in november of 2020, a powerful study, led by dr. judith steene at boston children’s hospital, detailed their focused analysis that was entirely directed towards the tau protein (wesseling et al., 2020). they deployed a previously published mass spectrometry-based assay, that they term “flexi-tau” (mair et al., 2016), which allowed them to generate a high-resolution quantitative proteomic map of 95 post-translational modifications on multiple tau isoforms from 91 human post-mortem brains. they then used unsupervised analyses to predict sequential addition of modifications. they found that while there is a great degree of heterogeneity, there appears to be a minimal set of modifications that develop in a processive fashion and are associated with tissue fractions associated with disease stage as well as tau seeding activity. the analysis also highlighted a specific alternatively spliced tau isoform with four microtubule binding domain repeats (4r), but lacking n-terminal domain inserts (0n). the 0n4r isoform is highly overrepresented in aggregates isolated from early disease stages alongside modifications associated with increased negative charge in the proline-rich region and decreased positive charge in the microtubule binding domain. these findings are promising because they give us the most comprehensive picture of tau secondary structure and provide a pathway towards continuing to advance the biomarkers that are rapidly evolving and identify the critical modifications that are responsible for the pathogenicity of tau paving the way towards new therapeutics. 5. plasma biomarkers for alzheimer disease obtaining a definitive diagnosis of a neurodegenerative disease continues to require an autopsy, the perennial gold standard, but continued advances in blood-based biomarkers are continuing to challenge this and offer a low-cost, convenient alternative. over the past decade, substantial progress has been made in our ability to assess molecular changes in the central nervous system using non-invasive amyloid positron emission tomography (pet) scans and cerebrospinal fluid (csf) markers. yet these modalities are expensive, inconvenient and come with a mild degree of risk. the long-sought development of a blood-based biomarker for alzheimer disease (ad), had been considered unrealistic because the levels of brain proteins in the blood had been thought to be simply too low to be reliably and reproducibly detected. further, there was concern that the extent to which blood levels of various factors reflect ongoing disease processes might be too disconnected or tangential to be clinically useful. so, it comes as no surprise that the research community has been stunned by the rapid progress in blood-based biomarkers in neurodegeneration. this year a milestone was achieved, with the “precivityad™” mass-spec amyloid-β assay, which was developed at washington university saint louis in the laboratory directed by dr. randall bateman, receiving approval under the clinical laboratory improvement amendments (clia) as well as an fda breakthrough designation. however, this is only half of the story. the other essential neuropathological hallmark of ad, the neurofibrillary tangle composed of abnormal tau, is also required for a diagnosis. remarkably, similar rapid progress is being made on this front with a series of groundbreaking studies that were published over that past year, continuing the momentum. in previous years, the first papers emerged describing highly sensitive immunoassays identifying p-tau phosphorylated at threonines 181 or 217 as early promising biomarkers. in 2020, phospho-threonine 231 (p-tau231) was reported to be an excellent early marker for tau pathology in csf (suárez-calvet et al., 2020). this was followed up with results from a study using an ultrasensitive single molecule array (simoa) for the quantification of p-tau231 in blood plasma (ashton et al., 2021). the study included a total of 588 subjects and successfully differentiated ad from amyloid-β negative cognitively normal individuals. plasma p-tau231 also differentiated ad patients from non-ad neurodegenerative disorders and amyloid-β negative mci patients. in samples taken from patients with post-mortem autopsy confirmation, plasma p-tau231 was extremely accurate in identifying ad neuropathology in comparison to non-ad neurodegenerative disorders (auc = 0.99). plasma p-tau231 was highly correlated with other ad biomarkers, i.e., csf p-tau231, tau pet ([18f]mk-6240) and amyloid-β pet ([18f]azd). the elevations in p-tau231 were a very early change, preceding amyloid-β pet and plasma p-tau181. plasma p-tau231 had the power to resolve even subtle differences, differentiating subjects across even early braak stages. together, we are witnessing impressive progress in blood-based ad biomarkers with the potential to detect the earliest disease stages, prior to significant irreversible brain destruction, and enable the next generation of clinical trials. 6. cryo-electron microscopy: ultrahigh resolution structure of tau in corticobasal degeneration in 2017, the nobel prize in chemistry was awarded to jacques dubochet, joachim frank and richard henderson for cryo-electron microscopy (cryo-em). the approach, which involves cooling samples to cryogenic temperatures and embedding them in an environment of vitreous water, has been in development since the 1970s, but recent technical advances in detectors and software algorithms have enabled solving molecular structures at near-atomic resolution. the power of cryo-em was first demonstrated that year to the neurodegenerative disease research community when the approach was applied to paired-helical filament tau fibrils in alzheimer disease, illuminating at near atomic resolution the c-shaped conformation of the core (fitzpatrick et al., 2017). this report was quickly followed by the solving of the structure for the j-shaped tau filament core in pick disease which contains additional residues making it slightly longer than in ad (falcon et al., 2018). next came chronic traumatic encephalopathy, which has a similar c-shape to that in alzheimer disease (falcon et al., 2019). also described was an additional non-proteinaceous hydrophobic density that has yet to be defined. these findings support the notion that there exists a spectrum of hitherto unrecognized tau structural features and conformations, often termed “strains” in a nod to the prion literature that has informed much of the research on protein misfolding, that may be distinct in different disease states. now, we are continuing to see additional insights with two papers describing the structure of the tau filament core in corticobasal degeneration as well as another which is the first cryo-em structure of α-synuclein (see next section). in a paper published in cell in february 2020, a team led by anthony fitzpatrick at columbia university detailed their study showing the cryo-em structure of filaments from corticobasal degeneration (arakhamia et al., 2020). in additional analyses, they further integrated these cryo-em structures and those from ad with mass spectrometry data to localize post-translational secondary modifications. a separate study, published in nature, led by sjors scheres and michel goedert at the mrc laboratory of molecular biology in cambridge, england, u.k. (zhang et al., 2020). this team also showed a similar structure in cbd (figure 4). together, in addition to providing a validated atomic level resolution c-shaped structure of the tau filament in cbd, these studies both revealed a density buried in the structure that represents a molecule of unknown identity, akin to what was identified in cte. the fibrils in cbd are composed entirely of tau with 4 microtubule binding domain repeats (4r), which differs from pick disease which is composed of 3r tau as well as ad and cte which are mixed 3r and 4r. additional future studies directed towards further delineation of the diversity of tau filament structures has the potential to pave the way towards improved diagnostics and advancing our understanding of the diversity of conformers in tauopathy that may be associated with different clinical symptomatology. figure 4. cryo-em of tau filaments in corticobasal degeneration. a. negative-stain electron micrographs of type i and type ii tau filaments extracted from the frontal cortex of cbd. b. cryo-em maps of type i and type ii tau filaments from the frontal cortex of cbd. c. top, the microtubule-binding repeats (r1–r4) of tau and the sequence after r4 that is present in the core of cbd filaments (all shown in different colors). bottom, atomic model of the cbd type ii tau filament. the extra density is shown in light blue, with k290, k294 and k370 indicated. images courtesy of drs. wenjuan zhang, sjors scheres and michel goedert mrc laboratory of molecular biology, cambridge). 7. cryo-electron microscopy: ultrahigh resolution structures of α-synuclein conformers this year we also saw reports of the first ultrahigh resolution cryo-em structures of α-synuclein fibrils published in nature also from sjors scheres and michel goedert at the mrc laboratory of molecular biology (schweighauser et al., 2020). the synucleinopathies are a group of neurodegenerative disorders that include parkinson’s disease (pd), pd dementia, diffuse lewy body disease (dlbd) and multiple system atrophy (msa). co-first authors schweighauser and shi et al., studied three cases of dlbd and five with msa. in msa, they found two types of filament, that they termed type 1 and type 2, each with filaments with different protofibrils that make up the core. the conformation of α-synuclein filaments derived from dlbd brains by cryo-em precluded 3d imaging, so 2d class averaging was used to show that there were differences in the structure from msa. overall, these structural differences are of great interest as accumulating evidence suggests that α-synuclein might behave in a prion-like manner, with templating and propagation of abnormal structures/conformers. this knowledge may be helpful in understanding the pathogenesis and natural history of the synucleinopathies, and especially in the development of specific α-synuclein pet tracers, which have been so far elusive. 8. characterizing α-synuclein strains the α-synucleinopathies, including parkinson disease, diffuse lewy body disease, and dementia with lewy bodies, while linked by aggregation of α-synuclein, have been hypothesized to diverge based on the prion-like properties unique disease conformational structural folding that templates replication of the abnormality. these strains are hypothesized to have different properties and may influence neuropathological features and symptomatology. this year, a study was published that took us one step closer to understanding the pathobiology and diversity of α-synuclein strains. in an elegant set of experiments, lau et al. recreated an array of α-synuclein fibrils with different confirmations in vitro using recombinant proteins by altering buffer conditions (lau et al., 2020). they generated a host of fibrils that varied in their biophysical properties and compared them with those derived from human post-mortem brain samples. then, the fibrils were inoculated into a transgenic mouse line that overexpresses human mutant α-synuclein and observed them. over time, the mice developed a range of traits that were strain specific, including aggregate morphology, incubation periods and behavioral changes, that could be propagated serially (figure 5). in the brains of these animals, distinct neuroanatomical vulnerability was observed that was dictated by strain type. these findings give us an additional model for testing questions related to how these different α-synuclein conformers target specific cells and brain regions leading to neurodegeneration. figure 5. distinct midbrain pathology in transgenic α-synuclein (m83) mice inoculated with salt (s) fibrils or no salt (ns) fibrils. a. the ns fibril-injected mice show the “lewy body-like” α-synuclein pathology. b. the s fibril-injected mice exhibit the “ring-like” α-synuclein pathology. both images show stains with the ep1536y antibody that recognizes synuclein phosphorylated at serine 129. images courtesy of dr. joel watts (university of toronto). 9. huntington’s disease and chorea abnormalities in proteostasis are a key feature of essentially all neurodegenerative disorders. this change is associated with endoplasmic reticulum (er) stress, which then triggers the unfolded protein response (upr). xbp1 is a key mediator of the er stress response thought to be a master regulator of the upr that drives the adaptation response to recover proteostasis through a number of mechanisms. in a series of compelling experiments, a team from the university of chile showed that treatment with igf2, a factor that they previously implicated as a downstream effector of xbp1 in animal models deficient in xbp1, may be an important contributor to this finding (garcía-huerta et al., 2020). treatment with igf1 led to a marked reduction in the burden of intracellular aggregates of mutant huntingtin in cellular models, including an induced pluripotent stem cell (ipsc)-derived model of medium spiny neurons from hd patients. when the autophagy and the ubiquitin proteasome were assessed, they surprisingly found that these pathways were not responsible. remarkably, they found igf2 signaling enhanced secretion of soluble mutant huntingtin into exosomes/microvesicles. these findings were not limited to cell culture but were recapitulated when igf2 was infused into the brain of hd mice. finally, these findings were validated in human tissue samples, with a reduction in igf2 in post-mortem hd brain and blood. these findings take us one step closer to understanding the role of the upr in hd and suggest a mechanism that could be targeted for therapeutics. 10. chronic traumatic encephalopathy: what about the astrocytes? slightly over ten years ago, chronic traumatic encephalopathy (cte) was propelled to the fore of neurodegenerative disease research where it has remained. since that time, broad consensus has emerged among neuropathologists that cte has a distinct neuropathological presentation that is distinguishable from other tauopathies, with a unique pathognomonic perivascular lesion that was codified in consensus criteria (mckee et al., 2016). in this report, the pathognomonic lesion was described as consisting of “p-tau aggregates in neurons, astrocytes, and cell processes around small vessels in an irregular pattern at the depths of the cortical sulci”. while this working definition was considered sufficiently precise, confusion emerged and persisted in the literature around the perivascular astrocytes in aging related tau astrogliopathy (artag) (kovacs et al., 2016) that can generally be differentiated based on the absence of neurons and localization in the white matter or subpial compartments. clarification was not sufficient to put the astrocyte question to rest (mckee et al., 2020). setting aside artag, there is a tremendous burden of p-tau positive gray matter astrocytes in cte. a study led by john trojanowski at the university of pennsylvania and william stewart at the university of glasgow published in brain communications sought to neuroanatomically map these astrocytes and found a preferential accumulation in the sulcal depths, the precise region that is biomechanically vulnerable in cte and where tau pathology first emerges (arena et al., 2020). this is in contrast to neuronal tau pathology which they found was more diffuse. this seems to suggest that astrocyte pathology is an early event. in another study published in brain pathology led by ann mckee and john crary (writer of this review), a spectrum of cases that also included very early cte lesions was able to pinpoint abnormal tau in early pathognomonic lesions to perivascular neurons (figure 6), but not astrocytes, which appeared to accumulate later in the disease progression (cherry et al., 2020). this tau pathology consisted predominantly of tau isoforms containing four microtubule binding domain repeats (4r tau), an isoform that has been proposed to be especially toxic given its increased propensity towards aggregation. together, these findings point to a complex picture of evolving tau pathology in neurons and astrocytes early in cte and we expect that research in the coming years around the role of astrocytes in early cte will intensify. figure 6. immunofluorescence microscopy in a sulcal region from an individual who passed away in their 20s with chronic traumatic encephalopathy (cte). a. low power image highlighting two perivascular lesions. b. high power image showing staining with markers for astroglia (gfap, blue), neurons (map2, green) and abnormal hyperphosphorylated p-tau (at8; green). white arrows mark colocalization. in these lesions, there is only map2 overlap with p-tau. no gfap/at8 colocalization was seen in this case. scale bar is 50 um. images courtesy of dr. jonathan cherry and ann mckee (boston university and boston va). acknowledgement dr. crary receives research funding from the nih (p30ag066514, r01ag054008, r01ns095252, r01ag062348, r01ns086736, u54ns115266, u54ns115322). we 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(2020). novel tau filament fold in corticobasal degeneration. nature, 580(7802), 283–287. https://doi.org/10.1038/s41586-020-2043-0 copyright: © 2021 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. top ten discoveries of the year: neurodevelopmental disorders feel free to add comments by clicking these icons on the sidebar free neuropathology 1:13 (2020) review top ten discoveries of the year: neurodevelopmental disorders mara dierssen centre for genomic regulation (crg); the barcelona institute of science and technology, and universitat pompeu fabra (upf), 08003 barcelona, spain corresponding author: mara dierssen · systems biology program · crg-center for genomic regulation · c/ dr. aiguader, 88 · prbb building · 08003 barcelona · spain mara.dierssen@crg.eu submitted: 25 february 2020 accepted: 12 april 2020 copyedited by: cathryn cadwell published: 15 april 2020 https://doi.org/10.17879/freeneuropathology-2020-2672 keywords: in utero diffusion magnetic resonance imaging, in utero diffusion tensor imaging, rna editing, retrotransposons, brain organoids, single cell omics abstract developmental brain disorders, a highly heterogeneous group of disorders with a prevalence of around 3% of worldwide population, represent a growing medical challenge. they are characterized by impaired neurodevelopmental processes leading to deficits in cognition, social interaction, behavior and motor functioning as a result of abnormal development of brain. this can include developmental brain dysfunction, which can manifest as neuropsychiatric problems or impaired motor function, learning, language or non-verbal communication. several of these phenotypes can often co-exist in the same patient and characterize the same disorder. here i discuss some contributions in 2019 that are shaking our basic understanding of the pathogenesis of neurodevelopmental disorders. recent developments in sophisticated in-utero imaging diagnostic tools have raised the possibility of imaging the fetal human brain growth, providing insights into the developing anatomy and improving diagnostics but also allowing a better understanding of antenatal pathology. on the other hand, advances in our understanding of the pathogenetic mechanisms reveal a remarkably complex molecular neuropathology involving a myriad of genetic architectures and regulatory elements that will help establish more rigorous genotype-phenotype correlations. abbreviations 3d three-dimensional, adar adenosine deaminase acting on rna, asd autism spectrum disorder, adhd attention deficit hyperactivity disorder, ca3 cornu ammonis area 3, cen central executive network, cns central nervous system, cnvs copy number variations, dg dentate gyrus, dlpfc dorsolateral prefrontal cortex, dmn default mode network, dsm the diagnostic and statistical manual of mental disorders, dti diffusion tensor imaging, edqtl editing quantitative trait loci, eeg electroencephalography, fa fractional anisotropy, fxs fragile x syndrome, gaba γ-aminobutyric acid, go gene ontology, gwas genome-wide association study, herv human endogenous retroviruses, hpscs human pluripotent stem cells, hescs human embryonic stem cells, hipscs human induced pluripotent stem cells, ipscs induced pluripotent stem cells, l1hs human-specific line-1, line-1 long-interspersed nuclear element-1, mpfc medial prefrontal cortex, mri magnetic resonance imaging, pgc psychiatric genomics consortium, rs-fmri resting-state functional mri, scrna-seq single-cell rna-sequencing, snrna-seq single-nucleus rna-sequencing, tbss tract-based spatial statistics, tes transposable elements, tsa tract-specific analysis introduction human brain structural and functional development occurs over a protracted period compared to many other mammals and primates (watson et al., 2006). this coordinated development provides the architecture for the expansion of behavioral and cognitive abilities, especially rapid in the first years, but also especially vulnerable to genetic and/or environmental insults leading to developmental brain disorders. classically those include intellectual disability, autism spectrum disorder (asd), attention deficit hyperactivity disorder (adhd), specific learning disorder, motor disorder, or epilepsy. however, beyond the traditional concept of neurodevelopmental disorders, studies in humans provide clear evidence that mental disorders such as schizophrenia, drug abuse, or neurodegenerative disorders such as alzheimer’s disease also have a strong developmental component that might be identified by specific neuropathological features (thibaut, 2018). understanding the role that development plays in the expression of these disorders is often overlooked, but definitively needs more attention to fully understand the impact of early life events on the complex neurobiological derangement. from the clinical point of view, there has been a recent move at the diagnostic level from a categorical toward a spectrum-based view. for example, the definition of autism has been highly debated, in several revisions of the diagnostic and statistical manual of mental disorders (dsm; dsm-iii, dsm-iiir, dsm-iv, and dsm-v) criteria (rodgaard et al., 2019). this has led to a steady increase in the heterogeneity of some conditions which could affect the results of autism research. the recent advances in genomic medicine and the development of improved cellular models, will certainly help overcome these difficulties. on the mechanistic side, advances in directed differentiation of human induced pluripotent stem cells (hipscs) and other neural cell preparations (van den ameele et al., 2014), coupled with the application of advanced histological, imaging, molecular, cellular, and genomic techniques, is providing insights into cellular and molecular processes in human neurodevelopmental disorders and also in a wide array of neurological and psychiatric disorders (brennand et al., 2011; lancaster et al., 2013; mariani et al., 2015; pasca et al., 2015). moreover, the recent advancements in single cell functional genomic techniques have enabled comprehensive and unbiased characterization of the molecular processes in human postmortem cns tissues and neural cell culture systems, and will allow us to explore neurodevelopmental neuropathology at a resolution that was not possible before. even so, our understanding of the spatiotemporal landscape of the rna species, and epigenetic features in the developing human brain, and their pathogenicity is still incomplete (qureshi & mehler, 2012). finally, some findings suggest common pathophysiological mechanisms that can be considered as a continuum of developmental brain dysfunctions. these findings revealed molecular pathways that are commonly altered in different forms of developmental brain disorders, both of genetic and environmental origin and new players are starting to be recognized such as genomic regulatory elements. 1. understanding neurodevelopmental disorders through imaging the prenatal brain one of the most crucial questions in neurodevelopmental disorders is what we can really detect from the wide range of developmental processes that take place (and can go wrong) during this critical period, and the extremely rapid pace of structural and functional brain development (geng et al., 2017). the dynamic morphological changes the fetal brain undergoes during early development result from neurogenic events, such as neuronal proliferation, migration, axonal elongation, retraction, and myelination. in the critically sensitive mid-gestational phase of development, structural and functional assessment of the fetal brain opens a window into prenatal diagnostics and prognostics, and would help establish biomarkers for prenatal diagnoses (batalle et al., 2017). conventional t1 and t2 weighted sequences provide anatomic detail of the normally developing brain and can demonstrate lesions, including those associated with preterm birth, hypoxic ischemic encephalopathy, perinatal arterial stroke, infections, and congenital malformations. specialized imaging techniques can also be used to assess cerebral vasculature (magnetic resonance angiography and venography), cerebral metabolism (magnetic resonance spectroscopy), cerebral perfusion (arterial spin labeling), and function (functional magnetic resonance imaging [mri]) (counsell et al., 2019). recent advances in in utero diffusion mri provide unique opportunities to noninvasively study the microstructure of tissue during neurodevelopment and possible mechanisms of how pathologies, maternal, or environmental factors that may interfere with brain development can be potentially detected. one important aspect is that postmortem histologic studies of early childhood cortical development are consistent with imaging studies and provide insights into the neurobiological process that underlie change observed in imaging studies (christiaens et al., 2019). abnormalities in processes, such as white matter and cortical connectivity in preterm babies have already been suggested to derive into late language development and impaired cognitive performance in children. in utero diffusion tensor imaging (dti) provides new windows to monitor the emergence of the connectome. these advances have led to emerging fields of research, such as developmental pathoconnectomics (jakab, 2019) that aim to unravel the development of disrupted brain connectivity. a wealth of quantitative tools, most of which were originally developed for the adult brain, can be applied to study the developing brain in utero and postnatally including measures of tissue microstructure obtained from diffusion mri, morphometric studies to measure whole brain and regional tissue volumes, and automated approaches to study cortical folding. however, the differences in functional organization between the brains of infants and adults call for infant-specific functional atlases for better definition of regions of interest and interpretation of results. thus, the creation of resources such as a dti atlas of the fetal brain is required for reliable detection of major neuronal fiber bundle pathways and for characterization of the fetal brain reorganization in utero. such tools are also useful for detection of normal and abnormal fetal brain development providing normative quantitative and qualitative data. the paper by khan et al. (khan et al., 2019) presents the first dti atlas of the fetal brain computed from in utero diffusion-weighted images. it was built from 67 fetal dti scans acquired from healthy fetuses each scanned at a gestational age between 21 and 39 weeks, addressing a wider gestational age range and larger number of sampled areas than prior studies. the atlas computation method represents the first comprehensive approach to compute motion-robust diffusion tensor maps from noisy in utero fetal measurements and combine individual dti maps into a kernel-regressed template at any given gestational age which would enable statistical voxel-wise analysis, tract-based spatial statistics (tbss), or tract-specific analysis (tsa) based on dti data (pecheva et al., 2017; khan et al., 2018). the neurodevelopmental trends characterized by the atlas in the fetal brain were qualitatively and quantitatively compared with prior ex vivo and in utero studies, and with observations reported in gestational age-equivalent preterm infants. the atlas correctly detected the early presence of limbic fiber bundles followed by the appearance and maturation of projection and association fiber bundles (characterized by an age-related increase in fractal anisotropy) during late 2nd and early 3rd trimesters and the appearance and maturation of projection throughout gestation. during the 3rd trimester association fiber bundles become evident. in parallel with the appearance and maturation of fiber bundles, from 21 to 39 gestational weeks a gradual disappearance of the radial coherence of the telencephalic wall was qualitatively identified. the results presented confirm previous advanced fetal connectome imaging studies indicating increased vulnerability of the human brain during late gestation for pathologies that might lead to impaired connectome development and subsequently interfere with the development of neural substrates serving higher cognition. the atlas complements previous initiatives including recent development of detailed atlases of the fetal (wright et al., 2015) and neonatal (makropoulos et al., 2016) brain that allow robust automated or semi‐automated segmentation of brain regions (makropoulos et al., 2014) and precise delineation of cortical sulcal and gyral development (garcia et al., 2018). together, all these tools allow characterization of the normal trajectories of fetal brain growth and creation of population centile charts (https://www.developingbrain.co.uk/fetalcentiles/ [gousias et al., 2013]). comparison with these typically developing growth charts therefore provides an ideal approach with which to assess, quantify, and identify deviations in regional and whole brain volumes and also in the ontogenesis, architecture, and temporal dynamics of the human brain connectome, and would lead to a more precise understanding of the etiological background of neurodevelopmental and mental disorders. these tools can be used as a reference for dti-based studies on in utero fetal brain development, for groupwise dti studies to investigate normal and abnormal brain development and to enable multi-modality imaging and computer-aided diagnosis from in utero dtis. all these works certainly highlight the significant progresses we have made. however, a lack of specificity between mri signal and neuropathological substrate is reported, as illustrated recently in one study showing that fractional anisotropy (fa) correlated with astrocyte density, a cell type typically not considered in mri studies (stolp et al., 2018). this indicates that the anatomical and histological significance of many “signals” in the brain remain to be investigated and further studies are needed to determine the precise cellular and molecular substrates of abnormal connectivity and gray matter microstructure observed on mri. the new methods of three-dimensional (3d) multiscale histological imaging developed in the last years, including clarity (chung & deisseroth, 2013), map (ku et al., 2016) or shield (park et al., 2018), enable rapid identification of multi-scale functional networks and interrogation of their system-wide, multifactorial interactions and thus, may help in the future for integrative and comprehensive understanding of large-scale complex biological systems. 2. predictive value of childhood neuroimaging as discussed, in utero mri detects fetal brain abnormalities more accurately than ultrasonography and provides additional clinical information in around half of pregnancies. however, it is less accurate when used to predict abnormal developmental outcome, although still better than ultrasonography as shown by hart and colleagues (hart et al., 2020). in their work published in lancet child and adolescent health, they studied the ability of in utero mri to predict developmental outcome and whether performing postnatal neuroimaging after age 6 months changes its diagnostic accuracy. the study was performed in a cohort of children assessed with the bayley scales of infant and toddler development, the ages and stages questionnaire, or both and contrasted with ultrasonography findings. participants' development was categorized as normal, at risk, or abnormal. however, the authors did not find statistically significant differences in infants with abnormal outcome. the authors suggest that although in utero mri remains the optimal tool to identify fetal brain abnormalities, it is not accurate in predicting developmental outcome (hart et al., 2020). further work is needed to determine how the prognostic abilities of in utero mri can be improved to identify putative infant brain markers that might be associated with neurodevelopmental disorders (batalle et al., 2018). instead, some works suggest that mapping the neurodevelopmental trajectories in childhood has the potential to enhance the early identification of risk. whitfield-gabrieli and colleagues analyzed the data from a longitudinal study of 94 children, who underwent resting-state functional mri scans at ages 7 and 11 (whitfield-gabrieli et al., 2019). they explored how specific patterns of brain resting state functional connectivity change during typical development, and how these changes related to behavior. the interesting feature of this study is that children were initially recruited as typically developing, but some began to exhibit clinical symptoms over time. the aim of the study was to identify predictors of anxiety and depression behaviors in children with no familiar risk for these disorders. specifically, the authors explored whether dysregulated top-down control mechanisms can be detected even before behavioral symptoms are evident and can predict individual children’s trajectories of attentional and internalizing problems, given that in adults with depression, anxiety, and adhd a number of studies have shown attenuation or failure of top-down control. the strength of coupling between regions involved in top-down control and their targets can be measured with resting-state functional magnetic resonance imaging (rs-fmri; liu et al., 2018; finn et al., 2015) and reliably characterizes the functional organization of the brain at a systems level (castellanos et al., 2013). the authors focused on functional connectivity involving the default mode network (dmn), a resting-state network associated with internal mentation and self-referential processing, whose key nodes include the medial prefrontal cortex (mpfc). in neurotypical adults, the dmn is negatively correlated (i.e., anticorrelated) with the central executive network (cen; fox et al., 2005), associated with externally focused attention and goal-directed behavior, of which the dorsolateral prefrontal cortex (dlpfc) is a key node. the magnitude of the anticorrelations between the mpfc and the dlpfc is significantly correlated with superior cognitive performance such as working memory capacity (e.g., hampson et al., 2010; keller et al., 2015). in typically developing children, the magnitude of anticorrelations between the mpfc and dlpfc increases with age along with the improvement of top-down control mechanisms. in their study whitfield-gabrieli and colleagues detected individual differences in functional connectivity of the dlpfc that predicted subsequent appearance of symptoms associated with adhd and depression. specifically, weaker positive functional connectivity between the dlpfc and the mpfc at age 7 was associated with a decrease in adhd symptoms by age 11, whereas weaker positive functional connectivity between the dlpfc and the subgenual anterior cingulate cortex was associated with an increase in mood-related symptoms by age 11. in fact, brain connectivity at age 7 predicted mood-related difficulties at age 11 better than baseline clinical symptoms themselves. a limitation of the work is the lack of information about which children eventually developed psychiatric disorders in this sample later on. however, these results suggest the potential utility of connectivity patterns as a biomarker of symptom trajectories. 3. the neurodevelopmental neuropathology of schizophrenia during the last year studies performed in humans have provided clear evidence that mental disorders such as schizophrenia have a strong developmental component that might be identified by specific neuropathological components. earlier neuroimaging works reported gray matter deficits in schizophrenic patients, mostly localized in frontal and temporal lobes, which are present prior to the onset of psychosis and worsen during the first few years of illness (glahn et al., 2008). more recently, widespread white matter decline was shown in whole-brain mri of schizophrenia patients, identifying it as a dysconnectivity syndrome, instead of just a cortical lesion syndrome (fornito et al., 2015). several theories of schizophrenia suggest that structural white matter pathologies may follow developmental (mcgrath et al., 2003; murray et al., 2017), maturational (van haren et al., 2008; french et al., 2015), and/or degenerative (cropley et al., 2017) trajectories. cetin-karayumak et al. (cetin-karayumak et al., 2019) have addressed this question and also studied whether structural white matter pathologies vary among fiber tracts across the brain. to this aim they analyzed the largest sample of harmonized diffusion mri data to comprehensively characterize age-related white matter neuropathology, as measured by fa. the analysis comprised diffusion scans of 600 schizophrenia patients and 492 healthy controls at different illness stages and ages (14–65 years), gathered from 13 sites. the authors cross-sectionally determined the pattern of age-related fa changes associated with schizophrenia. in whole-brain white matter, fa was up to 7% lower across the lifespan and reached peak maturation earlier in patients (27 years) than controls (33 years), and three distinct patterns of neuropathology could be identified. they detected tract-specific early developmental abnormalities in limbic fibers that do not progress over time, suggesting that limbic connections are selectively vulnerable to early developmental anomalies. instead, long-range intra-hemispheric association tracts (including language tracts) displayed abnormal maturation with shorter maturational windows and faster declines consistent with accelerated ageing processes in schizophrenia. finally, the authors report accelerated aging in callosal fibers that exhibited severe deficits from the outset of illness, which became more pronounced with increasing age (reaching a 10% reduction after sixth decade). this reduced anisotropy of the corpus callosum is a well-replicated diffusion imaging finding in schizophrenia, consistent across heterogeneous patient populations and maturational phases. the study makes a timely and important contribution to the field showing that white matter neuropathology in schizophrenia involves lifelong dynamic tract-specific changes. the findings support a developmental perspective, suggesting that widely distributed white matter deficits emerge early or display perturbed maturation. in addition, callosal and long-range association (but not limbic) fibers undergo accelerated aging processes. this work provides an initial benchmark for tract-specific trajectories of white matter abnormalities. 4. disease-in-a-dish developmental neuropathology in recent years there has been a growing emphasis on developing patient-specific cellular models that can be manipulated by the experimenter to understanding the role of different factors in shaping individual brain development and functioning. human pluripotent stem cells (hpscs), including human embryonic stem cells (hescs) and human induced pluripotent stem cells (hipscs), have been revealed as invaluable tools for modeling human disorders, especially those with complex genetic origins (takahashi et al., 2007; takahashi & yamanaka, 2006). hpscs have the potential to differentiate into any cell or tissue type. induced pluripotent stem cells (ipscs) reprogrammed from patient somatic cells also offer an opportunity to recapitulate disease development in relevant cell types, and they provide novel approaches for understanding disease mechanisms. stem cells can be used to generate organoids, organ-like 3d tissue cultures containing multiple cell types that represent accessible systems for modeling organogenesis and developmental disorders (lyon, 2019). hpsc-derived brain organoids self-assemble to form an organized architecture, composed of progenitor, neuronal and glial cell types, resembling the fetal human brain (jo et al., 2016; kadoshima et al., 2017; lancaster et al., 2013). until recently, these in vitro systems had strong limitations. first, organoids develop without the presence of normal embryonic surrounding thus lacking the developmental and patterning cues, which are essential for organ development. additionally, most protocols depend on the ability of stem cells to self-organize into distinct brain structures which can cause inconsistency in producing the desired tissues, resulting in heterogeneity or “batch-effects” in different batches of organoids, which can vary in quality and brain regions they generate, making it difficult to identify real phenotypes. human brain organoids are prone to high organoid-to-organoid variability (quadrato et al., 2016; yoon et al., 2019; grenier et al., 2020). furthermore, the absence of vascularization is probably responsible for the shortage of progenitor populations, making it difficult to replicate cortical plate formation. all of this has raised doubts as to whether developmental processes of the human brain can occur outside the context of embryogenesis with a degree of reproducibility that is comparable to the endogenous tissue (jabaudon & lancaster, 2018). the last years have been exciting for the field, because some of these limitations are starting to be overcome. recently, mansour et al. (mansour et al., 2018) showed that intracerebral transplantation of brain organoids in mice results in impressive growth of blood vessels into the human tissue, with clear benefits for cell survival and maturation compared with organoids kept in vitro. also, real and colleagues (real et al., 2018) have shown the potential of these methods to model human neuropathology. they transplanted human ipsc-derived cortical neurons from two persons with down syndrome into the adult mouse cortex and observed that those consistently organized into large (up to ~100 mm3) vascularized neuron-glia territories. down syndrome transplants showed increased synaptic stability and reduced oscillations, thus recapitulating in part the patients’ phenotypes. finally velasco and colleagues (velasco et al., 2019) showed that an organoid model of the dorsal forebrain can reliably generate a rich diversity of cell types appropriate for the human cerebral cortex. using single-cell rna-sequencing analysis of 166,242 cells isolated from 21 individual organoids, the authors show that the organoid-to-organoid variability is comparable to that of individual endogenous brains, and 95% of the organoids derived from different stem cell lines generate a virtually indistinguishable compendium of cell types, showing consistent reproducibility in the cell types produced. the authors thus demonstrated that establishment of terminal cell identity is a highly constrained process and that reproducible developmental trajectories of cellular diversity of the cns does not require the context of the embryo. 5. growing networks in a dish most of the current organoid protocols or medium formulations favor progenitor cells, thus not enabling them to mature and mimic some aspects of the human brain development such as dynamic changes in cellular populations during maturation or the formation of long-range connectivity. in a recent work, trujillo and colleagues (trujillo et al., 2019) developed cortical organoids that spontaneously displayed periodic and regular oscillatory network events that are dependent on glutamatergic and γ-aminobutyric acid (gaba)-ergic signaling. they could record consistent increases in electrical activity over the span of several months reflecting the formation of a spontaneous network that displayed periodic and regular oscillatory events. these nested oscillations exhibited cross-frequency coupling, subsequently transitioning to more spatiotemporally irregular patterns, resembling features observed in preterm human electroencephalography (eeg). these results suggest that the development of structured network activity in the human neocortex takes place even in the absence of external or subcortical inputs, and open opportunities for investigating and manipulating the role of network activity in the developing human cortex. two independent groups (cullen et al., 2019; kirihara et al., 2019) have developed a human stem cell-derived model of cerebral tracts and have described the phenotype and connectivity of constrained 3d human axon tracts derived from brain organoids. in the work of kirihara and colleagues they used a microfluidic device, in which two spheroids of cortical neurons derived from hipscs extended axons into a microchannel between the spheroids and spontaneously formed an axon fascicle, mimicking a cortico-cortical tract that connected the two spheroids reciprocally. the axon fascicle was able to communicate electrically between the spheroids with distinct response kinetics. this cerebral tract model should provide a promising platform to study the mechanisms underlying cerebral tract development and related diseases. using their model of cerebral tracts, the authors could recapitulate the agenesis of corpus callosum (edwards et al., 2014), by knocking down l1cam gene (demyanenko et al., 1999; siegenthaler et al., 2015) in the spheroids. axons from the l1cam knockdown cells exhibited significantly lower ratio of axons assembled into a bundle than the control cells, suggesting that this method can be used to model developmental disease related to cerebral tracts. microfluidic devices, involving a two-compartment system connected by narrow grooves, have also been used to reconstruct dentate gyrus (dg)–cornu ammonis area 3 (ca3) circuitry in which dg neurons are cultured in one compartment and ca3 neurons are cultured in the other compartment (sarkar et al., 2018). in this system, axonal growth is allowed through the narrow grooves connecting the two compartments whereas cell migration is restricted. rabies virus infection of the ca3 neurons permits the detection of presynaptic neurons that monosynaptically connect to the postsynaptic ca3 neurons. these engineered neural tissue connectivity models represent a first step toward potentially reconstructing brain circuits by physically replacing neuronal populations and long-range axon tracts in the brain, and might reveal how long-range connections are altered in the brains of people with neurodevelopmental disorders. 6. genes involved in early cortical patterning are at the heart of mental comorbidity over the past decade, genetic studies have been quite successful at identifying rare genetic variations, including inherited and de novo mutations and copy number variations (cnvs), related to specific developmental disorders. however, evidence is mounting to suggest that genetic risk variants identified among individuals with different brain disorders may converge on common genetic pathways. the remarkably complex architecture that embraces genetic mutations of distinct types (chromosomal rearrangements, copy number variants, small indels, and nucleotide substitutions) with distinct frequencies in the population (common, rare, de novo) creates difficulties in establishing rigorous genotype-phenotype correlations (cardoso et al., 2019). moreover, the cumulative effect of multiple common genetic variants, i.e., polygenic risk, is now being recognized as an important indicator of neurodevelopmental and psychiatric disorders (cross-disorder group of the psychiatric genomics consortium, 2013). large-scale genome-wide asd and cross-disorder association studies with enough statistical power to estimate small effects from common genetic variants are only now emerging, and they require combining data sets from multiple, large population samples. this need has been long recognized as testified by initiatives such as the psychiatric genomics consortium (pgc), the largest consortium in the history of psychiatry (sullivan et al., 2018). now, in the largest-ever study of its kind, published in cell, lee et al. (cross-disorder group of the psychiatric genomics consortium, 2019) identified more than 100 genetic variants that affect the risk for more than one mental health condition, indicating that distinct psychiatric diseases share a common genetic structure, as shown. using genome-wide association to analyze genetic data from 494,162 healthy controls and 232,964 individuals diagnosed with at least one of eight common psychiatric disorders, the researchers identified 109 gene variants that affect the risk for more than one psychiatric disorder. certain disorders shared many variants, allowing the researchers to divide the conditions into three groups of genetically-related conditions: disorders characterized by compulsive behaviors (anorexia nervosa, obsessive-compulsive disorder and, to a lesser extent, tourette syndrome); mood and psychotic disorders (bipolar disorder, major depression and schizophrenia); and early-onset neurodevelopmental disorders (asd, adhd and tourette syndrome). importantly, genes associated with multiple disorders showed increased expression beginning in the second trimester of pregnancy and play an important role in brain development. another research team has found the first common genetic risk variants for autism robustly associated with asd (grove et al., 2019) in a genome-wide association meta-analysis carried out in a danish population resource of 18,381 individuals with asd and 27,969 controls. the meta-analysis identified five genome-wide-significant loci. moreover, the study identified several asd gene variants that had especially widespread influence on the risk for a number of psychiatric disorders. concretely, the authors report seven loci shared with other traits, obtained by combining the dataset with genome-wide association study (gwas) results from phenotypes with significantly overlapping genetic architectures (schizophrenia, major depression, and educational attainment). the researchers also uncovered, for the first time, genetic differences between the different clinical subgroups of autism. this will pinpoint genes that separate the diagnostic groups and enable more precise diagnosis, and could orient the search for behavioral, imaging and electrophysiological markers of atypical development in the infant brain. these genetic findings provide an entirely new insight into the cross-disorder biological processes, particularly relating to neuronal function and corticogenesis, and help to triangulate on families of genes active during early cortical patterning (morgan et al., 2019; figure 1), associated with the establishment and maintenance of neuronal connectivity (parikshak et al., 2015) or with the foundation of inhibitory/excitatory balance in early childhood (marin, 2012).   figure 1. left panel: large-cohort genome-wide studies are allowing to triangulate families of genes involved in early brain maldevelopment. middle: early cortical patterning is at the heart of mental comorbidity. the advances in single-cell omics will help understand the pathogenesis of developmental lesions. right panel: critical steps of brain connectivity development have possible links to in utero mri-detectable phenomena. for example, emergence of long-range connectivity may be detected by in-utero diffusion tensor tractography as increasing integration demonstrated using whole-brain diffusion tractography. 7. mechanistic role of rna editing in schizophrenia and autism neuropathology we have progressively realized in recent years that the molecular neuropathology landscape of brain disorders is much more complex than anticipated and new players, such as epigenetic regulation, alternative splicing, post-transcriptional and -translational modifications, and somatic mosaicism of dna, not only in protein-coding sequences but also in large non-coding regions, contribute to developmental neuropathology. transcriptomic analyses of postmortem brains have begun to elucidate some of these new mechanisms in asd and schizophrenia. however, a crucial pathway involved in synaptic development and neuronal function, rna editing, has not yet been studied on a genome-wide scale. rna editing is a major rna processing mechanism, which refers to the alteration of rna sequences through insertion, deletion or substitution of nucleotides. it is thought to constitute one of the molecular mechanisms connecting environmental stimuli and behavioral outputs (lapp & hunter, 2019). adenosine to inosine (a-to-i) editing is the most common form of rna editing (mallela & nishikura, 2012; figure 2), affecting the majority of human genes, and is highly prevalent in the brain (liscovitch et al., 2014; suarez et al., 2018). editing sites in coding regions can be conserved across species and are commonly located in genes involved in neuronal function. site-specific a-to-i rna base conversions, carried out by adenosine deaminase acting on rna (adar) enzymes, exhibit precise regional specificity in the brain and modulate complex behavior in model organisms. examples of transcripts edited by adar are mrnas encoding glutamate receptors, serotonin receptors, and potassium channels and other neuronal signaling functions in the cns (rosenthal & seeburg, 2012; meier et al., 2016; streit & decher, 2011). these sites are tightly and dynamically regulated throughout preand post-natal human cortical development (figure 2). most editing sites reside in non-coding regions, and particularly within sequences called transposons via rna intermediates (retrotransposons, see below) with a possible pathophysiological role (krestel & meier, 2018). aberrant rna editing has been reported in several neurological disorders, including major depression, alzheimer’s disease, and amyotrophic lateral sclerosis.   figure 2. elevated adar2 expression in neuronal nuclei increases a-to-i rna editing during neuronal maturation. left panel: adar enzyme acting on double-stranded rna. adar rna-binding domains act on rna binding sites and convert adenine to inosine. right panel: as neurons mature, a-to-i rna editing increases gradually together with expression of importin-a4. as a result, adar is elevated in the nucleus leading to increased a-to-i rna editing in mature neurons. credits: ilario de toma, adapted from behm et al., 2017 and lorenzini et al., 2018. in asd, recent studies in postmortem samples have revealed transcriptome dysregulation affecting neuronal and glial coding and non-coding gene expression, neuronal splicing including microexons, and microrna targeting (irimia et al., 2014; salloum-asfar et al., 2019; gandal et al., 2018). tran et al. (tran et al., 2019) have now tested the largest cohort of post-mortem asd brain samples, spanning multiple brain regions for abnormalities in rna editing. a previous study had analyzed a few known rna editing sites in synaptic genes and reported altered editing patterns in a small cohort of asd cerebella (eran et al., 2013), but it was not known whether global patterns of rna editing contributed to the neuropathology of asd. now tran and colleagues report global patterns of dysregulated rna editing, with hypoediting across brain regions and involving many synaptic genes in asd brains. the set of genes harboring at least one differential editing site in frontal cortex exhibited significant gene ontology (go) enrichment for ionotropic glutamate receptor activity, glutamate gated ion channel activity, and synaptic transmission. the authors also identified a core set of down-regulated rna editing sites, enriched in asd susceptibility genes. interestingly, a set of these hypoedited sites are related to fragile x syndrome (fxs) proteins. concretely, fmrp and fxr1p interact with adar and modulate a-to-i editing. the authors detected convergent dysregulated patterns of rna editing in fxs and asd patients, consistent with the findings that genes harboring asd risk mutations are enriched in fmrp targets. their findings were corroborated across multiple datasets, including dup15q cases associated with intellectual disability. regarding schizophrenia, breen and colleagues (breen et al., 2019) have analyzed the global landscape and genetic regulation of rna editing across several hundred schizophrenia and control postmortem brain samples from the dorsolateral prefrontal cortex and anterior cingulate cortex. in schizophrenia, rna editing sites in genes encoding ampa-type glutamate receptors and postsynaptic density proteins were less edited, whereas those encoding translation initiation machinery were more edited. these findings were cross-validated in hundreds of non-overlapping dorsolateral prefrontal cortex samples. furthermore, ~30% of rna editing sites associate with cis-regulatory variants (editing quantitative trait loci or edqtls). fine-mapping edqtls with schizophrenia risk loci revealed co-localization of eleven edqtls with six gwas loci. the findings demonstrate widespread altered rna editing in schizophrenia and its genetic regulation, and suggest a causal and mechanistic role of rna editing in schizophrenia neuropathology. overall, both papers provide global insights regarding rna editing in asd and schizophrenia pathogenesis and reveal novel mechanisms underlying these disorders. 8. incomplete silencing of full mutation alleles in males with fragile x syndrome is associated with autistic features the paper by tran et al. (tran et al., 2019) convincingly demonstrated that rna editing acts as a molecular link between fxs and asd. fxs is caused by loss of the fmr1 product (fmrp), but also by mosaicism for active and inactive fmr1 alleles, including alleles termed premutation (55–199 cggs). importantly, both premutation and active full mutation (≥ 200 cggs) alleles often express elevated levels of mrna that are thought to be toxic. two studies published in 2019 report that incomplete silencing of toxic full mutation rna may be associated with autistic features in fxs males. baker and colleagues (baker et al., 2019) studied whether complete fmr1 mrna silencing from full mutation alleles and/or levels of fmr1 mrna (if present) in blood were associated with intellectual functioning and autism features in fxs. the study cohort included 98 participants (70.4% male) with fxs (full mutation-only and mosaic). fmr1 mrna was analyzed against control fmr1 mrna and correlated with intellectual disability and autistic features. fmr1 was completely turned off in some of the patients, while it was partially turned on in the majority of participants. females with fxs had significantly higher levels of fmr1 mrna that were not associated with intellectual functioning nor autistic features. in fxs males decreased levels of fmr1 mrna were associated with decreased intellectual functioning, but not autism features. the authors show for the first time that people with a partially turned-on fmr1 had intellectual disability and more traits of asd, whereas people with the gene completely turned off had intellectual disability and much less severe autism. the authors conclude that abnormally elevated levels of fmr1 mrna may lead to fmr1 mrna-related cellular “toxicity”. these findings may explain why severity of fragile x is not the same between affected individuals. based on this study, some months later field et al. (field et al., 2019) described the case of two young brothers with expanded fmr1 alleles, who were ‘high functioning’. the two brothers presented autistic features and language delay, but a higher non-verbal iq in comparison to typical fxs. both had low-level methylation mosaicism not detected by standard testing in blood, and their fmr1 mrna levels were increased ~5-fold compared to typical developing controls, and significantly above the levels reported from baker’s study described above. the authors speculate that the active unmethylated full mutation and/or premutation alleles lead to the expression of toxic expanded mrna in some cells, in conjunction with possible reduced fmr1 mrna and fmrp levels in other cells with fmr1 methylation. both mechanisms may contribute to the elevated asd symptoms. this hypothesis is also in line with the findings reported by baker et al. showing that males who expressed full mutation fmr1 mrna, had significantly more severe asd symptoms compared to males who had completely silenced fmr1. 9. transposable elements in neurodevelopmental disorders transposable elements (tes) constitute about half of the human genome and are becoming increasingly important to the field of neuroscience as their roles in mammalian development, immune response, and contributions to behavioral and cognitive domains continue to be uncovered (international human genome sequencing consortium, 2001; nandi et al., 2016). transposons are mobile dna elements present in virtually all eukaryotes that can replicate and mobilize from one chromosomal loci to another through either a dna or rna intermediate (levin & moran, 2011). retrotransposons are often called ‘jumping genes’, because the messenger rna transcribed from them can undergo a process called reverse transcription to produce an identical dna sequence that then reinserts into the genome at a different site. they parallel viruses in many ways in their structure and function, as they ensure their own survival by way of reintegration. their propensity to self-propagate has a myriad of consequences and yet their biological significance is not well-understood. although retrotransposons comprise about 42% of the human genome, most carry mutations that render them functionally inactive (cordaux & batzer, 2009). retrotransposons are emerging as potent regulatory elements within the human genome. transcription of those that remain functional must be prevented by proteinor rna-based regulatory mechanisms to prevent the jumping of retrotransposons, which can cause either genetic mutations or genomic instability and might lead to cancer (scott & devine, 2017). moreover, retrotransposons can be reactivated during ageing (de cecco et al., 2013). human endogenous retroviruses (herv) and long-interspersed nuclear element-1 (line-1) are two main classes of retrotransposons, mobilized through a “copy and paste” mechanism. line-1 somatic retrotransposition has been well-demonstrated to occur in neuronal lineage, however the significance of retroelement activity to normal brain function remains uncertain. furthermore, the contribution of these endogenous retroelements to the etiopathogenesis of neurodevelopmental disorders is a topic of recent exploration. roughly 130 pathogenic variants caused by retrotransposon activity have been documented, but the majority of these deleterious events are isolated cases. as such, they are not part of routine clinical sequencing, and thus represent a largely unassessed category of genetic variation in many disorders. human-specific line-1 (l1hs) is the most active autonomous retrotransposon family in the human genome. mounting evidence supports that l1hs retrotransposition occurs postzygotically in the human brain cells, contributing to neuronal genomic diversity. in a paper in 2018 jacob-hirsch et al. (jacob-hirsch et al., 2018) already reported that the number of retrotranspositions is higher in brain tissues than that in non-brain samples and even higher in pathologic vs. normal brains. their findings documented that l1hs elements integrate preferentially into genes associated with neural functions and diseases. the authors propose that pre-existing retrotransposons act as “lightning rods” for novel insertions, which may safeguard from deleterious events and thus, uncontrolled retrotransposition may breach this safeguard and increase the risk of harmful mutagenesis in neurodevelopmental disorders. zhao and colleagues (zhao et al., 2019) profiled genome-wide l1hs insertions among 20 postmortem tissues from rett patients and matched controls. they identified and validated somatic l1hs insertions in both cortical neurons and non-brain tissues, with a higher jumping activity in the brain and concluded that mecp2 dysfunction might alter the genomic pattern of somatic l1hs in rett patients. now gardner et al. (gardner et al., 2019) have identified retrotransposition-derived events in 9738 exome sequenced trios with developmental-affected probands. they encountered 9 de novo retrotransposons and 2 de novo gene retro-duplications. from those, 4 transposons were the likely cause of the symptoms of four patients, three of whom had not had previous diagnoses. these studies open a possibility of “diagnostic retrotransposition events” and are another step along the path to understanding the causes of developmental disorders. 10. single-cell genomics identifies cell type–specific molecular changes in autism i have presented several efforts and advances to disentangle important elements in the molecular neuropathology of neurodevelopmental disorders. one limitation, however, is that most postmortem studies apply bulk omic approximations to a complex tissue, the brain, in which diverse human cells may contribute differently to neurodevelopmental disorders. in asd, for example, previous bulk gene expression studies identified common genes and pathways dysregulated in the neocortex of autism patients that did not explain its clinical and genetic heterogeneity. direct assessment of specific cell types in the brain affected by neurodevelopmental disorders has not been feasible until recent advances in techniques for isolating thousands of intact cells and efficiently sequencing each of them. single-cell rna-sequencing (scrna-seq) has evolved over the past few years as a high-throughput method for transcriptome profiling of thousands of cells (tasic, 2018) and has identified diverse cell types in many brain regions, including neocortex (tasic et al., 2018), hypothalamus (campbell et al., 2017), and retina (shekhar et al., 2016). this will allow scientists to explore neurodevelopmental neuropathology at a resolution that was not possible before. an international consortium called the human cell atlas (https://www.humancellatlas.org/) is an effort to identify every human cell type, where each type is located in the body, and how the cells work together to form tissues and organs. another effort, a collaboration of 53 institutions and 60 companies across europe, called the lifetime initiative (https://lifetime-fetflagship.eu/), is proposing to harness single-cell technologies to understand what happens cell by cell as tissues progress toward diseases. transcriptomic profiling of complex tissues by scrna-seq has unfortunately also limitations, the most important being that neurons, being complex arborizing cells, are vulnerable to mechanical dissociation and sorting. this makes scrna-seq hard to apply to adult brain tissue to tease out individual cells from the elaborately tangled circuitry of the brain. few studies have analyzed neurodevelopmental disorders at a single-cell resolution. a turning point came last year, when velmeshev and colleagues (velmeshev et al., 2019) used newer techniques to extract rna from cell nuclei isolated from samples of brain tissue and then analyzed signature patterns of gene expression of single brain cells, including neurons and glia, from patients with autism. single-nucleus rna-sequencing (snrna-seq) affords some advantages over scrna-seq. in contrast to whole cells, nuclei are more resistant to mechanical assaults and can be isolated from frozen tissue (krishnaswami et al., 2016), so that snrna-seq provides less biased cellular coverage than scrna-seq, since it suffers less cell isolation-related transcriptional artifacts (some cell types are more vulnerable to dissociation process and are thus underrepresented in the final scrna-seq data set (lake et al., 2016)). velmeshev et al. applied this single-nuclei sequencing approach to snap-frozen post-mortem samples of prefrontal cortex and anterior cingulate cortex, two brain regions previously shown to be altered in patients with autism. snrna-seq analysis of more than 100,000 cell nuclei identified a common set of changes in genes involved in synaptic communication as well as neural outgrowth and migration. previous studies suggested convergence of asd on specific cell types during fetal development (parikshak et al., 2013; willsey et al., 2013). furthermore, in patient samples, specific sets of genes enriched in upper-layer cortico-cortical projection neurons and microglia correlated with clinical severity. they also found changes in glial cells that could impact their role in pruning and maintaining healthy neural circuits. many of the differentially expressed genes are known to be widely expressed across the entire brain, but they appeared to be significantly altered only in projection neurons of asd. these findings suggest that molecular changes in cell types with shared developmental lineages exhibit convergent transcriptional changes in adult asd patients, and that the expression of synaptic and neurodevelopmental genes in layer 2/3 cortical neurons is especially affected. this implies that disturbances of gene regulatory programs during development cascade into molecular pathology in specific mature neural cell types, such as upper-layer projection neurons and microglia, which correlate with the clinical severity of asd. one limitation of the study is the small cohort size, since the authors compared brain samples from only 15 people with autism and 16 people who died in the same age range (4-22 years) of non-neurological causes. since almost half of the patients with autism also suffered from seizures, the researchers examined brain samples from a cohort of patients with sporadic epilepsy, to identify brain changes more likely to stem from seizures than primary asd–associated gene expression changes. another general limitation is that, because cells must be removed from the brain, single cell omic techniques alone do not reflect how those cells interact with their neighbors or circuit level changes. future studies involving larger patient cohorts, including whole-exome sequencing and improved single-cell technologies, are needed to allow for precise identification of asd-driven changes and their association with genetic variants. the authors provide an interactive web browser to interrogate their transcriptomic data: https://autism.cells.ucsc.edu. colophon neurodevelopmental disorders are inherently complex, involving multiple components of an intricate network. the multi-dimensional nature of the developmental disorders suggests that no unifying “cause” but instead multi-level perturbation causes complex neurodevelopmental disorders with a host of systemic comorbidities and striking heterogeneity. many research groups are still trying to decipher the genomic complexity of disorders such as autism, and the list of autism “risk” genes grows each year. a number of new unexpected molecular players such as rna editing and retrotransposons have been identified, and their impact on brain development is being examined. because a deep understanding of these underlying mechanisms could prove seminal for personalized medicine, researchers have designed new cellular models with reasonable construct face and predictive validity. of course, other factors such as the contribution of individual lifestyles, cannot be dismissed as they are fueling a large spectrum of gene-environment interactions that have a key role in the genotypes /phenotypes relationships. a more thorough understanding of the intricate processes underlying normal and abnormal human cns development is needed to answer many fundamental questions in biology and medicine and will lead to meaningful clinical biomarkers of developmental neuropathology in humans. funding and acknowledgements this work was supported by the fondation jérôme lejeune, paris, mineco (saf2016-79956-r, h2020 sc1 go-ds21848077, eu (jpnd heroes (the crossroad of dementia syndromes). the crg is a center of excellence severo ochoa sev-2016-0571. the ciber of rare diseases is an initiative of the isciii. the laboratory of mara dierssen is supported by diue de la generalitat de catalunya (grups consolidats 2017 sgr 926). we also acknowledge the support of the spanish ministry of science and innovation to the embl partnership, the centro de excelencia severo ochoa and the cerca programme/generalitat de catalunya. references baker, e.k., arpone, m., aliaga, s.m., bretherton, l., kraan, c.m., bui, m., slater, h.r., ling, l., francis, d., hunter, m.f., elliott, j., rogers, c., field, m., cohen, j., cornish, k., santa maria, l., faundes, v., curotto, b., morales, p., trigo, c., salas, i., alliende, a.m., amor, d.j. & godler, d.e. 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(2019) somatic line-1 retrotransposition in cortical neurons and non-brain tissues of rett patients and healthy individuals. plos genet, 15, e1008043. copyright: © 2020 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. friedreich cardiomyopathy is a desminopathy feel free to add comments by clicking these icons on the sidebar free neuropathology 2:34 (2021) original paper friedreich cardiomyopathy is a desminopathy arnulf h. koeppen1,2, rahman f. rafique1, joseph e. mazurkiewicz3, steven pelech4,5, catherine sutter4, qishan lin6, jiang qian7 1 research service, veterans affairs medical center, albany, ny, usa 2 departments of neurology and pathology, albany medical college, albany, ny, usa 3 department of neuroscience and experimental therapeutics, albany medical college, albany, ny, usa 4 kinexus bioinformatics corporation, vancouver, bc, canada 5 department of medicine, university of british columbia, vancouver, bc, canada 6 rna epitranscriptomic & proteomics resource, university at albany, albany, ny, usa 7 department of pathology, albany medical college, albany, ny, usa corresponding author: arnulf h koeppen, md · research service (151) · va medical center · 113 holland ave · albany, ny · 12208, usa arnulf.koeppen@med.va.gov submitted: 11 november 2021 accepted: 08 december 2021 copyedited by: vanessa s. goodwill published: 13 december 2021 https://doi.org/10.17879/freeneuropathology-2021-3679 keywords: friedreich ataxia, cardiomyopathy, proteomics, desmin, αb-crystallin, desminopathy abstract heart disease is an integral part of friedreich ataxia (fa) and the most common cause of death in this autosomal recessive disease. the result of the mutation is lack of frataxin, a small mitochondrial protein. the clinical and pathological phenotypes of fa are complex, involving brain, spinal cord, dorsal root ganglia, sensory nerves, heart, and endocrine pancreas. the hypothesis is that frataxin deficiency causes downstream changes in the proteome of the affected tissues, including the heart. a proteomic analysis of heart proteins in fa cardiomyopathy by antibody microarray, western blots, immunohistochemistry, and double-label laser scanning confocal immunofluorescence microscopy revealed upregulation of desmin and its chaperone protein, αb-crystallin. in normal hearts, these two proteins are co-localized at intercalated discs and z discs. in fa, desmin and αb-crystallin aggregate, causing chaotic modification of intercalated discs, clustering of mitochondria, and destruction of the contractile apparatus of cardiomyocytes. western blots of tissue lysates in fa cardiomyopathy reveal a truncated desmin isoprotein that migrates at a lower molecular weight range than wild type desmin. while desmin and αb-crystallin are not mutated in fa, the accumulation of these proteins in fa hearts allows the conclusion that fa cardiomyopathy is a desminopathy akin to desmin myopathy of skeletal muscle. introduction heart disease in friedreich ataxia (fa), hereafter called fa cardiomyopathy, is the most common cause of death in this otherwise rather neurological disease. fa cardiomyopathy was introduced as an integral part of fa by russell (1946), and there is no doubt that the cardiac lesion is due to frataxin deficiency (perdomini et al., 2014). under the microscope, sections of fa heart show multiple abnormalities, including fiber hypertrophy (koeppen et al., 2015), fiber necrosis, fibrosis, inflammatory infiltration, chaotic disorganization of intercalated discs, and accumulation of iron in a small percentage of cardiomyocytes without iron excess in the whole heart (ramirez et al., 2012; kruger et al., 2016). the reason for the highly variable pathological phenotype of fa as it affects heart, central and peripheral nervous systems, eyes, and endocrine pancreas has not been fully established. the current hypothesis is that structural and signaling proteins are affected downstream from cellular frataxin deficiency. on antibody microarrays and western blots, desmin and its chaperone, αb-crystallin, are upregulated in fa cardiomyopathy. desminopathy is well known from the study of desmin-related skeletal myopathies, in which cardiomyopathy is frequent (dalakas et al., 2000; goldfarb et al., 2004; hnia et al., 2015). desmin mutations cause aggregation in skeletal muscle, and some experiments have suggested that posttranslational modifications of desmin, such as phosphorylation and proteolytic fragmentation, make the protein amyloidogenic (agnetti et al., 2014; kedia et al., 2015). based on the observations detailed in this report, the authors propose that fa cardiomyopathy is a protein aggregation myopathy involving desmin and αb-crystallin. in its biogrid data base of protein, genetic, and chemical interactions (version 4.3.196), uniprot (2021) lists 96 desmin-interacting proteins. among them, αb-crystallin is prominently recognized as a desmin partner or “chaperone”. indeed, αb-crystallin mutations cause “desmin” myopathy (goldfarb and dalakas, 2009). while desmin is an abundant cytoskeletal protein, it also has signaling properties, and the term “mechanochemical” may be applicable (hnia et al., 2015). material and methods autopsy specimens: the institutional review board of the veterans affairs medical center in albany, ny, usa has approved this investigation. heart autopsy specimens were obtained from 35 patients with fa through a national fa tissue repository in albany, ny. thirty-three patients had homozygous guanine-adenine-adenine (gaa) trinucleotide repeat expansions, and two were compound heterozygotes (becker et al., 2017). thirty-four patients had clinical heart disease or fa cardiomyopathy by histology. one fa patient had no heart disease during life or under the microscope. normal hearts were made available by national disease research interchange (philadelphia, pa, usa). at the time of autopsy, a transverse slice weighing approximately 50 g, was made midway between the apex and atrioventricular groove, and immediately frozen at -20°c or, if available, at -80°c. the remainder of the heart was fixed in cold phosphate buffered formalin (ph 7.4). samples of formalin-fixed left ventricular wall (lvw) were embedded in paraffin and sectioned at 6 µm for routine stains, immunohistochemistry, laser scanning confocal congo red fluorescence microscopy, and double-label immunofluorescence with antibodies to desmin and αb-crystallin, or desmin and atp synthase f1 subunit β (atp5b), a mitochondrial marker. antibody microarray: the principles of this proteomic method, advantages, and pitfalls were described previously in detail (yue and pelech, 2018). tissue samples were homogenized by sonication at ice temperatures in a lysis buffer containing (concentrations in parentheses) 3-morpholinopropane-1-sulfonic acid buffer ph 7.2 (20 mm), ethylene glycol-bis (β-aminoethyl ether), n,n,n’,n’-tetra acetic acid (2 mm), ethylenediaminetetraacetic acid (2 mm), sodium fluoride (50 mm), β-glycerophosphate (60 mm), sodium pyrophosphate (25 mm), sodium orthovanadate (5 mm), phenyl arsine phosphate (50 nm), triton x-100 (1%), and sodium dodecylsulfate (0.05%). the lysis buffer also contained a protease inhibitor cocktail of aprotinin (0.5 µm), benzamidine (3 mm), 4(2-aminoethyl) benzene sulfonyl fluoride hydrochloric acid (1 mm), leupeptin (10 µm); and dithiothreitol (1 mm) to disrupt disulfide bonds. protein concentrations were determined by bradford’s method (1976). proteins were cleaved at cysteine residues by the addition of tris (2-carboxyethyl) phosphine hydrochloride (10 mm) to the lysis buffer, followed by 2-nitro-5-thiocyanatobenzoic acid (100 mm) after sonication. pooled lysates were biotinylated (yue et al., 2017) and layered over 2 microarrays displaying, respectively, 900 and 1150 antibodies to a total of 2,050 antibodies (kinexus bioinformatics, vancouver, bc, canada). proteins bound to the antibody spots were detected by incubation with a 1:1 mixture of cy3and alexa 546-labeled anti-biotin. the fluorescent signals from the scanned microarrays were captured in a tif file image. signals were then quantified with imagene 9.0 microarray image analysis software (biodiscovery, el segundo, ca, usa) and normalized according to the sum of all the recorded signals per field. the values for duplicate measurements were averaged, and the mean value for the difference in duplicates from the average was typically ~10%. processing lysates of fa and control hearts in triplicate allowed statistical determination of significant upor down-regulation of proteins in fa by the student t-test. frataxin assay by enzyme-linked immunosorbent assay (elisa): details of frataxin elisa were published before (koeppen et al., 2015), but the amplifying steps with biotinylated anti-rabbit igg and horse radish peroxidase-labeled streptavidin were omitted. the reason for the change was the presence of biotin in non-fat dry milk that was used as a suppressor protein. briefly, heart lysates obtained by the method of condò et al. (2006) were diluted 10-fold in phosphate-buffered saline (ph 7.2) (pbs) to reduce the concentration of nonionic detergents to less than 0.1%. the diluted lysates were centrifuged through membrane filter units with a molecular mass cutoff of 30 kda (millipore sigma, burlington, ma, usa). wells of polystyrene elisa plates were coated with a layer of monoclonal anti-frataxin (abcam, cambridge, ma, usa, cat. no. ab268063), followed by aliquots of filtered lysates (corresponding to 4 mg original tissue) or human recombinant frataxin (10 pg to 1 ng; abcam, cat. no. ab95502). the sequence of subsequent additions (washing steps omitted) was polyclonal anti-frataxin (abcam, cat. no. ab175402) and horseradish peroxidase-labeled goat anti-rabbit gamma globulin (sigma, st. louis, mo, usa, cat. no. a4914). the chromogenic solution contained ortho-phenylenediamine (2 mm) and 0.01% hydrogen peroxide in sodium phosphate-citric acid buffer (ph 5). absorbances at 492 nm were determined in an elisa plate reader manufactured by molecular devices (san josé, ca, usa). frataxin levels were expressed as ng/g wet weight. coimmunoprecipitation: pooled tissue lysate of 6 fa hearts, corresponding to 150 mg wet tissue in a volume of 150 µl, were mixed with 5 µg rabbit polyclonal anti-desmin and 50 µl of a slurry of protein a on sepharose beads (thermo scientific, rockford, il, usa, cat. no. 29333). after overnight incubation at 4 ͦc, the sepharose particles were collected by brief centrifugation and washed with phosphate-buffered saline (pbs). bound proteins were released from protein a-sepharose by laemmli’s sample buffer and heating at 100 ͦc for 15 min. after cooling, the remaining sepharose particles were precipitated by brief centrifugation, and 24 µl aliquots were electrophoresed on 10% polyacrylamide gels. at the end of the electrophoresis, proteins were transferred by electroblotting onto polyvinyledenefluoride (pvdf) membranes. the pvdf membranes were cut into longitudinal strips for the detection of established or putative desmin-associated proteins (frataxin, αb-crystallin; α-actinin, ankyrin 1, α-spectrin, plectin, and zasp [cypher]). sources of antibodies were as follows: anti-desmin (polyclonal, invitrogen, rockford, il, usa. cat no. pa5-16705); anti-αb-crystallin (monoclonal, santa cruz biotechnology, dallas, tx, usa. cat. no. sc-137129); anti-α-actinin (monoclonal, abcam, waltham, ma, usa, cat. no. ab62298); anti-ankyrin 1 (monoclonal, santa cruz biotechnology, cat. no. sc-12719); anti-α-spectrin (monoclonal, santa cruz, cat no. sc-48382); anti-plectin (monoclonal, santa cruz biotechnology, cat. no. 37649); anti-zasp (cypher) (monoclonal, santa cruz biotechnology, cat. no. sc-374359); and anti-frataxin (monoclonal, abcam, branford, ct, usa; cat. no. ab268063). mass spectrometry: protein bands of interest on coomassie blue-stained sds-page gels were manually excised and minced. the pieces were dehydrated with acetonitrile for 10 min, vacuum dried, rehydrated with 5 mm triphosphine hydrochloride in 25 mm ammonium bicarbonate (ph 8.5) at 37 °c for 1 h and then alkylated with 100 mm iodoacetamide in 25 mm ammonium bicarbonate (ph 8.5) at room temperature for 1 h. the pieces were washed twice with 50% acetonitrile in 25 mm ammonium bicarbonate (ph 8.5) for 15 min, dehydrated with acetonitrile for 10 min, dried, and digested in sequencing grade modified trypsin (25 ng/µl; sigma, st. louis, mo, usa, cat. no. t8658) in 25 mm ammonium bicarbonate (ph 8.5) at 37 °c overnight. following digestion, tryptic peptides were extracted three times with 50% acetonitrile containing 5% formic acid for 15 min each time while being vortexed. the extracted solutions were pooled and evaporated to dryness under vacuum. liquid chromatography-tandem mass spectrometry (lc-ms/ms) was performed on an integrated micro-lc-orbitrap velos system (thermo), comprising a waters caplc microscale chromatography system (waters corp., milford, ma) with an auto-sampler, a stream-select module configured for pre-column plus analytical capillary column, and an orbitrap velos mass spectrometer fitted with an h-electrospray ionizer probe operated under xcalibur 2.2 control. injected samples (30 µl) were first trapped and desalted isocratically on a home-packed c18 pre-column cartridge (everest c18, 5 µm particle diameter; column dimensions: internal diameter, 500 µm; length, 15 mm [grace, deerfield, il]) for 6 min with 0.1% formic acid delivered by an auxiliary pump at 40 μl/min. the peptides were then eluted from the pre-column and separated on an ace c18 capillary column (15 cm x 500 μm internal diameter) packed with ace c18-300 particles (5 µm resin, advanced chromatography technologies, aberdeen, scotland). the c18 column was connected in-line with the mass spectrometer and eluted at a flow rate of 20 µl/min with a 40-min gradient of 5-to-80% acetonitrile in 0.1% formic acid. tandem spectrum data was processed using mascot 2.7 (matrix science, boston, ma). the lists of peaks were used to query a human protein database downloaded from national center of biotechnology information by setting the following parameters: peptide mass tolerance, 10 ppm; ms/ms ion mass tolerance, 0.1 da; allowing up to one missed cleavage; considering variable modifications such as methionine oxidation, cysteine carboxyamidomethylation, and deamidation. only significant hits as defined by mascot probability analysis was considered for a positive protein identification. immunohistochemistry: to visualize desmin, its chaperone protein αb-crystallin, and known desmin-linked intermediate filament proteins (α-spectrin; ankyrin-1; plectin; zasp [cypher]; and α-actinin), 6-µm thick paraffin sections were rehydrated by sequential incubation in a series of xylene, absolute ethanol, 95% ethanol, 80% ethanol, and deionized water. endogenous peroxidase activity was suppressed by incubation in methanol containing 3% aqueous hydrogen peroxide. antigen retrieval, background suppression, incubation with polyclonal or monoclonal antibodies, secondary biotinylated anti-rabbit igg (for rabbit polyclonal primary antibodies) or anti-mouse igg (for primary mouse monoclonal antibodies), and chromogenic solutions of diaminobenzidine and hydrogen peroxide were as described (koeppen et al., 2020). double-label fluorescence and immunofluorescence: to examine possible amyloidogenicity of desmin in fa cardiomyopathy, immunofluorescence of desmin and fluorescence of congo red were combined. slide processing for immunofluorescence was as described before (koeppen et al., 2020), and the fluorophore for desmin was alexa fluor 488-labeled goat anti-mouse igg (jackson immunoresearch, west grove, pa, usa, cat. no. 115-545-003). after washing, the sections were incubated in a solution of high ph congo red, as described by the supplier (ihc world, woodstock, md, usa), washed in water, covered by pbs containing 50% glycerol (by vol), and cover-slipped. excitation by laser light at 488 nm was suitable for the fluorescence of both alexa fluor 488 (desmin) and congo red. images were collected in a zeiss lsm 880 laser scanning confocal microscope (oberkochen, germany) set to a band pass of 507-544 nm for alexa fluor 488 and 560-600 nm for congo red. double-label immunofluorescence of the pairs desmin/αb crystallin and desmin/mitochondrial atp5b followed a published protocol (koeppen et al., 2020). atp5b, the β subunit of atp synthase f1, was detected by a polyclonal antibody (santa cruz biotechnology, dallas, tx, usa, cat. no. sc 33618) and cy3-labeled secondary goat anti-rabbit igg (jackson immunoresearch, cat. no. 111-165-003). results antibody microarray of heart lysates: each dot in the panels of figure 1 represents a unique validated antibody, and the legend describes how fluorescent signals of fa and controls were superimposed and analyzed. thirteen proteins were upregulated to variable degrees on the 900-antibody microarray. when expressed as percent change from control (%cfc), the following proteins stood out: adducin, %cfc 210; αb-crystallin, %cfc 194; insulin receptor substrate, %cfc 324; serine/threonine kinase type 4, %cfc 103; ribosomal protein s kinase alpha 3, %cfc 149. the 1150-antibody microarray of kinexus (yue, et al, 2018) yielded modest upregulation of 23 other proteins that were not further analyzed. the up-regulation of αb-crystallin prompted a more detailed study of this protein and its partner, desmin, by sds-page, western blots, mass spectrometry, and double-label immunofluorescence (figs. 2-8). figure 1. antibody microarray of pooled heart lysates of 9 fa patients (left upper panel) and 9 normal controls (left lower panel). in the smaller panels, pseudocolors represent high fluorescence (red) and lower fluorescence (blue). intermediate fluorescence intensity is represented by green and yellow, in that order. the larger image was created by overlaying the fa and control images (fa on top). all spots of the control image were made blue, and all fa spots were made red. using adobe photoshop technology, the fa images on top were rendered 50% transparent so that the bottom spots are white. if fa intensity was higher than that of the matching control, the spot appears as red. if the composite signal is blue, the signal in fa was lower than that of the control. pooling of lysates was intended to control for between-samples variability, and performance in triplicate allowed statistical analysis by t-testing. the next step in the experiment was sds-page and western blotting of proteins in pooled and individual lysates. frataxin levels: the frataxin level in 12 fa heart lysates (mean ± standard deviation) was 23.3±3.5 ng/g wet weight. in 4 control heart lysates the level was 522.5 ± 35 ng/g wet weight. the difference was significant at p=0.022. western blots: figure 2 shows a western blot of desmin and frataxin in pooled fa heart lysates. the pooled lysates represent equal additions of the first 6 fa cases shown in figure 3. the polyclonal antibody to desmin revealed a low intensity band at 53 kda which represents the canonical protein. the strongest desmin signal occurred from a fragment of approximately 45 kda. mass spectrometric analysis of this fragment (fig. 2) confirmed its derivation from desmin. while pooling of 12 heart lysates was useful in the control of inter-sample variability in fa, the western blot in figure 3 shows that the desmin fragment is shared by all fa samples. the qualitative intensity of the desmin band did not correlate with the length of the gaa trinucleotide repeat expansions in the shorter allele of the frataxin gene (fig. 3). a comparable desmin isoprotein is also present in one normal control (fig. 2, left panel, subject f,62). figure 2. western blot of desmin and frataxin in 6 pooled fa heart lysates and mass spectrometry of a proteolytic desmin fragment. lane 1: prestained proteins of known mass. lane 2: western blot of frataxin (fxn). pooling of heart lysates allows the detection of residual frataxin in fa. lane 3: western blot of desmin. the canonical desmin migrates at 53 kda (des), but a second, strongly reactive desmin band (arrow) is located at approximately 45 kda. lane 4: mass spectrometry of the truncated desmin fragment in lane 3 (arrow). the amino acids in red were identified by liquid chromatography-tandem mass spectrometry. the sequences represent signature peptides of desmin. figure 3. western blots of desmin in 6 normal control and 12 fa heart lysates. all wells received a total protein load of 50 µg. the arrows show the location of canonical desmin at 53 kda, a desmin fragment, and gapdh (as a loading control). the pvdf membranes were cut horizontally to generate two segments of the blot and allow visualization of gapdh. sex and ages are shown for the lanes of control lysates (left); in the right panel, of fa lysates, the entries include sex, age, and gaa trinucleotide repeats. in 8 of 12 fa lysates, the blots show prominent expression of a proteolytic desmin fragments at approximately 45 kda. figure 4. desmin co-immunoprecipitation. desmin in pooled lysates of 6 fa hearts was precipitated by anti-desmin, and the protein-antibody complex was recovered on protein a sepharose. desmin and other proteins were released from protein a by heating in laemmli’s sample buffer containing β-mercaptoethanol. aliquots were electrophoresed by sds-page, electroblotted onto pvdf membranes, and visualized by immunochemistry with antibodies to the stated proteins. the western blot shows desmin bands at approximately 53 kda and between 40 and 45 kda, respectively. a distinct band of αb-crystallin is also present. other lighter bands at 45 kda, 25 kda and 15 kda are considered preparative artifacts. immunohistochemistry: in normal hearts, antibodies to desmin and αb-crystallin revealed reaction product in intercalated discs and z-discs. anti-αb-crystallin also showed m bands on longitudinal sections of heart fibers that were desmin negative (fig. 5f). in fa, desmin and αb-crystallin reaction product showed abnormally wide, thickened, and hyperconvoluted intercalated discs and aggregates in the sarcoplasm (fig. 5). desmin and αb-crystallin reaction product in the two compound heterozygous fa cases showed a similar degree of protein aggregate myopathy. reaction product for β-spectrin, ankyrin, plectin, and zasp (cypher) was less informative, though α-spectrin immunohistochemistry displayed the chaotic disorganization of intercalated discs. α-actinin reaction product showed the striation of cardiomyocytes by reacting with z-discs but was not useful to assess the state of intercalated discs. figure 5. immunohistochemistry of desmin and αb-crystallin in left ventricular wall. a-b, desmin; c-f, αb-crystallin. a and c, normal left ventricular wall; b, d-f, fa. in normal myocardium, desmin (a) and αb-crystallin (c) are localized in intercalated and z discs. in fa, desmin reaction product reveals accumulation in or near disorganized intercalated discs (b, arrow). αb-crystallin immunohistochemistry shows wide and fragmented intercalated discs (d and e). enlargement of the square in e displays αb-crystallin reaction product in m bands (f, arrow, m). m bands are desmin-negative. bars; a-e, 10 µm; f, 3 µm. fa patients: b, f, 67 gaa 621/766; d-f, f, 69, gaa 560/560. double-label laser scanning confocal immunofluorescence: figure 6 shows the colocalization of desmin and αb-crystallin in z-discs and intercalated discs. in fa, intercalated discs are wider at approximately 50 µm than normal (approximately 23 µm) and have undergone chaotic disorganization (fig. 6d). a more advanced fiber destruction is illustrated in figure 7. two adjacent fibers display accumulation of desmin and αb-crystallin, obliterating the contractile apparatus of the cardiomyocytes. a search for amyloid by polarizing light for apple-green birefringence and congo red fluorescence did not reveal amyloid deposits. figure 8 shows double-label immunofluorescence of desmin and atp5b. all fibers in fa contain clusters of mitochondria, visualized by green fluorescence of atp5b, and one cluster is surrounded by a rim of desmin. in normal cardiomyocytes, mitochondria occur in rows of delicate granules between heart fibrils (fig. 8, top row). figure 6. double-label laser scanning confocal immunofluorescence microscopy of desmin and αb-crystallin. desmin immunofluorescence (a, d) is alexa fluor 488 green, αb-crystallin (b,e) cy3 red. merged images appear in c and f. the top panel (a-c) represents a normal control, the bottom panel (d-f) fa (man, 24, gaa 700/1050). desmin and αb-crystallin fluorescence colocalize at intercalated discs (arrows) and z discs. in fa, an intercalated disc has undergone chaotic modification. bars, 10 µm. figure 7. desmin and αb-crystallin overload in fa cardiomyopathy (same patient as in fig. 6). desmin is shown as alexa fluor 488 green, αb-crystallin as cy3 red, the merged image on the right shows extensive colocalization of these protein and destructive aggregation in two fibers (arrows). bars, 10 μm. figure 8. double-label laser scanning confocal immunofluorescence microscopy of desmin and atp5b. top row, normal control; bottom row, fa (same patient as in fig. 5). desmin is shown as cy3 red, atp5b as alexa fluor 488 green. in the normal control, mitochondria appear as rows of punctate green organelles aligned along fibrils. in fa, green mitochondria appear in clusters (arrows). the upper cluster is surrounded by a desmin deposit. bars, 20 µm. discussion frataxin levels in fa hearts: the detection limit of the described elisa is 10 pg. we followed biochemical tradition by expressing frataxin levels as mass per gram wet weight, which is at variance with the current practice of fa investigators to express frataxin as ng/total protein. the data must be interpreted with some caution because we assumed that the wet weight of normal and fa heart tissues was close to its water content. tissue lysates contained inhibitory substances that interfered with elisa. filtration through membranes with a molecular mass cutoff of 30 kda removed the interference but may have introduced an additional error in the calculation of corresponding wet weight. desmin in fa cardiomyopathy: uniprot (2021) identifies 36 natural variants of human desmin, of which some are pathogenic mutations. none are listed that could account for the deletion of an estimated 80 amino acids that would explain the high signal desmin isoprotein of 45 kda in fa hearts (figs. 2 and 3). clemen et al. (2008) attributed pathogenicity in patients with skeletal muscle desminopathy to the presence of a low-abundance truncated desmin. it is unknown whether this pathological mechanism is analogous to the prominent desmin band on our western blots of fa patients (fig.3). desmin is not mutated in fa, and fa patients have no skeletal myopathy. arbustini et al. (1998) presented western blots of heart and muscle lysates from patients with desmin-related cardiomyopathy that showed two isoforms of desmin of about the same intensity at masses of 55 and 53 kda, respectively. a truncated muscle desmin of the cases of skeletal myopathy reported by clemen et al. (2008) migrated at 54 kda, rather than the 45 kda in the lysates of fa hearts reported here (figs. 2 and 3). we assume that the truncated desmin in fa cardiomyopathy derives from normal desmin by proteolysis (fig. 2). the functional role of desmin-containing intermediate filaments in skeletal and cardiac muscle is complex. authors who published three-dimensional renditions of desmin in skeletal muscle (goldfarb and dalakas, 2009; clemen et al., 2013) and heart muscle (capetanaki et al., 2015) emphasized the localization of the protein at z discs and intercalated discs (heart only), but also near the nuclear and sarcoplasmic membranes, and surrounding mitochondria. the role of αb-crystallin in possible proteolysis and aggregation of desmin in fa cardiomyopathy is uncertain, but αb-crystallin is viewed as essential for the proper assembly of mature desmin filaments (ghosh et al., 2007; sharma et al., 2017). peculiarly, mutations of αb-crystallin cause myopathy with a phenotype that is 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microarrays for biomarker discovery and tracking cellular signaling. adv proteom bioinformat 2018:1-24. https://doi.org/10.29011/apbi-107.100007 copyright: © 2021 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. neuropathological findings in possible normal pressure hydrocephalus: a post-mortem study of 29 cases with lifelines feel free to add comments by clicking these icons on the sidebar free neuropathology 3:2 (2022) original paper neuropathological findings in possible normal pressure hydrocephalus: a post-mortem study of 29 cases with lifelines joni j. hänninen1,2, madoka nakajima3, aleksi vanninen1,2, santtu hytönen4, jaana rummukainen5, anne m. koivisto6,7,8, juha e. jääskeläinen2, hilkka soininen9, anna sutela10, ritva vanninen10, mikko hiltunen11, ville leinonen2, tuomas rauramaa1 1 the department of pathology, kuopio university hospital and the institute of clinical medicine – pathology, university of eastern finland, kuopio, finland 2 the department of neurosurgery, kuopio university hospital and the institute of clinical medicine – neurosurgery, university of eastern finland, kuopio, finland 3 the department of neurosurgery, juntendo university faculty of medicine, tokyo, japan 4 the national institute for health and welfare, forensic medicine, kuopio, finland 5 the department of pathology, kuopio university hospital, kuopio, finland 6 the department of neurology, kuopio university hospital and the institute of clinical medicine – neurology, university of eastern finland, kuopio, finland 7 the department of neurosciences, medical faculty, university of helsinki, helsinki, finland 8 the department of geriatrics/rehabilitation and internal medicine, helsinki university hospital, helsinki, finland 9 the department of neurology, kuopio university hospital, kuopio, finland 10 the department of radiology, kuopio university hospital and the institute of clinical medicine – radiology, university of eastern finland, kuopio, finland 11 the institute of biomedicine, university of eastern finland, kuopio, finland corresponding author: tuomas rauramaa · department of clinical pathology · kuopio university hospital · p.o. box 100 fin-70029 kys · kuopio · finland tuomas.rauramaa@kuh.fi submitted: 09 april 2021 accepted: 19 january 2022 copyedited by: elisabeth g. hain published: 26 january 2022 https://doi.org/10.17879/freeneuropathology-2022-3331 keywords: hydrocephalus, normal pressure hydrocephalus, post-mortem, neuropathology, neurodegeneration abstract aims: there are very few detailed post-mortem studies on idiopathic normal-pressure hydrocephalus (inph) and there is a lack of proper neuropathological criteria for inph. this study aims to update the knowledge on the neuropathology of inph and to develop the neuropathological diagnostic criteria of inph. methods: we evaluated the clinical lifelines and post-mortem findings of 29 patients with possible nph. pre-mortem cortical brain biopsies were taken from all patients during an intracranial pressure measurement or a cerebrospinal fluid (csf) shunt surgery. results: the mean age at the time of the biopsy was 70± 8 sd years and 74± 7 sd years at the time of death. at the time of death, 11/29 patients (38%) displayed normal cognition or mild cognitive impairment (mci), 9/29 (31%) moderate dementia and 9/29 (31%) severe dementia. two of the demented patients had only scarce neuropathological findings indicating a probable hydrocephalic origin for the dementia. amyloid-β (aβ) and hyperphosphorylated τ (hpτ) in the biopsies predicted the neurodegenerative diseases so that there were 4 aβ positive/low alzheimer’s disease neuropathological change (adnc) cases, 4 aβ positive/intermediate adnc cases, 1 aβ positive case with both low adnc and progressive supranuclear palsy (psp), 1 hpτ/psp and primary age-related tauopathy (part) case, 1 aβ/hpτ and low adnc/synucleinopathy case and 1 case with aβ/hpτ and high adnc. the most common cause of death was due to cardiovascular diseases (10/29, 34%), followed by cerebrovascular diseases or subdural hematoma (sdh) (8/29, 28%). three patients died of a postoperative intracerebral hematoma (ich). vascular lesions were common (19/29, 65%). conclusions: we update the suggested neuropathological diagnostic criteria of inph, which emphasize the rigorous exclusion of all other known possible neuropathological causes of dementia. despite the first 2 probable cases reported here, the issue of “hydrocephalic dementia” as an independent entity still requires further confirmation. extensive sampling (with fresh frozen tissue including meninges) with age-matched neurologically healthy controls is highly encouraged. introduction normal pressure hydrocephalus (nph) has a classic triad of clinical symptoms: impaired cognition, urinary incontinence and gait disturbance [1]. nph is considered idiopathic (inph) in the absence of known predisposing factors such as neurotrauma or hemorrhage [1, 2]. the surgical insertion of a shunt in order to bypass cerebrospinal fluid (csf) usually eases the symptoms [3], but unfortunately the improvements often decline over the years [4]. a considerable number of the patients with inph seem to develop cognitive impairment, eventually reaching dementia, commonly due to concomitant alzheimer’s disease (ad), frontotemporal lobar degeneration (ftld), vascular degeneration, or compellingly due to inph by yet unknown neurodegenerative mechanisms [5, 6]. in the norwegian population, the prevalence of inph is estimated to be approximately 22 cases per 100,000 people [7] and it is more common in the older age groups [8]. recent studies reporting familiar aggregation indicate that inph may be an independent neurodegenerative disorder that potentially has its own specific molecular biological basis and etiology [9]. in a study of 10 patients – 9 of whom are included in this study – it was shown that signs of other diseases such as ad, corticobasal degeneration (cbd) and vascular lesions can be identified in the post-mortem brains of the patients suspected to have nph [10]. the number of detailed post-mortem studies on inph is very small and there is a lack of defined and universally accepted neuropathological criteria for inph. the kuopio nph registry (http://www.uef.fi/nph) consists of data on the patients with presumed nph. they underwent intraventricular pressure (icp) monitoring together with right frontal cortical biopsies from 1991 up until 2010. from 2010 onwards, the shunt surgery has included the cortical brain biopsy. this study contains 29 patients with full post-mortem neuropathological examinations and with clinical follow-ups after the procedures with the biopsies. materials and methods kuopio nph registry the neurosurgical department of kuopio university hospital (kuh) alone provides full-time acute and elective neurosurgical services for the kuh catchment population in eastern finland. the medical evaluation of the kuh neurosurgery for possible nph has contained a clinical examination by a neurologist and a neurosurgeon, a computed tomography (ct) or a magnetic resonance imaging (mri) scan, and a 24-h icp monitoring together with a right frontal cortical biopsy up until 2010. in 2010, a 3-step prognostic test protocol was launched. first, csf tap tests are performed on all patients with suspected nph, where at least a 20% improvement in the gait speed in a 10-m walk is considered a positive result. in the second phase, those with negative tap tests undergo lumbar infusion tests, where a conductance of ≤ 10 is considered a positive result. in the third step, the participants with negative findings in both of the tests mentioned above undergo a 24-h monitoring of the icp or undergo a shunt surgery based on a clinical and radiological evaluation. brain biopsies are acquired from the patients who undergo the icp monitoring or the csf shunt surgery [11]. kuopio nph registry includes (i) the clinical baseline and the follow-up data and (ii) other hospital diagnoses, medications and the causes of death from the national registries, as well as the autopsy findings. in this study, nph is defined as a state with the typical clinical and imaging findings, and it is classified as inph when no predisposing factors are found. the shunt response may be present or absent. basic study cohort from 1991 up until 2017, 879 patients with possible nph had been studied with the diagnostic protocol described above including the right frontal cortical biopsies. final study cohort the construction of this study is outlined in figure 1. the patients in this study cohort were followed up until their deaths. all clinical data available from the hospitals in the kuh catchment area was collected. overall, 565 patients had died and 92 of them were autopsied, 29 of whom had had a full neuropathological examination. out of the 29 patients, 23 were shunted according to the diagnostic procedure. the development of the clinical symptoms was documented by neurologists, general practitioners, or both. the mini-mental state examination (mmse) was tested on 26 patients at the baseline. all available data was re-evaluated by 2 neurologists subspecialized in memory disorders (a. m. k. or h. s.), blinded for the cortical biopsy and the autopsy findings. dementia was diagnosed according to the diagnostic and statistical manual of mental disorders, fourth edition (dsm-iv) criteria [12]. the cognitive status was evaluated at the time of the biopsy and retrospectively at the time of death and classified as normal, mild cognitive impairment (mci), moderate or severe dementia (bedridden). figure 1. flow chart of 879 consecutive patients with possible nph. nph = normal pressure hydrocephalus. radiological evaluation all available ct and mri scans were re-evaluated separately by 2 experienced neuroradiologists (a. s. and r. v.), blinded for the biopsy and the neuropathological findings. in the incongruent evaluations, a consensus reading was performed. cortical and subcortical (lacunar) infarctions and cerebral white matter changes (wmcs) were especially looked for. the wmcs were categorized according to the modified fazekas scale: no wmc (grade 0), punctuate (grade 1), early confluent (grade 2) and confluent (grade 3) [13]. biopsy the biopsy procedure has been described previously [14]. briefly, 1 to 3 cylindrical right frontal cortical brain biopsies of 1-3 mm in diameter and 1-8 mm in length were obtained with biopsy forceps or a needle before inserting the intraventricular catheter for the 24-h icp monitoring or the shunt. the samples were fixed in buffered formalin overnight and then embedded in paraffin. apolipoprotein e genotyping dna was extracted and the apolipoprotein e (apoe) genotypes were determined as described previously [15]. autopsies and the neuropathological examination the main findings and the causes of death were assessed from the original autopsy reports. the brains were stored in 10% buffered formaldehyde for at least 1 week before being cut into 1-cm-thick coronal slices and assessed macroscopically by a neuropathologist. the brain specimens were taken from 16 standard regions and embedded in paraffin. the included brain regions were: the frontal cortex, the temporal cortex, the cingular gyrus at the level of the mammillary bodies, the parietal cortex, the motor cortex, the occipital cortex, the anterior and the posterior hippocampus with the entorhinal cortex, the basal forebrain including the amygdala, the striatum, the thalamus, the midbrain, the pons, the medulla, the cerebellar vermis and the cortex [16]. additional samples were taken if necessary for the diagnostics. histology and immunochemistry the 7-μ m sections were deparaffinized and rehydrated through the standard procedures. all sections were stained by using haematoxylin-eosin (h&e) and a selection of the sections also with immunohistochemical (ihc) methods. the antibodies used are described in table 1. shortly after the pretreatment, the sections were incubated with normal goat serum for 30 min at room temperature to block non-specific reactions. after unmasking the epitope, the antibodies were added to the dilution. the sections were then incubated overnight at 4 °c. during the next day, the sections were incubated with a biotinylated second antibody for 30 min, followed by streptavidin enzyme conjugate (labsa zymed laboratories, south san francisco, ca, usa) for 30 min at room temperature. the reaction products were visualized by using 3-amino-9-ethyl-carbazole (aec) or 3-3’-diaminobenzidin (dab). all immunostained sections were counterstained with harris’ haematoxylin, dehydrated, and mounted in depex (bdh chemicals, hull, uk). an amyloid-β (aβ) staining was performed on the biopsies and 5 neuroanatomical regions (the frontal cortex, the hippocampus and the basal forebrain including the amygdala, the substantia nigra and the cerebellar cortex). hyperphosphorylated tau (hpτ) was performed on the biopsies and 5 neuroanatomical regions (the temporal cortex, the occipital cortex, the anterior and the posterior hippocampus, the basal forebrain including the amygdala). an α -synuclein staining was performed on the biopsies and 7 neuroanatomical regions (the cingular gyrus, the parietal cortex, the posterior hippocampus, the substantia nigra, the pons, the medulla oblongata and the basal forebrain including the amygdala). transactive response dna-binding protein 43 (tdp43) was used on the biopsies and the posterior hippocampal sample. p62 was used on selected cases. all sections were evaluated with light microscopy by an experienced neuropathologist (t.r., j.r.). the pathology observed while applying the ihc techniques was assessed as described earlier [17–22]. in the biopsy samples, hpτ was classified as being present or absent and the type of the specific lesions was noted. the aβ pathology was classified as being present in mild, moderate or extensive extent, and the vessel wall positivity was noted, suggesting cerebral amyloid angiopathy (caa). alzheimer’s disease neuropathological change (adnc) was assessed according to the national institute on aging–alzheimer's association (nia-aa) criteria [23]. table 1. immunohistochemistry. hpτ = hyperphosphorylated τ, tdp43 = transactive response dna-binding protein 43, ac = autoclave, cb = citrate buffer, fa = formic acid, dab = 3-3’-diaminobenzidin, aec = 3-amino-9-ethylcarbazole (biocare medical). the statistical methods spss statistics (version 22.0 spss inc., chicago, il, usa) was used for all statistical analyses. spearman’s rank correlation coefficient was used for correlating aβ in the biopsies and the autopsies. ethical issues the study was approved by the ethics committee of the hospital district of northern savonia. all patients recruited after 2008 provided a written informed consent. patient treated in neurosurgery prior to that, were studied according to the permissions from the finnish national supervisory authority for welfare and health, and the finnish ministry of social affairs and health. results demographics: the patient demographics, the clinical findings including the brain imaging and the cortical biopsies, are described in table 2. more than half of the patients were female (17/29, 59%). the mean age at the time of the biopsy was 70± 8 sd (standard deviation) years and 74± 7 sd years at the time of death. the patients with clinically severe dementia died at the average age of 76± 5 sd years. the age at death did not differ between the cognitively normal/mci or the moderately demented patients. the follow-up time from the brain biopsy to the autopsy was 47± 44 sd months, ranging from less than 1 month to 165 months. table 2. patient characteristics at the time of the biopsies and the final clinical diagnoses. * no amyloid-β. mmse = mini-mental state examination, aβ = amyloid-β, hpτ = hyperphosphorylated τ, inph = idiopathic normal pressure hydrocephalus, ct = computerized tomography, mri = magnetic resonance imaging, wmc = white matter change (fazekas grade [13] at least 1), pv = periventricular, na = not available, ad = alzheimer’s disease, hta = arterial hypertension, chd = coronary heart disease, t2d = type 2 diabetes. clinical findings: at the time of the clinical work-up and the cortical biopsy, the leading symptom was gait difficulty in 75% of the subjects with normal cognition or mci (n=20). in the moderately demented group, half of the subjects (n=3/6) presented with cognitive impairment as the leading symptom, followed by gait difficulty (n=2) and tremors (n=1). in the severely demented group, 2 out of the 3 patients presented with cognitive impairment and 1 with gait difficulty as the leading symptom. in this study, inph is defined as a state in which the clinical findings, the imaging and the post-mortem findings are typically consistent with nph, the predisposing factors are excluded, and the shunting procedure has been performed with a positive shunt response. idiopathic nph was seen in 70% of the subjects with normal cognition or mci and 33% of the clinically demented subjects. secondary nph (snph) was seen in 3/20 (15%) of the subjects in the group with normal cognition or mci. the most common clinical diagnosis was hypertension (18/29, 62%), present in all groups. other common clinical diagnoses were coronary heart disease, type ii diabetes (t2d), ad, psychiatric disorders, cancer and asthma. two of the patients in the group with normal cognition or mci had clinically cognitive impairment related to alcohol, 1 patient presented with parkinsonism and 1 with epilepsy. regarding the psychiatric morbidities, 1 patient had a history of schizoaffective disorder, 2 patients had schizophrenia and 2 had a long history of psychiatric symptoms and 1 of maniac episodes. no specific neuropathological findings were seen in this group. shunting: one patient in the group with normal cognition or mci was not shunted and 15/19 (79%) of the shunted patients had positive responses. in the moderately demented group, 3/6 of the patients were shunted and 2 of them responded positively. one patient in the severely demented group (1/3) was shunted without a response. imaging: radiologically, cortical and lacunar infarctions were found in the normal or mci and the moderately demented groups. the findings are summarized in table 2. the infarctions were of grades 1 and 2. both periventricular and deep white matter changes were present in all groups. the changes ranged from grades 0 to 3. cortical biopsies: aβ aggregates were seen in 10 patients: 4/20 (20%) in the group with normal cognition or mci, 3/6 (50%) and 3/3 (100%) in the moderately and the severely demented groups, respectively. hpτ was not seen in the group with normal cognition or mci. one patient in the moderately demented group had hpτ immunopositive lesions (without aβ) and 2 out of the 3 patients in the severely demented group had hpτ positivity. prominent hpτ pathology was seen upon the neuropathological examinations in all the 3 cases with hpτ in the biopsies. the presence of aβ in the biopsies correlated with the extent of aβ (thal phase) [22] defined at the neuropathological examinations (spearman’s ρ 0.775, p = 0.01). table 3. causes of death. at the time of death, 11/29 patients (38%) displayed normal cognition or mci, 9/29 (31%) moderate dementia, and 9/29 (31%) severe dementia. the causes of death are described in table 3. the most common cause of death was due to cardiovascular diseases (10/29, 34%), followed by cerebrovascular diseases (n=5/29, 17%) or acute subdural hematoma (sdh) (3/29, 10%), pneumonia (5/29, 17%) and metastatic carcinoma (4/29, 14%). two patients died of postoperative intracerebral hematoma (ich) indicating the 0.2% (2/879) risk of fatal ich, related to the combined procedure of the brain biopsy and the insertion of the intraventricular catheter in the nph population. table 4. post-mortem neuropathological findings. caa = cerebral amyloid angiopathy, apoe = apolipoprotein e, na = not available, adnc = alzheimer’s disease neuropathological change (nia-aa [24]), part = primary age-related tauopathy, psp = progressive supranuclear palsy, cbd = corticobasal degeneration, aβ = amyloid-β deposition, ich = intracerebral hematoma, sah = subarachnoidal hematoma, sdh = subdural hematoma. neuropathology: the neuropathological findings are described in table 4. enlarged ventricles were seen to variable degrees in all cases. the neuropathological examination revealed that in the group with normal cognition or mci, almost half (5/11, 45%) of the patients had cerebrovascular atherosclerosis and more than a half (5/9, 55%) of the moderately demented patients had notable cerebrovascular atherosclerosis. all of the severely demented patients had cerebrovascular atherosclerosis to variable degrees. one patient in the least cognitively affected group had a ruptured basilar artery aneurysm resulting in subarachnoid hemorrhage (sah), 1 had sdh and 1 patient had a hippocampal infarct. in the moderately demented group, 4 out of the 9 patients had acute sdh and 75% of these were traumatic in origin, and 1 patient had traumatic sah. in the severely demented group, 1 patient had a multifocal metastatic (including meninges) carcinoma explaining the dementia, and 1 patient a temporal cavernoma with an ipsilateral hippocampal infarct. multifocal brain infarcts sufficient to be causative for the cognitive decline were seen in 3 patients, 2 demented (occurred after the shunting procedure) and 1 with mci (occurred during the year of death). in these patients, small lacunar infarctions were seen in the ct during the evaluation prior to the icp measurement or the shunting. gliosis in the subependymal region was seen in all cases to variable degrees and at least mild meningeal thickening was seen in 60% of the patients. the most common secondary findings are summarized in table 5, among the pooled findings from previous literature reviewed in 2012 [10]. table 5. neuropathological findings in nph. nph = normal pressure hydrocephalus, na = not available, ad = alzheimer’s disease. for adnc evaluation, nia-aa criteria were used [23]. four of the subjects with normal cognition or mci had adnc and 6 had primary age-related tauopathy (part) of braak stage iii at maximum. one patient with low adnc had also progressive supranuclear palsy (psp). in the moderately demented group, 3 had intermediate adnc and 4 had braak i part. in the severely demented group, 6 patients had adnc and 2 had part. in addition, single cases of psp, cbd, α -synucleinopathy and aβ depositions of thal stage 3 (without hpτ) were displayed. cerebral amyloid angiopathy (caa) was seen in 1 out of the 11 subjects with normal cognition or mci, 22% of the moderately demented patients and 44% of the severely demented patients. the apoe genotype was found in 15/29 patients. at the time of biopsy, in the group with normal cognition or mci, 8/10 (80%) had genotype ε 3/3, 1 ε 3/4 and 1 ε 2/3. among the moderately demented patients, 2/3 (67%) had the ε 3/4 and 1 had the ε 3/3 genotype. in the group of the severely demented patients, 1 had ε 3/3 and 1 ε 4/4 genotype. neuropathological wmcs in the form chronic vascular type were seen to a variable extent and did not correlate to the cognitive status. in figure 2, we describe a model for the neuropathological evaluation of the nph cases. figure 2. our suggested model for neuropathological evaluation of nph cases. (i/s)nph = (idiopathic/secondary) normal pressure hydrocephalus, ihc = immunohistochemistry, aβ = amyloid-β , hpτ = hyperphosphorylated τ, tdp43 = transactive response dna-binding protein 43, ad = alzheimer’s disease. figure 3. radiological and post-mortem sectional findings. a: coronal mri demonstrating ventricular enlargement pre-shunting, b: same case post-mortem section after shunting (time interval 2 years), c: coronal mri demonstrating ventricular enlargement pre-shunting, d: intracerebral hematoma after the shunting procedure (time interval 3 months). we present examples of radiological and post-mortem sectional findings in figure 3. table 6 is the case-by-case representation of the patients. two subjects with disproportionate neuropathological findings to the degree of cognitive impairment at death are highlighted, representing cases of probable “hydrocephalic dementia”. table 6. case-by-case representation of the patients. probable nph-dementia subjects are highlighted. + = yes, = no. mci = mild cognitive impairment, mmse = mini-mental state examination, aβ = amyloid-β, hpτ = hyperphosphorylated τ, n/a = not available, (i/s)nph = (idiopathic/secondary) normal pressure hydrocephalus, ct = computed tomography, mri = magnetic resonance imaging, wmc = white matter changes (fazekas grades [13]), ad = alzheimer’s disease, tia = transient ischemic attack, ich = intracerebral hematoma, t2d = type 2 diabetes, mcc = morbus cordis coronarius, af = atrial fibrillation, caa = cerebral amyloid angiopathy, apoe = apolipoprotein e, adnc = alzheimer’s disease neuropathological change, part = primary age-related tauopathy, psp = progressive supranuclear palsy, cbd = corticobasal degeneration, sah = subarachnoid hemorrhage, ich = intracerebral hemorrhage. click here for high resolution. discussion hakim and adams [24] described the clinical triad of nph, including the gait difficulty, the cognitive decline and the urinary incontinence, more than 50 years ago. due to its relative rarity, inph is still a rather poorly understood disease and the number of studies with full systematic neuropathological examinations is limited. in this study, we describe the post-mortem findings in 29 patients with presumed nph. idiopathic nph differs from most neurodegenerative diseases presenting with dementia, as the symptoms can be alleviated surgically [3, 25]. idiopathic nph mostly affects the elderly population. the median age of the patients studied here at the time of the diagnosis is comparable to previous studies [7, 26]. in this study, all patients were followed from the time of the shunting or the icp monitoring up until their deaths, and cortical biopsies were taken from all subjects during the operation. overall, in previous studies, the neuropathological findings have been reported to be highly variable and no specific findings of “hydrocephalic dementia” have been described up to date [10, 27, 28]. del bigio describes 5 patients with inph, 3 with snph and 20 patients in whom the ventriculomegaly could not be explained by the other disease processes identified. neuropathologically, these groups did not differ [2]. the classical hakim’s triad is only seen in full in 50% of the nph cases [1]. in our study, gait difficulty was the most common leading symptom in the group with normal cognition or mci. in the moderately demented group, half of the subjects presented clinically with cognitive impairment as the leading symptom and in the group of the severely demented patients, cognitive symptoms were more common than gait difficulty or incontinence. previous studies have shown that more than 20% of the initially shunt-responsive inph patients develop ad later in life [5, 10]. the outcome of the shunting is more favorable if the procedure is done at the earlier stages of the disease [29, 30]. therefore, the candidates for the shunting procedure need to be carefully selected as comorbid ad can make the shunting outcome less favorable [5, 31]. the correlation of aβ seen in the biopsies and the overall aβ load justify the use of the biopsies in the clinical evaluation, although many neurodegenerative diseases generally cannot be ruled out by examining a small cortical biopsy representing a small fraction of the brain tissue. however, hpτ in the biopsies predicted the neuropathological diagnosis of tauopathy or ad. clinically, it is especially important to rule out other types of dementia in patients with cognitive impairment as the leading symptom [33]. the shunting procedure itself may results in complications such as postoperative ich. in our study cohort, the neuropathological examination revealed that 2 patients had psp and 1 cbd. in previous studies, it has been shown that psp and parkinson’s disease can clinically mimic inph [28, 33]. in this study, the most common neuropathological finding in the demented group was adnc or early ad/part, followed by multifocal infarcts. in the study published by del bigio and coworkers [34], significant co-morbidity was frequent in nph. it is noteworthy that mixed diagnoses and co-pathologies are common especially in the elderly age groups [35]. mccarty et al. pointed out that a feature of nph, disproportionately enlarged subarachnoid-space hydrocephalus (desh), is often misdiagnosed as cortical atrophy [36]. in a recent study, it was shown that schizophrenia occurs 3 times more frequently among the inph patients when compared to the general population in finland [37]. studies using different imaging modalities have revealed that there are several progressive structural changes in the brains of the patients suffering from schizophrenia. the most common findings are the enlargements of the lateral ventricles and the reduction of the grey matter volume [38, 39] and the smaller hippocampi [40]. at the microanatomical level, various cytoarchitectural alterations such as a decreased number of neurons and increased neuronal densities in various neuronal populations have been reported [41]. in our study, we did not use a stereological methodology to quantify the cytoarchitectural alterations. overall, our cases with comorbid psychiatric diseases did not differ from the other nph cases. among our study subjects, acute sdh was a relatively common cause of death. one possible explanation for this is that gait disturbance is common in nph and this might lead to falls resulting in sdh. another explanation may be that the csf shunt can be a risk factor for sdh [42]. it should also be noted that atrophic brains may be more prone to develop sdh. three of the patients in this study died of postoperative ich, indicating the 0.2% (2/879) risk of fatal ich related to the combined procedure of the brain biopsy and the insertion of the intraventricular catheter in the nph population. previous studies have shown that inph patients often have cardiovascular and cerebrovascular diseases [10, 43–45]. in a recent study by israelsson and coworkers, it was shown that inph patients are more prone to vascular risk factors such as dyslipidemia and obesity [46]. in functional imaging studies, cerebral blood flow has been shown to be reduced in people with inph [47, 48]. in our study cohort, cerebrovascular atherosclerosis was a common finding to variable degrees and cardiovascular diseases were the most common cause of death. cerebral infarcts were seen in all groups, and some of these were multifocal, suggesting that dementia could be attributed to these lesions in some cases, i.e. representing “vascular dementia.” the etiology and the pathophysiology of inph are still poorly understood. the components of the nph clinical triad are likely to result from subcortical disconnections and disrupted axons. this differs from white matter damage in pediatric hydrocephalus where axonal stretch and local ischemia occur in a simple tissue environment. secondary changes occur in the neuronal cell bodies and at the synaptic level, but the neurons are viable. the csf shunting may reverse the dysfunction up to some degree, but the axonal damage cannot be restored [34, 49]. the subependymal gliosis and the fragmented ependymal lining seen in most of the cases in this study to variable extents may be reactive changes to the chronically increased csf pressure, rather than causative alterations. similarly, the white matter rarefaction may result from the csf leakage into the brain tissue and white matter changes are common in vascular pathologies in any case. another common finding was the meningeal thickening. this can be a secondary alteration to the edema caused by chronic hydrocephalus or the result of mild meningitis in the past, among other possible etiologies. the meningeal tissue assessed here represents only a fraction of the whole meninges and therefore, there is a risk of sampling bias. there has been a recent interest in the role of the disrupted glial-lymphatic, the “glymphatic circulation” in inph [50, 51]. aquaporin 4 (aqp4) may be one of the key molecules involved in the glymphatic system [52]. however, the concept of the cns glymphatic system is currently evolving and needs to be studied further [53]. a recent study by eide and hansson revealed that astrogliosis is common in the cortical inph biopsies and that the expression of aqp4 and dystrophin protein 71 is reduced in the astrocytic perivascular endfeet [54]. the expression of the perivascular aqp4 water channel has been shown to be altered in the inph patients [55]. other interesting structures possibly involved in inph are the ependymal cilia [56–58]. however, the number of studies assessing these structures is very limited. the csf is mostly produced by the choroid plexuses and flows through the arachnoid villi and the granulations into the blood. it has been suggested that the chronic increase in the icp causes the downregulation of the csf regulation [59]. in this study, we did not study these structures in detail, but hypothetically the function of these structures could be altered in inph patients and therefore should be further studied. our study has several strengths and limitations. the cases selected for this study presented with clinically presumed nph. first of all, we have studied only selected cases (only 29 out of the 565 deceased were available for the neuropathological examination) and therefore, there is a risk of selection bias. however, to the best of our knowledge, this is the most comprehensive study of its kind in the literature. the cognitive status of the patients at death was retrospectively assessed based on the clinical records and there is a chance that this could be biased. however, it is unlikely that cases with full-blown dementia were missed. another strength is the availability of the follow-up data with the cortical biopsies. in conclusion, in this study we report the neuropathological findings on nph in a post-mortem series, which is the largest to the best of our knowledge. the neuropathological diagnostic criteria of inph are still lacking, and despite the first probable cases reported here, the issue of “hydrocephalic dementia” as an independent entity still requires further confirmation. the neuropathological findings in our cohort of possible nph patients were highly heterogeneous, which is in line with previous literature. esiri and rosenberg have recommended that the principal goal in the neuropathological work-up in hydrocephalic dementia is the exclusion of the defined entities [60]. here, we described a model for the neuropathological evaluation of the nph cases. future studies may eventually reveal that inph belongs to the family of “proteinopathies” with the dysfunctional protein yet to be discovered. the use of more extensive sampling (with fresh frozen tissue including meninges) than in the protocol used here and the use of age-matched healthy controls is highly encouraged. special interest should be directed towards the demented patients with neuropathological 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dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. deposits of disease-associated alpha-synuclein may be present in the dura mater in lewy body disorders: implications for potential inadvertent transmission by surgery feel free to add comments by clicking these icons on the sidebar free neuropathology 1:6 (2020) original paper deposits of disease-associated alpha-synuclein may be present in the dura mater in lewy body disorders: implications for potential inadvertent transmission by surgery ellen gelpi md1,2, naomi p. visanji phd3, selma hönigschnabl md4, angelika reiner md4, peter fischer md5, anthony e. lang md3, herbert budka md1, gabor g. kovacs md3,6,7 1 division of neuropathology and neurochemistry, department of neurology, medical university of vienna, vienna, austria 2 neurological tissue bank of the biobanc-hospital clinic-idibaps, barcelona, spain 3 edmond j. safra program in parkinson’s disease and the morton and gloria shulman movement disorders clinic, toronto western hospital, toronto, ontario, canada 4 institute of pathology, danube hospital of vienna, austria 5 department of psychiatry, medical research society vienna d.c., danube hospital, vienna, austria 6 laboratory medicine program & krembil brain institute, university health network, toronto, ontario, canada; 7 department of laboratory medicine and pathobiology and tanz centre for research in neurodegenerative disease, university of toronto corresponding author: professor gabor g kovacs md phd frcpc · laboratory medicine program · toronto general hospital · university health network · 200 elizabeth street · toronto on · canada · m5g 2c4 gabor.kovacs@uhnresearch.ca submitted: 09 january 2020 accepted: 06 february 2020 published: 12 february 2020 https://doi.org/10.17879/freeneuropathology-2020-2616 keywords: alpha-synuclein, dura mater, lymphatic drainage, prion, propagation, transmission abstract deposition of alpha-synuclein in the brain is a hallmark of lewy body disorders. alpha-synuclein has been considered to show prion-like properties. prion diseases can be transmitted by the transplantation of cadaveric dura mater causing iatrogenic creutzfeldt-jakob disease. recent observations of amyloid-β deposition in dural grafts support the seeding properties of amyloid-β. here we assessed the presence of alpha-synuclein in dura mater samples as a potential transmissible seed source. we immunostained 32 postmortem dura mater samples; 16 cases with lewy-body disorder (lbd) showing different pathology stages and 16 non-lbd cases for phosphorylated (ser129) and disease-associated (5g4) alpha-synuclein. disease-associated alpha-synuclein aggregates were identified in intradural nerve fibres and associated with a vessel in a single lbd-braak stage 4 case. we conclude that alpha-synuclein is detectable, although rarely, in dura mater samples in patients with lbd. the risk of potential transmissibility of dural alpha-synuclein deserves assessment by complementary experimental studies. introduction neurodegenerative diseases are considered protein-misfolding disorders. as described in prion diseases, the host physiological protein adopts an abnormal conformation (seed), capable of inducing misfolding and aggregation of the neighboring molecules (e.g. for prion protein: the physiological prpc converting to disease-associated prpsc, and prpsc recruiting more prpc). in prion disease, strong clinical, epidemiological and experimental evidence has demonstrated human and interspecies transmissibility of prpsc(1). for several other proteins associated with neurodegenerative conditions, such as amyloid-β (aβ), tau, alpha-synuclein, and tar dna binding protein (tdp-43), properties similar to those of prp have been shown predominantly in an experimental setting (2). the potential of aβ peptides to be transmitted between humans has been a topic of great interest for public health. young adults with iatrogenic creutzfeldt-jakob-disease (cjd) following cadaveric pituitary-derived growth hormone in childhood were found to have aβ deposits in the brain parenchyma and blood vessels (3, 5, 6), as well as in iatrogenic cjd after dura grafting (4). two studies have reported the presence of aβ deposits in dura mater samples of postmortem brains of patients with alzheimer’s disease (ad) and other neurodegenerative pathologies (7, 8). additionally, neurosurgical procedures and dural grafting during childhood or teenage years have been related to increased cerebral amyloid angiopathy (caa) decades later (9, 10). these findings indicate that dura mater grafts could be also a source of aβ seeds capable of inducing the aggregation of aβ in the cns of the graft recipients. lewy-body disorders (lbd) and multiple system atrophy (msa) are characterized by the accumulation of alpha-synuclein. a growing body of experimental evidence supports prion-like propagation in alpha-synucleinopathies (11-13). observations in a small number of subjects with parkinson`s disease (pd) treated with fetal mesencephalic dopaminergic grafts in the striatum, who years later were found to have alpha-synuclein positive lbs in grafted neurons, first suggested the potential of alpha-synuclein propagation from host to graft cells (14, 15). while experimental studies have also successfully demonstrated the transmission potential of msa alpha-synuclein in genetically modified mouse models (16), observations in humans argue against a human-to-human iatrogenic transmission of msa and pd (17). given the potential implications for public health, we posit that thorough assessment of the potential transmissibility of all neurodegenerative proteinopathies is essential (18). therefore, in this study, we assessed human postmortem dura mater samples, some of which have been previously shown to harbor aβ deposits (7), for the presence of alpha-synuclein aggregates, which may represent seeds with the potential of propagating further synucleinopathy. material and methods formalin-fixed and paraffin-embedded postmortem tissue samples of the dura mater from the left temporal and posterior region as well as cross-sections from the area of the superior sagittal sinus and confluence of sinuses (7), were cut into 5-micrometer thick sections. this study was approved by the ethics committee of the medical university of vienna. brains were previously evaluated neuropathologically and a final primary diagnosis and concomitant pathologies were recorded. fixation time of dura mater samples ranged from 3 to 10 years. immunohistochemistry of the dura mater samples was performed on an automated immunostainer (dako autostainer link 48, glostrup, denmark) using two different anti-alpha-synuclein antibodies: clone 5g4 (1:4000), roboscreen, leipzig, germany; specific for disease-associated forms and the phospho-specific anti-alpha-synuclein phosphorylated at serine 129 (wako) (1:2000). in addition, we performed immunohistochemical staining to depict the nerve fibres within the connective tissue of the dura mater: anti-neurofilament protein (nfp), clone 2f11 (1:800), dako; pgp 9.5 (1:100), dako, rabbit polyclonal; anti-tyrosine hydroxylase, clone 1b5 (1:200), novocastra, newcastle, uk; and anti-phosphorylated neurofilaments, clone smi31 (1:5000), former covance research products, princeton, nj, usa, and for blood and lymphatic vascular endothelia (anti-cd34, clone qbend/10 (1:100), novocastra; and podoplanin (1:3000), rabbit polyclonal). dako flex system (peroxidase/dab) was used for visualization of antibody reactions. samples were assessed blind to the neuropathological diagnosis. the presence or absence of each specific immunoreactivity was recorded. in addition, double immunofluorescence combining pgp 9.5 and 5g4 antibodies and immunofluorescence staining with nfp and 5g4 on adjacent tissue sections was performed in one selected case. results we analyzed postmortem dura mater samples from 32 donors (50% females) aged between 84 and 87 years (mean 84.9) from the vienna transdanube aging (vita) study (19). table 1: demographic and neuropathological features of the study cohort abbreviations: ad: alzheimer’s disease; pd: parkinson’s disease; copd: chronic obstructive pulmonary disease; ad-np: alzheimer’s disease neuropathological changes; caa: cerebral amyloid angiopathy; agd: argyrophilic grain disease; hs: hippocampal sclerosis; part: primary age-related tauopathy; svd: small vessel disease; cbd: corticobasal degeneration. n.a.: not available; n.e.: not evaluable due to unsteady immunoreactivity these cases were analyzed in a previous study for the presence of aβ deposits neuropathological diagnoses included 16 cases with alpha-synuclein pathology (table 1; six cases with amygdala predominant lewy body pathology, one msa case, single cases of braak stage 1, stage 2, stage 3, stage 5 and stage 6, and four cases with braak stage 4 of pd-related pathology) (20); and 16 cases without lb pathology but other neuropathological conditions. nerve fibers were detected mainly by nfp immunohistochemistry in all cases in variable distribution: large nerves surrounding large vessels (fig. 1a, b) or smaller nerve branches in between both dura layers (fig. 1c, d). ). these patterns were also observed by immunofluorescence (fig. 2). no immunoreactivity for tyrosine-hydroxylase could be detected. cd34 and podoplanin also gave negative results. figure 1 a-d: histologic appearance of dura mater consisting of two lamellae of connective tissue. within the connective tissue there are large and small vessels, which are surrounded by nerve fibres (a-d, neurofilament immunohistochemistry). nerve fibres are also identified between the lamellae and within the connective tissue (arrows). e-i: immunohistochemistry for phosphorylated alpha-synuclein at serine 129 and for oligomeric forms with the 5g4 antibody did not show abnormal alpha-synuclein aggregates in most of the studied cases (fig. 1e, f). in one case (case #12 table 1), coarse (g) and punctate (h) aggregates within nerve fibres and perivascular (i) aggregates were identified with the 5g4 antibody. detailed immunohistochemical analysis of dura samples identified pathological alpha-synuclein aggregates with the 5g4 antibody in one case. these aggregates consisted of small punctate deposits along nerve fibres (fig. 1h, 2b, d), coarse aggregates (fig. 1g, 2f) and perivascular deposits (fig. 1i, 2a). we observed a variable staining intensity of axons among the different cases and also within the same case, which was not related to the presence/absence of alpha-synuclein. this variability might have been related to the fixation time or tissue quality, among other potential variables, so we could not reliably assess axonal preservation in the patient with alpha-synuclein aggregates. we found no tortuous axonal profiles or axonal swellings. this individual was an 85-year-old male with coronary disease with severe and generalized atherosclerosis, an old myocardial infarction, as well as pulmonary adenocarcinoma. no parkinsonism or dementia were clinically reported. neuropathological examination revealed a vascular encephalopathy and braak stage 4 lewy-body pathology, with only mild neurofibrillary pathology in the limbic system and mild neuritic plaque / aβ pathology (table 1, case 12). in the remaining cases included in our study, with or without cns lewy body pathology, there was a complete lack of alpha-synuclein immunoreactivity in dural samples applying both antibodies. figure 2: immunofluorescence images a, b: double immunofluorescence for pgp 9.5 depicts delicate perivascular fibres (red) which focally also accumulate alpha-synuclein (5g4, green). other regions show nerve fascicles with variable density of axonal profiles: some have abundant axons (b, pgp 9.5, red) and show only tiny alpha-synuclein accumulations (5g4, green, white arrow). c-f: in adjacent tissue sections some nerve fascicles have only few axons (c, nfp) while others show an intermediate density of axons (e, nfp red) with relatively abundant and coarse alpha-synuclein aggregates (f, 5g4). discussion in this study, we found alpha-synuclein aggregates in dura mater samples from one patient with lewy-body pathology (braak stage 4) (20). alpha-synuclein aggregates were not identified in the dura mater of 31 other cases with different stages of lbd or in other neurodegenerative conditions. these findings might have implications for public health as well as for the understanding of alpha-synuclein propagation. concerning public health, individuals with symptomatic or prodromal alpha-synucleinopathies may undergo neurosurgical interventions contacting the dura mater (for example, the widespread use of deep brain stimulation in pd). while there is still no epidemiological evidence of human-to-human transmission of alpha-synuclein (17), the application of appropriate prevention measures will be increasingly required if experimental studies strengthen evidence of the potential transmissibility of synucleinopathies. this further emphasizes the need for effective biomarkers for the early diagnosis of alpha-synuclein related diseases (21-24). furthermore, there is a clear need for the detailed mapping of tissues that may harbor potential seeds. indeed, with respect to the present findings, since dura mater transplants are in direct contact with brain tissues, they might provide an increased potential to propagate seeds to the recipient’s cns. concerning protein propagation, the dura mater has been a source of disease transmission in human prion disease (1) and is also a source for aβ seeds (9, 10). recent studies support the notion that these seeds are able to propagate pathological proteins, even without inducing the classical alzheimer´s phenotype. however, they can lead to unexpectedly increased frequency of cerebral amyloid angiopathy and associated brain hemorrhages (9, 10). propagation of human alpha-synuclein has been considered since the detection of alpha-synuclein aggregates in grafted fetal mesencephalic neurons in pd patients (14, 15). this has subsequently been demonstrated in experimental studies (13) including inoculation of human substantia nigra extracts containing lewy bodies into wild-type mice and macaque monkeys (26). in contrast, the inoculation of extracts of peripheral alpha-synuclein from the autonomic sympathetic stellate ganglion did not induce pathological conversion of endogenous alpha-synuclein, alpha-synuclein propagation, or neurodegeneration in mice (27). thus, central and peripheral nervous system-related alpha-synuclein might have different propagation potential and therefore different transmissibility. the dura mater is thoroughly innervated by afferent fibres arising from the ipsilateral trigeminal ganglion and nerve (including its ophthalmic, maxillary and mandibular divisions) and by sympathetic fibres arising from the ipsilateral superior cervical ganglion. the posterior cranial fossa dura mater receives sensory meningeal branches from the vagus and glossopharyngeal nerves. the sympathetic ganglion is upstream from the stellate ganglion, which has been shown to harbor prominent alpha-synuclein or lewy-type pathology in postmortem studies of patients with pd and dementia with lewy bodies (23, 24), along with the dorsal motor nucleus of the vagal nerve in the cns (20). therefore, it might be reasonable to consider that these sympathetic and parasympathetic fibres innervating the dura mater might also accumulate alpha-synuclein. although in that particular case where we found alpha-synuclein in the dura mater, we did not have the opportunity to stain the trigeminal ganglion or nerve nor the stellate ganglion, we have previously evaluated the trigeminus nerve using immunostaining for alpha-synuclein. in that study we found alpha-synuclein immunoreactivity in two out of four pd/lbd cases where a total of six trigeminal nerves (in two cases both sides available) were examined (28). alternatively, we have previously reported that the presence of lymphatic vessels in dura mater might be a drainage route for molecules from the brain parenchyma in neurodegenerative disorders, including alpha-synuclein in lbd (25). although we found discrete perivascular alpha-synuclein aggregates in dura mater samples, we were not able to confirm whether these represent lymphatic vessels, possibly due to the prolonged fixation time of this dura mater sample (<6 years), and therefore this question deserves further study. the present study has some limitations. first, as mentioned above, the dura mater samples were obtained after a prolonged fixation time, which may influence immunohistochemical results, not only for cd34, podoplanin and th, but for phospho-synuclein as well (29). therefore, the interpretation concerning the perivascular association of alpha-synuclein aggregates needs some caution. we could also not reliably assess axonal preservation in the case harboring alpha-synuclein aggregates. the prolonged fixation also does not allow complementary molecular/biochemical studies. while the identification of characteristic pathological aggregates of alpha-synuclein with only one antibody is limiting, the 5g4 antibody has shown to be a robust marker of early alpha-synuclein pathology, even in brains with prolonged fixation time (30). second, the level of pathological forms of alpha-synuclein detectable by conventional immunohistochemistry may be low. moreover, the analysis of the supratentorial, rather than the infratentorial dura could also have been a limiting factor due to differences in their innervation; however, the supratentorial dura mater also receives sympathetic and parasympathetic innervation, and thus could also be exposed to transport of misfolded proteins. the negative findings in the other lbd cases of the series might reflect a sampling limitation, as it was not feasible to examine the whole dura mater. alternatively, the lack of staining could also reflect the diverse innervation of the dura and different pathogenic capacities of central and peripheral alpha-synuclein, or it could also indicate that the dura mater is not a primary vulnerable region for alpha-synuclein aggregation and could be considered to be a low risk tissue for the accumulation of potentially transmissible seeds. it must be emphasized that this study raises a potentially important issue but is unable to provide a conclusion on the risk of transmission of alpha-synuclein via dura mater grafts or neurosurgical procedures. this observation would ideally be complemented by experimental studies using prospectively collected freshly harvested, short fixed and frozen dura samples, including a larger cohort of patients of different age groups. conclusion disease-associated alpha-synuclein is detectable by conventional immunohistochemistry in postmortem supratentorial dura mater, here in a single patient with lewy-body-type alpha-synucleinopathy in the cns. this suggests propagation of alpha-synuclein from the brain and may indicate that dura mater could be a potential risk tissue for inadvertent transmission of alpha-synuclein by dura mater grafts or surgery. further experimental and biochemical studies are needed to assess the risk of alpha-synuclein transmission via dura mater and its relationship with human disease. acknowledgements we are grateful to susi schmid, irene ebner, gerda ricken and sigrid klotz from the division of neuropathology and neurochemistry, department of neurology, medical university of vienna, for excellent technical support. we are indebted to brain donors and their family of the vienna transdanube study (vita) for generous brain and sample donation for research. this work has been supported by the bishop karl golser award to ggk. references 1. brown p, brandel jp, sato t, et al. iatrogenic 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staining of common neurodegenerative disease markers. j neuropathol exp neurol. 2010; 69:40-52. 30. kovacs gg, wagner u, dumont b, et al. an antibody with high reactivity for disease-associated α-synuclein reveals extensive brain pathology. acta neuropathol. 2012; 124:37-50. copyright: © 2020 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. loss of ramified microglia precedes axonal spheroid formation in adult-onset leukoencephalopathy with axonal spheroids. feel free to add comments by clicking these icons on the sidebar free neuropathology 1:27 (2020) original paper loss of ramified microglia precedes axonal spheroid formation in adult-onset leukoencephalopathy with axonal spheroids. murad alturkustani 1,2,3, qi zhang 2,3, basma alyamany 2,3, lee-cyn ang 2,3 1 department of pathology, king abdulaziz university, jeddah, saudi arabia 2 london health sciences centre, london, ontario, canada 3 western university, london, ontario, canada corresponding author: murad alturkustani · pathology department · king abdulaziz university · abdullah sulayman, po box 80205 · jeddah · saudi arabia alturkustani.murad@gmail.com submitted: 22 august 2020 accepted: 9 september 2020 copyedited by: deanna c. fang and henry robbert published: 5 october 2020 https://doi.org/10.17879/freeneuropathology-2020-2971 additional resources and electronic supplementary material: supplementary material keywords: adult-onset leukoencephalopathy with axonal spheroids, leukodystrophy, microglia, axonal spheroids, white matter, hdls, alsp abstract two different pathological mechanisms have been suggested to underlie adult-onset leukoencephalopathy with axonal spheroids (alas). pathological studies have suggested that alas involves primary axonopathy with secondary demyelination. however, the identification of mutations in colony stimulating factor 1 receptor (csf1r), important for microglial survival, has suggested that alas is a microgliopathy. this study examines the correlation between microglial changes and axonopathy in alas. a total of 6 alas cases were studied. white matter lesions were classified into three evolving stages: 1) numerous axonal spheroids among well-myelinated fibers; 2) moderate loss of myelinated fibers with or without axonal spheroids; and 3) a leukodystrophy-like pattern of severe confluent axonal and myelin loss. axonal spheroids and ramified microglia were semi-quantified and the lesions were assigned a score of 0–3. we found a strong correlation between the preponderance of axonal spheroids and ramified microglial loss. all areas with a predominance of axonal spheroids showed a near-complete absence of ramified microglia, which was also apparent in small cortical and white matter lesions. in contrast, some areas with no ramified microglia showed no axonal pathology. our findings support the suggestion that ramified microglia loss precedes axonal spheroids formation. this observation will help to better understand the pathogenesis of alas and suggests a protective role of microglia. introduction two different pathological mechanisms have been suggested to underlie adult-onset leukoencephalopathy with axonal spheroids (alas). pathological studies have proposed a primary axonopathy with secondary demyelination (1, 2), while genetic/molecular studies have identified a mutation in a gene important for microglial function and, hence, implied that alas is a microgliopathy (3). moreover, a recent pathological study has identified a microglial morphological alteration (i.e. “dysplastic” microglia) as the pathological correlate for microgliopathy (4). alas is also known by other names in the literature with most common ones being hereditary diffuse leukoencephalopathy with spheroids (hdls) and adult-onset leukoencephalopathy with axonal spheroids and pigmented glia (alsp). recent advances in our understanding of the biology and functions of microglia have demonstrated both its beneficial and harmful effects on various diseases (5). investigating the relationship between microgliopathy and axonopathy in the pathogenesis of alas may provide better insight into the role of microglia in this disease. recent attempts to explain the pathogenesis yielded different conclusions: 1) generalized colony stimulating factor 1 receptor (csf1r) signaling impairment in cd68-immunopositive microglia is followed by multifocal axonal degeneration (6); 2) increase in ionizing calcium-binding molecule 1 (iba1) and cd68-immunopositive cell numbers precedes axonal pathology (7); 3) uneven distribution of iba1-immunopositive cells in the white matter precludes any firm conclusions regarding the role of these cells in the pathology (4); and 4) csf1r mutations lead to aberrant microglia density and distribution, and regional loss of microglia (8). in this study, we investigated the relationship between axonal pathology and microglial changes in alas using the pathological staging system of alas proposed by alturkustani et al. (1). this system focuses on the specific stages of the lesions rather than the overall pathological stage of the entire case at a point in time (7) and should provide a more precise correlation. materials and methods a formal autopsy consent was provided in all cases and the western university research ethics board approved the study. a total of 6 alas cases were studied. four cases were described before (cases 1–4) (1). two additional cases of alas were referred to the london health sciences center laboratory in 2013–2017. the brains were fixed and prepared as described before (1). representative samples were submitted for microscopic examination and staining, and immunostaining procedures were performed as described previously (1). these procedures include luxol fast blue (lfb) with hematoxylin and eosin (he) staining. for immunohistochemistry, the primary antibodies used were anti-amyloid precursor protein (app; chemicon, temecula, ca), anti-phosphorylated neurofilament (smi31; covance, berkeley, ca), and anti-glial fibrillary acidic protein (dako, carpinteria, ca). mouse monoclonal anti-hla-dr antibody (1:200; clone cr3/43; dako, carpinteria, ca) was used to label microglial cells. anti-ionized calcium binding adaptor molecule 1 (iba1) antibody (1:500, 019-19741, wako) was stained in dr. v. wee yong’s laboratory (calgary, canada) following their previously published protocol (9). the white matter lesions were classified into three evolving stages, as discussed in our previous work (1): 1) numerous axonal spheroids within well-myelinated fibers; 2) moderate loss of myelinated fibers with or without axonal spheroids; and 3) a leukodystrophy-like pattern with severe confluent axonal and myelin loss. digital images of lfb-he, app, hla-dr, iba1, and smi31 staining slides were obtained using the aperio system. particular areas of interest were demarcated on the computer screen of these digital images. at least five areas for each pathological stage from each case were examined. the pathological features, regardless of the size of the lesion, defined the pathological stages. any lesion can range from very small areas (usually in stage 1 pathology) to very large (common in stage 3 pathology). a single area in the white matter could contain more than one pathological stage. axonal spheroids and the hla-dr-expressing cells were semi-quantified (see supplemental figure 1) and lesions were assigned a score of 0–3. the scoring for axonal spheroids was as follows: 0 = absence of axonal spheroids; 1 = few axonal spheroids (1–2/high power field; hpf); 2 = moderate number of axonal spheroids (3–5/hpf); and 3 = frequent axonal spheroids (6 or more/hpf). for the hla-dr and/or iba1-expressing cells, we subdivided them into ramified-phenotype and amoeboid-phenotype. their numbers were scored in the following manner: 0 = no hla-dr staining; 1= few cells (1–9/hpf); 2 = moderate number of cells (10–19/hpf); and 3 = frequent cells (20 or more/hpf). several morphological shapes of microglia have been identified (10). however, for the sake of simplicity, in this study we divided microglia into two different types: 1) ramified microglia with a small cell body with branching processes radiating from the cell bodies, including a broad spectrum of reactive microglial shapes with varying lengths and thicknesses of processes and different cell body dimensions; and 2) amoeboid microglia with a macrophage-like or amoeboid phenotype characterized by a round cytoplasm with no processes. the white matter from the same autopsy tissue in the areas adjacent to the affected white matter and separate from it was used as control tissue to confirm the reliability of the immunostains (i.e. internal positive control) used in this study. as the autopsy brain tissue from relatively normal cases was not expected to show axonal spheroids, we did not use such controls in our study. results the clinical information for the first four cases has been discussed before (1) and is summarized in table 1. in general, clinical presentations were dependent on the location of the white matter areas affected, which were preferentially frontotemporal. case 5 was a 57-year-old female who had a rapid 2-year progression of neurobehavioral symptoms (self-neglect and apathy) and seizures until death. she had a sister with cognitive impairments, but slower progression. both patients had a previously unreported variant of csf1r (c.2563 c>t) mutation (11). their mother had a history of early-onset dementia. case 6 was a 38-year-old female with a rapid decline in cognition and shortand long-term memory impairment. she was placed in a nursing home as she required assistance for daily life activities and died two years after the initial presentation. her first-degree relatives had a positive history of dementia. she had another novel variant of csf1r (c.2377a>g) mutation. the csf1r gene mutational analysis for the first 4 cases was unsuccessful as the quality of the dna extracted from fixed tissue was inadequate, as discussed before (1). both mutations were analyzed via the varsome website (https://varsome.com/variant/hg19/nm_005211.3%3ac.2563c%3et and https://varsome.com/variant/hg19/nm_005211.3%3ac.2377a%3eg). various software tools including dann (score 0.9991 and 0.9989, respectively), mutationtaster (disease causing), fathmm (damaging), fathmm-mkl (damaging), metasvm (damaging), and metalr (damaging) predicted pathogenicity, which was supported by locations within the protein kinase functional domain. antibodies against hla-dr and iba1 are considered the most sensitive markers for microglia as both stain all the morphological forms of activated microglia, phagocytic, and dystrophic microglia (5). although iba1 may be considered to be a more sensitive marker for these microglia by some authors (5), we found that hla-dr is more sensitive for microglia staining in the white matter and comparable to iba1 staining in the gray matter. iba1 immunostaining also showed background staining, requiring careful examination to distinguish the immunopositive cells from the background (see supplemental figure 2). this resulted in an inferior quality of images for analysis. the following results were confirmed by both hla-dr and iba1 immunostains but are better demonstrated with the hla-dr images, which we will utilize in the rest of this article. hla-dr and iba1 immunostaining showed a wide variation of microglial morphology in both gray and white matter in all cases. microglial processes showed variations in number (single to multiple), thickness, and length. some of the processes showed discontinuous staining and some areas showed only fragmented processes, which could be considered dysplastic (4) or dystrophic (5). microglia with fragmented processes were observed along with those with intact processes in areas of normal-appearing white matter. this wide morphological variation made it difficult to correlate between microglial morphology and axonal spheroids. therefore, as indicated in materials and methods, we only considered microglia with the amoeboid phenotype (i.e. globular cytoplasm with no processes) as a separate form of microglia. all other morphological variations of microglia, including those with fragmented processes, were considered ramified/reactive microglia. using this simplified categorization of hla-dr and iba1-expressing cells (ramified vs. amoeboid phenotype), we consistently detected the following changes in all 6 cases (table 2): the white matter lesions classified as stage 1 pathology showed well-myelinated areas with moderate to frequent (score 2–3) axonal spheroids and a consistent loss of hla-dr-expressing cells with ramified morphology (score 0) in close proximity to the axonal spheroids (figure 1a–f). some areas had a few (score 1) hla-dr-expressing microglia with amoeboid phenotype, while others showed none (score 0). this may indicate that these areas had a slightly longer pathology duration than the areas without these cells. the white matter adjacent to the affected areas was unremarkable, with no axonal spheroids, no pigmented microglia, or other abnormality (figure 1g). however, hla-dr immunostaining showed denser forms of “ramified” microglia (figure 1h). the remarkable association of axonal spheroids and the complete loss of ramified microglia was also evident in the affected cortex (figure 2a–d). this association was apparent even in a very small area of cortical involvement, while the immediately adjacent area showed ramified microglia (figure 2e–h). in stage 2 pathology, white matter showed reduced myelin staining (figure 3a–b), a variable number of axonal spheroids ranging from none to frequent (score 0–3), moderate to frequent amoeboid microglia (score 2–3), and none to few ramified microglia. according to the distribution of axonal spheroids, these areas were subclassified into three components: 1) areas with axonal spheroids (score 1–3), showing no ramified microglia (score 0), but moderate to frequent amoeboid microglia (score 2–3). these areas may represent an “active/ongoing injury.” the white matter surrounding these areas showed widespread dense ramified microglia (score 3), while cells with amoeboid-phenotype were present in the central area (score 2–3), and the surrounding white matter (figure 3c–f), used as an internal control for hla-dr immunostaining. 2) areas with no axonal spheroids (score 0), few-moderate amoeboid microglia (score 1–2), and no ramified microglia (score 0; figure 3g–h). these can be considered “susceptible areas” and can have a different degree of myelinated axons loss. 3) areas with no axonal spheroids (score 0), few-moderate amoeboid microglia (score 1–2), and none to few ramified microglial cells (score 1). these may represent areas of secondary axonal degeneration. figure 1. microglia and axonal spheroids in stage 1 pathology. (a) normal-appearing white matter with frequent spheroids inside the rectangular inset (lfb-he; scale bar: 500 µm). (b) higher magnification of (a) shows many spheroids and no pigmented microglia (lfb-he; scale bar: 50 µm). (c) app immunostaining highlights frequent axonal spheroids inside the rectangular inset (app; scale bar: 500 µm). (d) higher magnification of (c) (app; scale bar: 100 µm). (e) the affected area in (c) with many axonal spheroids in the white matter and a small area of adjacent cortex that shows a nearly complete loss of hla-dr-expressing ramified microglia (inside the polygonal inset) and only a few scattered hla-dr-expressing amoeboid-phenotype cells. the adjacent normal-appearing areas show an increased expression of hla-dr-positive ramified microglia (hla-dr; scale bar: 1 mm). (f) higher magnification of (e) (hla-dr; scale bar: 500 µm). (g) normal-appearing white matter adjacent to the affected area shows no recognizable abnormalities (lfb-he; scale bar: 50 µm). (h): hla-dr immunostaining shows a frequent expression of hla-dr-immunopositive ramified microglia (hla-dr; scale bar: 50 µm). all images in this figure are representative of case 2. figure 2. cortical involvement in alas. (a) focal involvement of the upper cortex with frequent axonal spheroids (inside ellipse) compared to the cortex on the right side. the underlying u-fibers are also affected (lfb-he; scale bar: 500 µm). (b) higher magnification of (a) from the cortical area inside the ellipse (lfb-he; scale bar: 50 µm). (a) and (b) represent an inset (higher magnification of the upper rectangle) in figure 4a. (c) hla-dr immunostaining shows a complete focal loss of staining in the area with frequent axonal spheroids (inside ellipse) (hla-dr; scale bar: 500 µm). (d) higher magnification of (c) to highlight the presence of axonal spheroids (arrows) associated with a complete absence of hla-dr-expressing ramified microglia (hla-dr; scale bar: 50 µm). (e): a small cortical area (inside the rectangle) with frequent axonal spheroids (app; scale bar: 500 µm). (f) higher magnification of (e) (app; scale bar: 50 µm.). (g) hla-dr loss is limited to the very small affected area (inside the rectangle), while the surrounding cortical areas show hla-dr-expressing ramified microglia (hla-dr; scale bar: 500 µm). (h) higher magnification of (g) (hla-dr; scale bar: 200 µm). all images in this figure are representative of case 6. figure 3. microglia and axonal spheroids in stage 2 pathology. (a) decreased myelin staining in the deep subcortical white matter (mostly inside the inset) compared to the surrounding subcortical u-fibers (lfb-he, scale bar: 500 µm). (b) higher magnification of (a) (lfb-he;, scale bar: 200 µm). (c) the area showing decreased myelin staining in (a) shows a complete loss of hla-dr-expressing ramified microglia (mostly inside the inset) but moderate numbers of hla-dr-immunopositive cells with amoeboid phenotype. the adjacent white matter shows frequent hla-dr-expressing ramified microglia (hla-dr; scale bar: 500 µm). (d) higher magnification of (c) (scale bar: 200 µm). (e) app-immunopositive axonal spheroids are limited to the area (mostly inside the inset) showing an extensive loss of ramified microglia and presence of hla-dr expressing cells with amoeboid phenotype (app; scale bar: 500 µm). (f) higher magnification of (e) (app; scale bar: 200 µm). (g): another area with decreased myelin staining due to a prominent loss of myelinated fibers but without axonal spheroids (lfb-he; scale bar: 50 µm). (h) scattered hla-dr-immunopositive cells with amoeboid phenotype in the same area (hla-dr; scale bar: 100 µm). all images in this figure are representative of case 5. in stage 3 pathology, the white matter showed a marked loss of myelinated axons and a relative preservation of the subcortical u-fibers (figure 4a), which was classified as a “leukodystrophy-like” pattern. although the white matter in this area as a whole was considered stage 3 pathology, hla-dr immunostaining highlighted different patterns of staining (figure 4b), which allowed us to appreciate variations in the preservation of the white matter and the distribution of hla-dr-expressing cells in stage 3. we could observe areas of compact neuropil in the background, few small myelinated axons, a moderate number of oligodendrocytes, and mild to moderate gliosis (figure 4c–e) along with rarefied areas showing a marked loss of oligodendrocytes and moderate gliosis (figure 4g). hla-dr immunostaining highlighted dense (score 3) ramified microglia in the areas showing higher white matter preservation and in the relatively preserved myelinated subcortical u-fibers (figure 4b, d, f), while rarefied areas showed no ramified microglia (score 0) and a few to moderate (score 1–2) amoeboid microglia (figure 4h). axonal spheroids were absent (score 0) in most areas, yet scattered and rare (score 1) in others. unfortunately, the imaging characteristics are not available for the radiological-pathological correlation. in general, stage 3 pathology, which denotes long-standing lesions, are prominent in frontal and temporal lobes, less prominent in parietal lobes, and rare in occipital lobes. discussion axonal spheroids and ramified microglia we found a strong association between the presence of axonal spheroids and a complete loss of ramified microglia in all pathological stages of alas lesions. this association was also apparent in very small areas with frequent axonal spheroids and in focal cortical lesions, strengthening the reliability of the findings. although this was a semi-quantitative study, our findings demonstrate a strong correlation between the complete absence of ramified microglia (score 0) and the presence of axonal spheroids in affected areas. this finding could be considered “quantitative”, as score 0 denotes a complete absence of the ramified microglia. in stage 3, the presence of ramified microglia was associated with areas of better white matter preservation. these findings provide a link between pathological features observed (i.e. axonopathy) and the genetic abnormality identified (i.e. microgliopathy) in alas, supporting the beneficial effects of the reactive ramified microglia in maintaining axonal integrity. csf1r and alas identifying csf1r mutations as the underlying genetic abnormality in alas suggested microgliopathy as the underlying abnormality (3, 12) in alas. as it is the most common mutation in alas, konno et al. proposed the term “csf1r-related leukoencephalopathy” for this entity (13). as the pathological features are the gold standard for alas diagnosis and not all alas cases have csf1r alterations, this term would be specific to cases with confirmed csf1r mutations only and would not be suitable for all alas cases. aars2 mutations have been described in cases suspected to represent alas, including one case with biopsy findings supportive of this diagnosis (14). however, we concur with konno et al. that “further studies, including detailed analyses of autopsied brains, are required to characterize aars2 associated leukoencephalopathy” (13). csf1r is a tyrosine kinase receptor whose signaling is fundamental for microglial survival (15). two different mechanisms have been proposed for csf1r mutations to affect the function of microglia in patients with alas: 1) haploinsufficiency mechanism that results in a loss of function of csf1r (8, 16, 17), and 2) a dominant-negative model (18). it is still unclear what the lost functions are or what is constitutive of the dominant-negative effect on the microglia. oosterhof et al. concluded that the haploinsufficiency resulted in the maldistribution of microglia that leads to their local loss (8). their recent work showed that homozygous mutations in csf1r could result in the complete absence of microglia in pediatric-onset leukodystrophy (19). our results confirmed the regional loss of ramified microglia in alas and found that the axonal pathology is mostly limited to these areas, supporting the importance of microglia in alas’ pathogenesis and suggesting a protective role of ramified microglia. figure 4. microglia and axonal spheroids in stage 3 pathology. (a) marked loss of myelinated fibers in the deep white matter with relative preservation of the subcortical u-fibers (lfb-he; scale bar: 5 mm). (b) variable degree of hla-dr expression in the subcortical deep white matter with multifocal areas containing hla-dr-expressing ramified microglia. these areas are found mainly in the subcortical u-fibers and small foci of the adjacent white matter. the deeper white matter shows only scattered hla-dr-expressing ramified microglia (hla-dr; scale bar: 5 mm). (c) higher magnification of the area within the lower rectangle outlined in (a). the deep white matter shows a variable loss of myelinated fibers. areas of prominent loss show marked rarefaction, while areas with better preserved myelinated fibers in the center show a more compact parenchyma (lfb-he; scale bar: 500 µm). (d) a better-preserved central area in (c) shows a marked expression of hla-dr ramified microglia, while rarefied areas show only scattered hla-dr-expressing ramified microglia and moderate to frequent hla-dr-expressing cells with amoeboid phenotype (hla-dr; scale bar: 500 µm). (e) higher magnification of the better-preserved area in (c). although there is a marked loss of myelinated fibers, a few scattered myelinated fibers remain, neuropil appears more compacted, and the loss of oligodendrocytes is mild to moderate (lfb-he; scale bar: 50 µm). (f) higher magnification of the central area in (d) (hla-dr; scale bar: 50 µm). (g) the rarefied white matter areas show only occasional myelinated fibers, the neuropil is rarefied and mainly formed by astrocytic processes, and oligodendrocytes loss is moderate to severe (lfb-he; scale bar: 50 µm). (h) hla-dr immunostaining of the area in (g) shows mainly microglia with the amoeboid phenotype (hla-dr; scale bar: 100 µm). all images in this figure are representative of case 6. morphological correlation of microglia and axonal degeneration tada et al. described “dysplastic” microglia in brain samples from six patients with confirmed csf1r mutations (4). these dysplastic microglia were interpreted as the morphological manifestation of the csf1r mutation, but no association with axonal spheroids was established. in this study, we observed various reactive microglia morphologies, including some that can be considered “dystrophic”/“dysplastic”. however, the significance of these changes was hard to interpret and to correlate with the pathology. a different sequence of events in the pathogenesis of alas has been suggested. riku et al. concluded that broad microglial impairment and low csf1r expression on microglia preceded multifocal axonal degeneration (6). meanwhile, oyanagi et al. concluded that microglia proliferation preceded axonal swelling and loss. this conclusion suggests that microglial proliferation rather than loss leads to pathology (i.e. microglia is harmful and not beneficial; 7), which stands in contrast to our conclusions. in this study, we concluded that microglial loss preceded axonal degeneration based on the following observations: 1) there is a consistent complete loss of ramified microglia (by both hla-dr & iba1 immunostains) in all areas with axonal spheroids. as we did not observe any area with axonal spheroids to have reactive ramified microglia, it is unlikely that axonal spheroids precede microglia loss. 2) there is a presence of “susceptible areas” in stage 2 (i.e. areas with no ramified microglia and no axonal spheroids). ramified microglia loss in these areas are significant as reactive microglia should be present in areas with white matter pathology. the presence of microglia with amoeboid phenotypes in these areas confirm the reliability of microglia immunostaining in these sections. 3) there is a presence of morphologically abnormal microglia in normal-appearing white matter. tada et al., using ultrastructural examination, considered these dysplastic (4). methodological differences between the studies might explain the difference in conclusions between this study and previous studies. in this study we described the correlation between the axonal spheroids and ramified microglia in each lesion based on its stage rather than the overall brain pathology used by oyanagi et al. the latter system is useful for the correlation of clinical findings and the duration of pathology, while the former is more suitable to evaluate the mechanisms of a multifocal disease showing lesions with different pathological stages. similar limitations may also apply to the conclusion drawn by tada et al. on the microglial distribution in alas patients. they described spatial differences in microglia distribution with areas showing reduced numbers of microglia and others showing microgliosis in both the cortex and the white matter. they concluded that this differential distribution interferes with any meaningful quantitative statistical study of microglia depending on the area examined (4). this conclusion is valid if all lesions are studied together as one stage but not specifically for the stage of the individual lesion as analyzed in this study. we found that this spatial distribution did correlate with the axonal pathology in alas. the second significant difference between the previous studies and our study is that the other authors did not distinguish between ramified microglia and those with amoeboid phenotype. many morphological variations of microglia have been identified (10). although morphology is not a reliable predictor of function, microglia with amoeboid phenotype is an exception as it is consistently associated with phagocytosis (10). therefore, we suggest that at least the microglia with amoeboid phenotype should be distinguished from other phenotypes. microglia functions and pathology microglia are regarded as immune cells of the central nervous system (cns) and play a role in tackling viruses, phagocytosis, and as antigen-presenting cells (10, 20). recently, additional functions have been demonstrated, such as active surveillance of synaptic integrity, regulation of brain development and potentially in blood vessel formation (10, 20). microglia have been considered both pro-inflammatory and anti-inflammatory as well as neurotoxic and neuroprotective depending on their functional profiles (10, 20). the beneficial effects of microglia described include limiting the effects of cerebral ischemia in animal models since their absence through csf1r blockade results in increased infarct size (21–23), while microglial repopulation reverses this effect (23). microglial loss due to absence of csf1r affects brain development and results in olfactory deficits in mice (24). most harmful effects of microglia are associated with the concept of neuroinflammation, especially in multiple sclerosis (25), and after traumatic brain injury (26, 27). however, the term and the concept of neuroinflammation should be used with extreme caution, as suggested by graeber and streit (20). microglia and axonal pathology there are only few studies that deal with the association between the axonal pathology and microglial reaction. in the experimental micropig model for mild traumatic brain injury, lafrenaye et al. found that activated microglia contacted the proximal segments of injured axons (i.e. axonal spheroids) within 6 hours of the induced injury (28). lafrenaye et al. suggested that microglia activation is beneficial and may enhance axonal regeneration through this contact (29). in a human study, microglia activation and microglia nodules were associated with axonal swelling in the perilesional white matter. these changes were demonstrated in the periplaque white matter lesions in multiple sclerosis, in white matter areas adjacent to infarcts, and in traumatic brain injury (30). in this study, the preferred hypothesis was that microglial reaction/nodules resulted in the axonal damage (30). however, no evidence of the timing of the events was provided to support this conclusion. both studies (29, 30) showed the close association of reactive microglia to the axonal spheroids in affected white matter areas. the interpretation is quite different as the latter study suggested that neuroinflammation (defined as a microglial reaction) resulted in the axonal pathology (30), while the former study found that microglia reaction is secondary to axonal injury and suggested a neuroprotective function to these activated microglia. our study described a different association between the axonal pathology and the ramified microglia, which, to our knowledge, has not been described before, and in which axonal spheroids were associated with loss of ramified microglia. the remarkable restriction of axonal spheroids to these areas, best appreciated in the small early lesions (i.e. stage 1 pathology), suggests that the loss of microglia is protective in preventing this pathology. pathogenesis of alas the focus on the pathogenesis of alas has changed in the literature since the recognition of csf1r mutations in alas. previous pathological studies focused on the role of axonal pathology in the pathogenesis. the most relevant conclusions were: 1) the pathological features of the affected white matter suggest that the main pathological process is axonal degeneration with secondary demyelination (1); 2) the recurring or ongoing nature of the pathology is supported by the gradual progression of the white matter pathologic processes (31, 32); and 3) a possible role of hypoxic-ischemic injuries in the pathogenesis of alas (32, 33). studies after the discovery of csf1r mutations in alas focused on the effect of these mutations on microglia and the morphological changes of microglia in alas. the most relevant conclusions from these studies include: 1) csf1r mutations can affect microglia through loss of function (haploinsufficiency model; 17) or dominant-negative effect (18); 2) demonstration of dystrophic microglia as the possible pathological correlate of these mutations (4); 3) change of microglia density precedes the white matter pathology, but with contradicting results. oyanagi et al. proposed that microglia proliferation preceded axonal degeneration (7), while oosterhof et al. found that microglia loss preceded it (8); and 4) csf1r haploinsufficiency may lead to the maldistribution of microglia and result in the regional loss (8). there are many different ways to associate the previous observations and conclusions about the lesions in alas with meaningful pathogenesis. we propose the following pathogenesis: microglia loss precedes the axonal pathology and most likely represents an early stage of the pathology. however, it is still unclear what causes axonal injuries to develop in these restricted areas (and how), and whether this progression occurs with time only (e.g. aging process) or if an additional etiology (e.g. hypoperfusion) is required. axonal injuries that result in axonal spheroids formation have many causes, including hypoxic-ischemic, metabolic, toxic, and traumatic causes. in previous studies of alas, evidence of oxidative stress caused by hypoxic-ischemic etiology was presented (32, 33) and this may represent possible additional etiological factors in the pathogenesis of alas. the effects of hypoxic-ischemic injury depend on the severity and the duration of the hypoxic-ischemic events. as there are no morphological features of moderate to severe hypoxic-ischemic injury in alas, and the axonal injuries are limited to areas of microglia loss, we concluded that this should be viewed as a mild form of injury. one possible cause of mild hypoxic-ischemic events that occur with aging is age-related cerebral hypoperfusion (34), which could potentially explain the delayed presentation and progression of lesions in alas. we emphasize that this is speculative, and not a conclusion of this study, but it might be an explanation of the observed correlation between the axonal pathology and the microglial loss. however, this proposed pathogenesis indicates that progression of alas is potentially preventable through: 1) replacement of the lost ramified microglia to prevent further damage through their protective function, and 2) improving or preventing the reduced cerebral blood flow associated with aging. the first suggestion supports the promising results from allogeneic haematopoietic stem cell transplantation (to repopulate the microglial niche) in patients with alas (35, 36). the second suggestion is currently unpractical, even in the animal model, but this may change in the future. limitation of the study the major limitation of this study was the inability to test for csf1r mutation from the paraffin sections in four of the six cases. however, the histopathological diagnosis of alas is reliable and considered the current gold standard as new mutations in different genes are being discovered in alas. the pathological features of all cases were similar and the characteristic three pathological stages were present in all cases. there was no difference in the morphological features between cases with confirmed csf1r alterations (case 5 and 6), and cases with no genetic confirmation (cases 1–4). the association of microglia distribution to the axonal spheroids and the pathological stages were similar in all cases, as well (table 2). our results and conclusions in this study (i.e. the correlation between microglia loss and axonal spheroids) are based on the pathological findings only, regardless of the underlying genetic alterations. as such, this limitation should not depreciate our results and conclusions. however, it restricted our ability to study the correlation between csf1r mutation and the morphological changes. conclusions the main finding of this study is a multifocal loss of ramified microglia in alas. this loss was seen in the affected areas of all pathological stages and best correlated with the presence of axonal spheroids. the presence of dense ramified microglia was associated with better preservation of the white matter. we concluded that ramified microglia most likely have a protective function for the white matter and that their absence contributes to axonal pathology observed in alas. these findings will contribute to a better understanding of the pathogenesis of alas and the protective roles of microglia, thus providing possible areas of intervention to prevent the progression of the disease. acknowledgments we are grateful for the contributions of professor v. wee yong, university of calgary and hotchkiss brain institute, alberta, canada (performing iba1 immunohistochemistry), the neuropathology technologists at the london health sciences centre, ontario, canada (technical support), dr. ashraf dallol, king abdulaziz university, jeddah, saudi arabia (interpretation of genetic data) and the department of pathology, western university, ontario, canada (research funding for lee-cyn ang). part of this work was presented as a poster at the xixth international congress of neuropathology, tokyo, september 23–27, 2018. references 1. alturkustani m, keith j, hazrati ln, rademakers r, ang lc. pathologic staging of white matter lesions in adult-onset leukoencephalopathy/leukodystrophy with axonal spheroids. j neuropathol exp neurol 2015:74;233-40. 2. alturkustani m, sharma m, hammond r, ang lc. adult-onset leukodystrophy: review of 3 clinicopathologic phenotypes and a proposed classification. j neuropathol exp neurol 2013:72;1090-103. 3. rademakers r, baker m, nicholson am, rutherford nj, finch n, soto-ortolaza a, lash j, wider c, wojtas a, dejesus-hernandez m, adamson j, kouri n, sundal c, shuster ea, aasly j, 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open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated charcot identifies and illustrates amyotrophic lateral sclerosis feel free to add comments by clicking these icons on the sidebar free neuropathology 2:12 (2021) flashback charcot identifies and illustrates amyotrophic lateral sclerosis charles duyckaerts1,2, thierry maisonobe1,3, jean-jacques hauw4, danielle seilhean1,5 1 raymond escourolle neuropathology department, la salpêtrière hospital, assistance publique des hôpitaux de paris, sorbonne université, paris, france 2 alzheimer-prions team, paris brain institute icm (inserm u1127, cnrs umr7225), sorbonne université-umrs1127), paris, france 3 clinical neurophysiology department, la salpêtrière hospital, assistance publique des hôpitaux de paris, sorbonne université, paris, france 5 académie nationale de médecine, paris, france 6 als: causes and mechanisms of motor neuron degeneration, paris brain institute icm (inserm u1127, cnrs umr7225, sorbonne université-umrs1127), paris, france corresponding author: charles duyckaerts · raymond escourolle neuropathology department · la salpêtrière hospital · assistance publique des hôpitaux de paris · sorbonne université · paris · france charles.duyckaerts@gmail.com submitted: 31 march 2021 accepted: 08 may 2021 copyedited by: lauren walker and henry robbert published: 18 may 2021 https://doi.org/10.17879/freeneuropathology-2021-3323 keywords: amyotrophic lateral sclerosis, charcot, history of medicine, pyramidal tract abstract jean-martin charcot described what he called amyotrophic lateral sclerosis in his 12th and 13th lessons published in 1873 by bourneville. he distinguished the symptoms that were related to the lesion of the anterior horn of the spinal cord and those that were due to the degeneration (that he named “sclerosis”) of its lateral column. he thought that “inflammation” progressed from the lateral column to the anterior horn (but the term inflammation is not to be taken in the current meaning): the lesion of the anterior horn was thus “deuteropathic”. an album containing drawings made by charcot is kept in la salpêtrière neuropathology department. four drawings are pasted on one of its pages, showing the degeneration of the pyramidal tract. they constitute the original of the engravings illustrating charcot’s 12th lesson. the illustration of the fascicular atrophy of the adductor pollicis presented in the album does not appear in the lessons, even though this alteration is widely discussed and linked to the lesion of the anterior horn, which was supposed to ensure the “nutrition” of the muscle. the technique used by charcot and his interpretation of the microscopic pictures, as exposed in his lessons, are discussed. introduction jean-martin charcot identified amyotrophic lateral sclerosis (als) on clinical-pathological grounds. the data, which he collected, and his conception are remarkably summarized in the second volume of his lessons on the diseases of the nervous system, edited by bourneville (charcot, 1873) and in his lessons on cerebral localization, edited by bourneville et brissaud (charcot, 1876). the neuropathology laboratory of la salpêtrière hospital keeps an album containing original drawings by charcot. one of its pages concerns an als case. this paper compares the two documents: the drawings and the description of the disease, both by charcot. the album the album bears the title “charcot museum, pathological anatomy”, handwritten in ink on the black hardcover (fig. 1). it contains 100 light-blue pages of a format known as “couronne” in france (37 x 47 cm), commonly used by the artists of the time. pages 15 to 100 are blank. they were numbered with stencils. “foxing” (i.e. brown spots and browning of the edges) suggests they are original. the first fourteen pages have been restored, as well as the binding, some twenty years ago, and were renumbered in pencil. the drawings, usually on white papers, are pasted on those pages. there are also a few engravings, some taken from charcot’s articles. all the drawings have the same style and, although not signed, are reputed to be by charcot himself. charcot had hesitated between medicine and the fine arts. he left numerous drawings of pathological anatomy and sketches of patients (see for instance, the case bachère, charcot, 1892, fig. 64, p 337, https://gallica.bnf.fr/ark:/12148/bpt6k55646784/f343.item.texteimage; or a case of parkinson’s disease ibidem, fig. 63, p 336; the illustration of “a labioglossal spasm”, charcot, 1892, fig. 19 and 20, p 211, https://gallica.bnf.fr/ark:/12148/bpt6k55646784/f217.image.r=fig; or the picture of the face in a case of parkinson’s disease, charcot, 1892, fig. 60, p 334, https://gallica.bnf.fr/ark:/12148/bpt6k55646784/f340.image.r=fig). fig. 1. hardcover of the album in which charcot’s drawings were collected. these drawings demonstrate his talent for illustrating the cases that he examined. the style of the drawings found in the album is identical to that of the drawings found in the notes that he prepared for his “lessons” (still visible at charcot library, now located at paris brain institute-icm, https://institutducerveau-icm.org/en/actualite/charcot-library): there is no reason to question their authorship. the drawings, initially made on loose sheets, were secondarily collected and pasted in the album that was passed down from charcot’s own laboratory to the current department of neuropathology. each page generally concerns a particular topic (microaneurysm, multiple sclerosis, myelitis, lateral funiculus in a foetus, syringomyelia, pott’s disease, vertebral metastases, anatomy of the posterior funiculi, lead paralysis, tumor of the spinal cord, als, cysticercosis). the album has never been the subject of a specific study, although some of its pictures have been published occasionally (the drawing of the central nervous system of the als case under investigation here has been published in seilhean, 2020). cases and samples three original drawings of the als case, p 12 of the album (fig. 2), made on white papers, are pasted on a grey (faded green) cardboard and concern a case, identified as “trouillet’s case” at its bottom (fig. 2 and fig. 4). they include sections of the medulla oblongata and spinal cord, shown in the superior part of the card (fig. 3) and labeled a, b, c, d, and two views of the adductor pollicis muscle, which occupy the inferior part and are labeled a and b (fig. 4). a microscopic view of “altered slender columns” of the spinal cord (fig. 5), possibly from another case, has been directly pasted on the right side of the page as shown in fig. 2. fig. 2. general view of page 12 of the album dealing with amyotrophic lateral sclerosis. three drawings are pasted on a grey (faded green) cardboard (arrows). they show sections of the central nervous system (at the top) and of the adductor pollicis (at the bottom). in the lower right corner of the card, “cas de trouillet” (trouillet’s case) can be read. an additional drawing on the right illustrates two microscopic aspects of an “altered slender column [of the spinal cord]”. the views of “trouillet’s case” are not dated but, as we found a great similarity between the drawings of the album and the engravings of the 12th lesson (charcot, 1873, pp 221, fig. 16-19), we are inclined to believe that they are anterior to 1873. we could not elucidate the identity of that trouillet, probably the doctor who referred the patient to charcot. charcot gave the names of the two als cases that he published previously, as was usual at the time. the first case, catherine aubel, was published in a paper entitled “two cases of progressive muscular atrophy with lesions of the grey matter and of the anterolateral fascicles of the white matter” (charcot and joffroy, 1869, first case, p 354 reproduced in charcot, 1873, p 402). the disease of the second case of that paper (named a.c.) is not reported in the lessons and is not identified as als by charcot. the second case identified as als, elisabeth p., was published with only gombault as its author (gombault, 1871). it is reproduced in the lessons (charcot, 1873, p 416) with charcot’s commentary “with the collaboration of gombault” as if he, charcot, known for his authoritarianism, was the first coauthor. trouillet is not mentioned in those two articles. as charcot indicated that his lesson was based on the study of five clinical-pathological cases (charcot, 1873, p 228, footnote 1) and as three cases are identified (catherine aubel, elisabeth p. and trouillet’s case), two remain unidentified and, as far as we know, unpublished. fig. 3. upper part: page 12 of the album. trouillet’s case (see fig. 2). four sections of the central nervous system. the section are labeled a, b, c, d by charcot himself. the legend reads: a: bulbe b: moelle cervicale c: moelle dorsale d: moelle lombaire which means: a: medulla oblongata; b: spinal cord at cervical level; c: at thoracic level, d: at lumbar level. lower part: below the original drawings of charcot found in the album, the engravings of lesson 12 (charcot, 1873, pp 221, fig. 16-19). the legend in french reads: fig. 19. cross section of the medulla through the middle part of the olive. a, a, sclerotic anterior pyramids fig. 16. – transverse section of the spinal cord through the middle part of the cervical enlargement. fig. 17. – transverse section through the middle of the dorsal region. fig. 18. – transverse section through the middle of the lumbar enlargement. preparation of microscopic sections by charcot (et bouchard) the technique that has been used to obtain the illustrated sections is not reported. we can rely on two articles in which charcot indicated how he proceeded (charcot and joffroy, 1869 reproduced in charcot, 1873, pp 402-416; charcot, 1865). the central nervous system was fixed for “nearly one month” with a “much diluted solution of chromic acid” (the fixation properties of formaldehyde were described by f. blum two decades later – blum, 1893). the microtome, as we know it today, was probably not in use in paris at the time. its invention by purkinje in 1841 (chvátal, 2017) or his in 1866 (dupont, 2018) is controversial but it is clear that it was commonly used much later. the manual of histological techniques by mathias duval (1878), for instance, does not mention microtomes (in the current meaning) and concludes: “a large number of instruments have been invented [to cut fixed pieces]. one comes always back to the simple razor which is more efficient than all complicated ‘discotomes’ [we put in quotes] if the habit required to use it skillfully has been acquired.” the fixed sample was usually stuck in elderberry pith and cut, freehand, with a razor blade. the sections were not dehydrated and mounted in balsam or resin, as they are today. the manual of mathias duval (1878) indicates that one common practice was to cover the section with a coverslip (much thicker than they are today) and to introduce between the slide and the coverslip, on one side, a drop of glycerol that would diffuse and push the water out on the other side. the coverslip could then be sealed with wax or bitumen of judea (duval, 1878). fig. 4. two drawings, labeled a and b, are visible in the lower part of the page. a: one aspect of the adductor pollicis. a tendon appears as an unstained mass in the center of the section. numerous atrophic fibers are visible. b: another aspect of the adductor pollicis. fascicular atrophy of the muscle fibers are clearly seen around an artery in the center of the section. the staining was obtained with a few drops of “a concentrated solution of ammoniacal carmine”. “[…] the sick parts take a violet color, darker when the alteration is more severe. the stain of the healthy part remains unchanged.” (charcot, 1865, p 31). “this procedure, to make more sensitive to the eye the alterations of sclerosis, which has been erased by the maceration in chromic acid, belongs to m. bouchard”, then an intern in charcot’s department and later professor of general pathology at paris medicine faculty. what did ammoniacal carmine stain in sections fixed by chromic acid? we have no definite answer to this question. astrogliosis is a good candidate. several methods were developed later to stain “fibrous nevroglia” with crystal violet or other phenyl methane dyes (such as weigert’s or holzer’s methods), but the intimate mechanism of the staining remained unknown (proescher, 1934). the lower square view (at the highest magnification) shows “altered slender columns [literally cords]”, most probably the lateral column, after treatment by acetic acid (as mentioned: “après acide acétique”), a practice that was usual at the time. mathias duval (1878) writes in his manual (p 211-212) “the most important [of the “insulating reagents”] and the most used is acetic acid: its special action is to swell and make the connective or laminous fibers disappear, and as there are few tissues where these fibers are not in a certain abundance and do not veil the other elements, there are few preparations in which acetic acid is not used.” charcot mentions that “we have taken for comparison some very nice preparations of healthy spinal cord which we owe to the kindness of mr. lockhart clarke” (charcot and joffroy, 1869, p 366). it is interesting to note that the technique used by lockhart clarke was different: the fixation of the tissue was obtained with spirit of wine; the section was then treated with one part of acetic acid and three parts of spirit of wine before being cleared in turpentine oil and observed under a coverslip: in other words, the fixation was alcoholic and there was no staining. lockhart clarke’s pictures of the spinal cord were of an exceptional precision and beauty (lockhart clarke, 1851, 1858). a case of lockhart clarke is cited by charcot as possible cases of als (turner et al., 2010; radcliffe and lockhart clarke, 1862). the method used to prepare the muscle samples that were drawn in trouillet’s case is also not indicated. charcot and joffroy examined the muscle directly and immediately (“in the fresh state”), dissociating the fibers with needles in the case of catherine aubel (charcot and joffroy, 1869, p 362). vulpian (1869), at the same time, mentions that he examined the muscle fibers after fixation in a dilute solution of chromic acid. in the case of trouillet, this fixation also must have been applied, as charcot’s careful drawings necessarily took a certain amount of time that an unmounted and unfixed preparation would not have allowed. fig. 5. microscopic view of an “altered slender column” of the spinal cord. at the top, one reads “350 to 400 d[iameters]” (the magnification) and below the lower view “after acetic acid” (“après acide acétique” – see text for explanation). the drawings the drawing of trouillet’s case shows in red the pyramids of the medulla oblongata and the lateral column of the spinal cord, over-colored by ammionacal carmine. charcot insisted upon the sparing of a fascicle of white matter, outside the pyramidal tract and just below the surface of the spinal cord: “on transverse sections, at the level of the cervical enlargement, the symmetrical alteration is greater in width than anywhere else. the area invaded by the sclerosis reaches anteriorly, and even exceeds the external angle of the anterior horn. posteriorly, it almost reaches the posterior grey matter. laterally, however, it is constantly separated from the cortical layer of the spinal cord by a spared bundle of white matter” (charcot, 1873, pp 220-221). the microscopic view shown on the right of the page (fig. 5) may have belonged to another case and illustrate “altered slender columns” [of the spinal cord] at a magnification of “350 to 400 d[iameters]”. it shows oligodendroglial nuclei and scanty myelin sheaths. the loss of fibers that it probably intended to show is impossible to ascertain without comparison with a normal case. two views of the adductor pollicis muscle are shown (fig. 4, a and b). the fascicular atrophy is evident on panel b. charcot reported “infiltration by leukocytes, predominant near the tendon” (fig. 4, a). the tendon is the unstained mass in the center. it is difficult to ascertain the presence of leucocytes. it could as well be atrophic muscle fibers. charcot’s interpretation charcot describes the effects of the lesions of the anterior horn of the spinal cord in his 11th lesson, and of the lateral column of the spinal cord in the 12th and 13th lessons, the association of both lesions affecting anterior horn and lateral column, defining als. the lesion of the anterior horn and muscle atrophy: according to charcot’s description, the “large cells” of the anterior horn appear too few and atrophic, and the “neuroglia” is “sclerotic” (charcot, 1873, p 223). the anterior roots and the nerves are atrophic. charcot had understood that the muscle atrophy, visible at clinical examination, was due to the lesion of the anterior horn and was related to atrophy of the muscle fibres, visible at microscopic examination, contrarily to what is observed in muscle atrophy due to inactivity. “motor inertia caused in the lower limbs by the suppression of cerebral action may be complete, absolute. muscles, however, in such cases, do not lack nutrition or only in the long run because of protracted inaction” (charcot, 1873, p 199). the anterior horn provides the “nutrition” for the muscle – “nutrition” is a term charcot uses repeatedly in the study of the lesions of the anterior horn. the atrophy of denervated muscles had been previously described among others by vulpian (1869), a colleague and friend of charcot. charcot thought that the lesion of the anterior horn was responsible for the fasciculations that he named “fibrillar twitching”. (he added a remark that experienced neurologists could still make today: “i could add that [such fibrillar twitching] is not only seen in progressive muscular atrophy, but also in healthy subjects. it may then constitute one of the symptoms of a peculiar form of hypochondria, rather frequent, incidentally, in medical students.”) he considered that the involvement of the anterior horn spared the “faradic contractility” (contrarily to the primary involvement of the muscle). in his view, the involvement of the anterior horn was “secondary” to the lesion of the lateral column (hence, the term “deuteropathic” applied to the “chronic spinal amyotrophy” observed in als). “the progression of the inflammatory lesion from the fascicles of the white matter to the grey matter very likely takes place […] through the nervous tubes that physiologically connect more or less directly the two regions”. what was the meaning of the word “inflammation” at that time? certainly not the meaning of “neuroinflammation” as we understand it today. cohnheim (1873) was just publishing his observation on the course of inflammation as observed in thin, transparent tissue. with this experimental model, he could observe diapedesis. metchnikoff had not yet discovered phagocytosis (1893, metchnikoff, 1968). it is also worth mentioning here that the microglia have been described much later by del rio hortega (1919). the use of the term inflammation by charcot does not correspond, from today’s perspective, to his neuropathological description of the lesions: he did not describe leucocyte infiltration of the white or grey matter of the spinal cord. how could an “inflammatory lesion” progress “through the nervous tubes”? in this context, was the term “inflammation” used as a synonym for “lesion”? the lesion of the “lateral fascicle”, named later “pyramidal tract”: although the plantar reflex had not yet been described by babinski (1896), charcot believed that the motor deficit was initially due to the lesion of the lateral fascicles, rather than to the lesion of the anterior horns. muscle stiffness was a sign of the involvement of the same fascicle. the autonomy of the “system of the lateral fascicles” or simply of the “lateral fascicle” was discussed at length. he uses embryology to support his reasoning (charcot, 1873, 12th lesson, pp 215-218), showing that both the lateral fascicle and “türck’s bundle”, localized in the anterior column and altered in some cases of als, developed late in the spinal cord, in an “autonomous” manner. the description of the anatomy is both more precise and functional in his lessons of 1876 (charcot, 1876; translated in english in 1883 charcot, 1883), probably in relation with his recent acquaintance with flechsig’s work, abundantly referred to but without quotation. flechsig, head of the histology department and later professor of psychiatry in leipzig, used the chronology of myelination during development to recognize the spinal cord fascicles. they are correctly identified in charcot’s figure 46 “after flechsig” (charcot, 1876, p 179): the term “system of the lateral fasciculus” (charcot, 1873, p 220 with figures on p 221) is replaced by that of the crossed “pyramidal tract” (charcot, 1876, p 179), due to türck, who, as a professor of neurology in vienna, developed the method of secondary degeneration to follow the spinal cord bundles (türck, 1851). charcot shows pictures of the spinal cord stained by osmic acid: “thus, on a plate of flechsig that i am passing before your eyes and relating to the spinal cord of a newborn, you can see the following particularities: all the parts tinted in black are the developed parts: the cylinder-axis is surrounded by myelin sheath…” the pyramidal tracts, still unmyelinated, remain white (charcot, 1876, fig. 48 and 49, p 188-189). charcot had understood that the “autonomous development of the pyramidal tracts” was related to a late myelination. the spared bundle of white matter, outside of the pyramidal tract, precisely illustrated in the album (fig. 3) is in his 1875 lessons (charcot, 1876) correctly identified as the dorsal spinocerebellar tract (flechsig’s bundle or direct spinocerebellar tract): “as for the base of the triangle [made by the pyramidal tract], directed outwards, it is separated from the pia-mater by a band of nervous substance forming a sort of mantle for it and consisting of the direct cerebellar fascicles. but this arrangement occupies only the upper half of the medullary cord; below the dorsal region, the cerebellar bundles end and, in the lumbar region, where there is no trace of them left, the crossed pyramidal bundles touch the pia-mater” (charcot, 1876, pp 191-192). charcot’s description of the motor symptoms, based on his clinical-pathological method (goetz, 2000) is now fully achieved and underlines the systematic aspect of the atrophy, and its functional significance. charcot underlined the absence of sensory deficit in als. he knew that the lesion of the posterior columns were linked with “locomotor ataxia” but there is a stark contrast, in the lessons dealing with “localization”, between the knowledge that charcot had of the pyramidal tract and the difficulties that he still met in understanding the somatosensory pathways. he could state, for example, that for sensitivity, there is “indifferent conduction through this or that element of the spinal cord” (charcot, 1876, p 282). in charcot’s view the typical progression of als started in the upper limbs on one side, affecting secondarily the lower limbs, sparing the nerves innervating the vesical and rectal muscles, and ending with “labio-glosso-pharyngeal” paralysis, the nerves located above the viith cranial nerve being unaffected. the various clinical aspects of als remained, at the time, to be described. a note on the historical context charcot used to quote rather extensively the literature, at least according to the standards of the time. his library, still visible today, testifies of the extent of his interests. he cited several cases of the literature from france, germany and the united kingdom in support of his analysis. the second volume of the lessons, however, was published in 1873, three years only after the siege of paris by the prussian troops. people caught in paris during the siege were hungry and ate dogs, horses, rats and even elephants and antelopes from the zoo (as told by victor hugo in his book “choses vues” – hugo, 1900 passim and pp 299, 308)! charcot had sent his wife and his three children to normandy, outside paris where he, personally, continued to work at the hospital (bonduelle et al. 1996, p. 296). if charcot used to remain on purely scientific grounds, it is not impossible that the comments on a paper by nikolaus friedreich (professor of pathological anatomy in würzburg, later professor of pathology and therapy in heidelberg) were tinged with a certain resentment: “such a neglect in matters of nosographic distinctions, especially in a question in itself rather obscure, is at least regrettable and can only maintain the confusion” (charcot, 1873, footnote 1, p 208). such a critical remark is counterbalanced by a good knowledge of the german literature. it is charcot who names the direct corticospinal tract türck’s bundle (referring to türck, probably türck, 1851) and underlines the importance of flechsig (unfortunately without quotation) in the functional understanding of the disease that he was identifying (charcot, 1876, p 191). flechsig published an early and impressive synthesis of his work in 1876 (flechsig, 1876); he was only 29. conclusion it may finally be considered that the wish that charcot expressed in his lesson has been fulfilled: “i must first declare that the reports on which my description is based, are still few, twenty at the most. but one must notice that it was also the case, some time ago, concerning progressive locomotor ataxia. the clinical picture drawn by duchenne (de boulogne) with the help of a small number of facts, nearly 20 years ago, has not aged. [...] may my description of amyotrophic lateral sclerosis experience the same fate!” (charcot, 1873, p 228) acknowledgement the help of véronique leroux-hugon, previous curator of charcot library, and of florian horrein, the current curator, is greatly acknowledged. charcot’s album belongs to the “musée de l’assistance publique-hôpitaux de paris”, which we thank, and kept in the neuropathology department of la salpêtrière hospital. references babinski j. (1896) sur le réflexe cutané plantaire dans certaines affections du système nerveux central. c r seances soc biol. 3: 207-208. blum f. (1893) der formaldehyd als hartungsmittel. z wiss mikrosk. 10: 314-315. bonduelle m., gelfand t., goetz c.g. (1996) charcot, un grand médecin dans son siècle. michalon publisher, paris. charcot j.-m. (1865) sclérose des cordons latéraux de la moelle épinière, chez une femme hystérique, atteint de contracture permanente des quatre membres. bull mém soc méd hôp paris. 2: 24-35. 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[the paper may be accessed on the internet: https://gallica.bnf.fr/ark:/12148/bpt6k432734s/f558.item ] copyright: © 2021 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. top ten discoveries of the year: neurovascular disease feel free to add comments by clicking these icons on the sidebar free neuropathology 1:5 (2020) review top ten discoveries of the year: neurovascular disease anna m. planas department of brain ischemia and neurodegeneration, spanish national research council (csic), barcelona, spain corresponding author: anna m. planas · department of brain ischemia and neurodegeneration · institute for biomedical research of barcelona (iibb) · spanish national research council (csic) · institute for biomedical research august pi i sunyer (idibaps) · rosselló 161 · planta 6 · 08036-barcelona · spain anna.planas@iibb.csic.es submitted: 09 january 2020 accepted: 25 january 2020 published: 30 january 2020 https://doi.org/10.17879/freeneuropathology-2020-2615 abstract the aim of this review is to highlight novel findings in 2019 in the area of neurovascular disease. experimental studies have provided insight into disease development, molecular determinants of pathology, and putative novel therapeutic targets. studies in genetic experimental models as well as monogenic forms of human cerebrovascular diseases identified pathogenic molecules that may also be relevant to sporadic cases. there have been advances in understanding the development of cerebral cavernous angiomas and arteriovenous malformations, and putative curative treatments have been suggested from experimental models. key pathogenic pathways involved in vessel calcification and stiffness have also been identified. at the cellular level, studies showed that proper function of endothelial and mural cells, particularly pericytes, is crucial to ensure full endothelial differentiation and blood-brain barrier integrity. moreover, recent discoveries support the existence of a homeostatic crosstalk between vascular cells and other neural cells, including neurons. cerebrovascular diseases are strongly associated with inflammation. beyond pathogenic roles of specific components of the inflammatory response, new discoveries showed interesting interactions between inflammatory molecules and regulators of vascular function. clinical investigation on cerebrovascular diseases has progressed by combining advanced imaging and genome-wide association studies. finally, vascular cognitive impairment and dementia are receiving increasing attention. recent findings suggest that high-salt intake may cause cerebrovascular dysfunction and cognitive impairment independent of hypoperfusion and hypertension. these and other recent reports will surely inspire further research in the field of cerebrovascular disease that will hopefully contribute to improved prevention and treatment. abbreviations apoe, apolipoprotein e; avm, arteriovenous malformations; bbb, blood-brain barrier; caa, cerebral amyloid angiopathy; cadasil, cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy; carasil, cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy; cbf, cerebral blood flow; ccm, cerebral cavernous angiomas; cdk5, cyclin-dependent kinase 5; dti, diffusion tensor imaging; endmt, endothelial-to-mesenchymal transition; gom, granular osmiophilic material; ipscs, induced pluripotent stem cells; pdgfb, platelet-derived growth factor subunit β; pfbc, primary familial brain calcification, fahr’s disease; sah, subarachnoid hemorrhage; smcs, smooth muscle cells; timp3, tissue inhibitor of metalloproteinase 3; tgfβ, transforming growth factor-β introduction in the next pages i will discuss a few topics in the field of cerebrovascular disease that i believe have made advances in 2019 supported by excellent studies covering genetic and sporadic forms of the diseases in humans and experimental animals. 1. cavernous angiomas originate from clonal expansion of mutated endothelial cells there have been recent advances in understanding the development of cerebral cavernous angiomas (ccm). new results suggest that small angiomas may derive from a clonal expansion of mutant endothelial cells that undergo endothelial-to-mesenchymal transition and express stem-cell like features (fig. 1). ccm are vascular lesions composed of clusters of capillary-venous blood vessels with thin walls and impaired endothelial tight junctions that usually contain stagnant blood. these abnormal vessels may rupture causing intracranial hemorrhage (fig. 1) or small subclinical bleeds, which may lead to seizures. most cases of ccm are spontaneous but 20% of cases may be attributed to autosomal dominant inheritance. the disease is caused by sporadic or inherited mutations in one of the following three genes: ccm1 (krit1), ccm2 (mgc4607), or ccm3 (pdcd10). in the affected vessels, endothelial cells suffer endothelial-to-mesenchymal transition (endmt), express stem cell markers, and undergo loss of typical endothelial cell properties, with increased cell migration, reduced adherence, and increased blood-brain barrier (bbb) permeability. in familial cases, the vascular lesions are discrete although the above genetic mutations affect all cells. furthermore, heterozygous mice do not develop the disease. the prevalent explanation is the two-hit hypothesis. endothelial cells carrying a germline mutation in one of the ccm genes need to suffer a second, likely a somatic, mutation that will trigger the pathology. malinverno et al. (2019) showed that only a few endothelial cells bearing a ccm3-/mutation, undergoing endmt, and expressing progenitor markers, are needed to generate cavernomas because the affected cells undergo clonal expansion. demonstration of clonal expansion was carried out using an elegant strategy by crossing the ‘r26r-confetti’ mouse, a multicolor reporter mouse model, first with cdh5(pac)-creert2 mice for an endothelial specific expression of cre, and then with ccm3fl/fl mice. endothelial deletion of ccm3 was accompanied by color-based clonal lineage tracing. the majority of small vascular lesions were composed of cells of the same color suggesting a clonal origin from proliferation of the affected endothelial cells. this study followed two previous studies (detter et al., 2018; manavski et al., 2018) where clonal expansion of mutant endothelial cell in ccm endmt was also demonstrated using a reporter confetti strategy. in contrast to the clonal origin of small lesions, large cavernomas are mosaics containing clonally dominant mutant cells and wild type endothelial cells suggesting recruitment of neighboring wild type cells to the cavernous lesion. those wild type endothelial cells also overexpressed endmt markers. transplantation of ccm3-/cells into the brain of wild type mice generated abnormal vessels and recruited endothelial cells from the host (malinverno et al., 2019). by means of several in vitro studies, these authors demonstrated that wild type endothelial cells acquire features of endmt after contacting ccm3-/cells. furthermore, by using genetic mouse models, malinverno et al. deleted ccm3 in endothelial progenitors. they hypothesized that the early steps of cavernoma formation are due to clonal expansion of resident endothelial progenitors. in this way they provided evidence supporting the view that endothelial progenitors suffering ccm3 mutation trigger cavernoma formation. hopefully these new discoveries will provide novel avenues to prevent or attenuate ccm. figure 1. cavernomas. schematic representation of cavernoma generation from vessels with mutated ccm genes (see text section 1). according to malinverno et al., (2019), the process involves endothelial-to-mesenchymal transition (endmt) and clonal expansion. susceptibility weighted imaging shows a cavernoma with hemorrhage in the right occipital lobe (arrow). the mri image was obtained in the comprehensive stroke center of hospital clinic of barcelona. 2. cerebral arteriovenous malformations: from origin to treatment failure to achieve full endothelial cell differentiation could underlie the pathology of arteriovenous malformations (avms) where recent studies identified β-adrenergic antagonists as putative curative treatments. avms involve the formation of arteriovenous shunts amongst other vascular abnormalities including vessel calcifications, and the presence of surrounding macrophages and gliosis. this condition impairs oxygen delivery to the parenchyma and predisposes to vessel rupture and hemorrhage. alterations in endothelial cell endmt are associated with this pathology. yao et al. (2019) recently investigated the presence of endmt markers in the endothelium of human avms. through complementary strategies they identified the expression of the stem cell marker sry-box 2 (sox2) and the mesenchymal marker n-cadherin in lesional endothelial cells, together with a reduction in the expression of typical endothelial markers. the authors also investigated a mouse model of avms, the matrix gla protein null (mgp-/-) mouse, which develops arterial calcifications, enlarged vessels, and arterial-venous shunts. cerebral endothelial cells of these mice also overexpressed sox2.they limited sox2 expression in endothelial cells by generating heterozygous cdh5-cre/sox2fl/wt mice with endothelial deletion of sox2 in heterozygosis. these mice were bred with mgp-/mice, which no longer overexpressed sox2 and, importantly, the pathological vascular features of avms were strongly attenuated. moreover, reduction of sox2 abolished the expression of endmt markers. chip-seq analysis showed alterations in histone methylation affecting the expression of several genes that participate in stem cell pathways and epithelial-mesenchymal signaling. the analysis identified overexpression of the gene jmjd5 in mgp-/cerebral endothelial cells as a downstream target of sox2. then, a human brain microvascular endothelial cell line depleted of mgp was generated using crisp/cas9 technology. this line showed higher expression of n-cadherin than wild type cells, suggesting endmt. in addition, these cells overexpressed sox2 and jmjd5. sox2 depletion reduced jmjd5 expression. further experiments suggested that sox2 also interacts with jmjd5 to regulate endmt. to identify drugs that could prevent avms by inhibiting sox2, a high-throughput robotic model was generated using a cell line that allowed screening of more than 3,000 products. the system identified a drug called pronethanol, which was shown to effectively reduce sox2 expression. furthermore, treatment of mgp-/mice with pronethanol for 14 days improved the cerebral vasculature in avm. interestingly, pronethanol is a non-selective beta-blocker with the drawback of causing neurological side effects in humans and carcinogenesis in mice. a number of ß-adrenergic drugs were subsequently tested. the results showed that ß-adrenergic antagonists decreased sox2 and endmt, and improved lumen formation. this study is a good example of the way mechanistic studies in human tissue, human cells, and animal models lead to the discovery of potential drugs to improve the disease. previous studies in mgp-/mice had shown the contribution of endmt and the emergence of multipotent cells to the process of ossification and vascular calcification (e.g. yao et al., 2013). like in the case of ccms, failure of brain endothelial cells to acquire or maintain their differentiation state generates aberrant vessels and vascular diseases. 3. arterial stiffness and calcifications: molecular insights and consequences vessel calcification and arterial stiffness are common in the aged population. arterial stiffness is caused by structural alterations reducing arterial wall distensibility and the arterial capacity to buffer pulsatile cardiac ejection. this condition is epidemiologically associated with hypertension and cognitive decline. arterial calcification is an important cause of arterial stiffness but the contribution of calcifications to cognitive decline over other associated features is unclear. muhire et al. (2019) showed in experimental animals that carotid calcification attenuated resting cerebral blood flow (cbf), impaired cerebral autoregulation, and induced cognitive deficits. the study used a mouse model of arterial stiffness induced by direct application of cacl2 on the carotid artery. the model is characterized by reduced arterial compliance and distensibility, increased thickness of the intima-media, and fragmentation of the internal elastic lamina, without increased systolic blood pressure. carotid stiffness increased bbb permeability in the hippocampus where the number of collagen iv+ vessels decreased, suggesting a pathogenic role of these calcifications. regarding the molecular mechanisms underlying vessel calcifications in brain arteries, a recent study pointed to the involvement of osteopontin and tgfβ signaling in the pathogenesis of calcifications (grand moursel et al., 2019). osteopontin is an extracellular glycoprotein with diverse features. it is cleaved by thrombin and cleaved forms can bind to several integrin receptors expressed by immune cells. given that vascular calcifications are detected in the cerebral cortex in severe forms of cerebral amyloid angiopathy (caa), the study investigated molecular modulators of arterial calcifications in post-mortem tissue of eight patients with hereditary cerebral hemorrhage with amyloidosis-dutch type (hchwa-d or d-caa). calcified vessel walls showed overexpression of medial and abluminal collagen type i. they also overexpressed osteopontin and tgfβ signaling factor phospho-smad2/3, and accumulation of these factors preceded overt vessel calcifications. the study found significant positive correlations between the level of osteopontin and phospho-smad2/3 and disease severity and suggested the involvement of these molecules in cerebral artery calcifications in caa. more severe brain vessel calcifications are found in certain rare genetic diseases. primary familial brain calcification (pfbc) is a heritable disease also termed fahr’s disease. it is a rare form of brain calcification affecting vessels of the basal ganglia and often manifests with neuropsychiatric symptoms. zarb et al. (2019) used a mouse model with mutations in platelet-derived growth factor subunit-β (pdgfb) gene causing reduced endothelial expression of this factor, resulting in vascular calcifications associated with pericyte loss and bbb alterations. interestingly, the mouse model recapitulated several behavioral alterations overlapping with pfbc, including sensorimotor deficits, hyperactivity, anxiety and impaired working memory. cells surrounding brain vessel calcifications expressed osteoblast, osteoclast, and osteocyte markers. the extracellular matrix contained bone matrix proteins. importantly, blood vessel ossification was also demonstrated in brain tissue from primary familial brain calcification patients. another consequence of vessel calcifications was activation of microglial and astroglial cells. given the reported astrogliosis in pfbc patients, astrocytes were investigated in the mouse model. notably, gfap+ astrocytes surrounding calcifications expressed oxidative stress and inflammatory neurotoxic markers, such as complement component 3 (c3) and lipocalin 2. 4. inflammation/innate immunity: role of complement activation inflammation is present in most brain diseases affecting the elderly, including cerebrovascular disease. recently, activation of the complement cascade was shown to play a pathogenic role in subarachnoid hemorrhage. moreover, interaction between initiator complement component c1q and apoe was reported. complement activation has been reported in cerebrovascular diseases and several lines of evidence strongly support the involvement of complement in secondary tissue injury following acute stroke. c3 is amongst key biomarkers associated with increased risk of major disability, mortality and vascular events following ischemic stroke (zhong et al., 2019). complement activation is also associated with poor functional outcome following aneurysmal subarachnoid hemorrhage (sah). a recent study showed that complement components c1q, c3/c3b/ic3b were more abundant in the brain of sah patients than controls (van dijk et al., 2019). a single nucleotide polymorphism in c5 correlated with poor outcome in sah patients, which showed increased levels of c5a in csf and plasma. in a mouse model of sah, blocking c5 activation, using mice deficient in c5a or after administration of c5a blocking antibodies, reduced microglial activation and neural cell death. interestingly, a recent study discovered molecular links between complement and apolipoprotein-e (apoe). apoe is a factor associated with alzheimer’s disease, cerebrovascular diseases, and atherosclerosis (yin et al., 2019). human apoe has four isoforms apoe, apoe2, apoe3, and apoe4. the apoe4 allele is an important risk factor for late onset alzheimer’s disease, whereas several studies indicate that altered apoe2 could be associated with white matter hyperintensities in small vessel diseases. yin et al. (2019) showed absence of apoe-mediated activation of the classical complement cascade in the choroid plexus. lipid deposits were noticed in choroid plexus of aged apoe-/mice regardless of whether they were fed a high fat diet or normal chow. knockin mice expressing the human form of apoe4 (apoe4-ki) also showed lipid deposits in the choroid plexus but only when fed a high fat diet. in contrast, apoe3-ki mice did not develop those lipid deposits irrespective of diet regimen. lipid accumulation was paralleled by leukocyte accumulation in the choroid plexus and csf. using laser-capture microdissection of choroid plexus followed by differential gene expression profiling, an interferon signature was detected in apoe4-ki mice. additionally, upregulation of complement genes was detected in apoe-/mice. accordingly, the choroid plexus of apoe-/mice showed accumulation of immunoglobulins, and complement proteins c1q, c3, c4, as well as c5 and anaphylatoxin c3a. through various in vitro strategies, the study demonstrated that apoe inhibits the classical pathway of complement activation through ca2+-dependent high affinity binding to the activated form of c1q, which was elicited by all apoe isoforms. notably, the formation of lipid droplets in the choroid plexus was found in human brains with various degrees of alzheimer’s disease pathology, and higher lipid droplet numbers were found in patients with dementia. lipid deposits were associated with c1q-apoe complex. furthermore, c1q and apoe colocalized in atherosclerotic plaques. overall, the study suggests that apoe may prevent complement activation. altered inhibitory effects of apoe on complement due to allelic alterations is proposed as a pathogenic mechanism in alzheimer’s disease and atherosclerosis. further studies will hopefully elucidate the role of this new molecular interaction between apoe and complement in cerebrovascular and neurodegenerative diseases. 5. cerebral amyloid angiopathy: microbleeds, microinfarcts and white matter hyperintensities progress has been made in understanding the neuropathology of caa as well as the neuropathological correlates of mri alterations in this condition. caa can lead to symptomatic lobar intracerebral hemorrhage. mri of caa patients may show microbleeds, microinfarcts, and white matter hyperintensities (reijmer et al., 2015). van veluw et al. (2019a) reported that microbleeds and microinfarcts represent different microvascular alterations in caa. the vessels showing microbleeds had accumulation of amyloid β (aβ) and fibrin/fibrinogen upstream and downstream of the microbleed, but not in the microbleed region devoid of smooth muscle cells (smcs). likewise, smcs were missing in the regions of microinfarcts, but in this case aβ was present in the injured core. the study suggested that removal of aβ in vessels that have lost smcs might increase the risk of bleeding, which, if confirmed, might potentially have future therapeutic implications. using high resolution mri in post-mortem brain tissue with correlative histopathological analyses, van veluw et al (2019b) provided relevant information on the neuropathological correlates of neuroimaging alterations. diffusion tensor imaging (dti) is the mri modality best suited to study white matter fiber track orientation and physical features that can assess microstructural integrity of the brain tissue. the post-mortem brain of caa patients was fixed with 10% formalin for at least 3 weeks. one brain hemisphere was scanned in a 3t mri scanner for 14 hours. image analysis included fiber tractography, and 3-mm thick regions of interest were highlighted in parts of the tracts later taken for histology. formalin fixation alters the mri diffusion properties, but fractional anisotropy measured in vivo was linearly related with fractional anisotropy acquired ex vivo in the same brains. the tissue was embedded in paraffin and 6-µm thick sections were obtained for histological analyses. the studies found reduced fractional anisotropy in caa patients compared to controls. in multivariate analysis, fractional anisotropy data was independently associated with tissue rarefaction as assessed with luxol fast blue & hematoxylin, lower myelin density as assessed with immunohistochemistry against myelin basic protein, and decreased axonal density determined by immunohistochemistry against neurofilament (nf200). another dti parameter, mean diffusivity, was increased in cca patients and was independently associated with myelin density (van veluw et al., 2019b). moreover, increased mean diffusivity in the frontal white matter was associated with caa severity in the frontal cortex. in contrast, the dti changes were not related to markers of gliosis or with oligodendrocyte counts. investigations on the neuropathological correlates of imaging alterations are important because mri is widely available and can be used to follow the progression of disease. 6. novel mechanisms underlying cadasil vasculopathy cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (cadasil) is the most common hereditary monogenic form of cerebral small vessel disease. cadasil leads to early onset hemorrhagic or ischemic stroke and vascular dementia. recent findings in cadasil unraveled novel molecular targets by describing attenuated vascular dysfunction due to inhibitors of endoplasmic reticulum stress and modulators of the cytoskeleton, or due to vascular endothelial growth factor supplementation. cadasil pathology develops due to mutations in the notch3 gene which result in dysfunction of vascular smcs. notch3 is involved in vasculogenesis and is mainly expressed in smcs in adults. however, no specific therapies have been developed so far, possibly because there are many aspects of notch3 function that remain currently unknown. a recent study (neves et al. 2019) identified molecular targets involved in vascular dysfunction in cadasil. notably, the study replicates in a cadasil mouse model, the tgnotch3r169c mouse, the same findings as in human cadasil. samples were obtained from peripheral biopsies of cadasil patients. molecular alterations were identified in peripheral arteries despite the fact that cadasil pathology manifests principally in cerebral vessels. peripheral vessels of cadasil patients showed impaired vascular reactivity and altered vascular structure with features suggesting reduced stiffness. alterations in smc proliferation, apoptosis, and cytoskeletal disorganization was also noted. vascular smcs of cadasil patients showed increased notch3 signaling, notch3 ectodomain (notch3ecd) accumulation, and expression of notch3 target genes relative to control patients. the study also showed nox5 upregulation linked to an exaggerated endoplasmic reticulum stress response and aberrant cytoskeleton-associated protein phosphorylation in cadasil. moreover, the study reports that inhibitors of notch3, nox5, er stress, and rhoa/rho kinase attenuate vascular dysfunction. this finding identifies putative pharmacologic therapeutic targets. vascular smcs in cadasil are surrounded by deposits of granular osmiophilic material (gom). however, the mechanisms underlying gom formation and their contribution to disease progression are unknown. a recent study investigated the course of gom deposit evolution in humanized tgnotch3arg182cys mice (gravesteijn et al., 2019) and in human cadasil tissue. gom deposits are electron dense and are composed of notch3ecd and extracellular matrix proteins, notably tissue inhibitor of metalloproteinase 3 (timp3) and clusterin, that accumulate in the basement membrane of vascular smcs and pericytes. the study proposes a five-stage gom classification system based on size, morphology, and electron density of the gom deposits in order to generate a more systematic, uniform, and unbiased way of analyzing gom accumulations. it appears that new gom deposits are continuously generated. however, gom in tgnotch3arg182cys mice did not reach the end-stage of gom accumulation as in human disease. a limitation of the study is that unlike other cadasil mouse models, the tgnotch3arg182cys mouse lacks histologic vascular pathology and functional deficits. the authors suggest that this phenomenon could be attributed to absence of end-stage gom accumulation in these mice. beyond mouse models, the cellular cadasil pathology has been investigated in patient-specific vascular mural cells generated from induced pluripotent stem cells (ipscs) obtained from skin biopsies of cadasil patients (kelleher et al., 2019). this study showed alterations in endothelial capillary structures caused by the aberrant notch3 expression in mural cells, and corrective effects of vascular endothelial growth factor supplementation. these novel advances are steps toward the identification of novel therapeutic targets in this monogenic form of small vessel disease. 7. genetic variants in small vessel disease and molecular target discovery in addition to studies examining monogenic forms of small vessel disease, there are efforts to study genetic variation in large cohorts of patients to identify genes involved in sporadic small vessel disease. genome-wide association studies correlating variants with mri features of sporadic small vessel disease may lead to the identification of new putative pharmacologic targets. small vessel disease is often diagnosed on mri, with characteristic findings including white matter hyperintensities and signs of lacunar infarcts (fig. 2). some recent studies investigated the relationship between genetics and white matter hyperintensities in small vessel disease. traylor et al (2019) carried out a genome-wide association meta-analysis of white matter hyperintensity volumes in 11,226 subjects, including 2,797 stroke patients. the study identified a locus at genome-wide significance in an intron of pleckstrin homology and rhogef domain-containing family g member 1 gene (plekhg1) associated with white matter hyperintensities. the association was validated in an independent cohort where this polymorphism was related with ischemic strokes. the strongest association was found with small vessel stroke. plekhg1 plays a role in vascular endothelial cell reorientation in response to mechanical stress. therefore, the polymorphism in this gene could generate some form of vascular alteration leading to the development of white matter hyperintensities. this study also validated 2 previously identified genes efemp1 and trim47/trim65. according to current knowledge, efemp1 encodes the fibulin3 protein, which is an extracellular matrix glycoprotein that inhibits timp3. timp3 contributes to cerebrovascular dysfunction in cadasil through accumulation in the vascular extracellular matrix. therefore, there seems to be some concordance between genetic alterations in sporadic small vessel disease and the monogenic form of cadasil. based on the concept that polymorphisms in genes involved in monogenic forms of small vessel disease may have some contribution in the sporadic form of the disease, mishra et al. (2019) carried out the first whole exome sequencing study on mri white matter hyperintensities in small vessel disease. this work used a composite extreme phenotype study focused on candidate genes with mutations causing mendelian heritable forms of small vessel disease, i.e. notch3, htra1, col4a1, col4a2 and trex1. the study identified significant associations of gene variants in htra1 and notch3 with the development of white matter hyperintensities. overall the study demonstrates shared mechanisms between monogenic forms and multifactorial forms of small vessel disease. the study suggested that the risk htra1 allele reduced htra1 expression. mutations in htra1 are associated with cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy, carasil, a condition inherited in an autosomal recessive pattern. htra1 is a serine-protease involved in cleaving extracellular matrix proteins, including fibulin3, and regulating important signaling pathways such as the insulin growth factor and tgfß pathway, which suffers an inhibitory effect. notably, htra1 is differentially expressed in astrocytes (chen et al., 2018) suggesting that dysfunction of this pathway may alter astrocyte function leading to compromised integrity of the neurovascular unit and the bbb. hopefully additional studies using ipsc from patients and genetically modified animal models further investigating molecular targets identified in prior genetic studies will lead to the discovery of treatments to prevent and attenuate the progression of small vessel disease. figure 2. lacunar infarcts typical of small vessel disease. a) human brain tissue showing a lacunar infarct (arrow) in the centrum semiovale. image obtained by dr. iban aldecoa, neurological tissue bank of the biobank of hospital clínic idibaps university of barcelona. b) mri showing a thalamic lacunar infarct (arrow) and c) corresponding ct-perfusion time-to-drain (ttd) image obtained in the acute phase showing a delay in blood flow in the lacunar infarction (arrow). images in b and c were obtained in the comprehensive stroke center of hospital clinic of barcelona. 8. cerebral hypoperfusion and vascular cognitive impairment: risks of high-salt diet chronic cerebral hypoperfusion has been associated with diffuse white matter damage and cognitive impairment of vascular origin. vascular pathology is also frequently observed in neurodegenerative dementias. hase et al. (2019) investigated the histopathologic features of white matter capillaries by studying approximately 0.7 million capillary profiles from the frontal lobe white matter of 153 subjects with different dementia types including alzheimer’s disease, dementia with lewy bodies, dementia in parkinson’s disease, vascular dementia, mixed dementia, post-stroke dementia and post-stroke no dementia. control samples were examined from old and young individuals devoid of brain pathology. all dementias showed higher vascular pathology scores than corresponding age-matched controls. in relation to these controls, white matter lesion scores were also higher in all dementias, with the highest values corresponding to vascular dementia and post-stroke dementia. the two latter conditions showed the highest abundance of string vessels, which were observed to a lesser extent in the other dementia types. the density of col4+ and glut1+ capillaries was lower in the white matter than in the cortex and was also lower in dementia patients versus aged controls, with the exception of individuals that had suffered a stroke. the capillary width in the white matter was 31% greater than in the cortex. notably, white matter capillaries were wider in all dementias compared to all controls irrespective of age. moreover, there was a moderate positive correlation between capillary width and white matter lesion scores. these results indicate adaptive compensatory mechanisms for chronic hypoperfusion in aging-related dementias. an emerging risk factor recently recognized as a driver of cerebral vascular dysfunction and hypoperfusion is high-salt intake (faraco et al., 2018). interestingly, the underlying mechanism is not dependent on salt-induced hypertension but on a complex innate immune response mediated by alterations in the gut immune system (faraco et al., 2018). last year, a study by the same team reported that chronic dietary high-salt intake in mice promotes cognitive impairment by inducing phosphorylation of the microtubule-associated protein tau rather than by insufficient cerebral perfusion (faraco et al., 2019). the presence of hyperphosphorylated tau is one of the hallmarks of alzheimer’s disease that has also been reported in patients with vascular cognitive impairment. mice exposed to high-salt diet for several months developed signs of cognitive impairment that correlated with the phosphorylation and aggregation of tau in the brain tissue. through different experimental strategies, the study demonstrated that high-salt diet impaired the production of endothelial nitric oxide following denitrosylation of calpain. deficient nitric oxide generation activated cyclin-dependent kinase 5 (cdk5) which caused tau phosphorylation. although these important findings were obtained in mice with unrealistically high levels of salt intake, the study identifies a causal relation between high-salt intake, vascular dysfunction, tau pathology, and cognitive impairment independent of cerebral hypoperfusion. 9. critical role of pericytes in bbb function and more pericytes are mural cells surrounding capillaries that play essential functions in development and maintenance of bbb integrity. pericytes facilitate molecular pathways critical in preventing vascular malformations, and it appears that these cells can generate factors that directly support neuronal viability. recent studies have provided valuable information regarding the biology of pericytes as well as their role in vascular function. using pdgfrb-creert2 mice with cre expression induced in mural cells by tamoxifen administration, diéguez-hurtado et al. (2019) deleted the expression of the rbpj gene encoding a transcriptional regulator of the notch signaling pathway. ablation of rbpj at early postnatal stages caused brain hemorrhages, avms at the pial surface, endothelial cell hyperplasia and apoptosis, enlargement of the subendothelial basement membrane, and reduced blood flow. these changes were accompanied by astroglial and microglia reactions, inflammation, edema, and some neuronal loss around severely affected blood vessels. rbpj deletion upregulated genes of the tgfß pathway. the study provides evidence showing that mice with rbpj gene deficiency in mural cells developed signs of ccms. interestingly, when rbpj was deleted in mural cells of adult mice, the above phenotype was no longer observed. however, induction of permanent brain ischemia in these mice caused increased edema, larger brain lesion volumes, and exacerbated inflammation and vascular abnormalities. beyond the critical role of pericytes in brain vascular function, nikolakopoulou et al. (2019) elucidated the role of pericytes in neuronal survival under conditions of vascular dysfunction. a mouse pericyte inducible cre-line was generated using a double gene promoter strategy including both pdgfrb and cspg4 promoters to increase the specificity of cre expression in pericytes over other cells. this line was crossed with mice expressing inducible human diphtheria toxin receptor under control of an upstream loxp-flanked stop sequence. injection of tamoxifen induced dtr expression in pericytes which then became susceptible to diphtheria toxin. ablation of pericytes in adult mice reduced tight junction proteins zonula occludens and occludin, reduced adherens junction ve-cadherin on cortical and hippocampal capillaries, decreased cbf, and increased bbb breakdown. pericyte loss was followed by vasogenic edema, confirming the involvement of pericytes in maintaining adult bbb function. furthermore, neuron loss in the cortex and hippocampus as well as behavioral deficits were detected several days after pericyte ablation. a brain-specific pericyte-secreted growth factor termed pleiotropin was hypothesized to prevent neuronal loss. in vitro, pleiotropin was able to protect neurons under hypoxic conditions. in vivo, silencing pleiotropin expression was not sufficient to cause neuronal loss. however, ischemia or excitotoxic lesions in mice with silenced pleiotropin expression caused larger infarctions and more neuronal degeneration. the study concluded that pericyte-dependent pleiotropin-mediated neurotrophic support promotes neuronal survival under circulatory stress. the expedient neuronal loss found in this model of pericyte ablation is intriguing because it was not found in previous pericyte-deficient models. the findings could be relevant to other neurodegenerative conditions if the dependence of neuronal survival on neurotrophic factors provided by pericytes under vascular stress is confirmed. 10. molecules derived from the vascular endothelium impact neuronal function in addition to pericytes, endothelial cells seem to signal to neurons. tan et al. (2019) showed that the endothelium secretes semaphorin 3g (sema3g) which regulates synaptic structure and plasticity in hippocampal neurons through actions on the neuronal neuropilin-2/plexina4 holoreceptor. this study suggests a direct link between endothelial-derived factors and neuronal function beyond the supply of oxygen and nutrients through blood flow and proposes a molecular mechanism by which endothelial dysfunction can alter neuronal circuits and cognition. the study shows that sema3g increases excitatory synapse density via neuropilin-2/plexina4 signaling and activation of rac1. to determine whether sema3g was derived from endothelial cells, mice bearing a sema3g deletion selectively in endothelial cells were obtained by crossing cdh5-cre mice with floxed sema3g mice. these mice showed less dendritic spine density in the ca1 and impaired memory. nonetheless, sema3g deletion with this strategy would cause sema3g deficiency during developmental stages which may cause indirect alterations and compensatory changes. generation of tamoxifen inducible cdh5-creert2/sema3gfl/fl mice allowed deleting sema3g in the endothelium of adult mice. this inducible deletion significantly impaired hippocampal long-term potentiation frequency and altered miniature excitatory post-synaptic currents in ca1 pyramidal neurons. furthermore, sema3g overexpression improved spine density loss and cognitive defects in cdh5-creert2/sema3gfl/fl mice. the results suggest that endothelium-derived sema3g is required for normal hippocampal synaptic plasticity in adulthood. this study raises many questions regarding the mode of transit of sema3g from the vascular endothelium to the neurons through the vascular and astrocytic basal laminae and astrocytic end-feet, and whether the source of sema3g is the capillary endothelium. the discovery of molecular interactions between endothelial cells and neurons suggest that endothelial cells influence neuronal function. therefore, alterations in the vascular endothelium might exert a direct effect on dendritic spine function and contribute to cognitive decline and memory loss. other recent studies further support the view that molecules generated by the cerebrovascular endothelium have a direct impact on neuronal function. liu et al. (2020) showed that mice with a selective deletion of cdk5 gene show a higher endothelial chemokine cxcl1 expression. therefore, dysregulation of cdk5 expression and/or function seems to be critical in different forms of cerebrovascular alterations. endothelial cdk5 knockout mice showed astrogliosis in the hippocampus and weakened astrocytic glutamate current mediated by the astroglial glutamate transporter glt1. the effects on astroglia were mediated by endothelial-derived cxcl1 acting on the astrocytic receptor cxcr2. these alterations had consequences for neurons since they reduced glutamate uptake and increased the excitability of hippocampal pyramidal neurons causing seizures in mice with endothelial-deficient cdk5. although current evidence is still scarce and based on experimental animal studies, the possibility that vascular endothelial cells generate molecules that signal to neurons and astrocytes and govern neuronal function warrants future studies. conclusion the above 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of cerebral microbleeds of different ages during neuropathology assessment feel free to add comments by clicking these icons on the sidebar free neuropathology 2:35 (2021) original paper ex vivo mri facilitates localization of cerebral microbleeds of different ages during neuropathology assessment sukriti nag1,2, er-yun chen2, ryan johnson2, ashish tamhane2, konstantinos arfanakis2,3, julie a. schneider1,2,4 1 department of pathology (neuropathology), rush university medical center, chicago, il, usa 2 rush alzheimer’s disease center, rush university medical center, chicago, il, usa 3 department of biomedical engineering, illinois institute of technology, chicago, il, usa 4 department of neurological sciences, rush university medical center, chicago, il, usa corresponding author: sukriti nag · rush alzheimer disease center · suite 1000 · rush university medical center · 1750 w harrison street · chicago, il 60612 · usa sukriti_nag@rush.edu submitted: 20 october 2021 accepted: 12 december 2021 copyedited by: christian thomas published: 20 december 2021 https://doi.org/10.17879/freeneuropathology-2021-3638 keywords: alzheimer’s dementia, cerebral microbleeds, ex vivo magnetic resonance imaging, hemosiderin, microhemorrhages abstract cerebral microbleeds (cmbs) identified by in vivo magnetic resonance imaging (mri) of brains of older persons may have clinical relevance due to their association with cognitive impairment and other adverse neurologic outcomes, but are often not detected in routine neuropathology evaluations. in this study, the utility of ex vivo mri in the neuropathological identification, localization, and frequency of cmbs was investigated. the study included 3 community dwelling elders with alzheimer’s dementia, and mild to severe small vessel disease (svd). ex vivo mri was performed on the fixed hemisphere to identify cmbs, blinded to the neuropathology diagnoses. the hemibrains were then sliced at 1 cm intervals and 2, 1 or 0 microhemorrhages (mh) were detected on the cut surfaces of brain slabs using the routine neuropathology protocol. ex vivo imaging detected 15, 14 and 9 possible cmbs in cases 1, 2 and 3, respectively. to obtain histological confirmation of the cmbs detected by ex vivo mri, the 1 cm brain slabs were dissected further and mhs or areas corresponding to the cmbs detected by ex vivo mri were blocked and serially sectioned at 6 μm intervals. macroscopic examination followed by microscopy post ex vivo mri resulted in detection of 35 mhs and therefore, about 12 times as many mhs were detected compared to routine neuropathology assessment without ex vivo mri. while microscopy identified previously unrecognized chronic mhs, it also showed that mhs were acute or subacute and therefore may represent perimortem events. ex vivo mri detected cmbs not otherwise identified on routine neuropathological examination of brains of older persons and histologic evaluation of the cmbs is necessary to determine the age and clinical relevance of each hemorrhage. introduction cerebral microbleeds (cmbs) are defined as rounded or ovoid, hypointense foci that are best demonstrated in susceptibility-weighted and gradient echo magnetic resonance imaging (mri). in most studies, the upper limit of the diameter of cmbs is 5-5.7 mm [1-3], although a diameter of 10 mm has also been reported [4]. neuroimaging studies show that cmbs tend to increase with age being observed in 5% of healthy adults [5, 6], and 7% of volunteers having a mean age of 62.1 ±7.4 years who were enrolled in the uk biobank study [7]. cmbs are reported in 18% of subjects between 60 to 69 years, while in those older than 80 years prevalence increases to 38% [6]. cmbs have also been associated with increased risk of stroke in the general population [8]. prevalence is higher in those with ischemic stroke and with non-traumatic intracerebral hemorrhage, being 34% and 60%, respectively [5]. in the latter study, cmbs were associated with hypertension or diabetes mellitus in otherwise healthy adults, while in adults with cerebrovascular disease they were only associated with hypertension. the frequency of cmbs is 84.9% in those with subcortical vascular dementia and in these subjects cmbs were related to cognitive impairment in multiple domains [9]. in most studies, cmbs were identified by in vivo mri alone [8-12], while in a few studies by neuropathology assessment alone in which they are referred to as microhemorrhages (mhs) [13]. the development of ex vivo mri led to studies correlating ex vivo mri findings with neuropathology in selected brain slabs [1, 2, 14-16]. in these studies, cmbs were considered to be markers of focal hemosiderin deposition that remained in macrophages for years following a mh [1, 11]. there is increasing evidence that the pathologic correlate of cmbs is more varied than just mhs since a significant association was observed between hemosiderin collections in the putamen and indices of small vessel ischemia such as microinfarcts, arteriolosclerosis and lacunes in any of the brain regions examined [2, 14]. the aim of the present study was to characterize the morphologic correlate of cmbs identified by ex vivo mri of a single cerebral hemisphere of 3 brains with and without mhs identified on initial neuropathology assessment. this was followed by detailed neuropathology assessment to determine the spectrum of pathology associated with the cmbs identified by ex vivo mri. since one in five patients with alzheimer’s disease (ad) has cmbs [17], and the frequency of cmbs is higher in association with cerebral amyloid angiopathy (caa) [2, 18], and arteriolosclerosis [1, 19], 3 community dwelling elders with a clinical diagnosis of alzheimer’s dementia and varying degrees of small vessel disease (svd) were selected for assessment. materials and methods the criteria for selection of the three autopsied cases for this study include a clinical diagnosis of alzheimer’s dementia and pathologic evidence of caa and arteriolosclerosis as well as absence of a clinical history of traumatic brain injury with loss of consciousness since diffuse axonal injury following head trauma is another potential secondary cause of cmb [11]. cases were from the memory and aging project (map), a large clinical-pathology study of aging and dementia with community dwelling participants. the protocols used in this study were approved by the institutional review board of rush university medical center. a signed informed consent was obtained from each participant for an annual clinical evaluation and for brain donation. participants underwent uniform clinical evaluations at baseline and annually thereafter for odor testing, parkinsonism signs and cognitive function as described previously [20]. ex vivo imaging following removal, the brain was hemisected and one hemisphere with visible gross pathology or one hemisphere with absence of pathology was selected arbitrarily and submerged medial side up in 4 % paraformaldehyde in a container which was rocked back and forth until no more air bubbles escaped from the lateral ventricles. the hemisphere was then placed medial side down and refrigerated. mri was done on average 1 month postmortem on hemibrains returned to room temperature, immersed in 10% formalin and held in place in a container using a plastic divider. ex vivo mri was done using a siemens 3tesla mri scanner and a three-dimensional multi-echo gradient echo (gre) sequence with 6 echoes (te = 5 + n×5 ms (n=0-5), tr = 35 ms), an acquired voxel-size of 1×1×1 mm3, and a scan time of about 8 min. microbleeds were identified by a trained reader who was blinded to clinical and pathology data. cmbs were diagnosed when a relatively round, small, hypointense region of few mm in diameter was observed on the last echo of the gre data. at least half of the hypointensity had to be surrounded by brain parenchyma to qualify. elongated, vessel-like hypointensities and potential mimics, including lesions in the subarachnoid space, calcifications, or vascular malformations were excluded. neuropathology assessment the mean postmortem interval for cases 1-3 was 22, 8 and 6 hours, respectively (table 1). a standard protocol was used to evaluate brains which included cutting each hemibrain into 1 cm coronal slabs guided by a plexiglass jig and brain blocks from eleven brain regions and any mh noted on the cut surfaces of the slabs were blocked for microscopy as described previously [21]. pathological diagnosis of ad, hippocampal sclerosis (hs), limbic predominant age-related tdp-43 encephalopathy neuropathologic change (late-nc), macroscopic and microscopic infarcts were made as described previously [21]. caa was assessed in meningeal and intracortical vessels in sections from the midfrontal, midtemporal, inferior parietal and occipital cortices which were immunostained for β-amyloid and graded as described previously [22]. arteriolosclerosis was assessed in the basal ganglia and graded using a semiquantitative scale from 0 (none) to 6 (severe) as described previously [21]. table 1: clinical and pathological findings in 3 cases with a clinical diagnosis of alzheimer’s dementia.   case 1 case 2 case 3 age, years 91.4 96.2 79.2 sex female female male education, years 16 12 16 blood pressure, mm hg (last valid) 96/54a 171/96b m mmse score (last valid) 11a 5b 9c total cmbs detected by ex vivo mri 15 14 9   post mortem interval, hours 22 8 6   pathological diagnoses from blocks taken routinely alzheimer’s disease + + cerad probable probable definite braak stage i iii iv thal stage 3 3 5 adnc low intermediate intermediate hippocampal sclerosis + late-nc stage 3 3 0 cerebral amyloid angiopathy, mild-severe mild mild severe arteriolosclerosis mild mod-severe mild atherosclerosis possible mild mild-mod macroscopic infarcts, chronic 4 1 microscopic infarcts, chronic 7 1 total mhs using the routine protocol (no ex vivo mri) 2 1 0 total mhs post ex vivo mri acute 5 2 5 subacute 9 0 0 chronic 1 12 1 additional pathologies in blocks taken post ex vivo mri microinfarct, chronic 1 1 microinfarct, subacute 0 1 0 abbreviations: adnc alzheimer’s disease neuropathologic change; cerad consortium to establish a registry for alzheimer’s disease; cmbs cerebral microbleeds; late-nc limbic predominant age-related tdp-43 neuropathologic change; m missing; mhs microhemorrhages; mod moderate. values obtained 3a, 7b and 4c months prior to death. microhemorrhages the ex vivo mri images were superimposed on photomicrographs of the brain slabs to identify the region of the suspected cmb. in this protocol, each 1 cm slab was further dissected to detect the cmbs observed in the ex vivo mr images and brain blocks containing these cmbs were obtained. in instances where a mh was not identified on the cut surface of the brain slab, a block was obtained in the region containing the cmb identified by ex vivo mri. all brain blocks were processed using standard techniques and embedded in paraffin. each block was serially sectioned at 6μm thickness to obtain 200-250 serial sections from each block. every 10th section was stained with hematoxylin and eosin (he) and the adjacent section was stained by the perl’s prussian blue stain for hemosiderin. in select cases, additional sections were stained by he and for hemosiderin. immunohistochemistry to detect β-amyloid was done in all cmb’s as described previously [22]. the size of mhs was measured using a 1 mm graticule at a magnification of x100. results the age of the three decedents was 91.4, 96.2 and 79.2 years and in keeping with the clinical diagnosis of alzheimer’s dementia, all cases had a low mini-mental state examination score which varied from 5-11 (table 1). blood pressure data was only available for 2 cases and only case 2 was hypertensive. on routine neuropathology assessment, (table 1) two of the three cases had intermediate likelihood of ad by the nia-reagan and adnc criteria while the third case with low likelihood of ad had hippocampal sclerosis with late-nc (stage 3) implying involvement of the amygdala, limbic and neocortical areas by tdp-43 neuronal and glial cytoplasmic inclusions. regarding vascular changes, cases 1 and 2 had mild caa while case 3 had severe caa. arteriolosclerosis was mild in cases 1 and 3 and moderate-severe in case 2. ex vivo imaging detected 15,14 and 9 possible cmbs in cases 1, 2 and 3, respectively (table 1). mr images showed hypointense, rounded areas in the gray or white matter which were interpreted as cmbs. (fig. 1a, e, 2a) the diameter of cmbs measured in the same echo as used for their visualization, varied from 1.56 – 4.20 mm. most of the cmbs observed in this study were located in the subcortical white matter near the grey/white junction of the frontal, temporal or occipital lobes, although, involvement of gray matter structures such as the midtemporal cortex, thalamus and caudate nucleus were also noted. figure 1: ex vivo mri scans, corresponding brain slabs and microscopy of acute and subacute hemorrhages are shown. (a) the mri scan shows a cmb in the white matter of the temporal lobe (arrowhead). (b) the corresponding brain slab was further sliced in the area of the cmb shown in the ex vivo mr scan and delineated by the rectangle to detect the mh which is shown in the inset (case 1, mh#6). (a, b) also present is another cmb at the interface of the cortex and white matter of the superior temporal gyrus (case 1, mh#8). (c) microscopy of a hematoxylin and eosin (he) stained section from mh#6 shows an acute hemorrhage with extravasation of red blood cells through a venular wall into the surrounding brain. (d) this mh shows no macrophages or hemosiderin by the perl’s prussian blue stain at this level or in serial sections. (e) ex vivo mri shows a cmb in the white matter of the inferior parietal lobule (arrowhead). (f) further slices of the corresponding brain slab in the area delineated by a rectangle shows a mh (case1, mh#14) in the white matter which is shown in the inset. (g) an he stained section of the mh shows significant extravasation of red blood cells through an arteriolar wall (arrowhead) into the neuropil. a few pigment-laden macrophages are identified at the periphery of the hemorrhage (inset). (h) the increased number of macrophages are clearly evident by the perl’s prussian blue stain. higher magnification of the hemosiderin positive macrophages is shown in the inset. c, d, g, h scale bar = 50 μm; insets g, h scale bar = 10 μm macroscopic findings the appearance of the mhs on macroscopic examination of the cut surfaces of brain slabs whether using the routine or the extended protocol was similar. mhs appeared as rounded, reddish areas, 1-2 mm in diameter resembling the mh shown in the inset of fig. 1b. in a few cases, there was a focal area of bluish discoloration suggesting presence of a hemorrhage below the cut surface of the brain slab while in other cases no hemorrhage was noted on the cut surface of the slab and was only identified following serial sectioning in the regions showing cmbs by ex vivo imaging (fig. 1f). microscopic findings a total of 12 acute, 9 subacute and 14 mhs were identified by microscopy of the 3 cases which include the 3 mhs observed using routine neuropathology assessment. most of the hemorrhages (74%) were located in the white matter while the remainder were in grey matter structures such as one in the midtemporal cortex, one in the caudate nucleus and others in the thalamus (table 2). microscopy of all 35 mhs showed that their diameter varied from 0.2-0.8 mm. most mhs were associated with arterioles. among the acute hemorrhages, 3 were associated with venules and 1 with multiple capillaries. in the case of chronic hemorrhages, only one was associated with a venule, while another chronic hemorrhage was associated with both an arteriole and venule which were adjacent to each other, while others were associated with arterioles. on serial sectioning, mhs involved variable lengths of the vessel with some mhs involving short 125-300 μm segments of arterioles while others involved long segments of arterioles varying from 480-780 μm in length. there was no evidence of calcification or vascular malformations, such as a cavernous hemangioma in any of the sections examined. most blocks showed a single mh correlating with the cmb seen on ex vivo mri, however, there were 3 instances when the block showed additional mhs which were not identified by ex vivo mri (table 2). table 2: location and age of microhemorrhages (mh) detected without mri and pathological features of those detected following ex vivo mri. mh diagnosis by routine np cmbs diagnosed following ex vivo mri microhemorrhages (mh) histological findings other microscopic findings location, age location age perl’s no. of mhs case 1 1 superior frontal, wm acute 1 2 subcortical wm, between superior and middle frontal gyri acute 1 3 inferior frontal gyrus, wm subacute + 1 4 anterior temporal tip, wm subacute + 1 5 midtemporal gyrus, wm subacute + 2 6 midtemporal gyrus, wm acute 1 7 superior temporal gyrus wm, acute superior temporal gyrus, wm subacute + 1 8 superior temporal gyrus, wm subacute + 1 9 superior temporal gyrus, wm subacute + 1 10 postcentral gyrus, wm subacute + 1 11 inferior temporal gyrus wm, acute inferior temp gyrus, wm acute 1 12 temporal periventricular wm no mh 0 13 superior parietal lobule, wm acute 1 14 inferior parietal lobule, wm subacute + 1 15 anterior insula, wm chronic + 1 case 2 1 occipital wm, posterior no mh 0 dilated vein with luminal blood 2 occipital wm, anterior chronic + 1 3 midtemporal cortex, chronic midtemporal cortex chronic + 1 4 thalamus, (ventral posterior lateral nucleus) no mh 0 5 thalamus, (lateral, posterior nucleus) chronic + 2 subacute microinfarct-1 6 thalamus (dorsomedial nucleus) no mh 0 7 thalamus (dorsomedial nucleus near midline) no mh 0 dilated vessels with luminal blood 8 wm near ventro-lateral nucleus of thalamus chronic + 2 9 thalamus (ventro-lateral nucleus) chronic + 4 10 thalamus (ventro-lateral nucleus) chronic 1 chronic microinfarct-1 11 wm ventral to putamen (posterior) no mh 0 12 wm adjacent to lateral putamen chronic + 1 perivascular hemosiderin 13 wm ventral to putamen (anterior) acute 1 14 caudate nucleus acute 1 case 3 1 no cmb diagnosed superior frontal gyrus no mh 0 2 wm lateral to putamen no mh 0 dilated vessels with luminal blood 3 frontal lobe wm acute 1 4 parietal lobe wm acute 1 5 occipitotemporal gyrus,wm acute 1 6 occipitotemporal gyrus, wm acute 1 7 occipital lobe wm acute 1 8 occipital lobe wm no mh 0 9 occipital lobe wm chronic + 1 chronic microinfarct-1 abbreviations: cmbs cerebral microbleeds; mhs microhemorrhages; wm white matter. mhs were acute, subacute or chronic based on the cellular response present. acute mhs showed extravasation of erythrocytes through the walls of mainly arterioles and fewer venules and extended into the surrounding neuropil (fig. 1c). serial sectioning of the entire block did not show inflammatory cells or hemosiderin either on he or the perl’s prussian blue stain thus their age was not altered (fig. 1d). an additional finding in subacute mhs was the extension of red blood cells further from vessel walls than observed in the acute hemorrhages (fig. 1g). a few intravascular and a variable number of perivascular macrophages were present and many contained faint brown hemosiderin pigment. the adjacent perl’s-stained section accentuated the macrophage numbers and their hemosiderin content (fig. 1h). the neuropil surrounding the hemorrhage showed vacuolation and sparse gemistocytes. in chronic mhs, a cavity was present containing or surrounded by a variable number of macrophages which showed intense brown cytoplasmic staining due to hemosiderin (fig. 2c, d). the neuropil surrounding the hemorrhage showed fibrillary astrocytes. adjacent arterioles showed acellular mural thickening with absence of smooth muscle cells and failed to show β-amyloid positivity (fig. 2e). in all mhs, vessels failed to show mural β-amyloid positivity with the exception of one chronic mh (case 2, mh #3) in the midtemporal cortex which showed a disrupted vessel associated with hemosiderin-laden macrophages (fig. 2f) and focal β-amyloid positivity at the periphery of the vessel (fig. 2g). another pattern of chronic mh was the presence of scattered hemosiderin-laden macrophages in a focal area of the white matter (fig. 3a). serial sectioning showed that these areas extended for depths of 120-180 μm and were related to terminal arterioles and capillaries (fig. 3b). in some mhs, arterioles with sclerosed, hyaline walls and collapsed lumina were present surrounded by variable numbers of hemosiderin containing macrophages in the neuropil. in one instance, only perivascular collections of few macrophages were present in multifocal areas of the putamen. figure 2: ex vivo mri scan, corresponding brain slab and microscopy of chronic grey matter hemorrhages are shown. (a) ex vivo mri shows a cmb in the lateral thalamus. (b) the corresponding brain slab shows an area of reddish discoloration (arrowhead) which is shown at higher magnification in the inset (case 2 mh#8). (c) the corresponding he stained section shows a cavity containing hemosiderin-laden macrophages adjacent to an arteriole which shows focal mural disruption and double-barreling. a nearby arteriole shows perivascular hemosiderin-laden macrophages. (d) the adjacent section shows strong prussian blue staining of the hemosiderin collections. (e) immunostaining of the arteriole shown in c and d fails to show mural β-amyloid although the double barreling is highly suggestive of caa. (f) a focal hemorrhage in layer three of the superior temporal cortex shows disruption of a vessel associated with hemosiderin-laden macrophages which are also present in the surrounding neuropil along with gemistocytes (arrowhead) (g) the adjacent section shows focal β-amyloid immunostaining at the periphery of the vessel (arrowhead). c, d scale bar = 50 μm; e, g scale bar = 50 μm, f scale bar = 50 μm. in addition to the mhs which correlated with the hypointense areas detected by ex vivo mri, serial sectioning showed mhs measuring < 0.4 mm in their greatest dimension (fig. 3c, d). these mhs were much smaller than the cmbs identified by ex vivo mri and were present in 1-3 consecutive sections per block. these small mhs were not included in the total mh counts shown in table 1, since they did not correlate with any of the reported hypointensities observed by mri and since they were too small to be visualized by a 3t scanner. although 38 cmbs were described in the ex vivo mr scans, in 8 instances microscopy did not show mhs despite additional slicing of brain slabs and microscopy of serially sectioned blocks. on microscopy, 3 of these presumed cmbs showed 2-3 large dilated vessels with luminal blood (table 2). in 2 presumed cmbs, the area of hypointensity represented the site of entry of penetrating arterioles in the lateral and inferior portion of the putamen and in 2 presumed cmbs the area of hypointensity probably represented the depths of sulci and only in one case no pathology was detected. additional pathology identified by serial sectioning were chronic microinfarcts in 2 cases and a subacute microinfarct in 1 case (table 2) and microinfarcts measuring < 0.4 mm in their greatest dimension (fig. 3e, f). figure 3: microscopy of chronic hemorrhages and chronic microinfarcts. (a) an example of a focal chronic white matter hemorrhage (600 x 300 μm) showing scattered hemosiderin-laden macrophages (arrowheads). (b) the adjacent section shows a terminal arteriole (arrowhead) and capillary (below the asterisk) with all macrophages showing positivity by the perls’s prussian blue stain. serial sections show two small mhs (c, d) measuring 200 (c) and 300 (d) μm in their longest dimension and two chronic microinfarcts (e, f) measuring 300 (e) and 200 (f) μm which do not correlate with any cmbs observed in the ex vivo scans. the microinfarcts show disruption of the neuropil and many macrophages some of which appeared pale brown suggesting hemosiderin but were negative by the perl’s prussian blue stain. a-c scale bar = 50 μm; d-f scale bar = 50 μm. discussion this study demonstrates the utility of ex vivo mri in the identification of mhs by neuropathology assessment. routine macroscopic assessment showed a total of 3 mhs while both macroscopic and microscopic assessment after ex vivo mri showed about 12 times as many mhs. in cases 1 and 2, the number of mhs was increased from that seen on routine neuropathology assessment, while in case 3, no mhs were identified on routine neuropathology assessment but were detected by ex vivo mri followed by neuropathology assessment. thus, routine neuropathology assessment alone is unreliable in detecting the presence or the total number of mhs in a specific brain. the mhs identified in this study by microscopy were smaller than indicated by their mr appearance being < 1 mm in diameter. the finding that cmbs detected by mri appear larger on gradient-echo sequences compared with the actual tissue lesions is attributed to the “blooming effect” of the mr signal at the border of these lesions [11, 23]. also, further shrinkage of brain blocks during the processing schedule for paraffin embedding may contribute to the reduced diameter observed on microscopy. diameters measured in ex vivo scans were reported to be on average 1.6 ±0.75 times [2] or 4 times [16] higher than the diameter identified by pathology assessment. however, since the size of a cmb on mri depends on imaging parameters, such as field strength and pulse sequence, comparison of cmb size across studies is probably not valid due to the difficulty in implementing identical imaging parameters on different mri systems [11]. the localization of mhs in the current study in both the grey and the subcortical white matter near the grey/white junction is similar to previous observations [1, 2, 13]. in addition, involvement of mainly arterioles and fewer capillaries and intracerebral venules in mhs has also been reported in a prior study [13]. in the current study, 38 blocks were examined by serial sectioning and 35 mhs were identified in the 3 cases with some blocks having more mhs than identified by ex vivo mri. the very small mhs observed only by histology and not by ex vivo mri in the present and previous studies [1, 13], are probably beyond the sensitivity of a 3t mri scanner. ex vivo imaging using a 7t scanner and high resolution (200 um isotropic resolution) can detect 2.6 times as many cmbs as compared to an in vivo 7t mri scanner while ultrahigh resolution detects additional hypointensities which on microscopy show vessel pathology but no additional hemorrhages [16]. the criteria used in the current study for aging mhs as acute, subacute or chronic is supported by previous experimental studies of rodent [24, 25] and rabbit [26] brains with mhs in which the time course of the cellular reactions was documented. during the acute phase up to 48 hours, the hematoma is surrounded by edematous neuropil and no inflammatory cells. the initial peri-hemorrhage inflammatory response at 48 hours consists of few blood-borne neutrophils and brain-resident microglia (cx3cr1 positive) which migrate to the lesion site from the surrounding tissue. astrocytic activation is reported to occur a few days after the onset of the microglial response. conversion to hemosiderin begins at day 5 after the injection of either blood or red blood cells into brain [26]. by day 8, macrophages (brain-resident microglia and fewer blood-derived macrophages) are abundant and show evidence of proliferation at the lesion site. by 2 weeks, the inflammatory response resolves. therefore, subacute mhs are up to 2 weeks or few weeks of age. changes between few weeks and 3 months are not reported, however, at 3 months cavities are observed at the mh site with variable numbers of macrophages showing abundant hemosiderin. fibrillary gliosis is present and arterioles in the area showed mural thickening which is not related to amyloid as noted in the current and a previous study [13], while in another study mural β-amyloid was detected in a bleeding vessel [2]. therefore, chronic mhs are 3 months of age or older. the frequency of acute mhs in the current study was 34%, while other studies have reported percentages of 26% [2] and 38% [16]. since serial sections were done in the current study, the possibility of focal hemosiderin deposits along the vessel length, that could elicit mr changes was excluded. the acute mhs were likely agonal as suggested previously [2]. none of the other studies have characterized mhs as subacute and there seems little justification for characterizing all hemosiderin-positive hemorrhages with edema of the neuropil and lack of inflammatory changes as chronic [2, 16]. the histology of chronic mhs observed in the current study was similar to previous reports [1, 2, 11]. although cmbs are generally considered to be markers of focal hemosiderin deposits [1, 11], other vascular pathologies have been observed in the areas of mri hypointensity. two hypointensities observed by susceptibility-weighted imaging were found to be due to a dissection in the wall of a grossly distended vessel and in the second case, a microaneurysm was present [2]. the finding that a distended vessel correlates with a hypointensity explains why the three hypointensities (table 2, case 2 #1 and #7, case 3 #2), observed in the current study were related to a single or several large vessel profiles with luminal blood. in a single study of subjects aged 65 and older, hemosiderin deposits which were considered to be the residua of mhs in the putamen, were associated with putaminal microinfarcts suggesting that cmbs detected by mri may be a surrogate for ischemic pathology rather than exclusively a hemorrhagic diathesis [14]. supporting these findings is the observation that three of the hypointensities (table 2, case 2 #5 and #10, case 3 #9) observed in this study showed additional microinfarcts by microscopy. in a prior study, a pathologic correlate for 38% of cmbs was not detected [1], while in the present study, no pathologic correlate was found for 13% of cmbs. most of the mhs in this study and another large hospital-based autopsy study [27] were located in the white matter or near the grey/white junction where caa is uncommon, suggesting that caa may not be an etiological factor in these white matter mhs. other studies [13, 27, 28] have reported lack of β-amyloid at the site of mhs. however, the findings of focal β-amyloid immunostaining at the bleeding site of one cortical arteriole in the present study, arterioles in a prior study [2] and increased pittsburg compound b retention at the sites of caa-related cmbs [29] do not rule out caa in the etiology of mhs. possibly, alteration of arteriolar morphology by mural β-amyloid deposition, predisposes to vessel disruption and release of β-amyloid into the bloodstream hence the lack of β-amyloid immunostaining in vessels in remote chronic mhs. this concept explains the presence of β-amyloid upstream or downstream of a bleeding vessel but not at the hemorrhage site [28]. vascular risk factors such as hypertension are associated with cmbs in otherwise healthy adults [5, 7, 13, 19] and in adults with cerebrovascular diseases [5]. another vascular risk factor, severe arteriolosclerosis, is reported to correlate with increased numbers of cerebellar mhs [19]. many of these risk factors were present in the 3 cases in the present study as listed in table 1. further studies with larger samples are warranted to determine the interplay of the stated factors and possibly additional factors in the pathogenesis of mhs. cmbs are clinically important in assessing the risk of cognitive impairment and high cmb counts are associated with an increased risk for cognitive deterioration and dementia [30]. another study while agreeing that cmbs are associated with cognitive impairment in the form of poorer executive function and decline in visuospatial ability, did not observe significant differences in incident dementia rates [31]. in both these studies, cmbs were diagnosed by in vivo mri only. the limitations of using only in vivo mri to diagnose cmbs is that mhs of different ages as well as hemorrhagic microinfarcts cannot easily be distinguished. in the present study, ex vivo imaging following by histology facilitated the detection of mhs of different ages and microinfarcts. the limitation of a small sample size in the present study with only 3 cases of alzheimer’s dementia and varying degrees of svd was offset by neuropathology assessment of 35 cmbs by serial sections and prussian blue staining for hemosiderin. hypointensities produced by anatomical landmarks such as the depths of sulci or the entry sites of basal ganglionic vessels can be excluded by making the mri reader aware of these pitfalls. in summary, in spite of a few limitations, these data suggest that ex vivo imaging is useful to supplement routine neuropathology examination for clinical-pathological correlations in relevant autopsy cases and for research purposes. acknowledgements the authors thank the participants of the rush memory and aging project and the staff of rush alzheimer’s disease center. this work was supported by national institute on aging grants r01ag064233, r01ag067482, p30ag010161, p30ag072975 and national disorders and stroke grants uh2-uh3ns100599 and 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unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. treatment of autoimmune encephalomyelitis with a histone deacetylase inhibitor: analyzing the role of immune-response genes feel free to add comments by clicking these icons on the sidebar free neuropathology 1:19 (2020) original paper treatment of autoimmune encephalomyelitis with a histone deacetylase inhibitor analyzing the role of immune-response genes arathi k. jayaraman a, karen avgush a, rashad kulam a, advait soni a, areeb khan a, mourad kerdjoudj a, sundararajan jayaraman a,b a dept. of microbiology & immunology, the university of illinois at chicago, 909 s wolcott avenue, chicago, il 60612, usa b dept. of surgery, the university of illinois college of medicine at peoria, il 61603, usa corresponding author: sundararajan jayaraman · dept. of surgery · the university of illinois college of medicine at peoria · il 61603 · usa anue2468@uic.edu submitted: 28 may 2020 accepted: 08 july 2020 copyedited by: laura peferoen published: 14 july 2020 https://doi.org/10.17879/freeneuropathology-2020-2819 additional resources and electronic supplementary material: supplementary material keywords: autoreactive t cells, epigenetic regulation, gene expression, experimental autoimmune encephalomyelitis, trichostatin a, multiple sclerosis abstract we have previously shown that treatment of female nod mice with a potent nonselective histone deacetylase inhibitor attenuated experimental autoimmune encephalomyelitis, a model for progressive multiple sclerosis. herein we show that immunization with the mog35-55 peptide induced prolonged upregulation of genes encoding interleukin 17a (il-17a), aryl hydrocarbon receptor, and histone deacetylase 11 in the spinal cord whereas the subunits of il-27, il-27p28 and il-27ebi3 were significantly increased in secondary lymphoid organs after a lag period. interestingly, the nitric oxide synthase gene was prominently expressed in both of these anatomic compartments following immunization. treatment with the histone modifier repressed the transcription of all of these genes induced by immunization. moreover, the drug suppressed the steady-state levels of the migration inhibitory factor and cd274 genes in both the spinal cord and peripheral lymphoid tissues. at the same time, the cd39 gene was downregulated only in secondary lymphoid organs. paradoxically, the epigenetic drug enhanced the expression of declin-1 in the spinal cord, suggesting a protective role in neuronal disease. immunization profoundly enhanced transcription of the chemokine ccl2 in the secondary lymphoid tissues without a corresponding increase in the translation of ccl2 protein. histone hyperacetylation neither altered the transcription of ccl2 nor its cognate receptor ccr2 in the central nervous system and peripheral lymphoid tissues. surprisingly, the drug did not exert modulatory influence on most other immune response-related genes previously implicated in encephalomyelitis. nevertheless, our data uncover several potential molecular targets for the intervention of experimental autoimmune encephalomyelitis that have implications for the treatment of progressive multiple sclerosis. 1. introduction multiple sclerosis (ms), an inflammatory disease of the central nervous system (cns), manifests commonly as the relapsing-remitting disease. some patients develop primary and secondary progressive forms of disease (dendrou et al., 2015; lassmann, 2017). the term ‘progressive multiple sclerosis’ has been proposed to encompass the primary and secondary progressive multiple sclerosis since there are more similarities than differences between them (fox and chataway, 2017). although many drugs are effective in reducing relapses, they failed to reverse axonal degeneration and are sometimes associated with adverse side effects, including progressive multifocal leukoencephalopathy (wingerchuk and carter, 2014; tintore et al., 2019). thus, effective drugs for the treatment of progressive multiple sclerosis remain an unmet need. since the early description of the experimental autoimmune encephalomyelitis (eae) in monkeys (rivers et al., 1933), monophasic, self-resolving, ‘classic’ eae, as well as ‘atypical’ eae, have been induced in rats and mice using whole spinal cord homogenates or peptides derived from the cns components such as myelin oligodendrocyte glycoprotein (mog), myelin basic protein (mbp) and proteolipid protein (plp)(dendrou et al., 2015; lassmann and bradl, 2017). autoimmune-prone female non-obese diabetic (nod) mice immunized with the mog35-55 peptide consistently induced severe and long-lasting progressive eae (peae) in 100% of animals characterized by paralysis of fore and hind limbs with (slavin et al., 1998; hidaka et al., 2014; dang et al., 2015) or without discernible remissions (basso et al., 2008; jayaraman et al., 2017; 2018). regardless, peae induced in nod mice mimics features of progressive ms including the life-long disease, prominent demyelination, axonal loss, and astrogliosis (slavin et al., 1998; basso et al., 2008; hidaka et al., 2014; dang et al., 2015; jayaraman et al., 2017; 2018), and hence is ideal for testing the efficacy of new drugs to treat progressive ms. the eae model is amenable for the investigation of the genes critically involved in ms pathogenesis. previous studies analyzed the roles of various immune response-related genes in eae using gene knockout mice and specific neutralizing antibodies in wild-type mice. these studies investigated the role of t helper 1 (th1), th17, and th1/th17 (th17.1) subsets as well as lymphokines such as interferon-γ (ifnγ) (ferber et al., 1996; okuda et al., 1998; hidaka et al., 2014), interleukin-4 (il-4) (falcone et al., 1998; okuda et al., 1998; ponomarev et al., 2007), il-17a (evangelidou et al., 2014), il-10, colony-stimulating factor 1 (borjini et al., 2016), il-22 (kreymborg et al., 2007), transforming growth factor-β (tgf-β (okuda et al., 1998) and il-27 (li et al., 2005) in eae. similarly, the importance of the transcription factors t-bet (o’connor et al., 2013), gata-3 (fernando et al., 2014), and rorγt (martinez et al., 2014) respectively, involved in the transcription of ifn-γ, il-4, and il-17a, was also studied in eae models. moreover, the factors required for the induction of th1 cells such as il-12 (gran et al., 2004), il-18 (lalor et al., 2011), and il-23 (kreymborg et al., 2007; el-behi et al., 2011) critical for the generation of th17 cells were implicated in eae. the roles of the pluripotent cytokines, namely the tumor necrosis factor-α (tnf-α) (hidaka et al., 2014; borjini et al., 2016) and granulocyte-macrophage-colony-stimulating factor (gm-csf/csf2) (mcqualter et al., 2001) were also studied in eae. in addition to the adaptive immune system, the innate immune cells such as neutrophils (jayaraman et al., 2018), and the cns-resident microglia and astrocytes (tran et al., 1997) appear to contribute to eae. interestingly, the first described and phylogenetically conserved pluripotent cytokine, the migration inhibitory factor (mif) (jayaraman and muthukkaruppan, 1977), has been implicated in ms (niino et al., 2000) and eae (powell et al., 2005). other determinants critical for eae include the orphan receptor, aryl hydrocarbon receptor (ahr) (nakahama et al., 2017), the transcription factors eomesodermin (eomes) (raveney et al., 2015) and declin-1 (dec1/bhlhe40) (lin et al., 2016), the immunoregulatory molecule cd39 (mascanfroni et al., 2013), matrix metalloproteinases (mmp) (kandagaddala et al., 2012; rempe et al., 2016), and the chemokine ccl2 (mahad et al., 2003; moreno et al., 2014; hidaka et al., 2014). the caveat is the lack of consensus regarding the critical gene(s) involved in eae. variations including the genetic background of experimental animals (rats and mice), types of eae models studied (short-term, self-resolving ‘classic’ eae, ‘atypical’ eae, relapsing-remitting eae, and peae), the immunogens used for eae elicitation (whole spinal cord homogenates, peptides derived from mog, mbp and plp), tissues (cns vs. peripheral lymphoid cells) and the time points (peak vs. chronic phase) examined can contribute to the uncertainty of the results. hence, investigation of the roles of these various immune response-related genes in a well-characterized model is likely to yield crucial information on the impact of these genes on neurodegeneration. our previous work demonstrated that the treatment of mog35-55 immunized female nod mice with the most potent histone deacetylase (hdac) inhibitor trichostatin a (tsa) (de ruijter et al., 2003) improved the clinical symptoms of peae (jayaraman et al., 2017). protection from peae was accompanied by histone h3 hyperacetylation in the spinal cord (sc) and spleen, reduced influx of t cells, and neutrophils into the cns as well as diminished axonal damage of the neurons in the cns (jayaraman et al., 2017; 2018). to gain insights into the roles of various genes in the peae model, we studied the expression profiles of 41 genes encoding lymphokines, transcription factors, accessory cell-associated determinants, and chemokines in the cns and secondary lymphoid organs (slo) longitudinally during the prolonged course of the disease (27-weeks). surprisingly, only a small set of mostly non-overlapping genes were differentially upregulated in the cns and slo, which were substantially repressed by tsa treatment, indicating their possible roles in peae. these data suggest that similar perturbation of the epigenome of ms patients may facilitate the identification of molecular targets for the development of novel drugs to treat this debilitating disease. 2. material and methods 2.1. eae induction and treatment this study was approved by the institutional animal care and use committee of the university of illinois at chicago and conducted according to the national institutes of health guide for the care and use of laboratory animals (nih publications no. 8023, revised 1978). female nod/shiltj mice were purchased from the jackson laboratories (bar harbor, me) and immunized subcutaneously (s.c) on the flank with 100 µg of mouse mog35-55 peptide (tocris bioscience) emulsified in complete freund's adjuvant and pertussis toxin was administered intravenously (jayaraman et al., 2017; 2018). randomly chosen littermates were injected s.c on the flank with 500 µg of tsa (sigma chemical company, st. louis, mo) per kg body weight three times a week. controls received the same amount of the vehicle, dimethyl sulfoxide (dmso) (sigma) diluted in phosphate-buffered saline (pbs). the body weight, blood glucose levels, (jayaraman et al., 2010; 2013; jayaraman and jayaraman, 2018; patel et al., 2011) and clinical scores (jayaraman et al., 2017; 2018) were recorded three times a week. the eae score was assigned as follows: 0, healthy, 1, limp tail, 2, one hind limb weakness, 3, both hind limb weakness, 4, forelimb weakness, 5, paralysis, moribund or death (jayaraman et al., 2017; 2018). five mice per group were chosen based on our previous investigations (jayaraman et al., 2017; 2018). the data are presented as the mean ± sem for each time point of observation. 2.2. gene expression analysis we analyzed the expression levels of 41 genes in the entire sc and slo (spleen and the draining inguinal, popliteal, axillary and cervical lymph nodes) of mice that were immunized with mog35-55 and treated with dmso or tsa. we have investigated 60 mice (five mice treated with dmso and five mice with tsa at six-time points) for the expression of genes in the sc and slo concurrently. to analyze the effect of the drug on the basal level of gene transcription regardless of immunization, we treated separate groups of 10 unimmunized mice with tsa or dmso and analyzed one day later since tsa acts within hours of treatment (van lint et al., 1996; de ruijter et al., 2003). these data are indicated at the day 1-time point in all figures. on the other hand, treatment groups received dmso or tsa starting from the day of immunization. mice were perfused with pbs before the spinal cord was extracted to avoid peripheral blood contamination (jayaraman et al., 2017; 2018). total rna was isolated from individual mice using trizol (invitrogen, carlsbad, ca). since preliminary experiments indicated similar levels of expression of genes among different mice in each group, the rna from five mice per group at each time point was pooled from identical tissues to minimize individual variability, as described previously (jayaraman et al., 2013). the rna was treated with turbo dnase and converted to cdna using the high-capacity cdna reverse transcription kit (applied biosystems, carlsbad, ca), as described earlier (jayaraman et al., 2010; 2013; 2017; 2018; patel et al., 2011). real-time quantitative reverse-transcriptase mediated polymerase chain reaction (qrt-pcr) was performed using the applied biosystems viia7 real-time pcr system. the cdna equivalent to 100 ng of total rna was used along with the 2x sybr green master mix in the qrt-pcr assay. the primer sets were designed, and specificity validated using the primer3 and blast programs (ncbi.nim.nih.gov) and bisearch web server tool (bisearch.enzim.hu). the mif primer sets were purchased from the integrated dna technologies (coralville, ia), and the custom primers were synthesized at the same facility. whereas the primer sets for gapdh, il4, il17a, il18, ifng, nos2, tnfa, tbet, rorgt, and gata3 were described previously (jayaraman et al., 2010; 2013), other primer sets are listed in supplementary table 1. every cdna sample was analyzed in triplicate at each time point, and the expression level of any given gene was ascertained using gapdh as the normalizer (jayaraman et al., 2010; 2013; 2017; 2018; patel et al., 2011) since it was not altered by tsa treatment in vitro (van lint et al., 1996). the same cdna pool was analyzed for the expression levels of all 41 genes. the gene expression level was determined using the 2-∆∆ct method (jayaraman et al., 2010; 2013; 2017; 2018; patel et al., 2011). the data are represented as the mean ± sem of triplicate technical repeats per time point. the outliers that deviated >10% from other data points were omitted, and the samples reanalyzed for gene expression. 2.3. elisa sera were collected from naïve and immunized mice with or without tsa treatment and assayed for ccl2 using the elisa ready-set-go kit (ebioscience, san diego, ca). samples were pooled from five mice per group, analyzed in duplicate, and expressed per mg of protein. 2.4. statistics the statistical significance of clinical scores between control and tsa-treated groups was determined using the area under the roc curve. the difference in gene expression was calculated using an unpaired two-tailed student's t-test. elisa data were analyzed for significance between groups by two-way anova. the p-value of <0.05 was considered significant. graphpad prism 6.0 software (san diego, ca) was used for all statistical analyses. 3. results 3.1. tsa treatment reduced the clinical manifestation of peae we have reported earlier that after immunization with the mog35-55 peptide the bodyweight declined transiently between 13 and 16 days, which steadily increased after that regardless of treatment with dmso or tsa (jayaraman et al., 2017). since female nod mice develop type 1 diabetes in an age-dependent manner, we routinely monitored blood glucose levels three times a week throughout the experiments. as reported earlier (jayaraman et al., 2010; 2013; jayaraman and jayaraman, 2018; patel et al., 2011), mice that were 9-11 weeks old (fig. 1a, d-e) were normoglycemic at the time of immunization with mog35-55 emulsified in complete freund’s adjuvant and remained diabetes-free throughout the observation, 12-27 weeks of age (fig. 1a, d-e). however, in two experiments, 40 and 60% of 17-week old mice immunized and treated with dmso were found to be diabetic. tsa treatment reduced the diabetes incidence respectively to 0% and 20% when analyzed at 20 and 21 weeks of age (fig. 1b-c). these results are consistent with our previous observations that the administration of complete freund’s adjuvant alone could effectively prevent type 1 diabetes in younger female nod mice. in contrast, similar treatment of older (>13-weeks) mice did not have the same potency in preventing diabetes (jayaraman and jayaraman, 2018). these results indicate that whereas the development of type 1 diabetes is age-dependent, induction of peae by immunization with the mog35-55 peptide emulsified in complete freund’s adjuvant is independent of the age and the glycemic status of mice. fig. 1 tsa treatment improved clinical disease.   randomly chosen littermates of female nod mice were immunized with mog35-55 and treated with tsa or dmso three times a week for the indicated time interval. clinical scores of five mice/group were determined on indicated time intervals and depicted as mean ± sem. the statistical significance between dmso (blank bars) and tsa-treated mice (hatched bars) was determined using the area under the roc curve test and indicated. individual data for dmso (empty circles) and tsa-treated (empty squares) groups are also indicated. mice were killed on day 14 (a), 21 (b), 28 (c), 54 (d), and 115 (e), and their spinal cord and peripheral lymphoid tissues were harvested and used for gene expression analysis. all of the mice immunized with mog35-55 developed peae without discernible remission (fig. 1a-e), consistent with earlier reports (basso et al., 2008; jayaraman et al., 2017; 2018). the overall clinical severity was comparable in most instances except in a single experiment, probably due to the difference in experimental conditions (fig. 1c). treatment with tsa bestowed robust and irreversible protection from peae for the entire period of observation (115 days) even after the cessation of the drug administration on day 45 (fig. 1e), as reported earlier (jayaraman et al., 2017). interestingly, the administration of the drug for one (fig. 1a), two (fig. 1b, c), four (fig. 1d) or six-weeks (fig. 1e) afforded a comparable level of protection against the disease. in previous studies, we have demonstrated that chronic tsa treatment decreased the influx of neutrophils and cd4+ t cells into the sc. drug treatment also reduced the inflammation and particularly axonal damage in the spinal cord, indicating the neuroprotective effect of the hdac inhibitor (jayaraman et al., 2017; 2018). to understand the underlying mechanisms of drug-mediated protection against peae, in the current study, we have concurrently profiled the gene expression in the cns and slo at various time points shown in fig. 1a-e. 3.2. differential impact of the histone modifier on gene expression in the cns the neuronal disease induced in nod mice resembled the ‘classic’ eae except that it lasted longer than in most strains of mice (slavin et al., 1998; basso et al., 2008; hidaka et al., 2014; dang et al., 2015; jayaraman et al., 2017; 2018). since the sc is thought to be the primary target of the ‘classic’ eae and the brain damage was selectively observed in 'atypical’ eae (pierson and goverman, 2017), we profiled the gene expression in the entire sc. expression levels of genes encoding 11 lymphokines, four cytokines, seven accessory cell surface-associated determinants, seven transcription factors, and 11 histone deacetylases (hdacs) were investigated by qrt-pcr. unimmunized mice were separately treated with dmso or tsa to determine the impact of drug treatment on the constitutive level of gene expression. the data are indicated at the day-1 time point in all figures. mice immunized with mog35-55 were treated with dmso or tsa starting from the day of immunization. the entire data sets are presented in supplementary fig. 1-5. only the differences in gene expression that occur consecutively at more than one-time point but not those altered transiently or sporadically are highlighted herein. longitudinal analysis during chronic peae revealed that some immune response-related genes were induced upon mog35-55 immunization after a 7-14-day lag period in both the cns and slo. the th cell-associated il17a, il4, and il22 and the inflammatory cytokine gene nos2 were upregulated for a prolonged period between 21 and 54 days in the cns in different experiments (fig. 2a-d). the epigenetic drug suppressed the expression of these genes that were upregulated by immunization. surprisingly, the constitutive expression of mif, ahr, and cd274 was also notably repressed by tsa treatment during the chronic phase of the disease (fig. 2e-g). unexpectedly, the pan hdac inhibitor upregulated the expression of dec1 (bhlhe40), specifically during the late stage of peae (fig. 2h). fig. 2 differential impact of the histone modifier on gene expression in the cns.   total rna was extracted individually from five mice per group from the spinal cord (sc) at various time points. at each time point, rna pooled from five mice/group was analyzed in triplicate. the expression of indicated genes was normalized to the housekeeping gene, gapdh, and depicted as mean ± sem of triplicate samples. statistical significance (p<0.05) between dmso (blank bars) and drug-treated mice (hatched bars) was determined using an unpaired two-tailed t-test and indicated by asterisks. individual replicates for dmso (empty circles) and tsa-treated samples (empty squares) are also shown. unimmunized mice (10/group) were treated with dmso or tsa and analyzed one day later for the effect of drug treatment on the basal level expression of genes irrespective of immunization. although previous studies implicated many other genes in eae, those encoding il-23, ifn-γ, il-18, il-27p28, il-27ebi3, il-12p35, il-10, gm-csf, tnf-α, and tgf-β were not transcriptionally upregulated in the cns of peae mice (see supplementary fig. 1-2). similarly, the mrna of the accessory cell surface-associated determinants, mmp9, mmp12, and cd74, remained stable in the cns following immunization (supplementary fig. 3). moreover, the transcription factor genes such as tbet, gata3, rorgt, eomes, and foxp3 also remained unchanged in the sc (supplementary fig. 4). notably, the histone modifier failed to alter the transcription of these genes. thus, our comprehensive analysis uncovered the selective repression of genes in the cns without affecting the levels of many other genes previously implicated in neuroinflammation. 3.3. the different patterns of gene regulation in the slo by tsa treatment the expression of the genes encoding the subunits of the heterodimeric lymphokine il-27 such as il-27p28 and il-27ebi3 peaked on day 21 and subsided steadily thereafter in the slo (fig. 3a-b). treatment with tsa reduced the expression of these genes and the inflammatory gene nos2 (fig. 3c). interestingly, the transcription of mif, cd274, and cd39 in the slo was also downregulated by the histone modifier (fig. 3d-f). surprisingly, the genes encoding the lymphokines such as il-4, il-10, il-17a, il-12p35, il-18, il-22, il-23, ifn-γ, tgfβ, gm-csf and tnf-α (supplementary fig. 1-2), as well as the macrophage-associated determinants, mmp9, mmp12, arg-1, and cd74 were neither upregulated during pathogenesis nor repressed by tsa treatment in the slo (supplementary fig. 3). similarly, the transcription of tbet, rorgt, gata3, eomes, dec1, ahr, and foxp3 remained mostly unaffected by the hdac inhibitor treatment (supplementary fig. 4). thus, the antigen-induced transcription of il27p28, il27ebi3, and nos2, as well as the constitutive expression of mif, cd274, and cd39, were selectively impeded by tsa treatment in the slo. these data collectively demonstrate the differential influence of epigenetic modulation on gene expression in the cns and slo in the peae model. fig. 3 different patterns of gene regulation in the slo by tsa treatment.   expression levels of indicated genes were determined in control dmso (blank bars) and tsa-treated mice (hatched bars) using the total rna derived from the spleen and draining lymph nodes (sp) from individual mice at the indicated time points. at each time point, rna pooled from five mice/group was analyzed in triplicate. individual data points for dmso (empty circles) and tsa-treated (empty squares) are shown. gene expression was normalized to the housekeeping gene, gapdh, and depicted as mean ± sem of triplicate samples. statistical significance (p<0.05) between control and drug-treated mice was determined using an unpaired two-tailed t-test and indicated by asterisks. unimmunized mice (10/group) were treated with dmso or tsa and analyzed one day later for the effect of drug treatment on the basal level expression of genes irrespective of immunization. 3.4. epigenetic regulation failed to influence the prominent chemokine system since previous work indicated the nonredundant roles of ccl2 and ccr2 in recruiting the inflammatory cells to the cns (mahad and ransohoff, 2003; moreno et al., 2014), we surmised that tsa-induced neuroprotection could also accompany modulation of these genes. immunization of nod mice with mog35-55 did not transcriptionally upregulate ccl2 in the cns (fig. 4a). on the other hand, ccl2 transcription increased dramatically in the slo between days 14 and 28 (fig. 4b). however, tsa treatment failed to modulate the transcription of ccl2 in the sc or slo. the gene encoding the cognate receptor of ccl2, ccr2, was not distinctively upregulated in either the sc or slo nor perturbed by the histone modifier (fig. 4c-d). in contrast to the robust increase in the ccl2 mrna level in the slo, the amount of ccl2 protein did not increase in circulation during the pre-symptomatic period (up to 16 days) as assessed by elisa, which remained unaffected by tsa treatment (fig. 4e). these data indicate that protection from peae afforded by the cns-permeant tsa (jayaraman et al., 2017; 2018) was not associated with the transcriptional regulation of the prominent chemokine system, ccl2: ccr2 either in the cns or slo. fig. 4 epigenetic regulation failed to influence the prominent chemokine system.   expression levels of indicated genes were determined in dmso (blank bars) and tsa-treated mice (hatched bars) using the total rna derived from the spinal cord (sc) (a, c) and peripheral lymphoid tissues (sp) (b, d) at various time points. at each time point, rna was extracted from five individual mice/group, pooled, and analyzed in triplicate. gene expression was normalized to the housekeeping gene, gapdh, and depicted as mean ± sem of triplicate samples. individual data points are shown for dmso (blank circles) and tsa-treated (empty squares) groups. ccl2 protein was estimated in duplicate from pooled sera of five mice per group using elisa (e). unimmunized mice (10/group) were treated with dmso or tsa and analyzed one day later for the effect of drug treatment on the basal level expression of genes irrespective of immunization. statistical significance (p<0.05) between control and drug-treated mice (n=five/group) was determined using an unpaired two-tailed t-test and indicated by asterisks. 3.5. tsa treatment repressed the transcription of hdac11 selectively in the cns whereas tsa can inhibit the activity of hdac class i, iia, and iib isoenzymes with varying potency in vitro (bradner et al., 2010), its ability to regulate hdac genes in vivo has not been determined. to address this issue, we analyzed the mrna levels of class i (hdac1, hdac2, hdac3, and hdac8), class iia (hdac4, hdac5, hdac7, and hdac9), class iib (hdac6 and hdac10), and class iv (hdac11) hdacs using validated primer sets and qrt-pcr. data shown in fig. 5 indicate that immunization of nod mice with mog35-55 steadily increased the transcription of the class iv hdac11 in the sc, which remained at high levels between 21 and 54 days postimmunization. importantly, tsa treatment reduced the transcript level of hdac11. although hdac1, hdac4, hdac5, hdac6, hdac8, and hdac9 were modestly increased in the cns with different kinetics after immunization, they were relatively insensitive to tsa treatment. surprisingly, the expression levels of hdac genes, including the hdac11, did not increase significantly in the slo of mog35-55 immunized mice nor repressed by tsa treatment (supplementary fig. 5). these results indicate that mog35-55 immunization leads to increased transcription of hdac11 in a cns-specific fashion, which is rendered sensitive to the action of the histone modifier. these results indicate the possibility that hdac11 could represent a novel target for the manipulation of peae. fig. 5 tsa treatment repressed the transcription of hdac11 selectively in the cns.   expression levels of indicated hdac genes were determined in dmso (blank bars) and tsa-treated mice (hatched bars) in the spinal cord as shown. at each time point, rna was isolated from five individual mice/group, pooled and analyzed in triplicate. gene expression was normalized to the housekeeping gene gapdh and depicted as mean ± sem of triplicate samples. individual data points are shown for dmso (blank circles) and tsa-treated (empty squares) groups. statistical significance (p<0.05) between control and the drug-treated group was determined using an unpaired two-tailed t-test and indicated by asterisks. ns, not significant. unimmunized mice (10/group) were treated with dmso or tsa and analyzed one day later for the effect of drug treatment on the basal level expression of genes irrespective of immunization. 4. discussion a cardinal assumption has been that the t lymphocytes play a crucial role in the induction and manifestation of multiple sclerosis, and therefore, induction of antigen-specific t cell tolerance is a reasonable approach to treat this debilitating disease. many attempts, including immunization with various neuronal peptides and t cell vaccinations, have failed to induce t cell tolerance and ameliorate multiple sclerosis symptoms (wingerchuk and carter, 2014; dendrou et al., 2015; steinman, 2015; lassmann, 2017). however, the induction of antigen-specific t cell tolerance using immunomodulatory drugs remains unexplored in patients with ms and other autoimmune disorders. recently, we have demonstrated that treatment with the potent hdac inhibitor tsa not only reduced the frequencies of th1, th17, and th1/th17 cells in the slo and their influx into the cns but also induced mog35-55 peptide-specific t cell tolerance (anergy) in nod mice (jayaraman et al., 2017). although anergy was originally reported in a mouse th1 clone that was suboptimally activated without co-stimulation in vitro (schwartz et al., 1989; jayaraman et al., 1992), the underlying mechanisms have not been fully deciphered. our investigation unraveled a good correlation between tsa-mediated neuroprotection and downregulation of selected immune response-related genes both in the cns and slo. whereas these data may not directly impinge upon histone modifier-facilitated mog35-55 peptide-specific t cell tolerance, they highlight the possible impact of differential gene expression on peae. the epigenetic approach unraveled an inverse relationship between the expression levels of a small set of genes and neuroprotection. in the cns of peae nod mice, il17a expression was upregulated for over a month (fig. 2), unlike its expression at the peak of the monophasic eae (kreymborg et al., 2007; evangelidou et al., 2014; borjini et al., 2016). prolonged expression of nos2 in the cns (fig. 2b) is congruent with the association of inos-positive macrophages and glial cells in demyelinating pathology (tran et al., 1997). in addition to the nos2 (fig. 3c), the subunits of il-27, namely il-27p28, and il-27ebi3, were prominently upregulated at the mrna level in the slo (fig. 3b-c). this is consistent with an encephalitogenic role of il-27 suggested by the suppression of the ongoing eae following administration of the neutralizing antibody against the il-27p28 subunit (goldberg et al., 2004). besides, tsa treatment reduced the steady-state expression of mif in both the cns and slo (fig. 2e, fig. 3e), consistent with a proposed pathogenic role of mif in monophasic eae (powell et al., 2005). we have previously shown that tsa treatment diminished the numbers of splenocytes expressing the co-inhibitory ligand cd274 (pd-l1) (jayaraman et al., 2018). consistently, the cd274 mrna level was also repressed in the cns (fig. 2g) and slo (fig. 3f) of tsa-treated peae mice, suggesting a role for this co-inhibitory ligand in encephalomyelitis, as proposed (jayaraman et al., 2018). also, the expression of ahr, uniquely required for the generation of t cells responsible for the late-onset eae (nakahama et al., 2013) was diminished in mice protected by tsa treatment (fig. 2f). collectively, the tsa-mediated downregulation of both inducible and constitutively expressed genes appears to be inversely proportional to the severity of the neuronal disease. however, it remains to be determined whether the expression levels of these genes may serve as biomarkers for the diagnosis of ms. nevertheless, the information uncovered in the nod mouse model may provide a framework for potential ms treatment, typically diagnosed as clinically isolated syndrome followed by years of asymptomatic period (dendrou et al., 2015). the upregulation of the transcription of hdac11 in a cns-specific fashion in mice immunized with mog35-55 (fig. 5) represents the first report of differential expression of hdac genes in vivo. although the non-selective hdac inhibitor tsa did not diminish the hdac11 enzymatic activity in vitro (bradner et al., 2010), our data demonstrated the control of hdac11 by tsa at the transcriptional level. since hdac11 gene expression was determined in the sc devoid of peripheral blood contamination, its reduction appears to be a direct effect of tsa on the cns resident cells. although we have not identified the cellular source of hdac11 in the spinal cord, previous work suggested that hdac11 knockdown increased il10 expression in peripheral antigen-presenting cells resulting in immunosuppression in vitro (villagra et al., 2009). in contrast, tsa-induced tolerance in mog35-55-specific t lymphocytes (jayaraman et al., 2017) did not accompany the upregulated transcription of il10 in the cns or slo (see supplementary fig. 1). although the deletion of hdac1 selectively in t cells was reported to prevent eae (göschl et al., 2018), immunization with mog35-55 neither upregulated the expression of this gene, nor the epigenetic drug influenced its transcription in the slo or cns (fig. 5, supplementary fig. 5). thus, the histone hyperacetylating drug appears to primarily target the hdac11 gene in the cns of peae mice and the elucidation of the genes that are under the control of hdac11 may provide novel insights into the mechanisms of peae. to our surprise, the epigenetic drug treatment failed to validate the purported roles of the genes critical for the development of encephalitogenic th1 and th17 subsets, including ifng, il12, il18, il23, tbet, rorgt, gata3, and eomes. the disruption of the ifn-γ gene failed to influence eae development (ferber et al., 1995), indicating a lack of ifn-γ-expressing cells in neurodegeneration. consistently, the reduction of clinical symptoms by tsa treatment did not accompany the transcriptional repression of ifn-γ (supplementary fig. 1). besides, the drug-mediated tolerance induction was evident without repressed transcription of tbet, il18, and il12 genes critical for th1 cell development (o'connor et al., 2013; lalor et al., 2011; gran et al., 2004) (see supplementary fig. 3). moreover, the transcription of il23 (kreymborg et al., 2007; el-behi et al., 2011) and rorgt (martinez et al., 2014), respectively, involved in th17 and th2 cell generation was also not diminished by tsa treatment. yet, the histone modifier reduced the overall numbers of th17 and th1/th17 cells in the slo (jayaraman et al., 2017). furthermore, the drug treatment failed to decrease the transcription of csf2 (see supplementary fig. 2) despite the diminished numbers of gm-csf-co-expressing th1 and th17 cells found in the slo of tsa-treated mice (jayaraman et al., 2017). these data indicate that t cell tolerance induction by epigenetic modulation of the genome does not involve selective suppression of genes required for the generation of functionally distinct th cell subsets. further work is necessary to decipher the underlying mechanisms of t cell tolerance. another deviation from the conventional idea of immunoregulation is the lack of the modulation of il10 and foxp3 both in the cns and slo of tsa-treated mice (supplementary fig. 4). although foxp3+ t regulatory cells are considered critical for immunoregulation (hori et al., 2003), their role in eae is less compelling (jayaraman et al., 2017; danikowski et al., 2017; jayaraman and prabhakar, 2019). whereas tsa treatment increased foxp3 expression and promoted t regulatory cell function via the upregulation of hdac9 in a different experimental model (tao et al., 2007), our results contradict these findings. it is also noteworthy that tsa treatment downregulated foxp3 expression and lowered the numbers of cd4+cd25+ t regulatory cells (liu et al., 2010). nevertheless, our data indicate that tsa-mediated protection from peae in autoimmune-prone nod mice is independent of il-10 and foxp3+ t regulatory cells. differential rna display (van lint et al., 1996) and microarray analysis of gene expression (jayaraman et al., 2013) indicated that histone hyperacetylation by tsa treatment could have positive, negative, or no effect on gene transcription. our qrt-pcr analysis showed that out of 41 genes interrogated, 11 were consistently downregulated over multiple time points following tsa treatment (vide supra). rarely, tsa treatment can also increase the transcription of genes due to the transcriptional suppression of repressor complexes that control gene expression. thus, dec1 was selectively upregulated in the cns by the histone modifier, suggesting a role in protection against peae (fig. 2h). this is in contrast to the finding that the genetic deletion of dec1 (bhlhe40-/-) afforded resistance to eae induction (lin et al., 2016). like an earlier study (hidaka et al., 2014), we found that the expression of the ccl2 gene was dramatically increased in the slo of mog35-55 immunized nod mice (fig. 4). however, tsa treatment failed to influence its expression. moreover, there is a disconnect between the ccl2 mrna expression in the slo and the release of the ccl2 protein into the peripheral blood. it has been proposed that ccl2 expressed by the cns resident astrocytes may serve as a target for ms treatment (mahad and ronsohoff, 2003; moreno et al., 2014). however, ccl2 and ccr2 genes are refractory to histone hyperacetylation, and therefore do not appear to be essential for peae induction and manifestation. this observation does not come as a surprise since the level of ccl2 was also reported to be lower in relapsing-remitting ms patients (narikawa et al., 2004; moreira et al., 2006), and the ifn-beta 1a therapy reduced relapses while increasing the level of ccl2 in ms patients (szczuciński and losy, 2004). thus, these data do not support the hypothesis that the ccl2: ccr2 system may impact neurodegeneration. it is noteworthy that tsa administration induced neuroprotection and gene regulation similarly in prediabetic, 9-13 weeks old, and aged (17-weeks old) mice (jayaraman et al., 2017; fig. 1-5). the administration of the epigenetic drug for one to six weeks bestowed comparable neuroprotection and gene regulation irrespective of the age of the mice. circumstantial evidence also indicates the lack of influence of diabetes on peae induction. as expected, the prediabetic (9-13-weeks old) mice were normoglycemic at the time of immunization with mog35-55 and remained non-diabetic as long as 27 weeks of age. the lack of diabetes in immunized mice could be due to the action of the microbial products present in the complete freund’s adjuvant. in fact, we have shown that the administration of complete freund’s adjuvant alone is sufficient to prevent prediabetic nod mice from developing overt diabetes (jayaraman and jayaraman, 2018). although a fraction of the older (17 weeks old) mice developed diabetes, it did not impede peae induction (fig. 1b-c) nor the modulation of gene expression in response to tsa treatment (fig. 2-5). collectively, these data demonstrate that the induction of peae and epigenetic regulation of gene expression is unrelated to the age or the glycemic status of mice. conclusions the analysis of the 41 genes using qrt-pcr unraveled differential regulation of gene expression in the cns and slo by an epigenetic drug. this study has highlighted the roles of il4, hdac11, cd274, and cd39 in the cns in addition to validating previously implicated genes, il17a, il22, inos, ahr, and mif in neuroinflammation. on the other hand, in the slo, the roles of il22, il27, nos2, and mif were confirmed while indicating the participation of cd274 and cd39 in peae. some of these genes are induced by immunization, while others are constitutively expressed. surprisingly, many other genes previously implicated in eae were refractory to histone hyperacetylation mediated transcriptional regulation. hence, it will be difficult to conclude whether these genes do not contribute to neuroinflammation. nevertheless, our data suggest that the drug-induced histone hyperacetylation is a promising strategy to treat demyelination and axonal damage by modifying the expression of selected genes. although the therapeutic potential of hdac inhibitors for the treatment of multiple sclerosis has been entertained (faraco et al., 2011), direct evidence is lacking (göbel et al., 2018). the data generated in the nod mouse model provide a framework for a similar pharmacological approach to treat ms patients. author contributions aj conducted experiments, collected data, edited the graphic and the manuscript. ka, rk, as, ak, and mk performed experiments, collected data, and approved the manuscript. sj conceived the project, designed and implemented experiments, analyzed the data, and wrote the paper. funding this research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. acknowledgments mark holterman and bellur 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article, unless otherwise stated. hydrophilic polymer embolism identified in brain tumor specimens following wada testing: a report of 2 cases feel free to add comments by clicking these icons on the sidebar free neuropathology 2:23 (2021) case report hydrophilic polymer embolism identified in brain tumor specimens following wada testing: a report of 2 cases vanessa s. goodwill md*1, michael g. brandel md, mas*2, jeffrey a. steinberg md2, thomas l. beaumont md, phd2, lawrence a. hansen md1 1 department of pathology, university of california, san diego, usa 2 department of neurosurgery, university of california, san diego, usa corresponding author: vanessa goodwill, md · uc san diego health, department of pathology · east campus office bldg (ecob), ste. 1-200 · 9444 medical center drive · la jolla, ca 92037 · usa vgoodwill@health.ucsd.edu submitted: 28 july 2021 accepted: 28 august 2021 copyedited by: deborah mcintyre published: 2 september 2021 https://doi.org/10.17879/freeneuropathology-2021-3457 keywords: embolism, vascular access devices, brain infarction, glioma, vasculitis abstract hydrophilic polymers are commonly used as coatings on intravascular medical devices. as intravascular procedures continue to increase in frequency, the risk of embolization of this material throughout the body has become evident. these emboli may be discovered incidentally but can result in serious complications including death. here, we report the first two cases of hydrophilic polymer embolism (hpe) identified on brain tumor resection following wada testing. one patient experienced multifocal vascular complications and diffuse cerebral edema, while the other had an uneventful postoperative course. wada testing is frequently performed during preoperative planning prior to epilepsy surgery or the resection of tumors in eloquent brain regions. these cases demonstrate the need for increased recognition of this histologic finding to enable further correlation with clinical outcomes. introduction intravascular devices, such as catheters, guidewires, and stents are commonly coated with hydrophilic polymer material. this hydrophilic polymer material has significant procedural benefits, such as acting as a lubricant to decrease vascular trauma, and reducing vascular spasm.1 over the past several decades there has been a drastic increase in the prevalence of minimally invasive endovascular medical procedures. as a result, there has been growing recognition of the risk of embolization of hydrophilic polymer coating material throughout the body.2,3 wada tests (also known as intracarotid sodium amobarbital procedure, isap) are commonly performed before ablative procedures or resections for epilepsy or tumor to assess hemispheric dominance for language. there is a low risk of complications following wada testing including seizures, status-epilepticus, vascular spasm, and transient encephalopathy.4 hydrophilic polymer emboli, however, have not previously been reported following pre-operative wada testing. here, we describe two patients who underwent primary brain tumor resection following wada testing. in both resection specimens, intravascular hydrophilic polymer material with associated foreign body giant cell reaction was identified, and in one case there was evidence of associated acute ischemic change in surrounding neurons. to our knowledge, the current work represents the first report of this histologic finding after wada testing. case summaries case 1 clinical findings: a 55-year-old right-handed man presented with new-onset generalized tonic-clonic seizures. magnetic resonance imaging (mri) revealed a non-enhancing infiltrative and expansile t2-hyperintense mass centered within the right superior temporal gyrus (fig. 1a-b). the patient underwent bilateral wada testing. vascular access was obtained via the right femoral artery using the modified seldinger technique. a 5-french angled glide diagnostic catheter was introduced over a 0.035 terumo glidewire into the descending thoracic aorta and then the cervical internal carotid artery under direct fluoroscopic visualization. a codman prowler select plus infusion microcatheter with a synchro 2 microwire was used to catheterize the internal carotid artery just distal to the posterior communicating artery. an angiographic run was performed to confirm catheter position prior to brevital (methohexitol) infusion (fig. 1c). the wada test demonstrated clear left hemisphere language dominance and greater left hemisphere memory support. figure 1: case 1 imaging and pathology. (a) axial t2-flair and (b) t1 post-contrast sequences of a brain mri demonstrating a non-enhancing infiltrative and expansile t2-hyperintense mass centered within the right superior temporal gyrus. (c) a pa view of digital subtraction angiogram demonstrating a microcatheter within the right internal carotid artery. (d-e) the tumor resection demonstrates a moderately to highly cellular infiltrating glioma, with significant nuclear atypia and scattered mitoses (arrow). (f-g) parenchymal vessels within the resection display giant cell vasculitis and contain basophilic granular foreign material consistent with hydrophilic polymer. five weeks later, the patient underwent a right temporo-parieto-occipital craniotomy for tumor resection. his postoperative course was complicated by a seizure upon awakening with right frontal venous infarct and layering remote cerebellar hemorrhage. he experienced weakness in his left upper and lower extremities and was discharged to a rehabilitation facility with antiepileptic and steroid medications. at 5 months follow-up, the patient had recovered his motor function with no additional seizures, was able to walk over a mile per day without assistance and was tolerating adjuvant chemoradiation. subsequently, post-treatment mris demonstrated a heterogeneously enhancing, centrally necrotic mass in the right temporo-parietal tumor bed with no associated hyperperfusion, characteristic of evolving pseudoprogression. these findings were associated with marked hemispheric cerebral edema refractory to steroid treatment and requiring bevacizumab (fig. 2). figure 2: case 1 post-treatment imaging findings. an 8-month post-operative mri following chemo-radiation showed evidence of pseudoprogression with significant associated cerebral edema. axial t1 post-contrast (a) shows a heterogeneously enhancing, centrally necrotic mass, with restricted diffusion on dwi (b), and no associated hyperperfusion (c). pathology: evaluation of the primary resection specimen revealed a moderately to highly cellular diffusely infiltrating glioma. there was significant pleomorphism and focally frequent mitoses, but no microvascular proliferation or necrosis, meeting histologic criteria for anaplastic astrocytoma, who grade iii (fig. 1d-e). molecular analysis by next-generation sequencing (ngs) and microarray revealed features of primary idh-wildtype glioblastoma, including egfr amplification, and the tumor was upgraded to who grade iv.5 additionally noted on pathologic examination were scattered foci of giant cell vasculitis and intravascular foreign material. the foreign material was granular, blue-gray and non-polarizable, morphologically consistent with hydrophilic polymer material. no surrounding ischemic changes were identified (fig. 1f-g). case 2 clinical findings: a 24-year-old right-handed female with no significant past-medical history presented after suffering a generalized tonic-clonic seizure at work. mri showed a large, non-enhancing infiltrative t2 hyperintense mass centered in the right hippocampus and inferior temporal lobe with regional mass effect (fig. 3a-b). she underwent stereotactic biopsy at an outside facility, which demonstrated a who grade ii diffuse astrocytoma. figure 3: case 2 imaging and pathology. (a) axial t2-flair and (b) t1 post-contrast mri brain sequences demonstrate a non-enhancing infiltrative t2 hyperintense mass centered in the right hippocampus and inferior temporal lobe. (c) pa view of a digital subtraction angiogram demonstrates a microcatheter within the left internal carotid artery. (d) the tumor resection shows a moderately cellular glioma infiltrating white matter and (e) cortex with perineuronal satellitosis. (f) a hypereosinophilic ischemic neuron (arrow and inset) is seen next to a vessel occluded by hydrophilic polymer material with associated inflammation. (g) some vessels contain multinucleate foreign-body giant cells consuming basophilic polymer material. the patient underwent bilateral wada testing with the same microcatheterization technique described in case 1 (fig. 3c). injections of both sodium methohexital (brevital) and amobarbitol (amytal) were completed, and language representation was identified in the left hemisphere. three weeks later, the patient underwent right frontotemporal craniotomy for tumor resection. she had an uncomplicated postoperative course and was discharged home on postoperative day 3 at her baseline functional status, with no neurologic deficits. she remains neurologically intact and seizure-free 9 months postoperatively. pathology: histologic examination of the resection specimen showed a paucicellular infiltrating glioma composed of atypical astrocytes with enlarged, irregular, and hyperchromatic nuclei. no mitoses, necrosis, or microvascular proliferation were detected (fig. 3d-e). ngs was performed and an idh1-r132h mutation was identified, along with mutations in tp53, and atrx. the final diagnosis was diffuse astrocytoma, idh-mutant, who grade ii. also noted multifocally were a few small parenchymal vessels containing non-polarizable granular blue-gray foreign material consistent with hpe. surrounding these vessels was foreign-body giant cell reaction, and a few hyper-eosinophilic acutely ischemic neurons (fig. 3f-g). discussion hydrophilic polymer embolization in cerebral vessels was first reported in 1997 by barnwell et al. in four cases involving use of a microcatheter with hydrophilic coating for cerebral angiography (three autopsy specimens after endovascular thrombolysis for acute ischemic stroke, and one biopsy specimen of a previously-embolized meningioma).6 emboli were described as basophilic and granular, identical in histologic appearance to hydrophilic polymer material seen on sections of microcatheter submitted for histologic analysis. hpe have since been identified widely throughout the body, including the lungs, skin, heart, and central nervous system (cns).3,7–11 hpe may be discovered incidentally or in association with a wide range of complications. reported sequelae of hpe throughout the body include vasculitis, thrombosis, infarction, and even death.12 additionally, there is a significant range in the time-course of presentation and duration, spanning acute, subacute, and delayed symptomatology. one study described evidence of persistent inflammation over three years after the intravascular procedure.13 hpe has been reported following various neuro-interventional procedures such as aneurysm coil embolization, aneurysm flow diversion, intra-arterial thrombolysis, and diagnostic cerebral angiography, and may result in a variety of cns complications.12,14,15 mehta et al. reported a series of 32 cases of hpe within the cns, with findings including vasculitis, granuloma formation, chemical meningitis, ischemia, and hemorrhage.8 these findings were associated with a 38% incidence of stroke and 28% rate of death. the high rate of morbidity and mortality secondary to hpe in this study, however, may be an overestimate as patients were identified in part by their adverse clinical outcome, and types of catheterization also included central venous access and hemodialysis catheterization. here, we report two cases of hpe identified in brain tumor resection following pre-operative wada testing. to perform a wada test, sodium methohexital (brevital) or amobarbital (amytal) is injected into right or left internal carotid artery, which results in ipsilateral hemispheric anesthetization. at our institution, a neuropsychologist conducts a baseline evaluation prior to wada testing, and then repeats this assessment during injections of each hemisphere. in addition, an epileptologist interprets the continuous eeg and compares background activity in each hemisphere to activity during injection to confirm appropriate post-anesthetic changes. a variety of microcatheters are utilized at our institution, including the prowler select plus which is a braid/coil microcatheter with a radiopaque tip and hydrophilic coating. to our knowledge, no prior reports have described an association between this microcatheter and hpe. the rate of complications following cerebral angiography is between 0.3-1.3%, and only a fraction of these are neurologic complications with lasting effects.16,17 the complication rate for wada testing is likely around 2%, although some studies have reported up to 10%.4 of particular interest, our patient from case 1 suffered immediate post-craniotomy vascular complications possibly induced or exacerbated by hpe-associated vasculitis and thrombosis, although there were no immediate or delayed complications from the wada test on its own. although this patient had recovery of neurological function and was ambulatory and independent with activities of daily living, he had early radiologic evidence of pseudoprogression complicated by cerebral edema refractory to steroid treatment and requiring bevacizumab. though one can only speculate, as no additional tissue resection was performed following chemo-radiation, it is possible hpe associated vascular changes in this patient may have also contributed to his significant refractory cerebral edema and extent of pseudoprogression. the patient from case 2 has suffered no adverse clinical sequela to date despite histologic evidence of vasculitis and surrounding acute ischemia. hpe-induced vasculitis and thrombosis may contribute to surgical and post-operative vascular complications in patients undergoing craniotomy for tumor resection or during epilepsy surgery. as the clinical sequelae of hydrophilic polymer emboli can be quite severe based on the findings of several case series,8,12 clinicians and surgeons should be made aware of their presence when identified histologically. therefore, we recommend including the presence of hpe and any associated vascular or ischemic changes as a separate diagnostic line in pathology reports when identified in biopsy or resection specimens to allow for clinical correlation in the event of any unforeseen vascular complications (see example below, case 2). right temporal brain mass, resection: integrated diagnosis: astrocytoma, idh-mutant, who grade ii. histological diagnosis: diffuse astrocytoma. histological grade: who grade ii. molecular information: molecular alterations including: activating mutation of idh1 (r132h), and inactivating mutations of tp53 and atrx. hydrophilic polymer emboli with associated giant cell vasculitis and focal acute ischemic changes (see comment). comment: hydrophilic polymers are applied as surface coatings on vascular devices, and embolization of this material has been reported following various vascular procedures. hydrophilic polymer emboli may be an incidental finding, but can also result in a range of vascular complications. clinical correlation is suggested. furthermore, the incidence of hpe following neuro-interventional procedures is likely under-recognized. published studies are limited by selection bias; however, and neither the overall rate of hpe after cerebral angiography nor the true rate of clinical complications in the setting of cerebral hpe can be identified from the current literature. as such, it is important to recognize hpe histologically so that prospective clinical outcome studies of patients undergoing neuroendovascular procedures prior to cerebral tissue biopsy may be performed. disclosures the following authors have nothing to disclose: vanessa s. goodwill, michael g. brandel, jeffrey a. steinberg, thomas l. beaumont, lawrence a. hansen. references 1. koga s, ikeda s, futagawa k, et al. the use of a hydrophilic-coated catheter during transradial cardiac catheterization is associated with a low incidence of radial artery spasm. int j cardiol. 2004;96(2). 2. mehta ri, mehta ri. hydrophilic polymer embolism: an 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(https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. specific immune modulation of experimental colitis drives enteric alpha-synuclein accumulation and triggers age-related parkinson-like brain pathology feel free to add comments by clicking these icons on the sidebar free neuropathology 2:13 (2021) original paper specific immune modulation of experimental colitis drives enteric alpha-synuclein accumulation and triggers age-related parkinson-like brain pathology stefan grathwohla, emmanuel quansahb, nazia maroofa, jennifer a. steinerb, liz spychera, fethallah benmansourc, gonzalo duran-pachecod, juliane siebourg-polsterd, krisztina oroszlan-szovika, helga remya, markus haenggia, marc stawiskia, matthias selhausend, pierre maliverd, andreas wolferte, thomas emriche, zachary madajb, arel sud, martha l. escobar galvisb, christoph muellerf, annika herrmannd, patrik brundinb*, and markus britschgia* a roche pharma research and early development, neuroscience and rare diseases discovery and translational area, roche innovation center basel, f. hoffmann-la roche ltd, grenzacherstrasse 124, basel, switzerland b parkinson’s disease center, department of neurodegenerative science, van andel institute, 333 bostwick ave. ne, grand rapids, mi, usa c roche pharma research and early development, predi, roche innovation center basel, f. hoffmann-la roche ltd, grenzacherstrasse 124, basel, switzerland d roche pharma research and early development, pharmaceutical sciences, roche innovation center basel, f. hoffmann-la roche ltd, grenzacherstrasse 124, basel, switzerland e roche pharma research and early development, pharmaceutical sciences, roche innovation center munich, roche diagnostics gmbh, nonnenwald 2, penzberg, germany f institute of pathology, university of bern, murtenstrasse 31, bern, switzerland *corresponding authors: markus britschgi · roche pharma research and early development · neuroscience and rare diseases discovery and translational area · roche innovation center basel · f. hoffmann-la roche ltd · grenzacherstrasse 124 · 4070 basel · switzerland markus.britschgi@roche.com patrik brundin · van andel institute · 333 bostwick ave. ne · grand rapids · mi 49503 · usa patrik.brundin@vai.orgs submitted: 07 april 2021 accepted: 08 may 2021 copyedited by: bert m. verheijen published: 18 may 2021 https://doi.org/10.17879/freeneuropathology-2021-3326 additional resources and electronic supplementary material: supplementary material keywords: alpha-synuclein, experimental colitis, enteric nervous system, parkinson’s disease, substantia nigra abstract background: in some people with parkinson’s disease (pd), α-synuclein (αsyn) accumulation may begin in the enteric nervous system (ens) decades before development of brain pathology and disease diagnosis.   objective: to determine how different types and severity of intestinal inflammation could trigger αsyn accumulation in the ens and the subsequent development of αsyn brain pathology.   methods: we assessed the effects of modulating shortand long-term experimental colitis on αsyn accumulation in the gut of αsyn transgenic and wild type mice by immunostaining and gene expression analysis. to determine the long-term effect on the brain, we induced dextran sulfate sodium (dss) colitis in young αsyn transgenic mice and aged them under normal conditions up to 9 or 21 months before tissue analyses.   results: a single strong or sustained mild dss colitis triggered αsyn accumulation in the submucosal plexus of wild type and αsyn transgenic mice, while short-term mild dss colitis or inflammation induced by lipopolysaccharide did not have such an effect. genetic and pharmacological modulation of macrophage-associated pathways modulated the severity of enteric αsyn. remarkably, experimental colitis at three months of age exacerbated the accumulation of aggregated phospho-serine 129 αsyn in the midbrain (including the substantia nigra), in 21but not 9-month-old αsyn transgenic mice. this increase in midbrain αsyn accumulation is accompanied by the loss of tyrosine hydroxylase-immunoreactive nigral neurons.   conclusions: our data suggest that specific types and severity of intestinal inflammation, mediated by monocyte/macrophage signaling, could play a critical role in the initiation and progression of pd. introduction parkinson’s disease (pd) is a progressively debilitating neurodegenerative disease affecting 1% of the population above 60 years [1]. typical symptoms are motor impairments including muscle rigidity, tremor, and bradykinesia. neuropathologically, pd is hallmarked by loss of dopaminergic neurons in the substantia nigra (sn), a concomitant reduction of striatal dopaminergic signaling [2], and the presence of intraneuronal inclusions called lewy bodies and neurites [3]. lewy pathology is enriched in α-synuclein (αsyn), a presynaptic protein that tends to aggregate and become phosphorylated at serine 129 under pathological conditions [2]. rare point mutations in αsyn and gene multiplications also cause familial forms of pd and related neurological conditions, and certain single nucleotide polymorphisms close to the αsyn gene (snca) locus are associated with increased risk for sporadic pd [4]. these findings make αsyn a focal point of biomarker and drug development programs for pd. several years before the first appearance of motor symptoms, many patients exhibit a variety of non-motor symptoms including constipation, sleep disorder, depression, and hyposmia [5–7]. indeed, co-occurrence of some of these non-motor symptoms is coupled to elevated pd risk [8–11]. constipation is an important non-motor feature of prodromal pd, with 28-61% of patients having exhibited gastrointestinal dysfunction for several years during the prodrome [7,10,12]. notably, αsyn-immunoreactive inclusions have been found in neurons of the submucosal plexus in people with pd [3,13]. taken together, this converging evidence suggests an early involvement of the enteric nervous system (ens) in the pathogenesis of pd. already over a decade ago, braak and colleagues hypothesized that αsyn-immunoreactive inclusions first appear in the ens and then occur in the parasympathetic (e.g., vagal output neurons in the intestines) and sympathetic (e.g., in the celiac ganglion in the upper abdomen) nervous system and gradually engage the brainstem, including the vagal dorsal motor nucleus and midbrain areas [3,13]. several studies in preclinical models have demonstrated that αsyn pathology in the gut is associated with the development of αsyn pathology in the brain [14–21]. for a better understanding of pd pathogenesis and particularly events happening at preclinical stages of pd, it is critical to determine factors that regulate αsyn accumulation in the ens and to understand whether the process underlying αsyn accumulation in the gut can also lead to αsyn pathology in the brain. inflammation can potentially trigger αsyn pathology in the ens of the gut and in the brain. a recent finding in children with gastrointestinal inflammation suggests an immune regulatory function of αsyn [22]. immune pathways are indeed activated in the brain and colon of pd cases [23,24]. also, several genes associated with an increased pd risk have an immune system-related function [25], and it was recently proposed that pd heritability is not simply due to variation in brain-specific genes, but that several cell types in different tissues are involved [26]. further genetic evidence supporting a role for immune pathways in pd pathogenesis is provided by a genome-wide association study that identified common genetic pathways linking pd and autoimmune disorders [27]. most prominently, lrrk2, a major genetic risk factor for pd [28] also confers increased risk for developing inflammatory bowel disease (ibd) [29]. certain risk alleles are shared between pd and crohn’s disease [30], and lrrk2 is known to modulate the function of monocytes, macrophages and other immune cells [31,32]. intriguingly, ibd is associated with an increased risk for developing pd and specifically blocking the tumor necrosis factor (tnf) pathway reduces this risk [33–37]. recently, it was reported that experimental colitis in αsyn transgenic mice leads to enteric accumulation of αsyn and the development of pd-like brain pathology and symptoms within a few months [38]. converging clinical and nonclinical data suggest that the intestinal immune environment plays a role in triggering pd or facilitating the molecular events involved in the earliest phases of the disease process [39,40]. here, we tested the hypothesis that specific types and severity of intestinal inflammation are required to trigger the accumulation of αsyn in the ens and the subsequent development of αsyn pathology in the brain. experimental forms of colitis in wild type and αsyn transgenic mice demonstrated that the type and degree of inflammation regulates the amount of αsyn accumulation in the colon. macrophage-related signaling limited the extent of αsyn immunoreactivity as demonstrated in a genetic and a pharmacological immune modulation paradigm in the experimental colitis mouse model. most remarkable, when αsyn transgenic mice were exposed to experimental colitis at 3 months of age and then were allowed to age normally up to 9 or 21 months, the accumulation of aggregated αsyn in midbrain, including the sn, was much exacerbated in the 21-month-old group, but not in the 9-month-old group. these 21-month-old mice also exhibited loss of nigral tyrosine hydroxylase-immunoreactive neurons. together, our data provide experimental evidence in mice that certain specific forms of intestinal inflammation might be a relevant upstream trigger that plays a critical role in the initiation of pd pathogenesis and the disease progression. methods aim, design and setting we aimed to combine an αsyn transgenic mouse model of age-dependent development of αsyn pathology with well-established experimental colitis paradigms in order to explore the effect of type and severity of immune activation on the development of αsyn pathology in the colon and the brain. the design and setting of the different studies are illustrated in fig. 1. fig. 1 age-dependent increase of intracellular αsyn accumulation in enteric nervous system of hemizygous (thy1)-h[a30p]αsyn transgenic mice and setup of the experimental colitis paradigms. a confocal microscopy imaging of the inclusions of human αsyn (red, antibody clone 211; human αsyn specific) within the ganglia of the submucosal plexus (green, peripherin; blue, dapi/nuclei) of hemizygous (thy1)-h[a30p]αsyn transgenic mice. arrowhead points to one of the typical irregularly sized and shaped αsyn inclusion bodies visualized in 2d z-stacks of rotated confocal images. scale bar: 100 μm. b stereological quantification of normally occurring human αsyn inclusions in the myenteric and submucosal plexuses of 3and 12-month-old hemizygous (thy1)-h[a30p]αsyn transgenic mice (n = 4 per group; mean and s.e.m. are shown; student t-test between the two age groups in each region). c setup of experimental colitis paradigms employing dextran sulfate sodium (dss, per os in drinking water) or bacterial lipopolysaccharide (lps, intraperitoneal injection). except for the ‘chronic dss paradigm, constant dose’, all paradigms were started at the age of 3 months. the ‘chronic dss paradigm, constant dose’ was started in mice aged 5 months and the colon were analyzed right after. for some experiments, we used a ‘chronic dss paradigm, increasing dose’, to mimic better the chronic nature of ibd and the longer water intervals are generally more gentle for the mice from an animal welfare perspective as well. under this paradigm, we induced experimental dss colitis intermittently as indicated over 23 days, then let the mice recover and age for two more months up to the age of 6 months on normal drinking water, and analyzed their colon. in a separate experiment, we aged the mice further up to 9 or 21 months and analyzed their brain pathology. open arrows on time axis indicate that colon was analyzed and stars with closed arrows indicate that brains were analyzed. d hematoxylin staining of 35 μm thick colon sections of 3-month-old hemizygous (thy1)-h[a30p]αsyn transgenic mice. organizational layers of the intact colon (left panel). representative images of various severity degrees of dss-driven colitis from weak leukocyte infiltration (top panel of acute dss) to more extensive leukocyte infiltration with mucosal ulceration (lowest panel of acute dss). note the different appearance of enteric inflammation in acute lps-driven peripheral inflammation compared with dss, e.g., confined immune cell clustering and lymphoid hyperplasia, intact mucosal layer. scale bar: 50 μm (intact colon), 100 μm (acute dss), and 200 μm (lps). mice male c57bl/6j wild type mice (jackson laboratories, bar harbor, usa), hemizygous tg(thy1-snca*a30p)18pjk ((thy1)-h[a30p]αsyn) [41] and tg(thy1-snca*a30p)18pjk crossed with cx3cr1tm1litt ((thy1)-h[a30p]αsyn /cx3cr1-def; homozygous for cx3cr1-gfp knock-in allele; [42] transgenic mice were used for the study. (thy1)-h[a30p]αsyn transgenic mice express mutant human αsyn under the neuron selective thy1 promoter. (thy1)-h[a30p]αsyn transgenic mice were crossed to cx3cr1-def transgenic mice which express egfp replacing fractalkine receptor gene expression. all mice were maintained on a c57bl/6j background for more than 10 generations and under specific pathogen-free conditions. to the extent possible, littermates were used in the experiments. health status was monitored daily during experiments. the in vivo experiments were endorsed by a roche internal review board and approved by the local animal welfare authorities of the canton basel-stadt, basel, switzerland. experimental colitis paradigms in mice paradigms for the induction of inflammation were either 1 week (acute) or 3-4 weeks (chronic) with or without an incubation phase under normal conditions of 2-, 6-, or 18-months post application (fig. 1). acute systemic inflammation was induced by intraperitoneal (i.p.) lipopolysaccharide (lps) application [43] of 0.5 mg/kg in 100 µl injection volume on day 0 and 3 (sigma-aldrich chemie gmbh, steinheim, germany, lps 055:b5). acute colitis was induced by application of 36-50kda dextran sulfate sodium (dss) [44] (160110, mp biomedicals, llc, illkirch, france) at 1%, 2.5% or 5% in autoclaved drinking water for 5 continuous days respectively, followed by 2 days of water (1 dss application cycle). chronic colitis was induced by two different dosing protocols: i) in a constant dose of dss (1% or 2.5%) for 5 days and changed to 2 days with normal drinking water and repeated three times (4 application cycles in total); ii) in an increasing dose of dss starting at 1% for 5 days followed for 4 days on normal drinking water, then increased to 1.5% dss for the next 5 days followed by 4 days of water and a final cycle of 2% dss followed by aging the mice on normal drinking water until they were sacrificed. mice from the same littermate group were randomized per cage into ‘exposed to inflammation inducing agent’ (lps or dss, respectively) or ‘unaffected’ (vehicle for the lps paradigm or normal drinking water for the dss paradigms, respectively). for the long-term experiments with the two aging cohorts ‘9 months’ and ‘21 months’, respectively, all mice in that study were simultaneously exposed in one large cohort at the age of about 3 months to the increasing dose chronic dss paradigm (fig. 1) and dss exposure was stopped for all mice on the same day after the 23-day period. the mice were then kept and aged on normal drinking water and under normal housing conditions in the same room until the day they were perfused and tissue was collected. il-10 treatment and exposure measurement two different forms of mouse igg bound murine il-10 (migg(v1)-mil10 and migg(v2)-mil10) were diluted in pre-prepared sterile formulation buffer comprised of 0.5% mouse serum supplemented with 25 mm citrate, 300 mm arginine to a final concentration of 0.75 mg/ml and the ph adjusted to 6.7 on the day of application. each mouse was treated once with 150 µg i.p. concurrently with the initiation of the acute colitis paradigm with 5% dss. the concentrations of migg-mil10 fusion proteins in murine serum samples were determined by enzyme-linked immunosorbent assays (elisa) specific for the fab moiety of the administered migg-mil10 fusion protein. biotinylated migg-mil10-specific target molecules were used for capturing, goat anti-mig igg-hrp conjugate and peroxidase substrate abts were used for quantitative detection of migg-mil10 fusion proteins. immunohistochemistry mice were injected with a lethal dose of pentobarbital (150 mg/kg). upon full anesthesia, mice received transcardial perfusion with room temperature phosphate buffered saline (pbs). for biochemical and immunohistochemical analysis, one section of the proximal colon was either fresh frozen and stored at -80°c or post-fixed in 4% paraformaldehyde (pfa) solution for 24 h. following post-fixation, organs were incubated in 30% sucrose/pbs at 4°c for at least 48 h before further processing. subsequently, enteric tissue was cryotome-sectioned to 35 µm thick longitudinal sections (approx. 1 cm length). the brain was collected and post-fixed for 24 h in 4% pfa followed by 30% sucrose in phosphate buffer until cryo-sectioning of floating sections at 40 μm. histological analysis of mouse colon was performed using standard hematoxylin staining. immunohistochemical staining was accomplished using the vectastain elite abc kits and peroxidase substrate kit sk-4100 (vector laboratories, burlingame, ca, usa) or fluorescently labelled secondary antibodies (alexa 488, 555 or 647, life technologies, zug, switzerland). the following primary antibodies have been used for overnight incubation at a dilution of 1:1000; monoclonal antibody to human αsyn (clone 211, sc-12767, santa cruz biotechnology, heidelberg, germany; specific to human αsyn and binds to normal αsyn as well as abnormal αsyn inclusions which contain the respective epitope), monoclonal antibody generated towards rat αsyn, cross-reactive with murine and human αsyn (syn1/clone 42, bd transduction laboratories, allschwil, switzerland; used for wild type mice), polyclonal antibody to the peripheral neuronal marker peripherin (millipore corporation, billerica, ma, usa), and polyclonal antibody to macrophage marker iba-1 (wako chemical gmbh, neuss, germany). to detect αsyn phosphorylated at serine 129 (pser129-positive inclusions of pathological/abnormal αsyn) in the free-floating brain sections, monoclonal antibody (ab51253, abcam, cambridge, usa) was used at a dilution of 1:10000. prior to the pser129 staining, the free-floating brain sections were incubated for 10 min at room temperature in a phosphate buffered saline solution containing 10 μg/ml proteinase k (cat # 25530015; invitrogen, california, usa). tyrosine hydroxylase (th)-immunoreactive cells were detected using a polyclonal antibody (657012, millipore sigma) at a dilution of 1:1000. to measure the density of nissl-positive cells, the th-stained cells were counter-stained with cresyl violet. the slides were incubated in 0.1% cresyl violet solution for 9 min and then dehydrated in 95% and 100% ethanol and then xylene prior to cover slipping with cytoseal 60 mounting media (thermo fisher scientific). quantifications of the blind-coded th/nissl-stained slides were done using stereo investigator (version 2017.01.1; mbf bioscience, williams, vt, usa) on an imager m2 microscope (zeiss) coupled to a computer. we analyzed 5-7 nigral sections per animal, and a total of 7-8 animals per treatment group. we outlined the substantia nigra pars compacta and counted every th-immunoreactive and nissl-positive cell in that area (using a counting frame of 40 µm x 40 µm, grid size of 140 µm x 140 µm, a guard zone of 2 µm and optical dissector height of 20 µm) and then computed the number of cells per section, generating the average cell count per animal. we then calculated the average count of cells per treatment group and analyzed the data using unpaired student’s t-test after confirming normality and homoscedasticity in prism 7.0 (graphpad software). imaging and stereological quantification of αsyn deposits in enteric nervous system imaging and stereological quantification were performed on a zeiss axio imager z2 fluorescence microscope (carl zeiss ag, jena, germany). leica tcs sp5 confocal system using an hcx pl apo cs 40x 1.3 oil uv or an hcx pl apo lb 63x 1.4 oil uv objective was utilized for image recording. accumulation of αsyn in the ens (i.e., punctate intracellular bodies/features) was assessed on a random set of 3 adjacent 35 µm thick, αsyn-immunostained sections comprising the myenteric and submucosal neuronal plexuses. analysis was performed with the aid of a stereology software (stereo investigator 10, mbf bioscience, williams, vt, usa) as described previously [45]. in the myenteric plexus ganglion, volume was defined by multiple outlined plexuses containing a range of 5-20 neuronal cells and quantified by the optical fraction fractionator technique. in contrast to the myenteric plexus, the submucosa consists of compact plexuses with 1-5 cells including interconnecting neurites. therefore, the entire submucosa was set as region of interest, analyzed with the area fraction fractionator technique. results of the submucosal plexus are displayed by percent area containing αsyn deposits. for the il-10 experiment, αsyn positive inclusions from immunofluorescence images were counted for each image. inclusion body-like features were filtered based on having a size between 12 and 50000 pixels and a minimal intensity value greater than 300. the filtering step was performed to exclude small background features and macrophages (very large spots). the counts were then aggregated to the animal level by summing the inclusion feature counts of all images per animal and then normalizing for (i.e., dividing by) the number of images for a given animal. upon exploratory data analysis two mice were excluded: one mouse because it only had one image (technical outlier, missing data point; repeating the staining for this one mouse would have required re-staining the entire cohort in order to be consistent with staining conditions for quantification; this was unnecessary after statistical analysis) and another due to it being an outlier, based on its infiltration score and image data (i.e., in contrast to the other mice that had received dss, this mouse did not show signs of inflammation or colonic tissue damage that is normally induced by dss; it could not be determined if that mouse was correctly dosed with dss and thus it was excluded from the analysis). quantification of leukocytes infiltration to determine the leukocyte covered area in the colon after lps or dss application, three adjacent hematoxylin-stained sections were quantified. total area of colon sections and localizations of leukocyte assemblies within the tissue architecture were identified and outlined utilizing a stereology software (stereo investigator 6, mbf bioscience, williams, vt, usa). percentage of leukocyte covered area has been set in proportion to total area of the analyzed colon section, e.g., to at least the length of 1 cm of proximal colon. for the il-10 experiment, hematoxylin-stained colon slices were examined by an expert pathologist blinded to treatment conditions. a score of 0-3 was assigned to each section for each of the 3 layers lamina propria, submucosa and muscularis based on the degree of inflammatory infiltration. a score of 0 denoted no inflammation and a score of 3 indicated extensive infiltration. the mean of the values for all 3 layers was taken as the final measure of leukocyte infiltration per mouse. quantification of iba-1/αsyn-double positive macrophages the number of iba-1/αsyn-double positive cells was evaluated by quantification of 10 random regions in 2 adjacent sections of the proximal colon. the region of interest was set to contain the myenteric plexus/circular muscle layer and the submucosal plexus. scoring of pser129 pathology and brain heatmap we evaluated pser129 pathology on a full series of immunostained coronal sections from 10 mice per treatment group (i.e., water vs. dss-treated groups) on blind-coded slides using a previously described method [46]. we visualized pathology from one hemisphere of all brain sections (apart from the olfactory area) using a nikon eclipse ni-u microscope and assigned scores ranging from 0 to 4 to each brain area based on the relative abundance of proteinase k (pk)-resistant pser129-positive inclusions (i.e., cell bodies and neurites). in this case, 0 = no aggregates, 1 = sparse, 2 = mild, 3= dense, 4 = very dense. for the heatmap, we obtained the average score values of each brain area for each treatment group. the average data for each treatment group (n=10 mice/group) was then represented as a heatmap in a sagittal mouse brain background. to create the brain heatmap a postscript file downloaded freely from allen brain atlas (mouse, p56, sagittal, image 15 of 21; -> weblink*) was converted to an xml in r v 3.4.4, and the mean scores were manually assigned to respective brain regions. the remaining brain regions were estimated via the r package ‘akima’, using a pointwise bivariate interpolation algorithm for irregular data on the mean x and y coordinates for each brain region.   *(http://atlas.brain-map.org/atlas?atlas=2#atlas=2&structure=771&resolution=16.75&x=%7755.7470703125&y=3899.625&zoom=-3&plate=100883867&z=5) densitometry of pser129 αsyn brain pathology the density of pser129 pathology in 12 major brain areas (reticular nucleus, pontine reticular nucleus, periaqueductal gray, gray and white layer, reticular formation, substantia nigra, ventral tegmental area, thalamus, hypothalamus, central amygdala, pallidum and striatum) was determined in the water and dss-treated animals. a nikon eclipse ni-u microscope was used to acquire 20x magnification images (without condenser lens) from all the indicated brain areas, using the same exposure time for all images. in all cases, images were acquired on three sections separated by 420 μm intervals (localized between bregma). we then processed the acquired images using image j64 [47], created a mask (to exclude background) that redirects to the original image for analysis, measured the total area and the mean grey value of the area that had inclusions. for brain areas such as periaqueductal gray that do not fill the entirety of the field to be analyzed, we drew a contour of the area and the analysis was performed only within that contoured area. we subsequently calculated the grey value of the area per square pixels for each image (i.e., a.u./px2 = mean grey value x area stained/total area assessed). based on this, we calculated the average grey value per square pixels for each brain area for each animal (n = 6 mice/group), and then extended this calculation to determine the average grey value per square pixels for each treatment group and each of the twelve brain areas of interest. blinding of experimenters for histological and immunohistochemical analyses for analyses of colon and brain tissue on slides, a second individual assigned unique codes to stained slides. therefore, the experimenter conducted the analyses blinded to the identity of the mice. for randomization of treatment groups see above. mrna expression to assess mrna expression levels from the proximal colon, rna was extracted from fresh frozen tissue with magnalyser green beads (roche diagnostics, mannheim, germany) and qiazol lysis (reagent cat.no.79306, hilden, germany) purified on magnapure lc (hp kit no.03542394001, f. hoffmann-la roche ag, rotkreuz, switzerland) and amplified via real-time pcr (4 ng rna/reaction; lightcycler 480, roche diagnostics corporation, indianapolis, usa). amplification of mrna was performed by using taqman probes for human or murine specific α-synuclein and for selected cytokines/chemokines (applied biosystems europe b.v., zug, switzerland). target mrna was normalized to tissue specific murine gapdh levels and displayed as relative expression after 30 amplification cycles. statistics measurements for inflammation and αsyn accumulation in the ens were taken from distinct samples (e.g., in three to six technical replicates per mouse). data from each mouse was used only once, thus no repeated measure of the same sample was performed. statistical analysis of gut pathology and inflammation was performed using graphpad prism 6.04 or 7.0 software (graphpad software, inc. la jolla, ca, usa). the results are expressed as mean values ± standard errors of the mean (s.e.m.). student’s t-test (or welch’s t-test for unequal variances) was used to compare two groups and anova was used for multi-comparison of groups followed by tukey hsd post-hoc analysis. for the statistical analysis of the mrna expression, data quality was assessed by inspecting the distribution of cp values of reference endogenous genes across samples, by inspecting the level of cp variation between technical replicates and by exploring the samples multivariate signal distribution as in a principal component analysis. relative gene expression levels were expressed as 2-(cpgene – cpref). statistical analyses to assess the effect of the experimental conditions on the log2 gene expression levels were done with linear models using the limma package (bioconductor/r, [48]). these analyses were implemented in r v 3.1.1. for the statistical modelling of the effects of the il-10 treatment on αsyn counts, as well as infiltration scores, the levels for igg1(v1)-il10 and igg1(v2)-il10 treatment were compared to the positive (vehicle/dss) control. additionally, since levels of the control antibody treatment (igg1(v1)) were very similar to the positive control, the two groups were pooled in further contrasts in which effects of individual antibodies or control igg was assessed. for αsyn counts in the enteric nervous system, a linear model on the treatment groups with one-degree freedom contrasts was applied. for the infiltration score a kruskal-wallis test, with the same contrasts, was used. all statistical tests were two-tailed with a significance level of p <0.05. for the statistical analysis of the pser129 αsyn brain pathology, zero-inflated negative-binomial mixed-effects models with a random intercept for each sample and variance assumed to increase linearly with the mean (verified against a quadratic increase using akaike information criterion [aic] and bayesian information criterion [bic]) were used to analyze the dataset via the ‘glmmtmb’ package in r v 3.4.4. linear contrasts with false discovery rate (fdr) adjustments were then used to test our hypotheses and account for multiple testing (for brain area and experimental group). results experimental colitis exacerbates αsyn load in the submucosal plexus of αsyn transgenic and wild type mice during the process of further characterizing a (thy1)-h[a30p]αsyn transgenic mouse line [41], we detected by immunohistochemistry human αsyn accumulation in all innervated organs that were analyzed (suppl. fig. 1). to examine the localization of αsyn inclusions in nervous structures in the ens, we performed an immunofluorescent co-staining for human αsyn (clone 211) and peripherin, a specific marker for peripheral nerves. by applying confocal microscopy, we established a process and protocol to identify myenteric and submucosal plexuses in order to quantify the αsyn inclusions found in the nerve cells of the ganglia in the colon. the intracellular presence of the irregularly sized and shaped αsyn inclusion bodies was confirmed in 2d z-stacks of rotated confocal images (fig. 1a). we observed an age-dependent (mice aged 3 months versus 12 months) increase of baseline human αsyn inclusions in both plexuses (fig. 1b). given the clinical and epidemiological link between ibd and pd, we wanted to test whether different types and strengths of ibd-related experimental inflammation in the colon exacerbates this local accumulation of αsyn acutely (e.g., within a few days or weeks) and how the age of the αsyn transgenic mice may influence the outcome. administration of dextran sulfate sodium (dss) in the drinking water in acute or chronic dosing paradigms are well-established mouse models of experimental colitis mimicking aspects of ibd, i.e., by exhibiting infiltration of leukocytes into the submucosa with various degrees of destruction of the colonic mucosa and submucosa [49]. it is well-known that the effects induced by the dss paradigm can vary substantially based on the genetic background of the mice and due to different animal housing environments. thus, in order to establish the dss paradigm in our environment and with our mice, we first tested the effect of dss administration at different doses and durations in the (thy1)-h[a30p]αsyn transgenic mice (fig. 1c). we observed that leukocyte infiltration was appropriately modulated by the acute dose (1% or 2.5% dss for 5 days followed by 2 days drinking water) and chronic constant dose (1% or 2.5% dss alternating with normal drinking water for 28 days, respectively) dss paradigms (fig. 1d and 2a). in the same experiment, we wanted to test for an age effect on a potential aggravation of αsyn accumulation in the ens and applied the acute dose paradigm on 3-month-old mice and the chronic dose paradigm was started at the age of 5 months leading to a final age of 6 months at analysis of the mice. in the acute dss dosing paradigm with mice at the age of 3 months, 2.5%, but not 1%, dss triggered intracellular accumulation of αsyn in peripherin positive nerve cells of the submucosal plexus (fig. 2a, b). in the 28-day chronic constant dss dose paradigm in mice aged 6 months (fig. 1c and 2a), we observed that the 1% constant chronic dss dose showed a similar degree of αsyn inclusions as the 3-month-old mice which were on an acute 2.5% dss dose paradigm. in addition, mice previously exposed to 2.5% constant chronic dss dose presented on average with a slightly but robust increased percent area of αsyn inclusions compared with the 6-month-old mice that were on 1% dss (fig. 2a). together, this demonstrated that different dss paradigms can be established in (thy1)-h[a30p]αsyn transgenic mice and that different dss paradigms can induce robust elevation of αsyn inclusions. this first experiment also provided us with data points to estimate a potential effect size for triggering αsyn inclusions in the different dss paradigms. we observed that the younger (thy1)-h[a30p]αsyn transgenic mice showed a robust increase in αsyn inclusions and better signal-to-noise conditions of the autofluorescent colonic tissue than the older ones, experiments were henceforth continued with mice of the younger age. in this initial experiment, it was also interesting to observe that (thy1)-h[a30p]αsyn transgenic mice exposed to acute 2.5% dss colitis presented with several αsyn-positive cells with a morphology consistent with them being infiltrating leucocytes, which was confirmed by an iba-1 co-staining (suppl. fig. 2). this finding was relevant for the quantification of αsyn inclusions in the myenteric and submucosal plexus, i.e., such features were excluded from the quantification process. fig. 2 experimental dss colitis severity and duration-dependent aggravation of accumulation of αsyn inclusions in the colonic submucosal plexus of hemizygous (thy1)-h[a30p]αsyn transgenic and wild type mice. a administration of dss in drinking water induced a robust increase of leukocyte infiltration in the acute (1% or 2.5% dss for 5 days followed by 2 days of normal drinking water; one group was kept on normal drinking water) and chronic constant dss dose (1% or 2.5% alternating with normal drinking water) paradigm in hemizygous (thy1)-h[a30p]αsyn transgenic mice. the highest acute dose (2.5%) and the two constant chronic doses led to a very robust increase of αsyn inclusions in the submucosal plexus (stereological quantification of αsyn inclusions in the submucosal plexus of all 3and 6-month-old hemizygous (thy1)-h[a30p]αsyn transgenic mice; n = 5-7 per group; mean and s.e.m. are shown). b representative 2d z-stacks of confocal images of increasing abundance of αsyn inclusions (red, human-αsyn specific monoclonal antibody clone 211) in a ganglion of the submucosal plexus (green, peripherin) with cellular nuclei in blue (dapi) in the acute dss paradigm. arrow heads point to the typical irregularly sized and shaped αsyn inclusion bodies that accumulate in the highest dss dose. scale bar: 200 µm. c overview of colonic region of 3-month-old wild type mice exposed to water or acute dss (5%) with immunofluorescence analysis of murine αsyn load in the colon performed immediately after colitis. white dotted rectangles in the top row indicate the area that was zoomed-in in the lower panels. in the zoom-ins we show representative images of dapi and αsyn (red, rodent αsyn cross-reactive monoclonal antibody syn1/clone 42) inclusions with and without the peripherin channel (green). the white dotted circled area illustrates the peripherin-positive area that was analyzed for αsyn inclusion bodies (arrow heads in bottom row). scale bar for the lower three panels: 200 μm. d stereological quantification of murine αsyn inclusions in the submucosal plexus of wild type mice right after acute dss colitis (n = 5 per group). note the regularly arranged and smoothly distributed immunoreactivity for the physiological αsyn with barely any inclusion bodies in the intact enteric nerves of the water group. for both panels (a) and (d), statistical analysis for αsyn accumulation was omitted as the noticeable and very robust differences between the means are self-evident (error bars indicate standard error of the mean) and an indication for an estimation for significance would be irrelevant. wild type mice also express endogenous αsyn in innervated organs, but at much lower levels compared with the levels of human αsyn expressed by the hemizygous (thy1)-h[a30p]αsyn transgenic mice (suppl. fig. 1). to confirm that the finding in (thy1)-h[a30p]αsyn transgenic mice was independent of transgenic expression of human αsyn, we applied an acute 5% dss dose paradigm (5 days dss + 2 days water) in wild type mice (fig. 1c). we observed, in the submucosal plexus, small inclusion bodies of endogenous murine αsyn (detected by rodent cross-reactive αsyn-specific monoclonal antibody syn1/clone 42, fig. 2c, d). these features were close to undetectable in the water group that did not experience experimental colitis. a separate experiment also confirmed that the observed effects of elevated αsyn inclusions following acute dss (5% dose) could not be attributed to increased gene expression of murine or the transgenic human αsyn (suppl. fig. 3). together, these results confirmed the validity of this experimental ibd paradigm to test the effect of inflammation on αsyn accumulation in the ens in wild type and (thy1)-h[a30p]αsyn transgenic mice. because the (thy1)-h[a30p]αsyn transgenic mouse model is well-established to analyze human αsyn-related pathology, we focused for the remainder of the study on employing these transgenic mice. colitis induced by peroral dss but not by intraperitoneal administration of lps aggravates αsyn accumulation in colonic submucosal plexus of αsyn transgenic mice different inflammatory agents induce different types of immune stimulation and thus can influence the phenotype of experimental colitis. a well-established experimental immune trigger is the bacterial endotoxin lps. in order to explore the effects of different approaches to induce inflammation in or nearby the gut in (thy1)-h[a30p]αsyn transgenic mice, we compared the outcome of acute (5 days dss + 2 days normal drinking water) 5% dss per os with acute 0.5 mg/kg intraperitoneal lps administration (fig. 1c and 3). to maximize the inflammatory response, we administered both dss and lps at relatively high doses. at day 7, both agents had induced variable degrees of leukocyte infiltration in the submucosa of the colon while a marked destruction of the mucosa was induced when giving only dss (fig. 1d). as before, the dss-exposed mice presented with increased accumulation of αsyn in the ganglia of the submucosal plexus (fig. 3a). in contrast, we detected no change in αsyn load in the myenteric plexus, consistent with lack of leukocyte infiltration in this part of the colonic wall (fig. 3b). despite the high dose, lps-induced inflammation did not increase αsyn accumulation in the colonic nervous plexuses (fig. 3c, d). notably, lps and dss resulted in a differential expression of cytokines, and consistent with leukocyte recruitment, ccl2 was elevated in both (fig. 3f, g). in the lps paradigm, mrna for il-10 was markedly elevated, whereas dss strongly increased il-6 and also il-1β but not il-10. together these results indicate that, in our model, colonic inflammation induced by peroral dss but not intraperitoneal lps increases the accumulation of αsyn in the colon. fig. 3 colitis induced by peroral dss but not peritoneal lps enhances αsyn accumulation in the colonic submucosal plexus of hemizygous (thy1)-h[a30p]αsyn transgenic mice and can be increased by lack of monocyte/macrophage-related cx3cr1 signaling. mice received in an acute paradigm either peroral 5% dss in their drinking water or intraperitoneally 0.5 mg/kg lps. effects of dss and lps in the colon, respectively, were compared to effects induced by vehicle (see figure 1c for timelines). stereological quantification of αsyn inclusions in the submucosal plexus as % area (a, c) and in the mucosal plexus as particle load per ganglion (b, d) (two-way anova with tukey post hoc test; covariates genotype and treatment paradigm). e representative 2d stacks of confocal images of intracellular αsyn inclusions (red, human αsyn specific monoclonal antibody clone 211; arrow heads pointing to some selected inclusions) in a ganglion of the myenteric plexus (green, peripherin) with cellular nuclei in blue (dapi). scale bar: 50 μm. gene expression analysis of selected cytokines in the colon of (thy1)-h[a30p]αsyn transgenic mice that received either acute dss (f) or lps (g) compared to their respective vehicle or water controls. note the strong increase in il-6 and the lack of elevation of il-10 in the dss paradigm compared to the lps paradigm indicating a different inflammatory colonic milieu despite the abundant leukocyte infiltration in both paradigms. n = 5-8 per group; mean and s.e.m.; student’s t-test between inflammatory agent and vehicle for individual cytokines. lack of monocyte/macrophage related cx3cr1 signaling during dss colitis increases αsyn load in the submucosal plexus of αsyn transgenic mice given the role of monocytes/macrophages in ibd and in the related dss paradigm, we hypothesized further that modulating monocytes/macrophages may affect accumulation of αsyn in our dss model as well. in a first set of experiments we manipulated monocytes/macrophages genetically by crossing (thy1)-h[a30p]αsyn transgenic mice with mice that have a deletion for the fractalkine receptor cx3cr1 (cx3cr1-gfp knock-in mice) (fig. 3a, b). the cx3cr1-cx3cl1 axis plays an important role in maintaining the function of the lamina propria macrophage population of the gastrointestinal wall and lack of this signaling pathway in experimental colitis models may either aggravate or ameliorate the induced pathology [50–52]. in our experiment, the area covered by infiltrating leukocytes following exposure to dss was near the mucosa and submucosa and was not significantly higher in the cx3cr1-deficient αsyn transgenic mice than in the cx3cr1-competent mice (suppl. fig. 3a). however, a significantly higher level of αsyn accumulated in the submucosal plexus in αsyn transgenic mice lacking cx3cr1 compared to αsyn transgenic mice expressing cx3cr1 (p = 0.001, two-way anova with tukey hsd post-hoc analysis; fig. 3a). in the myenteric plexus, we found no marked increase in αsyn accumulation in neither the αsyn transgenic mice with normal cx3cr1 nor the αsyn transgenic mice deficient in cx3cr1, indicating as in the experiments above a possible prominent role for the localization of leukocyte infiltration in the process of αsyn accumulation in the submucosa (fig. 3b). collectively, our results in cx3cr1-deficient αsyn transgenic mice provide a potential association between monocyte/macrophage signaling and αsyn accumulation in ens in this experimental ibd model. systemic il-10 reduces dss-induced colitis and associated enteric αsyn accumulation in αsyn transgenic mice to continue testing the hypothesis that modulating monocytes/macrophages may affect accumulation of αsyn in our dss model we moved to a pharmacological modulation of this cellular subset. interleukin-10 (il-10) is an important regulator of monocytes/macrophages, and genetic ablation of il-10 signaling or blocking il-10 with specific antibodies has been reported to enhance dss colitis [53,54]. in the experiments with lps we had also noted an increase of il-10 compared with the dss paradigm and lps inflammation was in contrast to dss colitis not associated with increased αsyn accumulation in the ens (fig. 3). to mimic the effect of higher levels of il-10 in an acute model of dss colitis (5% dss for 5 days + 2 days normal drinking water, fig. 1c), we administered intraperitoneally recombinant murine il-10 (mil10) in this paradigm. the half-life of injected recombinant il-10 protein in blood is very short. to reduce the number of injections, we extended the half-life of mil-10 in circulation by engineering it onto two different murine igg variants (i.e., migg1(v1)-mil10 and migg1(v2)-mil10, respectively). as described above, dss induced a marked increase in leukocyte infiltration and αsyn accumulation, and we found both to be similar in the untreated and control igg treated group (fig. 4a, b). in contrast, both migg1(v1)-mil10 and migg1(v2)-mil10 significantly reduced leukocyte infiltration in mice treated with dss (p<0.0001, one-way anova with tukey hsd post-hoc analysis; fig. 4a, b). a significant down-regulatory effect of an il-10 treatment on dss colitis induced accumulation of human αsyn in the submucosal plexus was only observed with migg1(v2)-mil10 (p=0.02, one-way anova with tukey hsd post-hoc analysis; fig. 4b). this effect by migg1(v2)-mil10 on αsyn levels was accompanied by detectable serum levels of migg1(v2)-mil10 at the end of the in vivo phase, whereas migg1(v1)-mil10 was no longer detectable at that point (fig. 4c). this indicates that although both forms of il-10 have a down-regulatory effect on leukocyte infiltration, a sustained pharmacological exposure of il-10 may be required for reducing αsyn accumulation. these results highlight an important role for the il-10 pathway in keeping αsyn accumulation at a reduced level throughout the course of experimental ibd. together, our observations by genetic (i.e., cx3cr1-cx3cl1 axis) and pharmacological modulation (i.e., il-10) of dss colitis corroborate an important role for monocyte/macrophage pathways in the development of αsyn accumulation in the ens of the colon. fig. 4 systemic il-10 ameliorates dss colitis and slightly reduces associated local αsyn accumulation in (thy1)-h[a30p]αsyn transgenic mice. two different recombinantly engineered and murine igg1-fused forms of murine il-10 (migg1(v1)-mil10 and migg1(v2)-mil10) were administered (150 µg per mouse i.p.) at the beginning of the acute dss paradigm (5%) in (thy1)-h[a30p]αsyn transgenic mice. vehicle and the migg1(v1) alone served as untreated controls. a leukocyte infiltration was assessed by visual scoring and (b) inclusion features of αsyn were stereologically and semi-automatically quantified and result log scaled for statistical analysis. both the vehicle group and the migg1(v1) group had similar levels of leukocyte infiltration and αsyn inclusions and were merged for the statistical analysis to compare with the il-10 treated groups. both forms of il-10 ameliorated leukocyte infiltration whereas migg1(v2)-mil10 also blocked the appearance of αsyn inclusions significantly (n = 3-6 per group; mean and s.e.m.; one-way anova and tukey post hoc test). c persistent exposure migg1(v2)-mil10 versus migg1(v1)-mil10 (lower limit of detection is indicated at <0.234 μg/ml) as measured in serum at the end of the in vivo phase corresponds with beneficial treatment effects on αsyn readout observed above. the migg1(v1) was only measured in two mice. dss colitis-induced submucosal αsyn accumulation at a young age persists for months and is exacerbated by lack of cx3cr1 signaling in humans there is strong epidemiological evidence that ibd increases pd risk [33,35,37] and recent evidence in crohn’s disease [55] indicates that such gut inflammatory conditions are associated with αsyn accumulation in the ens [36]. in these experiments in mice, we have until here established and replicated, in different setups, a link between modulation of inflammation and induction of αsyn accumulation in the ens. because longer exposure to dss (i.e., over several weeks) mimics more closely the chronic nature of ibd, we elected to explore αsyn accumulation in the submucosal plexus of (thy1)-h[a30p]αsyn transgenic mice that were subjected to dss colitis in a 4-week chronic increasing dose paradigm. in addition, the dose increase with longer water intervals is more gentle for the mice from an animal welfare perspective. in order to allow for a full recovery from the chronic inflammation, we aged the mice for two months on normal drinking water and analyzed them at the age of 6 months (fig. 1c). at this point we wanted again to explore the effect of modulating monocytes/macrophages in this chronic setting and added an experimental arm with (thy1)-h[a30p]αsyn transgenic mice lacking cx3cr1. as expected, after 2 months of recovery, the area that is usually extensively covered by leukocytes in the submucosal plexus of the acute dss paradigm had returned to normal levels following the two-month recovery period (suppl. fig. 4a). remarkably, however, the area containing αsyn inclusions in the ganglia of the submucosal plexus was still almost doubled when compared to αsyn transgenic mice that were not exposed to dss, and this was exacerbated in αsyn transgenic mice deficient for cx3cr1 (suppl. fig. 4b). the finding in the αsyn transgenic mice suggests that accumulation of αsyn is not a transient effect or response. in addition, modulation of monocytes/macrophages by down-regulating the cx3cr1-cx3cl1 axis contributes to aggravation of this accumulation. experimental dss colitis-induced at a young age exacerbates αsyn brain pathology and dopaminergic neuron loss in old αsyn transgenic mice at this point, we have established and repeatedly demonstrated that modulation of inflammatory mechanisms in experimental colitis induced by acute and chronic dss administration is causatively linked to induction and persistence of intracellular αsyn inclusions in the ens of young adult mice. the previously highlighted hypothesis by braak and colleagues associates αsyn brain pathology in pd with αsyn pathology in the ens earlier in life [3,56]. to assess development of brain αsyn pathology and to link it to ibd risk, we exposed 3-month-old hemizygous (thy1)-h[a30p]αsyn transgenic mice to a chronic increasing dose dss paradigm or normal drinking water and after 23 days returned all mice to normal drinking water until sacrifice several months later (fig. 1c). we chose to use the αsyn transgenic model rather than wild type mice for this study because of two reasons: 1) we knew that the model as hemizygous transgenic mice exhibit some αsyn brain pathology that develops slowly under baseline conditions. importantly, the pathology is much less pronounced than in homozygous (thy1)-h[a30p]αsyn mice [57]; 2) at the time of the experiment, it was not clear whether wild type mice could develop αsyn brain pathology upon dss colitis. thus, we chose hemizygous (thy1)-h[a30p]αsyn transgenic mice to increase the chances for a successful outcome and potentially to aggravate the brain pathology from mild to strong. after exposing the mice either to normal drinking water or a chronic increasing dose dss paradigm, we aged them in two cohorts on normal water and housing conditions to either up to the age of 9 months or 21 months. at these two timepoints we analyzed various brain regions for αsyn inclusions that are generally considered pathological by being proteinase k (pk)-resistant and immunopositive for pser129-αsyn. when we examined the 9-month-old αsyn transgenic mice, we found that both experimental groups (i.e., those who were on dss and those who stayed on normal water throughout their entire life and thus never experienced dss colitis) exhibited extremely low levels of pathological αsyn aggregation in the brain (fig. 5 and suppl. fig. 5). our observation of the level of pathological αsyn aggregations in the brain of these 9-month-old hemizygous (thy1)-h[a30p]αsyn transgenic mice (fig. 5a) is indeed consistent with earlier descriptions of the model at the age of 11 months [57]. the 21-month-old hemizygous (thy1)-h[a30p]αsyn transgenic mice that only received water during their lifetime showed, in contrast to the 9-month-old cohort, more discernible pk-resistant pser129-αsyn immunoreactive features (fig. 5b) and the abundance of these features was consistent with previous observations in this transgenic line at the age of 24 months [57]. in marked contrast, the 21-month-old hemizygous (thy1)-h[a30p]αsyn transgenic mice that were exposed to dss at three months of age presented with pser129-positive αsyn pathology throughout various brain regions in a much more exacerbated fashion than mice that were aged up to 21 months without having experienced dss colitis at young age (fig. 5b-e). the degree and distribution of pk-resistant αsyn in the brain was similar to what was previously described for homozygous (thy1)-h[a30p]αsyn transgenic mice at the age of 8 to 9 months [57]. the significant aggravation of αsyn pathology in the substantia nigra (p ≤ 0.01 in a negative-binomial mixed-effects model adjusting for multiple comparisons performed over all brain areas) was accompanied by a significant loss of tyrosine hydroxylase (th) and nissl-positive cells at 21 months of age (p ≤ 0.05, student’s t-test; fig. 6). together, we found that experimental dss colitis at a young age caused an age-dependent exacerbation of pk-resistant pser129-αsyn pathology and a loss of nigral dopaminergic neurons in the brains of (thy1)-h[a30p]αsyn transgenic mice. fig. 5 a 23-days chronic dss colitis insult at young age causes an age-dependent accumulation of proteinase k-resistant pser129-αsyn in various brain regions of (thy1)-h[a30p]αsyn transgenic mice. a 23-days chronic increasing dose dss paradigm was performed with 3-month-old (thy1)-h[a30p]αsyn transgenic mice. after recovering and further aging, various brain regions were analyzed for proteinase k (pk)-resistant pser129-αsyn immunoreactivity in 9-month (a) and 21-month-old (b) mice, respectively. the dark brown features in (a) (barely any visible in both the water and the dss group) and (b) (strongly visible with typical neuritic and punctated inclusion-type morphology) indicate pk-resistant inclusions with pser129-αsyn immunoreactivity. densitometric quantification of pser129-αsyn immunoreactivity in different brain regions of 21-month-old mice (c, d) (n=6 mice per group). in order to visualize better the differences between the water versus the dss group at 21 months, brain regions with large increase were plotted on an y-axis up to 250 a.u./px2 and small increase on a y-axis up to 40 a.u./px2. the about <5 to 150 a.u./px2 average values in the dss group versus the about 0 to 30 a.u./px2 average values in the water group in several brain regions confirm the visual impression in panel (b). one 21-month-old mouse in the dss group was excluded from analysis due to presumed failed treatment; it is included in the graphs. statistical analyses were performed using negative-binomial mixed-effects models adjusting for multiple comparisons. e representative heatmap of the average distribution scores of pser129-αsyn immunoreactivity for each treatment group in varying brain regions in all the 9-month (a and supplemental figure 5) and 21-month-old (b) mice was generated in a sagittal mouse brain (n=10 mice per group). statistical analyses were performed using linear mixed-effects model adjusting for multiple comparisons. a.u./px2, = mean grey value x area stained/total area assessed. scale bars: 500 μm. discussion currently, there is no therapy for pd available to slow or stop disease progression and an obstacle in the quest to develop one is that we do not understand how the disease develops [58]. abnormal intraneuronal accumulation of αsyn (i.e., in lewy bodies and neurites) is a key neuropathological hallmark and the distribution of lewy pathology in postmortem brain is used for staging in pd [2,59]. accumulation of αsyn has also been observed in the peripheral nervous system in pd, some individuals at risk of developing the disease, and normal individuals [60–62]. similar to this finding in humans, αsyn-immunoreactive inclusions and signs of age-dependent αsyn-related pathological changes have also been detected in the ens of wild type rats [63,64] and several transgenic mouse models prior to development of brain pathology [21,65]. based on preclinical models employing injection of brain extracts or re-combinant αsyn fibrils to different brain regions and intestines [15–17,19,20,46,66,67] together with postmortem brain pathology [56,59,68], it has also been suggested that αsyn pathology propagates temporospatially from cell-to-cell in a prion-like manner [3,59,66,68,69]. however, the initial factors triggering αsyn aggregation in the tissue or organ of origin of the pathology are yet to be established [58] and the involvement of peripheral stimuli in the aggregation and pathogenic spread of αsyn is only beginning to unravel. in this study, we provide evidence that dss colitis, i.e., an experimental ibd-like inflammation, triggers αsyn accumulation in the ens of wild type mice and in a human αsyn transgenic mouse model of pd (fig. 2). we found aggravation of enteric αsyn accumulation in αsyn transgenic mice lacking cx3cr1 signaling and amelioration of inflammation and associated slight reduction of enteric αsyn load by systemic il-10, demonstrating that genetic and pharmacologic modulation of inflammation can influence the degree of αsyn accumulation in the ens (fig. 3 and 4). because il-10 and the cx3cr1-cx3cl1 axis are able to mediate this effect, this suggests that monocytes/macrophages may modulate the process in this model. we further observed that the aggravated αsyn accumulation in the ens persisted even after two months of recovery from dss colitis and was aggravated in the absence of cx3cr1 signaling. this indicates that the accumulation is persistent and this further establishes that monocytes/macrophages play a critical role in this process (suppl. fig. 4). remarkably, at 18 months but not 6 months post induction of dss colitis (thus, at age 21 months but not 9 months, respectively), αsyn transgenic mice had developed massively elevated αsyn brain pathology (fig. 5 and suppl. fig. 5). this elevated pk-resistant pser129-αsyn pathology in the midbrain, including the substantia nigra, and other brain regions coincided with an average decrease of 30-50% of thand nissl-positive cells in the nigra (fig. 6). we chose to perform the long-term experiments in αsyn transgenic mice rather than wild type mice while being aware of the caveats of employing genetic overexpression models, which use a neuron selective thy-1 promoter cassette such as these (thy1)-h[a30p]αsyn transgenic mice. however, these particular αsyn transgenic mice had previously been shown to slowly develop αsyn pathology in the brain on a homozygous genotype [41,57] making them ideal when asking the question of whether transient colonic inflammation can aggravate brain pathology in a genetically predisposed animal such as the hemizygous transgenic mice used in this study. others have recently demonstrated in a more aggressive αsyn transgenic mouse model that mild dss colitis can accelerate αsyn accumulation in the ens and brain [38]. in future long-term studies, we plan to address whether αsyn pathology develops also in the brains of wild type mice if they sustain transient experimental ibd at a young age. in our present study, experimental dss colitis in αsyn transgenic mice recapitulated the accumulation of enteric αsyn which is proposed to occur in humans several years before pd diagnosis [39]. additionally, the subsequent age-related development of αsyn pathology in the brain of αsyn transgenic mice together with the loss of nigral dopaminergic neurons mimicked a progression of the disease similar to what is considered to occur in pd. fig. 6 a 23-days chronic dss colitis insult at young age results in loss of tyrosine hydroxylaseand nissl-positive cells in the substantia nigra of (thy1)-h[a30p]αsyn transgenic mice at 21 months of age. (thy1)-h[a30p]αsyn transgenic mice were exposed to a 23-days chronic increasing dose dss paradigm at the age of 3 months followed by aging on normal drinking water up to the age to 21 months. these mice showed a significant loss of mean count of nissl-positive cells with tyrosine hydroxylase (th) immunoreactivity and cellular nissl staining in the substantia nigra compared to age-matched littermate mice in the group that did not experience dss colitis (water). a representative images of two levels of the substantia nigra in one mouse per group. b stereological quantification of cells positive for th or nissl (n=7-8 mice per group). statistical analyses of the th dataset were performed using student’s t-test, while welch’s t-test was used for the nissl dataset to adjust for unequal variances. scale bar: 500 μm. we established that a mechanism by which a specific type of peripheral inflammation promotes αsyn accumulation in the colon potentially involves monocytes and macrophages. both peroral dss and intraperitoneal lps administration provoked strong local immune reactions resulting in leukocyte infiltration into the submucosa of the colon. the inflamed region of the colon contains the submucosal plexus and is anatomically separated from the myenteric plexus by a thick circular muscle (fig. 1). this discrete localization of inflammation to the submucosa might explain why αsyn only accumulated in the nerves of the submucosal plexus and not in the myenteric plexus of our mice that received dss in both a strong acute and the two chronic paradigms. the mechanism underlying how intraperitoneally administered lps leads to submucosal leukocyte infiltration likely involves the monocyte attractant chemokine ccl2 (fig. 3), but the specifics remain to be clarified [70]. indeed, ccl2 was also upregulated in the colon of our dss model. however, in contrast to intraperitoneal lps, where infiltrating macrophages were present in discrete patches in the colonic wall, dss-related macrophage infiltration was distributed both in small groups and larger randomly distributed patches of cells across the entire colonic submucosa. also, perorally administered dss destroys the mucosa of the colon, similar to some forms of ulcerative colitis, resulting in the transient disintegration of the intestinal epithelial barrier. in our (thy1)-h[a30p]αsyn transgenic mice, the subsequent immune response to the infiltration of commensal bacteria evoked an elevated expression of cytokines such as il-1β and il-6, a phenomenon also observed in the colon of ibd patients [71,72]. this upregulation was absent in the lps paradigm in which the intestinal mucosa remained intact. by acting on tight junctions, il-1β and il-6 can increase intestinal barrier permeability (gut leakiness), facilitating the recruitment of additional immune cells to the site of the inflammation, eventually culminating in widespread immune activation [73,74]. consistent with the breach of barrier permeability in our mouse model, some pd patients exhibit increased colonic cytokines such as il-1β, il-6 and tnf, occurring together with increased intestinal permeability [23,75]. in this context, it is also notable that people with crohn’s disease present with increased enteric αsyn expression [55] and even more striking that ibd patients on anti-tnf therapy have a reduced risk of developing pd compared to ibd patients not given this treatment [35]. notably, mucosal macrophages with intra-lysosomal αsyn content were previously described in the intact human appendix [76]. these macrophages were in close proximity to the axonal varicosities of the vermiform appendix, which showed an enriched staining for αsyn in the mucosal plexus. furthermore, we recently found that the vermiform appendix contains aggregated and truncated αsyn that has the propensity to seed aggregation of recombinant αsyn in vitro [62]. our finding of lps not being able to induce an αsyn phenotype in the colon is in contrast to the reports by kelly and colleagues who injected wild type mice with lps and observed an immediate and progressive increase in αsyn immunoreactivity in the myenteric ganglia of the large intestine but not in the small intestine [77]. the low dose (0.5 mg/kg used in our study versus 2.5 mg/kg used by kelly and colleagues), coupled with the possible usage of different bacterial strains to generate lps affecting potency and pyrogenicity of lps (here strain o55:b5 was administered versus presumably strain o111:b4 used by kelly and colleagues and others [78], and potential effects of different environments and microbiomes [79] may contribute to this discrepancy. it will be interesting to study the mentioned parameters and their role in inducing αsyn pathology further in rodent models and explore how this could be translated to the even more heterogeneous human setting. similarly, others showed that employing a sub-chronic 0.5% dss paradigm for 3 weeks in wild type mice did not trigger an αsyn pathology in the colon [80]. while we have not tested this particular sub-chronic dss colitis paradigm and instead had administered 5% dss acutely to the wild type mice (fig. 2c), the data by garrido-gil and colleagues may corroborate our results which show in αsyn transgenic mice that a certain dss dose and severity of inflammation is required in order to induce accumulation of αsyn in the submucosal plexus (fig. 2a). what could be a functional role of the αsyn species found in abundance in the gut wall? monomeric and oligomeric αsyn species reportedly act as chemoattractants for neutrophils and monocytes, enhancing the maturation of dendritic cells in the ens [22,81]. with such a role in intestinal immunity, it is possible that the tissue destruction induced by dss in the present study led to release of αsyn, which perhaps served as a chemoattractant for monocytes. the increased abundance of αsyn and altered intestinal permeability, along with the dss-evoked inflammatory response may have provided an enabling milieu allowing further αsyn accumulation in the ens of the colon [77]. macrophages and other immune cells are also regulated by several genes including lrrk2, an established risk gene for pd and ibd. it will be interesting to explore how mutations in genes that control autophagy, including the lrrk2 gene, influence the handling of αsyn by macrophages that invade the inflamed colon in our dss colitis paradigm. despite the intriguing translational aspect of our finding in the dss paradigm, others have very recently reported that dss colitis in mice down-regulates the expression of enteric αsyn on protein levels in vivo [82,83]. this is in contrast to our immunofluorescence (i.e., increased accumulation of αsyn in submucosal plexus upon dss colitis; fig. 2, 3, and 4) and gene expression data (e.g., no change in endogenous and transgenic αsyn upon dss colitis; suppl. fig. 3) in the same paradigm and may reflect the well-known lab-to-lab variability that can occur for the dss models [84]. perhaps the most striking finding in our study was that a single period of dss-induced colitis at a young age led to an exacerbation of αsyn pathology in the brain of αsyn transgenic mice much later in life (fig. 5). how does severe αsyn inclusion pathology develop in the brains of these mice? one hypothesis is that the brain αsyn pathology observed in this study could be due to direct effects of peripheral immune activation on the brain and that certain peripheral triggers can directly affect microglial activity. for instance, short-chain fatty acids derived from gut microbiota appear to influence function and maturation of microglia in the mouse brain [85] and inflammatory mediators released by gut microbiota into the bloodstream have been suggested to induce brain pathology and behavioral changes in an αsyn transgenic mouse model [86]. moreover, rats and nematodes have been reported to develop αsyn inclusions after exposure to the bacterial amyloid protein curli, a protein which stimulates microgliosis, astrogliosis, and secretion of il-6 and tnf [87]. intriguingly, a recent study reported that peripherally applied inflammatory stimuli induce acute immune training (that exacerbates β-amyloid pathology) and immune tolerance in the brain that reprograms microglia, an effect which can persist for at least six months [88]. whether this is a relevant mechanism in the dss paradigm needs to be explored. another hypothesis is that the observed brain αsyn pathology may have accumulated as a consequence of the transfer of pathogenic αsyn seeds from the gut via the vagal nerve. several experimental studies have demonstrated that pathogenic αsyn seeds can be transferred from the peripheral to the central nervous system. aggregated recombinant αsyn injected intraperitoneally, intramuscularly or into the gastric wall of certain mouse models of pd results in αsyn inclusions in the brain [16,89]. data from animals injected with recombinant αsyn protein in the gut wall or viral vectors expressing αsyn into the vagal nerve suggest that pathogenic seeds can be transmitted via the vagal nerve [15,19,20,64,67,90–92]. a role for the vagal nerve in pd was also suggested by an epidemiological study indicating that vagotomy in a danish population is associated with decreased pd risk [93], although this association has been challenged [94]. in the present study, αsyn pathology was much more prominent in the reticular nucleus (including the vagal nucleus) and midbrain areas compared to the rostral areas at 18 months post dss colitis. although we did not conduct the definitive experiment of cutting the vagal nerve, our data are consistent with the growing body of evidence that the vagal nerve is involved in the accumulation of αsyn aggregates in the brain. that said, the innervation of the colon occurs via both parasympathetic (e.g., vagal output neurons) and sympathetic (e.g., in the celiac ganglion of the upper abdomen) nerves. the possibility of propagation of αsyn pathology via the two routes is supported by the observation that injection of recombinant αsyn fibrils to the duodenum of certain αsyn transgenic rats leads to accumulation of pathological αsyn in organs innervated by the parasympathetic and the sympathetic nerves [67] and an age-dependent propagation of αsyn pathology from gut-to-brain in wild type rats [64]. clinically, more relevant, αsyn accumulation is present in both parasympathetic and sympathetic vagal nerves in humans as well [95]. thus, propagation may occur through both vagal and spinal routes. aging is considered a major risk factor for neurodegenerative diseases, as failing cellular mechanisms are proposed to be unable to efficiently clear pathologically accumulating proteins and organelles [96]. intriguingly, in the current study, while both cohorts were exposed to the same peripheral dss colitis insult at 3 months of age, αsyn pathology only developed in the brain of aged (21-month-old) but not in the relatively younger (9-month-old) mice (fig. 5). while the mechanisms involved in this phenomenon are still unclear, a recent study in mice reports an age-dependent association of development of αsyn pathology in the brain and development of motor deficits after inoculating the gut wall with recombinant αsyn fibrils [20]. investigating this further, the study observed a reduction of the lysosomal enzyme glucocerebrosidase in the gut of aged but not young mice [20]. viral vector-based overexpression of glucocerebrosidase partially rescued the ens network connectivity with concomitant variable downregulation of pser129-αsyn levels [20]. although other age-related factors are likely involved, the finding of challis and colleagues, together with the well-known genetic risk for glucocerebrosidase and several other lysosomal genes in pd [97,98], suggest a critical role of the lysosomal pathway in the long-term persistence of pathology in the ens and the progression of αsyn pathology from the gut to the brain of aged mice. upcoming studies in human tissue may shed more light on the mechanisms on how autophagy-lysosomal pathways are regulated and how they, together with aging, may contribute to the pathogenesis in pd. in summary, we report that αsyn accumulates in the colon of αsyn transgenic and wild type mice subjected to experimental dss colitis and that this process can be modulated by genetically and pharmacologically modifying pathways related to monocyte/macrophage signaling. we further demonstrate that chronic but transient dss colitis in young αsyn transgenic mice leads to a markedly exacerbated accumulation of αsyn aggregates in the brain of aged mice. in the same aged mice, the numbers of thand nissl-positive neurons in the substantia nigra are reduced, suggestive of a neurodegenerative process. together, our findings are in consonance with studies demonstrating a link between ibd and pd [33,35,99] and suggest a critical role for specific types of intestinal inflammation and αsyn accumulation in the initiation and progression of pd. acknowledgments including sources of support we thank drs. l. ozmen and a. bergadano for their tremendous support in maintaining the mouse colony and we are grateful to the animal caretakers, veterinarians and many unnamed staff at roche for their valuable work with the mice in this study. in addition, at roche we thank dr. k.g. lassen for critical input to the paper, dr. c. ullmer for co-mentoring s.g. and providing scientific input, dr. l. collin for helping with confocal imaging and we are grateful to dr. t. kremer, n. haenggi, d. mona, a. girardeau, and j. messer for providing support in tissue dissections and g. walker and r. lauria for technical support. ms. e. schulz from vai assisted with immunostaining of the brain tissue. we thank the contract research organization frimorfo for carefully sectioning the brains for this study. we acknowledge drs. l. gaudimier (née chicha) and f. pan-montojo for scientific discussions early in the project, dr. w. zago from prothena for valuable scientific input throughout the project, and drs. m. and p. derkinderen for critical input on the link between ibd and the enteric αsyn accumulation in humans. p.b. acknowledges the van andel institute and the many individuals and corporations that supported financially the neurodegenerative research at van andel institute. research at van andel institute reported in this publication was also financially supported by roche through a research collaboration with p.b. s.g and n.m. were supported by a grant from roche under the roche postdoctoral fellowship (rpf) program. conflict of interest at the time of the study s.g. and n.m. were roche postdoctoral fellows employed by roche and l.s., f.b., g.d.p., j.s.p., k.o.s., h.r., m.h., m.se. m.st., p.m., a.w., t.e., a.h. and m.b. are or were fulltime employees or trainees at roche and they may additionally hold roche stock/stock options. s.g. and l.s. are currently employees of neurimmune ag, schlieren, switzerland. p.b. has received commercial support as a consultant from axial biotherapeutics, calico life sciences, curasen, fujifilm-cellular dynamics international, h. lundbeck a/s, and idorsia pharmaceuticals ltd. he has received commercial support for grants/research from h. lundbeck a/s and roche. he has ownership interests in acousort ab. the other authors declare that they have no competing interest with regard to this research. author contributions s.g., n.m. and l.s. planned and performed the in vivo experiments, colon immunostaining, analysis, and quantification; s.g. and n.m. drafted a first version of the manuscript; e.q. performed, imaged, quantitated pser129, th and nissl staining in the brain sections, and drafted a more advanced version of the manuscript with j.a.s., who also provided helpful discussion. both j.a.s. and e.q. were critical in revising the manuscript. f.b. and k.o.s. supported the image acquisition and image analysis for the colon samples; m.st. performed imaging and data analysis of experiments with wild type mice; g.d.p. and j.s.p. performed statistical analysis of the dss experiments; h.r. and m.h. performed mrna analyses; m.se. trained s.g. and l.s. on mouse necropsy and supported their work; p.m. performed expert pathology staging on leukocyte infiltration; t.e. and a.w. provided migg-mil-10 fusion proteins and measured serum exposure; z.m. performed statistical analysis for the pser129 αsyn immunohistochemistry data. a.s. contributed with scientific and veterinary expert input for implementation and analysis of the dss colitis model at roche. m.l.e.g. provided helpful discussion and project planning. a.h. co-mentored s.g. and n.m., performed expert pathology staging on leukocyte infiltration and contributed to experimental planning. c.m. trained s.g. on the colitis model and provided expert input on the experimental ibd model. m.b. and p.b. co-mentored roche postdoctoral 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dg, saad m, et al. large-scale meta-analysis of genome-wide association data identifies six new risk loci for parkinson’s disease. nat genet. 2014;46:989–93. 99. villumsen m, aznar s, pakkenberg b, jess t, brudek t. inflammatory bowel disease increases the risk of parkinson’s disease: a danish nationwide cohort study 1977-2014. gut. 2018;68:18–24. copyright: © 2021 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. neuropathology of covid-19 (neuro-covid): clinicopathological update feel free to add comments by clicking these icons on the sidebar free neuropathology 2:2 (2021) review neuropathology of covid-19 (neuro-covid): clinicopathological update jerry j. lou1,2*, mehrnaz movassaghi1*, dominique gordy1, madeline g. olson1, ting zhang1, maya s. khurana2, zesheng chen1, mari perez-rosendahl2, samasuk thammachantha3, elyse j. singer4, shino d. magaki1, harry v. vinters1,4, william h. yong1,2 1 department of pathology and laboratory medicine, david geffen school of medicine at ucla, usa 2 department of pathology and laboratory medicine, university of california irvine school of medicine, usa 3 department of pathology, prasat neurological institute, thailand 4 department of neurology, david geffen school of medicine at ucla, usa   * authors contributed equally and are listed in alphabetical order corresponding author: william h. yong md · department of pathology and laboratory medicine · university of california irvine school of medicine · usa and department of pathology and laboratory medicine, david geffen school of medicine at ucla · usa yongwh@hs.uci.edu submitted: 03 september 2020 accepted: 14 january 2021 copyedited by: calixto-hope g lucas, jr. published: 18 january 2021 https://doi.org/10.17879/freeneuropathology-2021-2993 additional resources and electronic supplementary material: supplementary material keywords: cns, covid-19, sars-cov-2, brain, pituitary abstract coronavirus disease 2019 (covid-19) is emerging as the greatest public health crisis in the early 21st century. its causative agent, severe acute respiratory syndrome coronavirus 2 (sars-cov-2), is an enveloped single-stranded positive-sense ribonucleic acid virus that enters cells via the angiotensin converting enzyme 2 receptor or several other receptors. while covid-19 primarily affects the respiratory system, other organs including the brain can be involved. in western clinical studies, relatively mild neurological dysfunction such as anosmia and dysgeusia is frequent (~70-84%) while severe neurologic disorders such as stroke (~1-6%) and meningoencephalitis are less common. it is unclear how much sars-cov-2 infection contributes to the incidence of stroke given co-morbidities in the affected patient population. rarely, clinically-defined cases of acute disseminated encephalomyelitis, guillain-barré syndrome and acute necrotizing encephalopathy have been reported in covid-19 patients. common neuropathological findings in the 184 patients reviewed include microglial activation (42.9%) with microglial nodules in a subset (33.3%), lymphoid inflammation (37.5%), acute hypoxic-ischemic changes (29.9%), astrogliosis (27.7%), acute/subacute brain infarcts (21.2%), spontaneous hemorrhage (15.8%), and microthrombi (15.2%). in our institutional cases, we also note occasional anterior pituitary infarcts. covid-19 coagulopathy, sepsis, and acute respiratory distress likely contribute to a number of these findings. when present, central nervous system lymphoid inflammation is often minimal to mild, is detected best by immunohistochemistry and, in one study, indistinguishable from control sepsis cases. some cases evince microglial nodules or neuronophagy, strongly supporting viral meningoencephalitis, with a proclivity for involvement of the medulla oblongata. the virus is detectable by reverse transcriptase polymerase chain reaction, immunohistochemistry, or electron microscopy in human cerebrum, cerebellum, cranial nerves, olfactory bulb, as well as in the olfactory epithelium; neurons and endothelium can also be infected. review of the extant cases has limitations including selection bias and limited clinical information in some cases. much remains to be learned about the effects of direct viral infection of brain cells and whether sars-cov-2 persists long-term contributing to chronic symptomatology. 1. introduction in late 2019, a novel infectious disease associated with pneumonia and acute respiratory distress emerged in wuhan, china. the implicated human coronavirus was genetically related to but distinct from the underlying viral agent behind the 2003 severe acute respiratory syndrome (sars) outbreak, sars coronavirus (sars-cov). the international committee of taxonomy of viruses designated this novel coronavirus as severe acute respiratory syndrome coronavirus 2 (sars-cov-2)1 and the world health organization termed the associated disease as coronavirus disease 2019 (covid-19).1 as unsuspecting asymptomatic patients readily spread the disease, the covid-19 pandemic has swept rapidly across the world resulting, by the end of december 2020, in over 79 million confirmed human infections and over 1.7 million deaths.2 these numbers are believed to be substantial underestimates of the true toll. covid-19 is best known for its pulmonary involvement, but other organs are often affected including heart, kidney and nervous system. this review will provide an overview of tissue-based covid-19 neuropathological analyses, almost entirely autopsy derived, as well as a brief discussion of sars-cov-2 virology to provide context for understanding its pathogenesis, and of diagnostic testing limitations that may bias the examined cases. the nervous system is affected both secondarily from systemic complications such as hypoxia and coagulopathy, and also likely from primary infection though much remains to be elucidated regarding viral invasion of the brain, spinal cord and other components of the nervous system. 2. sars-cov-2 virology sars-cov-2 is the latest coronavirus to emerge as a human pathogen. coronaviruses are single-stranded, positive-sensed ribonucleic acid (rna) viruses subdivided into four genera: alpha-coronavirus (α-cov), beta-coronavirus (β-cov), gamma-coronavirus (γ-cov), and delta-coronavirus (δ-cov).3 sars-cov-2 is a beta-coronavirus, typically 60 to 140 nm4–6 in size, that shares genetic similarities to sars-cov (~79% homology) and middle east respiratory syndrome coronavirus (mers-cov) (~50% homology).7 the sars-cov-2 genome encodes 16 non-structural proteins involved in viral replication and four structural proteins consisting of the envelope, membrane, nucleocapsid, and spike glycoprotein (figure 1).3,8,9 the virus is constantly evolving with numerous strains of sars-cov-2 identified, some preferentially localized (at least temporarily) to geographic regions such as europe or north america.10 the mutation rate of sars-cov-2 is estimated to be 0.84-1.12 x 10-3 substitutions per site per year,7,11 which is lower than that of human immunodeficiency virus (hiv)12 or influenza a.13 the genetic spectrum of disparate sars-cov-2 strains undergoing continued mutation could contribute to the variability of neuropathological findings discussed later. antibodies against nucleocapsid and spike proteins have been used for immunohistochemical studies. figure 1: severe acute respiratory coronavirus 2 (sars-cov-2) structure. there are four structural proteins: spike (s) protein (red), envelope (e) protein (violet), membrane (m) protein (blue), and nucleocapsid (n) protein (orange). 3. ace2 and other receptors mediate sars-cov-2 entry the entry of sars-cov-2 into human cells is commonly thought to be mediated by the interaction of the spike protein with the angiotensin converting enzyme 2 (ace2) receptor, an important regulator of the renin-angiotensin system (ras).14 gender, age, lifestyle, smoking, and other patient co-morbidities are implicated in the modulation of ace2 receptor expression in various tissues.15–21 ace2 receptor expression has been reported in the cerebrum, cerebellum, brainstem, retina, and olfactory mucosa.22–24 neurons, vascular pericytes and smooth muscle, and glia express the ace2 receptor.22,23,25 in addition to the ace2 receptor, in vitro studies show that sars-cov-2 may gain entry using other cell receptors, such as basigin (bsg; cd147),26 neuropilin-1 (nrp1),27 transmembrane serine protease 2 and 4 (tmprss2/4),28,29 and cathepsin l (ctsl).9 expression of ace2 receptor is highest in oligodendrocytes, tmprss2/4 in neurons, ctsl in microglia, and nrp1 in endothelial cells.25 in principle therefore, a broad range of cells in the central nervous system (cns) have a variety of receptors that may facilitate infection. 4. laboratory testing for covid-19 has significant limitations in part due to the natural course of disease wherein viral titers rise over time, laboratory testing for sars-cov-2 does not completely exclude patient infection, complicating the neuropathologist’s management of surgical or autopsy cases. there may also be a bias towards evaluating autopsies of covid-19 patients with severe disease and high viral loads. the analytical sensitivity of the commonly used covid-19 reverse transcriptase polymerase chain reaction (rt-pcr) assay is excellent with a limit of detection as low as 6.25 copies/µl in a nasopharyngeal sample.30 in a clinical setting, the sensitivity of rt-pcr may be 83.3% or significantly less, and may be affected by infection phase, sample type, collection procedures, and testing platforms.31,32 covid-19 rt-pcr testing may produce false-negative results in the initial phase of infection. in sequential testing of patients who have symptoms, suspicious chest computed tomography (ct) findings and an initial negative rt-pcr test, covid-19 rt-pcr positivity occurs at a mean of 5.1 ± 1.5 days after the initial test.32 a patient with a negative rt-pcr test result in a nasopharyngeal swab but with a positive sars-cov-2 cerebrospinal fluid (csf) test has been reported.33 while rt-pcr detection of genomic rna is not specific for viable virus, identification of sub-genomic rnas transcribed in infected cells have been used in clinical testing to document the presence of actively replicating virus in lieu of viral cultures.34 although chest ct scans may detect signs of covid-19 days before rt-pcr positivity, ct findings overlap with other viral pneumonias.35 serologic testing has uses for contact tracing, epidemiology, and vaccine studies but is more challenging to use for primary diagnosis given the latency for development of antibodies.36–40 the most common serological assays are the rapid lateral flow assay, enzyme linked immunosorbent assay (elisa), and virus neutralization assay. two-step elisa assays are quantitative and more reliable than flow assays that are easily scalable but are qualitative.40 the virus neutralization assay detects and quantifies antibodies that inhibit viral replication but is technically complex.37 5. systemic pathophysiology of covid-19 in the first stage of infection, sars-cov-2 targets nasal and bronchial epithelial cells as well as pneumocytes.28 as infection progresses, sars-cov-2 infects pulmonary endothelial cells, abrogating the epithelial-endothelial barrier.41 a subsequent ingress of neutrophils and monocytes is followed by pulmonary edema and hyaline membrane formation, a component of early acute respiratory distress syndrome (ards).41,42 in severe covid-19, coagulopathy can occur, reflecting microthrombi formation secondary to endothelial cell inflammation and cytokine storms.41,43 covid-19 associated hypercoagulability induces venous thromboembolism and arterial occlusion.41 the interplay of these systemic derangements likely contributes to the pathologic changes seen in the cns (figure 2). figure 2: flow chart modeling covid-19 pathogenesis and neurological dysfunction. adem= acute disseminated encephalomyelitis; ane= acute necrotizing encephalopathy; gbs= guillain-barré syndrome. 6. covid-19 neurological manifestations covid-19 associated neurological manifestations range from mild symptoms such as dizziness, headache, dysgeusia, or anosmia to severe disorders such as stroke, guillain-barré syndrome (gbs), acute hemorrhagic necrotizing encephalopathy, meningoencephalitis, and cerebral venous thrombosis. the frequency of reported neurological signs and symptoms is variable but substantial regardless. in an early chinese retrospective study, 36.4% of 214 covid-19 patients had neurological symptoms which included dizziness (16.8%), headache (13.1%), impaired consciousness (7.5%), dysgeusia (5.6%), and anosmia (5.1%).44 in western studies, dysgeusia and anosmia are reported in the majority of patients.45,46 a french study reports that 49 out of 58 (84%) covid-19 intensive care unit (icu) patients had neurological signs which included agitation (69%), confusion (65%), corticospinal tract signs (67%), and dysexecutive syndrome (33%).47 a study from a british referral center also describes cases of septic or para-infectious encephalopathy, autoimmune encephalitis including acute disseminated encephalomyelitis (adem), and gbs.48 7. specific neurological disorders associated with covid-19 olfactory and gustatory dysfunction in the aforementioned study from china, covid-19 patients had gustatory dysfunction and olfactory dysfunction at frequencies of less than 6% each.44 in a prospective european study, 88.8% of 385 covid-19 patients had gustatory dysfunction and 85.6% of 417 covid-19 patients had olfactory dysfunction.45 in a california study, 71% of 59 covid-19 patients recorded ageusia and 68% reported anosmia.46 olfactory and gustatory dysfunction in covid-19 typically resolves after 17 to 30 days from initial onset.49,50 although long term follow-up is generally lacking,51 one study reports resolution rates at day 30 in home-quarantined covid-19 patients of 87% for olfactory dysfunction and 82% for gustatory dysfunction.52 presence of sars-cov-2 virions in the olfactory neuroepithelium of the nasal mucosa as well as in the olfactory bulb has been documented53,54 though the exact basis for olfactory dysfunction and recovery remains to be resolved. stroke stroke is the most common debilitating neurological disorder associated with covid-19 and has a predilection for males and the elderly. two retrospective new york studies reported respectively that 1.6% of 1,916 patients with hospitalizations or emergency department visits for covid-19 and 0.9% of 3,556 hospitalized covid-19 patients had radiologically-confirmed ischemic infarcts.55,56 cryptogenic strokes were twice as common in hospitalized covid-19 patients compared to either contemporary or historical controls.56 a third new york retrospective study found 1.1% of 3,218 covid-19 patients had strokes; in the small subset with acute neuroimaging, 68.5% of strokes were ischemic (44.5% large vessel, 24% lacunar) and 24% were hemorrhagic.57 a retrospective chinese study of 214 covid-19 patients reported strokes in six patients (2.8%).44 a small american case series reported large vessel ischemic strokes in five covid-19 patients younger than 50, four of whom had no prior history of stroke.58 for comparison, the average incidence of stroke among patients admitted through a u.s. emergency department prior to the pandemic was approximately 3.2%.59 of patients who visited emergency departments or were hospitalized, only 0.2% of 1486 influenza patients had ischemic infarcts which was significantly less than covid-19 patients even after adjusting for age, sex and, race.55 in another new york study, 33 out of 755 (4.4%) covid-19 patients with neuroimaging had evidence of intracranial hemorrhage not associated with trauma, brain metastases, or tumor resection.60 parenchymal hemorrhages with mass effect and herniation were present in five patients, punctate hemorrhages in seven, small to moderate sized hemorrhages in 17, and large single site hemorrhages without herniation in four.60 twenty-six of the 33 intracranial hemorrhages occurred as a transformation of an ischemic infarct.60 of the five patients with parenchymal hemorrhages, four had high partial thromboplastin time or anti-factor xa in the 72 hours before the intracranial hemorrhage.60 advanced age, cardiovascular disease, cerebrovascular disease, diabetes, chronic respiratory disease, hypertension, obesity, smoking, and cancer are risk factors for severe covid-19 disease or poor outcome.61–67 in a canadian study, critically ill covid-19 patients with blood groups a and ab were more likely to require ventilation, continuous renal replacement therapy, and prolonged intensive care unit admission.68 in contrast, an earlier american covid-19 study did not show an association between specific blood groups and either ventilation or death.69 differences in cohorts including mortality rates may account for the contradictory findings.68 it cannot escape notice that a number of the aforementioned covid-19 prognostic factors are associated with atherosclerosis and arteriolosclerosis that, in the setting of covid-19 respiratory compromise and coagulopathy, may contribute to strokes. guillain-barré syndrome gbs is an uncommon, immune-mediated demyelinating disease of the peripheral nerves that often follows viral infections. common presenting symptoms in covid-19 patients include symmetrical flaccid quadriparesis, ataxia, facial weakness, respiratory failure, and lower paresthesia.70–72 rt-pcr of nasopharyngeal swabs tested positive for sars-cov-2 but csf was negative for tested patients.73–75 gbs symptoms tend to emerge between day five to 10 after covid-19 symptom onset.71 over 30 cases of gbs have been reported.73–76 one proposed mechanism is an autoimmune hyperreaction, triggering release of pro-inflammatory mediators such as interleukin (il)-6, that cause autoimmune demyelination or axonal damage. alternatively in some cases, sars-cov-2 may induce production of antibodies targeting gangliosides, leading to peripheral neuropathy.72,77 covid-19 associated miller fisher syndrome (mfs), a variant of gbs with ophthalmoplegia, areflexia, and ataxia, and polyneuritis cranialis (pnc), a gbs variant with multiple cranial neuropathies, have been reported.78 meningitis and encephalitis covid-19 associated meningitis has only infrequently been reported but one case highlights the need for csf testing if suspected.33 a 24-year-old japanese male presented with headache, generalized fatigue and fever. nine days after onset of typical covid-19 symptoms, the patient had altered mental status, transient generalized seizures, and neck stiffness. brain magnetic resonance imaging (mri) showed hyperintensities along the lateral ventricle and in the medial temporal lobe including the hippocampus. sars-cov-2 rna was not detected by rt-pcr in a nasopharyngeal swab but was in csf.33 autopsies performed on covid-19 patients have suggested meningoencephalitis in some cases and these are discussed in section 8.79 other disorders a brazilian study reported central venous thrombosis (cvt) in three previously healthy covid-19 patients younger than 41 years, underscoring the need for awareness of a hypercoagulable state in covid-19 even in this age group.80 acute hemorrhagic necrotizing encephalopathy, an uncommon complication characterized by multifocal symmetric brain lesions including bilateral thalamic lesions, has been associated with covid-19, though rarely.81,82 this complication may be caused by blood-brain-barrier disruption related to intracranial cytokine storms.81 covid-19 associated cases of adem-like disease and autoimmune encephalitis have been documented.72,83 8. neuropathological findings and neuropathogenesis of covid-19 to examine the spectrum of covid-19 neuropathology, we reviewed 20 papers encompassing 184 patients with tissue-based neuropathological analyses including 101 cases analyzed by rt-pcr for sars-cov-2 and 83 cases by immunohistochemistry (ihc) (table 1).25,53,79,83–99 all cases are autopsies except for four biopsies and one case of unspecified type. the range of histologic findings is broad and in part reflects the heterogeneity of neurological findings. the most frequent findings (table 1) include microglial activation with microglial nodules in a likely underestimated subset; lymphoid inflammation including perivascular lymphocytosis (figure 3a), parenchymal lymphocytic infiltration, and leptomeningeal lymphocytic inflammation; hypoxic-ischemic changes (figure 3b); astrogliosis; acute/subacute brain infarcts; primary hemorrhage; and microthrombi. it should be noted that the prevalence of detected lymphoid inflammation and microglial nodules is high in papers with immunohistochemical studies and low in those without. table 1: neuropathological findings in covid-19 brain tissue table 1: some histologic findings are likely to be under-reported as reviewed studies are variable in their focus. *only studies tabulating the prevalence of microglial nodules are included. studies mentioning microglial nodules in their case series but not enumerating the frequency are excluded. figure 3: representative covid-19 histopathology: a. mild perivascular lymphoid inflammation, 400x (arrow); b. eosinophilia in purkinje cells compatible with acute hypoxic-ischemic change, 200x (arrows); c. subacute infarct of anterior pituitary gland, 100x (arrows); d. alzheimer type ii astrocytes in basal ganglia, 400x (circle). sars-cov-2 is detectable in central nervous system tissue at least three routes of cns infection by sars-cov-2 have been proposed: retrograde transmission via olfactory sensory neurons, infiltration of immune cells, and entry across the blood-brain barrier.100,101 the prevalence of anosmia and ageusia in covid-19 patients led to the theory that sars-cov-2 enters the brain via infection of neurons in the olfactory neuroepithelium (which resides in the mucosa of the nasal cavity) and from there to the olfactory bulb and then to other brain regions. this olfactory route is used by other coronaviruses, such as sars-cov102 and mers-cov.103 covid-19 patients frequently display mri hyperintensity in the olfactory cortex.104 the ace2 receptor, used by sars-cov-2 for cellular entry, is expressed in sustentacular cells and stem cells of the nasal olfactory epithelium.23,105 while one study suggests olfactory neurons themselves do not express the ace2 receptor,23 another report contradicts this finding.106 besides technical sensitivity issues, it may be that the expression levels of the ace2 receptor and other receptors differ under inflammatory conditions as compared to physiologic conditions. viral particles have been detected by electron microscopy and ihc in the olfactory epithelium54 as well as by electron microscopy in olfactory neurons of the nasal mucosa at autopsy.53 morbini and colleagues report ultrastructural evidence of sars-cov-2 particles in the olfactory bulb of a covid-19 patient.54 a second theory posits that sars-cov-2 may infect immune cells that cross the blood-brain barrier and deliver virus into the brain. this mechanism is well described in hiv.107 white blood cells including lymphocytes and monocytes express the ace2 receptor.108,109 infection of immune cells by sars-cov-2 is an active area of study. finally, the third theory extends the well documented behavior of encephalitic blood-borne coronaviruses to enter through the blood-brain barrier via infection of vessel wall cells.110 histologically, on routine hematoxylinand eosin-stained slides, neither viral inclusions nor specific cellular changes recognizable as direct viral infection have been reported. however, sars-cov-2 has been detected in the brain by rt-pcr, ihc, and electron microscopy. as many as 54 out of 101 (53.5%) cases were positive for sars-cov-2 in the brain by rt-pcr and 23 out of 83 (27.7%) cases were positive by ihc (table 1). in some cases, copies of virus detected by rt-pcr were low in number and the detection of virus in blood or blood cells within intracerebral vasculature rather than brain cells was a possibility. however, ihc has confirmed the presence of viral antigens in autopsy brain cells. of note, antibodies against sars-cov-2 spike protein were more effective in detecting viral antigens than those targeted against nucleocapsid protein25,87,97 (supplementary table a). importantly, staining using both anti-spike and anti-nucleocapsid antibodies may be more sensitive than either alone as, in a few cases, nucleocapsid protein was detected while spike protein was not. whether this finding is a technical issue related to the quality of the antibodies or the intrinsic accessibility of the relevant epitopes or both is unresolved. staining of virus localizes in scattered cortical neurons and endothelial cells,87 as well as brainstem cranial nerve roots and isolated cells (cell type unclear) in the medulla oblongata; the images of the isolated cells had a striking lack of attendant chronic inflammation.25 this disconnect between viral infection and inflammation raises the question of immune evasion. one case exhibited viral staining around the edges of subcortical white matter microinfarcts.87 the immunostaining pattern consists of diffuse cytoplasmic and perinuclear positivity with small concentrated foci possibly representing viral inclusion bodies.87 meinhardt and colleagues, using in situ hybridization (ish), identified sars-cov-2 in the olfactory epithelium and mucus.53 lastly, viral particles compatible with sars-cov-2 have been identified by electron microscopy in olfactory bulb and frontal lobe tissue.54,96 to date, regions in which sars-cov-2 have been detected by ihc, rt-pcr, or electron microscopy relevant to the cns include cornea, conjunctiva, olfactory epithelium, olfactory bulb, olfactory tubercle, frontal lobe, cerebellum, medulla oblongata, cranial nerves and trigeminal ganglion. most investigations studied limited areas of brain or did not specify the origin of cerebral cortex tissue. additional studies are needed to better establish the frequency and extent of direct infection in the cns. as some patients have "long covid” wherein they have persistent symptoms for months, whether and how sars-cov-2 may persist in the brain will need to be evaluated in the future. chronic inflammatory and reactive glial changes microgliosis and astrocytosis are common in covid-19 brains including in the olfactory bulb.25 lymphoid inflammation, which tends to be minimal or mild in many cases, is not uncommon particularly in the medulla oblongata and if ihc is used for detection.25 in many studies that did not use ihc, lymphoid inflammation was reported as absent or infrequent; this is concordant with our experience and anecdotally with that of colleagues in asia and in the united states (personal communication). a substantial portion of these reactive changes may be secondary to systemic issues (e.g. sepsis) or other neuropathology (infarcts, hemorrhages, etc.) rather than a response to direct infection. microgliosis and chronic inflammation did not correlate with the severity of covid-19 disease nor were there any discernible neuropathological differences in patients from nursing homes, hospital wards, or intensive care units.25 furthermore, microglial activation, perivascular lymphocytosis, and leptomeningeal lymphocytic infiltration is observed to similar degrees in the control brains of septic (non-covid-19) patients when compared to covid-19 patients.86 some cases present findings compatible with viral meningoencephalitis including parenchymal lymphocytic clustering around microglial nodules, concomitant leptomeningeal lymphocytic inflammation, and even neuronophagy. in the study by matschke and colleagues, four out of 16 cases immunopositive for sars-cov-2 had 10 to 49 cd8+ cytotoxic t lymphocytes per high power field in at least three fields in the medulla oblongata.25 the observed lymphocytes tended to cluster near activated microglial nodules,25 as commonly seen in viral encephalitis. rt-pcr detected sars-cov-2 rna in three out of the four cases.25 three studies report neuronophagy in the medulla associated with histiocytic and lymphocytic parenchymal infiltration.25,84,89 von weyhern and colleagues report six cases of perivascular and parenchymal lymphocytosis with neuronal loss and axon degeneration in the brainstem, which the authors determined to be adequate to diagnose sars-cov-2 viral encephalitis.79 in the same study, five of the six cases had concomitant meningitis.79 respiratory and cardiovascular control centers of the medulla may be affected.53 while the majority of covid-19 patients with neurological manifestations do not have detectable sars-cov-2 rna in csf by rt-pcr,111–113 exceptions to this trend have been reported in at least four covid-19 patients.33,114–116 anti-sars-cov-2 antibodies were detected in the csf of one covid-19 patient suggesting an immune response to viral infection.87 it appears therefore that histologically documentable cases of sars-cov-2 encephalitis and/or meningitis do occur, with a tendency to involve the brainstem. as only a few series report the majority of brainstem microglial encephalitis cases, the question arises as to whether particular viral strains, therapeutic approaches, genetic background or detection methods are responsible for its apparent absence or paucity in the majority of studies. autoimmune mediated inflammation may also occur. autoimmune encephalitis, adem, and acute necrotizing encephalopathy have also been reported clinically though histopathologic evaluation has been limited. adem-like pathology has been reported in one autopsy case.83 microthrombi, infarcts, hemorrhages, and “neutrophilic plugs” sars-cov-2 may induce a cytokine storm, a severe hyperimmune reaction characterized by excessive and rapid release of cytokines such as il-6 and il-1β into the blood.117,118 il-6 activates the coagulation system and increases vascular permeability119 which, in combination with viral endotheliopathy,120 may account for the well documented covid-19 associated coagulopathy.41,121 sars-cov-2 has been detected within cerebral endothelial cells by ihc and electron microscopy.87,96 hypercoagulability, in turn, results in histologic findings of microthrombi, infarcts, and hemorrhages. also, il-1β plays a major role in triggering vascular “neutrophilic plugs” containing neutrophils and/or platelets as well as neutrophil extracellular traps, a mesh of deoxynucleic acid (dna)-rich material coated with antimicrobials that entrap and kill microbes.122 these “neutrophilic plugs” have been found in the brain as well as the lungs, heart, kidneys, and liver of covid-19 patients who come to autopsy.88 approximately 1.6% of cases have “neutrophilic plugs” in the brain (table 1) though this percentage is likely an underestimate as pathologists do not typically evaluate for these structures. pneumonia and ards with resultant hypoxemia as well as pre-existing arteriosclerosis are also likely contributory to the cerebral infarcts. in addition to cerebral infarcts, we have noted occasional cases of pituitary infarcts (figure 3c) in our covid-19 autopsies. numerous extramedullary megakaryocytes were present in subacute cerebral infarcts of one covid-19 patient.84 alzheimer type ii astrocytosis alzheimer type ii astrocytosis (figure 3d) characteristic of hepatic encephalopathy has been reported.89,97 the reports do not specify whether the frequency of the alzheimer type ii astrocytes reaches a threshold of five or more per 20 high power fields, the cutoff suggested by agarwal and colleagues for hepatic encephalopathy.123 in a paper by solomon and colleagues, four out of four cases with alzheimer type ii astrocytes had chronic liver disease or alcohol use disorder.97 the relative contribution of hepatic encephalopathy to cases currently ascribed as septic or para-infectious encephalopathy remains to be seen. 9. conclusions systemic dysfunction and viral infection of the cns can cause a wide range of covid-19 related neuropathological changes. the incidence of neuropathological findings should be viewed with caution given great variability in what tissues were studied and what types of ancillary studies were (or were not) performed. inflammatory changes were reported in a high percentage of cases in some series but not in others, at least in part due to the use of immunostaining; however, the lack of controls in most of these studies limit interpretation of these findings. selection or referral bias, the decedent’s co-morbidities, therapies given, patient’s genetic background, immune status, immunization status and perhaps viral strains of sars-cov-2 may also contribute to differences and will need to be dissected out in the future. whether or how much of the inflammatory changes seen are due to autoimmune phenomena versus direct viral infection or other causes remains to be resolved. the presence of microglial nodules and detectable virus are indicative of viral meningoencephalitis in some cases; a predilection for the medulla perhaps with compromise of respiratory and cardiovascular control centers may compound the covid-19 patient’s often already tenuous cardiorespiratory function. while there is evidence for an olfactory route of infection, it is unclear whether this is the major mechanism of cns infection given that the virus is rarely detectable in the olfactory bulb. cerebral endothelial infection is also present and a hematogenous route of infection is therefore plausible perhaps even common. data on where the virus is detectable in the brain is limited as studies have focused on the frontal lobe and brainstem. it would not be surprising to find a greater extent of infection than currently confirmed. it should be kept in mind that the sensitivity of virus detection is suboptimal given that most samples are derived from autopsies. lastly, it is notable that inflammation does not always coincide with virus localization, raising the concern that the virus may be evading the immune response in the cns. whether the cns is a potential long-term reservoir of virus and a contributor to “long covid” remains to be seen. lastly, increased risk for vascular dementia or 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link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. differential gene expression in the cortical sulcus compared to the gyral crest within the early stages of chronic traumatic encephalopathy feel free to add comments by clicking these icons on the sidebar free neuropathology 2:21 (2021) original paper differential gene expression in the cortical sulcus compared to the gyral crest within the early stages of chronic traumatic encephalopathy jonathan d. cherry*1,2,3,4, filisia agus2,5, erin dixon3,4, bertrand huber2,3,4,6, victor e. alvarez2,3,4,7, jesse mez2,3, ann c. mckee1,2,3,4,7, adam labadorf2,5,6, thor d. stein1,3,4,7 1 department of pathology and laboratory medicine, boston university school of medicine, boston ma, usa 2 department of neurology, boston university school of medicine, boston ma, usa 3 boston university alzheimer’s disease and cte centers, boston university school of medicine, boston ma, usa 4 va boston healthcare system, jamaica plain ma, usa 5 bioinformatics program, boston university, boston ma, usa 6 national center for ptsd, va boston healthcare system, boston ma, usa 7 va bedford healthcare system, bedford ma, usa * corresponding authors: jonathan d. cherry, phd · va boston healthcare system · 150 s huntington ave · boston, ma 02130 · usa jdcherry@bu.edu thor d. stein, md. phd · va boston healthcare system · 150 s huntington ave · boston, ma 02130 · usa tdstein@bu.edu submitted: 21 july 2021 accepted: 12 august 2021 copyedited by: lauren walker published: 17 august 2021 https://doi.org/10.17879/freeneuropathology-2021-3453 additional resources and electronic supplementary material: download supplementary material keywords: cte, rna-seq, repetitive head trauma, inflammation, tau, tbi abstract chronic traumatic encephalopathy (cte) is a progressive neurodegenerative tauopathy found in individuals with a history of repetitive head impacts (rhi). previous work has demonstrated that neuroinflammation is involved in cte pathogenesis, however, the specific inflammatory mechanisms are still unclear. here, using rna-sequencing and gene set enrichment analysis (gsea), we investigated the genetic changes found in tissue taken from the region cte pathology is first found, the cortical sulcus, and compared it to neighboring gryal crest tissue to identify what pathways were directly related to initial hyperphosphorylated tau (p-tau) deposition. 21 cases were chosen for analysis: 6 cases had no exposure to rhi or presence of neurodegenerative disease (control), 5 cases had exposure to rhi but no presence of neurodegenerative disease (rhi), and 10 cases had exposure to rhi and low stage cte (cte). two sets of genes were identified: genes that changed in both the sulcus and crest and genes that changed specifically in the sulcus relative to the crest. when examining genes that changed in both the sulcus and crest, gsea demonstrated an increase in immune related processes and a decrease in neuronal processes in rhi and cte groups. sulcal specific alterations were observed to be driven by three mechanisms: anatomy, rhi, or p-tau. first, we observed consistent sulcal specific alterations in immune, extracellular matrix, vascular, neuronal, and endocytosis/exocytosis categories across all groups, suggesting the sulcus has a unique molecular signature compared to the neighboring crest independent of pathology. second, individuals with a history of rhi demonstrated impairment in metabolic and mitochondrial related processes. finally, in individuals with cte, we observed impairment of immune and phagocytic related processes. overall, this work provides the first observation of biological processes specifically altered in the sulcus that could be directly implicated in cte pathogenesis and provide novel targets for biomarkers and therapies. introduction chronic traumatic encephalopathy (cte) is a progressive neurodegenerative disease found in individuals with a history of exposure to repetitive head impacts (rhi) typically received through playing contact sports such as american football, hockey, soccer, boxing, or rugby, or through military service-related injuries like blasts [3, 23, 24]. currently, cte can only be definitively diagnosed after death through neuropathologic autopsy. the pathognomonic lesion that defines the disease consists of perivascular hyperphosphorylated tau (p-tau) found in neurons and sometimes astrocytes, initially at the depths of the cortical sulcus in the frontal cortex [3]. through computational modeling and helmet sensor data, it was observed that the sulcus and blood vessels receive the greatest amount of damage after head trauma, directly linking rhi induced trauma to cte pathogenesis [11]. however, the specific biological response after trauma that drives cte pathology is still unclear. under normal circumstances, inflammation is a necessary part of the injury cascade. in the brain, neuroinflammation has been observed to be necessary for wound healing responses after damage, triggering an innate and adaptive immune response to fight infectious agents, and recruit beneficial immune cells to areas of need [6]. however, prolonged or chronic inflammation can be harmful and can result in tissue damage and long-term pathology. rhi has been shown to induce a chronic neuroinflammatory environment in both mouse and human studies [9, 10, 32]. additionally, previous work from our laboratory has demonstrated that elevated microglial recruitment via the chemokine ccl2, and glial activation was related to greater years playing contact sports and is a prominent feature in cte [4, 7]. we observed that microglia-mediated neuroinflammation may exist in a feedback loop with p-tau pathology. neuroinflammation induces p-tau deposition, which in turn induces more neuroinflammation leading to a vicious cycle, similar to the inflammatory cascade hypothesis in alzheimer’s disease (ad) [12, 16]. however, the concept of neuroinflammation is broad, encompassing inflammatory, anti-inflammatory, senescent, and immunoregulatory features that previous histology-based studies were unable to easily segregate [6]. therefore, there is a critical need to understand the individual inflammatory phenotypes that emerge during rhi to have more complete insight into the various mechanisms which may directly result in cte pathogenesis. to help fill in knowledge gaps, high level bioinformatic analyses are needed. previous studies have demonstrated that high throughput genomic techniques and bioinformatic pipelines are an optimal method to investigate large scale changes in the brain and determine specific pathways and effects in a more efficient manner [1, 17]. herein, we utilized bulk tissue rna-sequencing targeted to cortical anatomy, in order to investigate the spectrum of inflammatory and neurodegenerative phenotypes that occur as a result of rhi and during early stage cte. as the earliest neuropathologic feature of cte is p-tau pathology found at the depth of the cortical sulcus, we chose to examine what genetic changes occur specifically in the sulcus compared to the neighboring gyral crest. by directly comparing between the sulcus and crest within each case, we will have unique insights into which genes or biological processes are specifically altered in the sulcus as opposed to genes that are part of a more tissue wide response. we will utilize three separate groups, individuals without any rhi or cte (controls), individuals with rhi but no cte (rhi), and individuals with early stage cte (cte). comparison of these three groups will allow direct analysis of different stages of disease. furthermore, the results will provide new understandings and insight into what genes and biological processes might be directly related to initial cte p-tau deposition as opposed to a general cns response to trauma. finally, genes that are found to be specific to cte related pathology will become targets for future biomarker or therapeutic intervention studies. methods subjects a convenience sample of 15 brain donors with a history of rhi exposure from contact sports were selected from the understanding neurological injury and traumatic encephalopathy (unite) brain bank. selection was based on availability of frozen frontal cortex tissue and if the cases were male and between the ages of 35-65 years old. samples were excluded from the study if they carried a neuropathologic diagnosis of ad, neocortical lewy bodies, frontotemporal lobar degeneration (ftld), or motor neuron disease (mnd) based on established neuropathologic criteria for each disease [19, 20, 25, 26]. an additional 6 donors were obtained from the national post-traumatic stress disorder (ptsd) brain bank (national center for ptsd, va boston healthcare system, boston ma). these 6 donors were selected from a group that lacked a history of rhi, did not have evidence of a neurodegenerative disease at autopsy, and were not diagnosed with ptsd in life. samples were grouped into three categories: 6 cases with no history of repetitive head trauma or neurodegenerative disease (control), 5 cases with a history of repetitive head trauma but no neurodegenerative disease (rhi), and 10 cases with a history of repetitive head trauma and a diagnosis of low stage cte (cte). there was no difference in the mean age of death of each group: control51.3 years old ± 8.2, rhi51.4 years old ± 6.8, cte49.5 years old ± 8.1. all cases were male and had a history of playing american football. individual descriptive statistics for the age at death, years of exposure, sport played, and rna integrity number (rin), are provided in table 1. next-of-kin provided written consent for participation and donation. institutional review board approval for brain donation was obtained through the boston university alzheimer’s disease and cte center, human subjects institutional review board of the boston university school of medicine, and edith nourse rogers memorial veterans hospital (bedford, ma). neuropathological assessment post-mortem fresh frozen tissue from the dorsolateral frontal cortex was obtained using previously described procedures [4]. neuropathologic diagnosis was obtained from formalin fixed paraffin embedded tissue as previously described [21, 24]. briefly, 22 sections of paraffin embedded tissue were stained for hyperphosphorylated tau (p-tau), alpha-synuclein, amyloid beta, tdp-43, luxol fast blue, and hematoxylin and eosin using previously described methods [22]. a neuropathologic diagnosis of cte was made using national institute of neurological disorders and stroke (ninds) consensus criteria [3, 21]. cte cases could be subdivided based on the presence, extent, and severity of p-tau deposition through the brain. as the focus of the study was to examine early changes, only cases that met the “low stage cte” criteria were used for analysis. the low stage cte designation encompasses cte stage i&ii [2, 21] and shows strong agreement with the recent ninds consensus staging for low stage cte [3]. neuropathologic examination occurred blinded to clinical results. all evaluations were reviewed by four neuropathologists (va, bh, ts, am); discrepancies in the diagnosis were resolved by consensus conference. demographics, athletic history (type of sports played, level, position, age of first exposure to sports and years playing contact sports), military history (branch, location of service and duration of combat exposure), and traumatic brain injury (tbi) history (including number of concussions) were queried during a telephone interview as detailed previously [30]. rna-sequencing 50µg of fresh frozen tissue was taken from the dorsolateral frontal cortex grey matter at two locations for each case: the depth of the superior frontal cortical sulcus (defined as the bottom third of the sulcus) and the associated neighboring gryal crest at the level of the anterior caudate nucleus. frozen tissue was process using a mortar and pestle, and mrna was extracted and isolated using a maxwell rna extraction kit (promega) as per manufactures instruction. an ion apliseq transcriptome human gene expression kit (thermofisher scientific) was then used to convert mrna to cdna and establish human transcriptome cdna libraries as per manufactures instructions. human transcriptome libraries were then sequenced using an ion torrent s5 next generation sequencer (thermofisher scientific). samples were processed and run across two batches. batch one contained 6 control and 6 cte cases, and batch two contained 5 rhi and 4 cte cases and were compared separately to avoid batch effects. normalized counts for each sample were determined using the ion torrent suit software 5.10 and concatenated together into genes by sample count matrix. the normalized counts were then transformed with rlog for principal component analysis (pca). differential expression analysis genes with more than 50% zero counts within each group were filtered out. pairwise differential expression (de) analyses were conducted separately for cte vs rhi and cte vs control using deseq2 bioconductor package, modeling counts as a function of case status adjusting for age at death [18]. to analyze changes between the sulcus and crest within each group, a ratio between the sulcus and crest genes were calculated using the concatenated normalized counts matrix. genes that could not be detected in all samples (genes with 0 counts for all samples) were filtered out. a pseudocount of 0.01 was added to the counts to prevent infinity values from zero divisions. the ratio was then calculated as log 2 of sulcus counts divided by crest counts for each gene within each sample. to attenuate extreme ratios due to small count values in either tissue, the ratios were arcsin transformed by first dividing by 0.5 and then applying the arcsin function using the numpy python package. this procedure results in a log2 fold change of sulcus vs crest for each gene in each sample. to identify genes that exhibited significantly different expression between tissues within each case status, one-sample, two-tailed t-tests comparing with an expected mean log2 fold change of zero were performed for the fold changes of each gene. p-values were adjusted for multiple hypotheses using the benjamini-hochberg procedure. similarly, two-sample t-tests were run to compare the sulcus vs crest fold changes for cte vs rhi, cte vs control, and rhi vs control. gene set enrichment analysis (gsea) gene set enrichment analysis for all de gene lists were performed using the fgsea r package in bioconductor and gene ontology (go) geneset annotations from goatools python package [14, 29]. gsea statistics were calculated using gene list sorted by descending log2 fold change (from de) or log 2 ratio t-statistics, and significance was assessed for gene sets at fdr < 0.1. the resulting normalized enrichment scores (nes) were then used to create heatmaps. gsea biological process were grouped into 1 out of 11 biological categories by hand, based on expert opinion. categories were discussed and confirmed among 3 authors (jc, al, ts). any differences were resolved through consensus conference. the biological categories were: immune, vascular, neuronal, extracellular matrix (ecm), mitochondrial/metabolism, signaling, cytoskeleton, transcription/translation, endocytosis/exocytosis, protein processing, and any process that didn’t fit the previous 10 category was assigned to “other”. finally, gene ontology analysis from common or unique genes groups was carried out using metascape [33]. all code and supplementary information needed to reproduce this analysis is available at https://osf.io/exsfa/. results overall, 3 differential expression analyses were carried out. a summary of each comparison is outlined in figure 1. the first analysis was to directly compare the differentially expressed genes in the sulcus or crest to the matching region across control, rhi, or cte groups (figure 1a, comparison 1). the second analysis was to directly compare the sulcus vs. crest within each group (figure 1b, comparison 2). the final analysis was to compare how the sulcus vs. crest relative ratios of each gene changed across each disease group (figure 1c, comparison 3). figure 1. summary of differential expression analysis used for comparisonsrepresentative images demonstrating how which regions were being used for comparison. a) comparison 1 compares the differentially expressed genes in the sulcus and crest to the corresponding region between control, rhi, and cte. b) comparison 2 directly compares the sulcus to the crest within each group. c) comparison 3 uses the ratio created from comparison 2 and compares that sulcus vs crest ratio from rhi and cte groups to the control group to determine how the sulcal specific genes changes over the course of disease. differentially expressed genes had similar directions of effect in the sulcus and crest we first examined the differentially expressed (de) genes found in the sulcus and crest to identify the genes most affected in early cte compared with control and rhi groups (table 2) (figure 1a, comparison 1). the relative sulcus and crest de genes that were significantly altered (nominal p < 0.05) in at least one region were plotted against each other to examine if there were similar direction changes occurring in the sulcus and crest (figure 2). changes that were consistent across both sulcus and crest could be interpreted as a general frontal cortex wide response. first, we compared cte to control (figure 2a). we observed that 95% of genes had the same direction of effect in both the sulcus and crest. only 109 (5.0% total de genes) genes had an opposite direction of effect (figure 2a). when examining the changes between cte and rhi, we observed fewer genes were altered compared to cte vs control (table 2). almost all of the genes had the same direction of effect (figure 2b). only 2 genes (0.4% total de genes), jmjd6 and vat1l, were elevated in the sulcus but decreased in the crest. the genes with the largest change, in addition to specific genes of interest were highlighted. a full list of de genes for the cte vs control and cte vs rhi analysis can be found in supplementary file 1. figure 2. comparative analysis of the differentially expressed genes found in the sulcus and crestanalysis of de genes from the sulcus and crest plotted against each other comparing (a) cte vs control and (b) cte vs rhi. genes were plotted if they were significantly altered (p<0.05) in at least one region. each dot represents one gene. genes of interest and the top expressed have been annotated. gene set enrichment analysis (gsea) was then performed on each set of genes to characterize biological processes that are altered in cte (table 3). each gsea process was then assigned to 1 of 11 overall biological categories to better contextualize changes (figure 3). when comparing cte vs control (figure 3a), 81 processes were observed to have an fdr adjusted p < 0.1 in the sulcus or the crest. 41 processes were positively enriched and 34 were negatively enriched in the same direction in both the sulcus and crest. a complete breakdown of group assignments can be found in table 3. the majority of positively enriched processes were involved the immune category with 70.7% of total processes. conversely, only 14.7% of the negatively enriched processes were immune processes. neuronal processes were the largest group of negatively enriched processes with 35.5%. there were fewer positively enriched neuronal processes (4.9%). most signaling processes were negatively enriched (20.6%) compared to positively enriched (4.9%). mitochondria processes were only found to be negatively enriched, while vascular processes were only found in the positively enriched group (table 3). 6 processes were found to have a mixed direction of effect. of those 6 processes, 2 were immune and 4 were extra-cellular matrix (ecm) related (table 3). for cte vs rhi (figure 3b), 44 processes were observed to have an fdr adjusted p < 0.1 in the sulcus or crest. 36 processes were positively enriched, and 7 processes were negatively enriched in the same direction. the largest positively enriched category was immune processes with 38.9%. no immune processes were negatively enriched. ecm processes were the second largest positively enriched category with 30.6% of processes. no ecm processes were negatively enriched. neuronal processes were the majority of negatively enriched processes (85.7%) and were higher than positively enriched neuronal processes. the only other negatively enriched process was transcription/translation (14.3%). vascular, signaling, cytoskeletal, endo/exocytosis, and other processes were all similarly expressed. one process, “signaling receptor activity”, from the signaling category, was decreased in the crest and increased in the sulcus (figure 3). figure 3. heatmap of sulcus and crest gsea biological processesusing the de genes from the sulcus and crest, gsea biological processes were derived and plotted in a heatmap comparing (a) cte vs control and (b) cte vs rhi. processes were included if they were significantly enriched (fdr adjusted p < 0.1) in at least one region. each gsea biological process was assigned to 1 of 11 biological categories to better identify overall changes. [please click on the figure to download a high-resolution version] there were significant differences in the sulcus compared to the crest we next chose to directly compare the sulcus and crest within the controls, rhi, and cte groups to determine which genes were specifically altered in the sulcus compared with the crest by computing an arcsin transformed log2 ratio of sulcus vs crest normalized counts within individuals (see methods) (table 4) (figure 1b, comparison 2). alterations within each group could be related to the unique aspects of each condition. changes in the control sulcus are related to general anatomy, rhi sulcal changes are related to repetitive head trauma induced damage, and cte sulcal changes were related to p-tau deposition. within controls, we observed 671 genes had an fdr adjusted p < 0.1. of the fdr corrected genes, 579 were increased in the sulcus and 92 were decreased in the sulcus compared to the crest (figure 4a). for cases in the rhi group, 1926 genes were found to have a nominal p < 0.05. however, only 1 gene, manea, reached fdr corrected significance. of the nominally significant genes, 1127 were increased and 799 were decreased in the sulcus compared to the crest (figure 4b). in subjects with cte, 1147 genes reached fdr adjusted p < 0.1. 603 genes were elevated in the sulcus and 433 genes were decreased in the sulcus compared to the crest (figure 4c). the top 10 increased and decreased genes by log2 fold change were annotated in each plot. a full list of sulcus vs. crest gene changes can be found in supplementary file 2. figure 4. genes that are differentially expressed in the sulcus compared to the crestthe sulcus was directly compared to the crest across (a) control, (b) rhi, and (c) cte and sulcal specific de genes were calculated. each dot represents one gene. dots that are red represent genes that met fdr adjusted p < 0.1 significance. the top 10 positive and negatively enriched genes were annotated. gsea analysis clarifies biological response processes that are occurring in the sulcus in each condition to further explore how the sulcal specific response contributed to the larger biological response, gsea was performed using the genes that were significantly altered in the sulcus compared to the crest. using fdr adjusted p < 0.1, 301 significant processes were observed in controls, 78 in rhi, and 129 in cte. the observed processes represented biological changes specific to the sulcus compared to the crest in each relative group. we then wanted to compare enrichment of each biological process across groups and determine if there were similar or different directions of effect (figure 5). for added clarity and biological relevance, each process was assigned to 1 of 11 greater biological categories similar to figure 3. a heatmap was then created using processes that were significantly enriched in at least one category (figure 5). the immune category had 51 (13.7%) processes. the majority of processes were observed to be elevated in the sulcus compared to the crest across all 3 sample groups. however, among the cte group there was an increase in the number of processes that had a negative enrichment in the sulcus compare to the crest (figure 5a). next, there were 30 (8.0%) transcription/translation processes, the majority of which were positively enriched in the sulcus compared to the crest (figure 5b). the category with the most assigned processes was the neuronal category with 99 (26.5%) processes. almost all of the neuronal processes demonstrated a negative enrichment in the sulcus. of the few processes that did have a positive enrichment, there was a trend towards decreasing after rhi and during cte (figure 4c). next, there were 9 (2.4%) cytoskeletal processes. the ecm category had 34 (6.4%) processes. all the processes were positively enriched in control cases. however, there was a possible progressive increase in negatively enriched processes through rhi and cte (figure 5e). mitochondria had 24 (6.4%) associated processes. the rhi group only had 1 negatively enriched pathway while control and cte had a mixed split of positive and negative processes (figure 5f). the signaling category had 56 (15.0%) processes and was evenly split between positive and negative enrichment (figure 5g). the vascular category had 14 (8.0%) processes. the control group was entirely positively enriched in the sulcus but there was a switch to more negatively associated processes in rhi and cte (figure 5h). the endocytosis/exocytosis category had 13 (3.5%) processes and was primarily negatively enriched in the sulcus (figure 5i). the protein processing category had 16 (4.3%) processes and was a mix of positive and negative processes. there appeared to be an increase in negative processes associated with cte (figure 5j). finally, the 27 processes that did not fit into the previous 10 categories were placed into “other” (figure 5k). overall, throughout all 11 categories, there was a high agreement in the direction of effect in all cases. 73 (21.2%) processes did not have the same direction of effect. figure 5. heatmap of sulcal specific gsea biological processesusing the genes that were significantly altered in the sulcus compared to the crest, we used gsea to identify biological processes. each process was then assigned to a greater overall biological category: a) immune, b) transcriptional/translational, c) neuronal, d) cytoskeletal, e) extracellular matrix, f) mitochondria/metabolism, g) signaling, h) vascular, i) endocytosis/exocytosis, j) protein processing, k) other. [please click on the figure to download a high-resolution version] next, using the ratio that was created by directly comparing the differentially expressed genes in the sulcus to crest within each group, we compared how those ratios changed in rhi and cte relative to control (figure 1c, comparison 3). this analysis provided additional information into how genes which were increased in the sulcus compared to the crest change after exposure to head trauma and p-tau pathology. 87 processes were found to be significantly altered in rhi vs control, and 127 processes were altered in cte vs control. the same 11 greater biological processes were used to group gsea processes and all processes that were significant for at least one of the two comparisons were included in a heatmap (figure 6a-k). there were 54 immune processes altered (figure 6a). this was the largest number of altered processes compared to the other 10 categories. the majority of immune processes were observed to be decreased compared to controls. transcription/translation was the second largest with 46 processes being altered (figure 6b). the neuronal category had 31 processes and the majority were observed to be increased compared to control (figure 6c). cytoskeletal had 16 processes and had more processes that decreased compared to increased (figure 6d). ecm had 25 processes and all of them were decreased compared to control (figure 6e). mitochondria had 21 processes and the majority were increased compared to controls (figure 6f). signaling had 44, with the majority being decreased compared to control (figure 6g). vascular had 16 processes with all but one observed to decrease compared to controls (figure 6h). endocytosis/exocytosis had 9 processes with an even split of changes (figure 6i). protein processing had 22 processes with an even split of changes (figure 6j). finally, the 12 processes that didn’t fall in the previous 10 categories were assigned to an “other” category and were all decreased compared to control (figure 6k). overall, rhi and cte had similar changes compared to controls. for rhi-control and cte-control comparisons that did not have the same direction of effect, cte-control comparisons always had reduced enrichment of those processes. figure 6. heatmap of sulcal specific gsea biological processes that change in rhi and cte compared to controlthe rhi and cte gsea processes that were found to be specifically altered in the sulcus were compared to the control. each process was again assigned to a greater overall biological category: a) immune, b) transcriptional/translational, c) neuronal, d) cytoskeletal, e) extracellular matrix, f) mitochondria/metabolism, g) signaling, h) vascular, i) endocytosis/exocytosis, j) protein processing, k) other. [please click on the figure to download a high-resolution version] common and unique genes found in the sulcus across disease groups finally, using the differentially expressed gene results comparing the sulcus to the crest (figure 1b, comparison 2), we wanted to examine if there were any sulcal specific alterations that were unique or common across each group (figure 7). first, we examined what sulcal specific genes were commonly altered in all three groups. 333 genes were differentially expressed in the sulcus compared with the crest among all three groups (figure 7a). 331 of 333 genes had the same direction of effect among all three diagnostic groups (figure 7b). only 2 genes, kiaa2022 (up in rhi) and plekhh2 (down in rhi), had a conflicting direction of effect in rhi compared to control and cte. to better understand the biological relevance of those common genes, gene ontology (go) analysis was performed (figure 7c). the most highly significant go term of the common genes was “gliogenesis”, suggesting an altered glial response is an innate feature of the sulcus compared to other brain regions. when examining the rest of the top 20 significant processes, many processes were observed to encompass development, homeostasis, and organization. we then wanted to determine which differentially expressed genes in the sulcus compared with the crest were unique to each diagnostic group. a stringent criteria was used to avoid including any gene that was significant in one group but also had p values close to significant in the other two suggesting they could be common genes if power were increased. therefore, only genes that had a nominal p < 0.05 in one group and a p > 0.25 in all others were marked to be “uniquely” altered. 1074 genes were found to be uniquely altered in the control sulcus with 806 being increased and 268 being decreased compared to the crest (figure 7d). 168 genes had an fdr < 0.1. when observing the go processes of the unique control genes, developmental and immune regulation processes appeared to be the most prominent (figure 7e). 389 genes were found to be unique in the rhi group. 192 genes were increased, and 197 genes were decreased in the rhi sulcus (figure 7f). no rhi unique gene met fdr corrected significance. using go analysis, it was observed that many of the processes were metabolic or transcriptionally related (figure 6g). finally, 531 genes were uniquely altered in the cte sulcus. 239 genes were increased, and 292 genes were decreased in the sulcus relative to the crest (figure 7h). 136 genes met frd corrected significance < 0.1. go analysis of cte specific biological processes demonstrated immune and cell damage biological processes (figure 7i). figure 7. analysis of common and unique genes and biological process found in the sulcususing the genes that were found to be significantly altered in the sulcus compared to the crest, we compared which genes were common to all groups and which were unique to each condition. a stringent criteria was used to avoid including any genes that were significant in one group but also had p values close to significant others suggesting they could be common genes if power was increased. therefore, only genes that had a p < 0.05 in one group and a p > 0.25 in all others were marked to be “uniquely” altered. a) a venn diagram of the common and unique genes found among all three groups. b) a heatmap of the common genes showing similar directions of effect of the majority of genes. c) go analysis of the common genes. d) a plot of the unique sulcal genes found in control cases. e) go analysis of the unique control genes. f) a plot of the unique sulcal genes found in rhi cases. g) go analysis of unique rhi genes. h) a plot of the unique sulcal genes found in cte cases. i) go analysis of unique cte genes. in all plots, red dots denote genes that met fdr adjusted p < 0.1. go analysis was performed using any gene that met nominal p < 0.05 significance. the top 5 positive and negative genes for each group were annotated. [please click on the figure to download a high-resolution version] discussion building on previous work suggesting that altered neuroinflammation is an early event in cte, here we have demonstrated that there is a complex inflammatory response encompassing several unique biological categories occurring specifically in the sulcus and might be directly related to cte pathogenesis. when comparing the sulcus to the neighboring gyral crest we observed that although there was pronounced gene expression related changes present in both regions, there was a further amplified and specific sulcal alterations that might be related to head trauma and cte pathogenesis. when comparing what genes and biological processes were altered in the sulcus compared to the crest, several higher order biological categories were observed to consistently change across control, rhi, and cte. immune, transcriptional/translation, ecm, and vascular categories all were positively enriched in the sulcus while neuronal and endocytosis/exocytosis processes were commonly negatively enriched in the sulcus. these findings demonstrate that even in the absence of rhi exposure or cte pathology, the sulcus had a unique expression pattern compared to the gryal crest and might help partly explain the susceptibility to head trauma and tau deposition. when examining how the sulcal specific alterations might change in response to trauma and disease, in the rhi and cte group there was a complex mixed increase and decrease of inflammatory processes such as astrocyte development, scavenger receptor activity, regulation of immune response, leukocyte migration, and antigen processing compared to the controls. additionally, neuronal processes were elevated after rhi and during cte relative to control cases, possibly suggesting injured neurons might induce compensatory mechanisms in efforts to mitigate rhi induced damage. these complex alterations could also be observed in transcriptional/translational, cytoskeletal, ecm, mitochondria/metabolism, signaling, vascular, endocytosis/exocytosis, and protein processing processes as well. finally, we observed that each of the three groups had a set of genes and processes that were uniquely altered in the sulcus. go analysis demonstrated that within the control group, specifically altered genes were related to development and homeostasis; within rhi, altered genes were metabolic and mitochondria related; and within cte, altered genes were immune and inflammation related. overall, we have built on previous work and expanded our understanding of the complex neurodegenerative response that occurs after rhi and during cte. since p-tau deposition in the crest is less likely in early cte, genes that were found to be differentially expressed in both the sulcus and the crest are possibly less specific to cte sulcal p-tau deposition and more related to a general response to head trauma. consistent with previous reports on how repetitive head trauma affects the cns, the most common genes that were altered in both the sulcus and crest in cte were immune and inflammatory related (figures 2&3). we observed that there was a significant upregulation in complement genes c4a and c4b suggesting a persistent innate immune response and activation of the complement cascade was present in the frontal cortex. complement has a wide variety of functions, but in the context of neurodegeneration, it has been associated with increased synaptic pruning and synaptic loss [27]. it is possible the c4 activity is related to the psd-95 synaptic protein loss that has been reported in previous cte studies [5]. c4 has been suggested to play a role in synaptic pruning in schizophrenia [28], and c4 gene expression and protein levels have recently been linked to tau phosphatases and the development of ad suggesting a possible mechanism in the development of tau pathology [15]. additionally, microglia related genes, such as cxcr1, were elevated in cte, which is in agreement with previous studies examining microglia related changes in cte [4, 7]. astrocytic genes, gfap and aqp1, were also found to be increased in both the sulcus and crest. it has been suggested that there is impairment of aqp4 after trauma, and it is possible that aqp1 was upregulated in compensation [31]. interestingly, there also appeared to be a significant neutrophil response found across the sulcus and the crest. several neutrophil related genes such as cd177, s100a8, s100a9, bpi, and lcn2, and multiple neutrophil gsea processes were highly increased. however, it is possible that the observed neutrophil response could be related to various causes of death as opposed to contact sport exposure. future work will be needed to closely examine if neutrophil markers could be observed using biomarkers from living individuals to better explore the possible effects of tbi and a long-term neutrophil response. altogether, these results demonstrate that there is a prominent neuroinflammatory response found in the frontal cortex of subjects who experience repetitive head trauma. however, it is likely that changes that occur uniformly in both the sulcus and crest do not fully explain the mechanisms behind p-tau deposition and are only part of the equation of cte mechanisms. therefore, it was crucial that we expand our analysis to focusing on what changes could be observed uniquely in the sulcus and might directly relate to cte pathogenesis. interestingly, we found that multiple factors might be contributing to sulcal specific alterations. our findings suggested that sulcal specific changes were the result of either 1) anatomy, 2) repetitive head trauma, and 3) p-tau pathology. although the sulcus and crest are only separated by centimeters, our current result suggest that each region has a unique environment even in the absence on trauma or neurodegenerative disease. when comparing the sulcus to the crest across each group, there was high agreement in the direction of effect observed in all the processes between control, rhi, and cte. these results provide novel information on basic neuroanatomy of how closely related tissue regions might be significantly different and result in unique neuropathologic features. to our knowledge, this is the first study to report such findings. the largest category of processes was found to be neuronal, and was downregulated in the sulcus compared to the crest. there are two possible explanations for this, either the sulcus has less neurons and supporting cells or there is a persistent neuronal impairment found in the sulcus across all groups. regardless which is correct, it is possible that an impaired or reduced neuronal response might predispose the sulcus to elevated damage post head trauma. the next largest category of processes were immune related. interestingly, the majority of these processes were elevated compared to the crest. this suggests that the sulcus might already be “primed” to have an exaggerated immune reaction. the primed phenotype could be consistent with having more glial present or the current glia might be predisposed to have a more severe immune reaction. additionally, the sulcus was observed to be positively enriched in ecm and vascular processes compared to the crest, further demonstrating the sulcus is comprised of a unique environment that responds differently during pathology compared to other neighboring regions. although it is likely the physics of head trauma induced damage and sulcal force concentration are the main driver of sulcal specific pathology [11], the findings reported here could describe additional mechanisms explaining how the sulcus is preferentially affected after repetitive head trauma [3]. consistent with our hypothesis and previous observations, repetitive head trauma was also a significant mechanism driving sulcal specific genetic alterations. the cte and rhi group were observed to be very similar in many genes and biological processes that were altered in the sulcus compared to controls (figure 5). the changes that were observed to be consistent between the rhi and cte group could be interpreted as trauma related alterations, as a common feature of both groups is they share a history of repetitive head trauma received from playing american football. it was unexpected to observe more than half of the of immune biological responses in rhi and cte were decreased in the sulcus compared to control. previous work had suggested that neuroinflammation was a prominent feature of head trauma and likely related to initial p-tau deposition [7]. when examining which biological processes were altered in the sulcus, it was observed that the upregulated processes were more related to foreign object recognition, antigen presentation, and lysosomal function, while the downregulated processes were related to inflammatory cytokine production and other elements related to neurodegeneration. this is consistent with our previous study demonstrating that after repetitive head trauma there was relatively unchanged numbers of total microglia, but increased levels of cells positive for cd68, a lysosomal marker that is elevated during increased phagocytosis and innate immune activation [8] furthermore, tmem106b genotype, which is related to lysosomal and phagocytic alterations, has been also found to relate to cte severity [5]. overall, the surprising downregulation of multiple immune-related processes may suggest that an impaired immune response following rhi predisposes individuals to developing initial tau pathology cte. future studies examining cell type specific alterations and the effects of immune-related genetic polymorphisms will be necessary to test this hypothesis. although the cte and rhi group did overlap for many alterations, we observed that the predominant unique rhi genes and go processes were related to mitochondrial and metabolic activity. rhi is a group of cases that is defined by having exposure to repetitive head trauma, but no neurodegenerative pathology. therefore, it is intriguing to observe that there was a strong metabolic deficit present even before pathology occurs. mitochondrial activity is heavily tied to the health of the cell and any impairments could directly impact cell function. these results suggest that after a history of repetitive head trauma, mitochondrial dysfunction might be the main element driving early symptoms and pathology. additionally, there have been several reports of mitochondria related dysfunction occurring as a consequence of head trauma [13]. it is unclear if there are a specific cell type that is most affected or what might be upstream of the mitochondrial dysfunction, but these results point towards another possible therapeutic target to treat disease before p-tau deposition even begins. finally, we investigated the contribution of p-tau pathology to the sulcal specific genetic alterations. when directly comparing sulcal specific changes found in rhi and cte, very few differences were observed. this was unsurprising as our cte group was restricted to cases with low stage cte and had minimal tau pathology. therefore, it is likely that exposure to repetitive head trauma was a stronger driving force for sulcal specific changes than p-tau pathology in these individuals. it is also likely that many genes related to repetitive head trauma are part of the p-tau deposition mechanisms and are not necessarily unique to cte. rhi and cte might not be district pathologic groups, rather they exist in a spectrum. however, we were still able to identify unique genes found altered only in the cte sulcus and therefore, likely related to p-tau deposition. the main cte unique genes and go processes were related to immune and cell damage further highlighting the possible connection between p-tau deposition and the immune system. it is unclear if these unique cte genes are a consequence of p-tau deposition or act as a mechanism that, in addition to other trauma related pathways, could lead to p-tau deposition. however, these observed unique cte biological processes are novel targets for future biomarkers studies to help identify early stage disease. there are several limitations in this study. rna-sequencing was done on bulk tissue so no comments on the contribution of individual cells can be made at this time. future work utilizing single cell technologies will be needed to determine the specific effects of each cell on the sulcal environment during cte. furthermore, it is difficult to determine the full mechanistic effect in studies using post-mortem human tissue, as we can only look at a single point in time at the end of the donor’s life. mechanistic studies using mouse models or cell culture systems will be needed to determine if the observed genes are mechanistically related to pathologic or healthy physiologic responses. additionally, due to the inherent variation that is present in human studies, a larger sample size is needed to identify more subtle changes. as the present study only focused on early disease, future work is also needed to compare the changes found in low stage disease to high stage cte to determine if processes are similar. finally, as all of the observed changes were based on mrna, proteomic and histologic studies will be needed to verify if the results are also found at the protein level. in conclusion, these results provide clear evidence that there is a complex molecular response occurring in the sulcus compared to the gyral crest. even without exposure to repetitive head trauma, the sulcus had a persistent immune response. in response to rhi, there was immune alteration, metabolic/mitochondria dysfunction, vascular, and ecm impairment. during cte pathogenesis, the immune response found in low stage cte was not observed to be tissue destruction, cell loss, overt neuroinflammation, or other neurodegenerative related responses, rather, it was more focused on antigen presentation, phagocytosis, and wound repair responses. in addition to complex immune changes, early mitochondrial, vascular, ecm, and neuronal activity are all affected during repetitive head trauma induced damage and early tau deposition, and represent possible avenues to halt cte pathogenesis and prevent disease. these results help refine our understanding of the neuroinflammatory environment present during disease and will help guide the discovery of future therapeutic strategies and novel biomarkers. future studies will be crucial to further dissect and study the observed biological processes provided here to better understand the full spectrum of early disease. acknowledgements this work was supported by grant funding from: nih (u19-ag068753, ag08122, ag054076), nia (ag057902, ag06234, rf1ag054156, rf1ag057768), ninds (u54ns115266, u01ns086659, and k23ns102399), national institute of aging boston university ad center (p30ag013846; supplement 0572063345-5); department of veterans affairs 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biologist-oriented resource for the analysis of systems-level datasets. nat. commun 10: 1523 https://doi.org/10.1038/s41467-019-09234-6 copyright: © 2021 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. neuropathology through the ages my life between neurology and neuropathology feel free to add comments by clicking these icons on the sidebar free neuropathology 1:25 (2020) reflections neuropathology through the ages my life between neurology and neuropathology kurt a. jellinger neurobiology, vienna, austria corresponding author: kurt a. jellinger · institute of clinical neurobiology · alberichgasse 5/13 · a-1150 vienna · austria kurt.jellinger@univie.ac.at submitted: 12 august 2020 accepted: 12 august 2020 copyedited by: katy lawson published: 27 august 2020 https://doi.org/10.17879/freeneuropathology-2020-2945 additional resources and electronic supplementary material: supplementary material keywords: neuropathology, vienna, personal reflections introduction when the editors of free neuropathology, werner and tibor, approached me about writing an autobiography for the journal, similar in scope to sam ludwin’s excellent piece two years ago [1], i confess that i had reservations about writing an autobiography, because i never wanted to talk about myself. however, after a lengthy hesitation and eager discussions with my wife and my former scholar and friend, hans lassmann, i realized with a certain degree of doubt that, on the threshold of age 90 years, i could perhaps provide some thoughts about the bridges between clinical neurology and neuropathology for those who might be interested in the experiences of an old neuroscientist. thus, in lieu of concentrating on my career as neuropathologist, i wish to instead write about my life in the context of unifying these intrinsically linked but disparate areas within the rapidly progressing scientific world: neurology and neuropathology. this long, and not always easy, path through the wide field of practical and theoretical neuroscience, as well as other interests, was influenced by my late mother, my beloved wife elisabeth, colleagues, patients, friends, and students whom i met during my professional and private life. this essay is intended to encourage young colleagues to not concentrate exclusively on experimental neuropathology without also considering the practical part of the neurosciences since there is an urgent need to inspire young neurologists to pursue a double career as both a clinician and neuroscientist in order to promote progress in the neurosciences. why the appeal of clinical neurology and neuropathology? there are three principal reasons to try to unify these two closely connected fields of neuroscience. first and foremost is the aim to help patients suffering from disorders of the nervous system by trying to make an early diagnosis, provide effective treatment, and promote preventive measures. second, is the ongoing need to apply ever-evolving basic scientific approaches to diseases of the nervous system in order to support the clinical neurologist in elucidating the pathogenesis of disorders afflicting their patients. neuropathology should be pursued not just for its own sake but to aid the clinician with the mutual goal to improve diagnoses and enable successful therapeutic strategies for many hitherto incurable disorders. lastly, there is inherent fascination and fulfillment in using our knowledge to elucidate the structure and function of the nervous system in the healthy and diseased human individual. based on this knowledge, the neuropathologist is able to elucidate much of the background of disorders of the nervous system using immunohistochemical, molecular, biological, and ultrastructural methods not only in autopsy material but also tissues from living patients, including biopsies, cerebrospinal fluid, and blood. often, these images are comparable to modern art (fig. 1). the selective and interdisciplinary application of these methods may enable the neuropathologist to not only make a reasonable diagnosis of many, though not all, disorders of the nervous system but may also provide insight into the complex pathogenetic cascades that are responsible for the onset and progression of such disorders. in this respect, the neuropathologist is privileged to act as both a physician and a basic scientist. this retrospective will consider highlights and drawbacks in my 63 years of life between clinical neurology, neuropathology, and private activities. given my many years of work and limited space herein, may my friends, scholars, colleagues, and co-workers excuse that i regrettably could not consider all of them in this review. early life and schooling i was born may 28, 1931 in vienna, as the only child to rosa and alois jellinger, both with roots from upper austria and moravia (near olomouc). my father was an official in the government of lower austria; my mother, a trained dressmaker, was a housewife. in the prewar years, i had a wonderful and well-kept childhood filled with summer vacations in the beautiful surroundings of vienna and in the idyllic countryside, while in the city there were increasing tensions, poverty, and changes in the political atmosphere. i remember kurt schuschnigg's radio speech of march 11, 1938, with his concluding sentence “may god protect austria,” after which my father commented, “this will be the beginning of the end.” after austria's “anschluss” to the german reich, i attended a public primary school in vienna and later, during world war ii, a public secondary school (realgymnasium, also in vienna), where i received basic education in languages, natural sciences, geography, and history. in 1943, to escape from air raids on the city, many children were sent to live with farmers in the batschka, then southern hungary, now serbia. once there, we had a wonderful and peaceful time, though the atrocity of witnessing the pogroms of jewish people was never far from sight or mind. on our way home we passed the then peaceful city of budapest, which i would see again during the hungarian revolution in 1956. once returned to vienna, in 1944, we had to change school buildings due to bombing damages. air raids were still so frequent that i often did not make it home in the evenings but had to instead seek shelter along the way. in september of 1944, we lost our home due to an air raid and had to wait for a new flat. around this time, i attended one of the last performances of schiller's wallenstein trilogy of plays featuring the famous actor werner krauss in the vienna burgtheater. soon afterwards, all theaters were closed. on april 13, 1945, during the battle between german and russian armies in the city of vienna, i had my first medical “experience” by cleaning up brain tissue of russian soldiers who were killed by the projectile from a german tank. after my father died in a russian pow camp in 1947, my mother and i were left alone to overcome the difficult times after the end of the war. she worked in her former profession as a dressmaker and i became tutor of children. in 1949, i graduated high school summa cum laude. in that final year, our class contained 18 boys and, when we had our final exams, i got excellent marks (fig. 2). after graduation, our class, led by our latin teacher, traveled to rome and naples, where we enjoyed arts, cuisine, and life in italy. to this day, i remain grateful to my many teachers who encouraged me to liberally approach history and natural sciences. years later, one of my favorite high school teachers became a patient in my clinic with myasthenia gravis, which we treated with success. over the years, my classmates and i met several times at reunions to remember and discuss previous experiences. the last meeting was 2009 to celebrate the 50th anniversary of our graduation. fig. 1. (a) ms-like autoimmune encephalitis after repeated subcutaneous injections of lyophilized calf brain cells in male aged 51 years with hemi-parkinson syndrome. left hemisphere with prominent periventrivular demyelinated lesion and multiple demyelinated plaques in cortex and subcortical nuclei. from [2] (b) em image of focal (core) plaque in hippocampus showing dense accumulation of thick amyloid fiber bundles, surrounded by dystrophic neuritic endings and hyperintense structure of myelinated axon. (x 4000). photo: k.a. jellinger. (c) major histopathological changes in alzheimer’s disease. (c1) amyloid deposits in the neuropil (plaques) and vasculature (caa) (congo red stain). (c2) neuritic plaque with dystrophic neurites (bodian stain). (c3) neurofibrillary tangles (large arrows) and neuropil threads (small arrows) (bodian stain). from [3]. medical school and beyond in 1949, i entered the medical faculty of the university of vienna, where the circumstances of medical education were made far more difficult due to the sequelae of the war. for example, we worked in the anatomy theater in winter with glassless windows and without warm water. despite this, and with eight of us were working on a corpse, we succeeded thanks to the influence of our professor, alfred gisel, a wonderful teacher and enthusiastic anatomist, who, in addition, was medical chief of the austrian red cross. the curriculum had basic subjects like anatomy, chemistry, physics, and physiology for 2 years, followed by 3 years of theoretical and practical training, beginning with pathological anatomy, physiology, pharmacology, and clinical specialties. most of our teachers were outstanding, some of them scientists of high reputation, such as: prof. h. tuppy, chemistry (nobel price candidate); prof. f.t. brücke, pharmacology; prof. g. schubert, physiology; and prof. h. chiari, head of pathological anatomy. during my studies, and despite regular grants, i supported myself with work as a paramedic for physically disabled people, as a tutor for children and students, and as a writer of short stories for newspapers and magazines. our clinical teachers were very dedicated and regularly organized seminars and clinical practicums, but the majority of teaching was much less practical but more formal and didactic than in the new curricula. in december 1955, i finished my medical studies, but had to wait for my graduation due to administrative delay since mine was the first “promotio sub auspiciis praesidentis rei publicae,” the highest possible distinction for academic achievements in austria, in medicine since the end of world war ii. in the meantime, i worked as guest doctor without salary in the franz josef hospital in vienna, within the department of internal medicine and surgery, directed by prof. herbert kraus, later chairman of the first department of neurosurgery of vienna medical school. on april 14, 1956, i was promoted to doctor of medicine “sub auspiciis praesidentis rei publicae,” which i received in the uniform of an officer of the austrian red cross (fig. 3). hospital years and first experiences in 1956, once the soviet army left budapest subsequent to the bloody uprising of the hungarian people, i went with a red cross convoy to deliver medical supplies and blood bottles to the liberated citizens of budapest. we were lucky to return to vienna before the russian army reconquered budapest. i enjoyed clinical medicine but my major interest was neuropathology. on january 5, 1957, i began as a postdoctoral fellow (wissenschaftliche hilfskraft) at the neurological institute (ni) a.k.a. obersteiner institute of the vienna medical school headed by hans hoff, who was also chairman of the neuropsychiatric clinic. founded by heinrich obersteiner in 1882 as a box that was subsequently expanded by obersteiner's personal expenses, the ni became the first interdisciplinary institute of neurosciences and was visited by many scientists from japan, the usa, and other countries. obersteiner edited a series of publications under the title “arbeiten aus dem obersteinerschen institut” and books about basic neuroanatomy and pathology. together with the edinger institute in frankfurt, the ni became one of the leading centers of neurosciences in the world until the beginning of world war ii, when it was directed by oskar gagel, an experienced neurooncologist. the ni, together with the institute of histology, occupied the first floor in a building of preclinical institutes, next to the general hospital of the vienna medical school (allgemeines krankenhaus/akh). parts of this area had been bomb-damaged during the war. fig. 2. our graduation class 1949 together with h.r. klieba, principal of the realgymnasium wien viii, and dr. seifert, our class teacher. kj second left in the first row. my first venture into neuropathology was to save and reorganize the large collection of slides that included material from major brain diseases. it was through this work that i learned the basics of neuroanatomy and neuropathology. the ni had a huge library, originally compiled by obersteiner himself, which was a treasure trove of rare and antique books, including an original edition of thomas willis' “circulus arteriosus cerebri,” one of the first descriptions of brain vasculature. in 1958, prof. franz seitelberger became director of the ni (fig. 4). in the same year, i met and immediately fell in love with elisabeth, a beautiful and clever girl aged 21 years, born in brno (then cssr), who studied english, russian, and history of arts. we attended theaters, operas, concerts, and frequently met in the university library. elisabeth and i married on august 17, 1960. fig. 3. promotion to doctor of medicine 1956 in the uniform of a red cross officer, with dr. heinrich drimmel, federal minister of education and science. training progress during the following years, i was trained by franz seitelberger, an internationally renowned neuropathologist with particular interest in metabolic and neurodegenerative diseases of the nervous system, who later became dean and rector of the university of vienna. i was in charge of brain autopsies (600 to 1000 per year) and diagnostic workup of neurosurgical biopsies. my early scientific work concerned acute demyelinating encephalitis following injections of lyophilized calf brain cells in a patient suffering from parkinson's disease (pd) [2] (fig. 1a). this particular case, which closely resembled those of patients who received rabies vaccinations in japan, was later re-published as acute autoimmune demyelinating encephalitis [4]. other projects included studying the neuropathological side effects of the 1958 influenza epidemic and the neuropathology of spring-summer (tick-borne) encephalitis in austria, which we described in detail. in parallel to my work at the ni, i underwent specialization in neurology and psychiatry as a guest doctor at the university clinic of neurology and psychiatry in vienna, headed by hans hoff, between january 1958 and december 1963. among my tutors were klara weingarten, an excellent clinical neurologist, and franz gerstenbrand, later chairman of the clinic of neurology, medical university innsbruck. we examined patients, both clinically and neuropathologically, with chronic coma or apallic syndrome (now unresponsive wakefulness syndrome) following head injury and those who suffered hypoxic brain damage and found a close relationship between the pattern of brainstem lesions due to intracranial pressure, the state of consciousness, and clinical outcome. in addition to hospital work, i was assistant to prof. hoff's lectures in neurology, which was often difficult since he wanted the patients' histories and clinical statuses to be in exact accordance with his neurology book. i also heard lectures by prof. herbert reisner, an excellent neurologist and psychiatrist, who later became chairman of the clinic of neurology of the university of vienna. after training in neurology, i worked in the division of psychiatry, where i got experience in all kinds of psychiatric disorders. on free weekends, i worked as a voluntary health officer with the red cross. on january 1st, 1960, i was promoted from assistant to consultant (oberarzt) at the ni. i studied encephalitis and myelitis following poliomyelitis vaccination and movement disorders (progressive pallidum atrophy and striatonigral degeneration) with erwin neumeyer, who unfortunately died early. in 1961, elisabeth and i attended the international congress of neuropathology in munich where we met all the top scientists in this field. in 1962, after a lecture on chronic vascular myelopathy at the 25th reunion neurologique in paris, i became membre associé à titre ètranger (associate foreign member) of the french neurology association. during that time, many excellent neuroscientists visited the ni and lectured there, such as hugo spatz, hans jacob, wilhelm krücke, chief of the max planck institute of brain research in frankfurt, webb haymaker, chief of the armed forces institute of pathology (afip), and ludo van bogaert of bunge institute in antwerp, belgium, for whom i had the honor of translating his lectures. a number of postdocs and research fellows, particularly from japan, were trained in the ni, such as mansori tomonaga, professor at tokyo university (his charming wife was a wonderful soprano singer), riki okeda (who counted cells in the pons during the the night while listening to classical music), and many others (see [5]). it was an exciting time in the international neuroscience community! between february and august 1963, i worked part-time as guest at the institute of pathology, vienna medical university, chaired by hans chiari, where i performed general autopsies and brain cutting. i detected the first case of clinically-suspected creutzfeldt-jakob disease (cjd), which i “stole” in order to study it histologically, with the consequence that i was dismissed from the morgue. this was the end of my training in general pathology, but the relations with this department remained excellent. between 1963 and 1974, i additionally worked two afternoons each week as chief of the outpatient service of the anton proksch institute, a therapy facility for alcoholism and addiction, in order to supplement my low salary as a university assistant. with wichard kryspin-exner, later chairman of the department of psychiatry, medical university of innsbruck, a number of papers about the course and prevention of alcoholism were published. full-time neuropathology in the following years, in addition to routine postmortem neuropathology and diagnostic biopsy work, i examined vascular spinal cord pathology and commented on zülch's vascular “borderline zones.” other subjects included spinal cord injuries, in particular, cervical hyperextension trauma with vascular complications. in january 1963, i earned board certification as specialist (facharzt) in neurology and psychiatry and returned as a full-time consultant to the ni. fig. 4. briefing with prof. f. seitelberger (left) and dr. h. hoff (right) in the neurological institute, 1958. in 1965, a postmortem study of a series of anoxic-vascular brain lesions resulting from complications of open heart surgery caused quite an outcry in the surgical community, but, as a consequence, the incidence of complications was reduced due to changed modalities and techniques in cardiac and vascular surgery. at the international congress of neuropathology in zurich, i gave a lecture on the neuropathology of coma and postcomatose encephalopathies. then, i began experimental studies of spinal cord vasculature in cats and other animals, working together with the neurosurgeon heinrich brenner in the experimental laboratory of the department of surgery. these studies, together with large human postmortem material, were the basis for my phd thesis “zur onkologie und pathologie der rückenmarksdurchblutung” (“on the oncology and pathology of spinal cord blood circulation”), published in 1966 by springer verlag wien. on january 19, 1966, i was granted the venia legendi (dozentur, lecturer) in neurology, neuroanatomy, and neuropathology, and became chief of the division of neuropathology of the ni vienna. in addition to routine diagnostic work, i lectured about general and specific neuropathology, and performed regular brain cutting sessions for students and colleagues who were interested in neuropathology (fig. 5). in the same year i received the kardinal innitzer award for promotion of medical research. atypical encephalitides and spinal cord circulation disorders were presented at the 1st meeting of the cssr neuropathology meeting in prague in september 1966, where prof. bednar, chairman of die institute of pathology of prague medical school, was a charming host. 1967, with w. sturm, a series of radiation injuries of the cervical spinal cord with relation to the applied radiation doses was published. at that time of progressing international congresses, publications and oral presentations were increasingly presented in english, and i published many articles in acta neuropathologica, founded in 1961 by franz seitelberger, who was editor-in-chief until 1987. the focus of clinical work and research were brain tumors, which were discussed in weekly clinico-pathological conferences with the department of neurosurgery. tumor cell imprints for rapid diagnosis were introduced, which were confirmed by histology with up to 95% concordance. in 1970, invited by m. mossakowski, head of the polish academy of sciences, i lectured about various neuropathological subjects in warsaw, poznań, and kraków, and was impressed by the lifestyle of the political upper class. at the 6th international congress of neuropathology in paris, where i spoke about delayed radiation lesions of the spinal cord, elisabeth and i were overwhelmed by the charm of france's capital. in verona, the neuropathology of the “apallic syndrome” after head trauma was presented. in the same year, seitelberger and igor klatzo, from the nih, bethesda, usa, organized an international symposium on axons and axonal flow, where i discussed neuroaxonal dystrophy in humans. fig. 5. brain cutting with (left to right) hans lassmann, gernot wöber (neurosurgeon), kj, and georg spiel (later pediatric neurologist) in the neurological institute, 1966. note the numerous fixed brains in the glass jars in the back. in october 1971 herbert budka joined us and became my assistant for more than three years. we shared routine diagnostic neuropathology. herbert recently described the situation and further fate of the ni, and became director of the clinical institute of neurology, the successor of the ni after its transfer to the allgemeine krankenhaus (see [5]). in 1972, i lectured in baltimore, kansas city (j.j. kepes' invitation), and new york. heinz regele, later chief of pathology in linz, and i observed peculiar things that were described later by others. this included, in patients with brain death, finding cerebellar material around the cervical and thoracic spinal cord, which dropped down as sequelae of cerebellar incarceration due to increased intracerebral pressure, later reported as a characteristic finding in brain death (vita reducta). with lothar kucsko, a highly experienced pathologist and heavy smoker, a postmortem transorbital puncture of the brain in a patient with clinically-suspected cjd was performed. under the electron microscope, i saw ruptured cell membranes, which i initially interpreted as postmortem artifact, but peter lampert from the afip published these findings as typical lesions in cjd [6] so we were too late! in the course of routine examination of brain tumor biopsies, i detected a large b cell lymphoma in the parietal lobe of a famous conductor. erwin deutsch, chairman of the department of internal medicine, did not believe in my diagnosis, since whole body examination did not show any malignancy. so i sent slides to harry zimmerman, chairman of neuropathology at montefiore hospital in new york, one of the world’s best neuropathologists and mentor to generations of neuropathologists, who confirmed my diagnosis. this patient survived for more than 12 years. autopsy revealed a generalized immunocytoma, but no tumor residual in the brain. together with harry and seitelberger, in 1974, an international symposium on malignant lymphomas of the central nervous system (cns) in vienna was organized, where experts discussed the classification of primary lymphomas of the cns. in 1972, traumatic vascular diseases of the spinal cord were reviewed for the handbook of clinical neurology. with t. radaszkiewicz, an excellent expert in lymphomas who unfortunately died too soon, a large collection of primary and secondary lymphomas of the cns was published. in 1973, hanno bernheimer, oleh hornykiewicz (who died may 26, 2020), walther birkmayer, franz seitelberger, and i published a highly-cited paper (currently 3,005 citations) about dopamine in the syndromes of parkinson and huntington. it was one if the first studies that correlated the neuropathological findings in the striatonigral system (semiquantitative assessment of neuronal loss in substantia nigra) and the biochemical data (dopamine and its metabolites) in a large number of autopsy cases [7]. during my time at the ni and afterwards, i extended brain autopsy service to several institutions outside of vienna, including the neurological hospital in linz. since i had no car, i traveled by train. once, on the return trip to vienna, while transporting sacks with freshly-dissected brains soaked with formalin to be examined in the ni, an acute train stop resulted some brains falling out of the sacks. i was almost arrested as a suspected mass murderer but the awful smell of formalin explained the reality. later career in 1973, i was offered the position of chairman of the division of neuropathology at the institute of pathology in zürich, but as i was number two on the list, reinhard friede was instead appointed. in february 1974, i was offered the position of professor and chairman of the department of neuropathology at the medical university düsseldorf, germany, which after 2 years of unsuccessful negotiations and for family reasons, i had to decline. wolfgang wechsler, then at the max planck institute at cologne, got this position which he held until his retirement in 1998. in 1975, in collaboration with andras guseo, a young neurologist from székesfehérvár, hungary and guest in the ni, the patterns of inflammatory perivascular infiltrates and their impact on the type and prognosis of multiple sclerosis were published. this was the beginning of a fruitful cooperation with the group of hans lassmann, then chief of the neuroimmunology division of the austrian academy of sciences and later of the department of neuroimmunology at the institute for brain research in vienna. in the same year, together with felicia slowik, a young neurooncologist from budapest and guest in the ni, a large collection of biopsy and autopsy-proven meningiomas was examined with impact on the histological specificities and prognostic relevance of various subtypes. in between, i was offered the position of head of the division of neuropathology at miami school of medicine, fl. on this occasion, elisabeth and i toured through the usa and i lectured at various universities. we got a deep impression about the way of living in the usa, which differed considerably from ours. a few months later i was offered the position of head of the division of neuropathology at case western medical school of medicine, cleveland, oh (successorship of r. friede). however, due to personal and family reasons, i declined all these positions. since it was impossible to get an independent position in vienna,, i finally decided to accept the position as head of the department of neurology at lainz hospital, one of the oldest municipal and teaching hospitals in vienna. it was built between 1908 and 1913 together with a large geriatric centre on the occasion of the 60th anniversary of emperor franz josef's regency and was equipped with all facilities of modern medicine. during julius tandler's time, a famous anatomist and counselor of health of the city of vienna, lainz hospital became “a second university.” in 2006 it was fused with the nearby neurological hospital rosenhügel as a neurological center and is currently named clinic hietzing. on october 12, 1976, i was appointed chief of the department of neurology and became director of the ludwig boltzmann institute of clinical neurobiology in january 1977, as successor of walter birkmayer, one of the “fathers” of research in pd. the ludwig boltzmann society, named after the austrian physicist ludwig boltzmann, was founded 1960 as an austrian network of specialized research institutes in the fields of medicine, humanities, and social sciences, all of which were sponsored by the city of vienna and the austrian government. in 2002, the society was reorganized and the number of institutes greatly reduced. so, one career ended and a new area of neuroscience began, although many within the vienna neurological community reacted with skepticism whether a neuropathologist would be able to manage a new clinical department. and, indeed, the beginning was difficult. the department had 75 beds, part of them still occupied by geriatric patients, a waterbed ward, two trained nurses, and some auxiliary personnel but only a minimum of technical equipment. i had to find coworkers, some of whom needed a training position, which was not easy to organize. in addition to the clinical department, which occupied the ground floor of the building, the histological and neurochemical labs on the second floor had to be equipped and prepared for functioning. my first coworkers from the previous neurogeriatric department were m. podiwinsky, an elderly neuropsychiatrist, and roda weiss, a young armenian doctor born in odessa and married to a viennese lawyer. soon afterwards, herbert flament and hannes schmidt, two young neurologists in training from the nearby rosenhügel hospital, and two specialists, peter kothbauer, nephew of the former minister of health, ingrid leodolter, and edith sunder-plassmann, wife of a neurosurgeon, completed my first crew. kothbauer later had to leave the department. within a few years, i finally gathered a crew of 12 neurologists, most of them becoming excellent clinicians. for the neuropathological lab, mrs. veronika rappelsberger followed me from the ni. without her help, i would never have been able to do my routine work and research in neuropathology. i had to perform brain autopsies and biopsies for the whole hospital and geriatric center and biopsies for a large neurosurgical department. since i received no funding from the city, i had to pay my lab assistant from my own pocket. the chief of the neurochemistry lab was peter riederer, an experienced neurochemist who had graduated from the technical university vienna and, since 1971, had worked together with walter birkmayer in the ludwig boltzmann institute (lbi) of neurochemistry, the predecessor to the lbi of clinical neurobiology. later, a number of students and postdocs joined us. in addition to my new double functions, in 1976, i was appointed editor-in-chief of acta neuropathologica, after having acted as supplementary editor for several years. between 1976 and 1981 i also edited the series “current topics in neuropathology” with selected papers from meetings of the austrian society of neuropathology. in total, i edited or co-edited 10 books. with peter riederer, wolf-dieter rausch, a dedicated post-doc who is currently professor of neurochemistry at the veterinary university of vienna, paul kruzik, and others, we examined the biochemistry of hepatic encephalopathy and the mode of action of l-deprenyl in the human cns. peter left vienna in 1986 and became professor of neurochemistry and head of clinical neurochemistry at the psychiatric clinic of würzburg medical university until his retirement in 2008. we had a successful scientific cooperation and became close friends with him and his wife, inge. graduate engineer ildiko wichart, a young neurochemist from budapest, also joined our group and worked with us for many years even after her retirement. in addition to clinical service, with 1,700 to 2,000 in-patients and an outpatient service with more than 15,000 consultancies per year, our department had to perform the neurological and psychiatric consultant service for the whole hospital complex. the clinical crew published about brain tumors, intramedullary metastases, and lymphomas; while the neurochemistry group wrote about neurotransmitters in metabolic encephalopathies, hepatic coma, brain infarcts, and deprenyl in the human brain. in june 1976, i gave lectures about cns involvement by malignant lymphomas/leukemias and radiation-induced lesions of the cns in new york, philadelphia, and baltimore. in 1977, i received the award of the vienna university foundation, and in 1979 the award of the medical scientific foundation of the city of vienna. we organized the first neurooncology group in vienna, in which the preoperative examinations were done by my department, neurosurgery by the hospital rudolfstiftung (head h. brenner), biopsy diagnosis, combined radiation, and polychemotherapy according to the comt scheme, and postoperative care by our department. we introduced modern csf cytology, and studied cytological changes of gliomas caused by chemotherapy. the clinical crew was organized in a way that everyone had their own specialty: clinical neurology (edith sunder-plassmann, herbert flament, roda weiss, andrea vass, maria grisold, dieter volc); and neurophysiology (rembert vollmer, wolfgang grisold). with alexander meng, son of a chinese professor of pediatrics and a viennese mother, and a specialist in acupuncture and traditional chinese medicine (tcm), a successful multidisciplinary pain clinic was organized. later, we performed the first biomarker screenings (total tau and β-amyloid-42) in csf for the early diagnosis of alzheimer disease (ad). the department got the first transmission zeiss em 9 microscope in a city hospital, transferred from the lbi of hematology (chief alois stacher, both an experienced hematologist and dedicated politician). in 1978, our neurochemistry group studied the activity of tyrosine hydroxylase (th), the key enzyme of the dopaminergic system in the brain and adrenal medulla. we found that the th activity was not only reduced in the striatonigral system by up to 90% but also in the adrenal medulla by about 74% [8], a fact that questioned later research about transplantation of adrenal glands in patients with pd and would go on to save billions of dollars. in the background of this research, a laboratory for muscle diseases and electromyography was opened with wolfgang grisold, one of the best clinical neurologists and neurophsiologists. he later became chief of the neurology department of franz josef hospital, the lbi for neurooncology, and is currently secretary of the world federation of neurology. between 1975 and 1980, i wrote the chapter of pathology of the nervous system for several editions of the working book of pathology, edited by j.h. holzner, successor of m. chiari as chief of pathology of the vienna medical university. in 1978, reviews about vascular tumors and malformations were published, and lectures were given in new york, bethesda, and washington (international congress of neuropathology). again in collaboration with peter riederer, the first austrian-german brain bank was organized and we collected a host of neurodegenerative diseases, ad, pd, and normal controls, all of which was later transferred to peter's lab in würzburg. in 1980, with filippo gullotta, first chairman of the department of neuropathology at münster medical school, germany, and miroslaw mossakowski from warsaw, the first european congress of neuropathology in vienna was organized, bringing together many neuropathologists in our city. in the following years, many new associates joined our group. christian bancher was active in both clinical and neuropathological research, with a focus on neurodegenerative and senile brain diseases, and werner paulus, a postdoc from germany, worked on brain tumors, lymphomas, and iron and ferritin in pd brains. he later became chairman of the department of neuropathology at münster medical school and editor-in-chief of acta neuropathologica until 2019, and is the current editor-in-chief of free neuropathology since 2020. my working day was full and split. often, in the mornings, i went to pick up fresh brains from the nearby mortuary and carried them over the yard to the laboratory to dissect them for the brain bank, with one half being deep frozen for neurochemistry and the other fixed in formalin for histological examination. the first part of the day was dedicated to clinical work and, after the morning conference with my coworkers, i went on clinical rounds twice a week and every saturday. in between, i saw patients in the outpatient department or had conferences with other clinicians or neuroscientists. meals were eaten together with doctors and nurses in the kitchen of the ward. the afternoons were dedicated to neuropathology, brain cutting, and sign out (about 700 brains per year), or research in the em lab (fig. 1b). in addition, i prepared a catalogue of all in-patients and their diagnoses on cards intended to be computerized later, which unfortunately was not possible at that time. i routinely read the letters of dismission (discharge) of patients, since all of my associates had to prepare these letters spontaneously – “trust is good but control is better.” this usually lasted until night. besides the clinic and the institute, i ran a small private praxis once or twice a week, together with elisabeth, who was an ideal and helpful companion, loved by most of our patients. in addition, i served as court-certified expert witness for insurance companies or courts. so, my clinical duties were as broad as my scientific interests, which suited me very well and was tolerated by elisabeth with great patience. international activities: the roaring eigthies in 1980, i was invited to speak by prof. hoshino, chairman of the department of neurosurgery, teikyo university, tokyo, one of the founders of high dose irradiation of brain tumors. in addition to lecturing, i reclassified the tumor collection of this department. during my 3-week stay in japan, i got deep insights into the japanese lifestyle, culture, and cuisine, not only in big cities but also in the countryside. also around this time, i presented holoprosencephaly and agenesis of the corpus callosum at the meeting of the american association of neuropathology in vancouver, canada. in 1982, together with f. seitelberger, and h. lassmann, the 9th international congress of neuropathology in vienna was organized, one of the most important meetings of this specialty in vienna that was visited by about 700 specialists from all over the world. at a meeting about advances in neurotraumatology in milan, october 1982, i presented brain stem involvement in blunt head injuries and elisabeth and i were welcomed by our italian colleagues and we thoroughly enjoyed their hospitality and the flair of the city. again with peter riederer, metabolic brain diseases, dopa and tryptamine binding, selegiline effects, and lisuride in the treatment of pd were studied, thus broadening my experience beyond neuropathology. at a 1984 symposium on parkinsonism in bermuda, i was impressed by this wonderful island. between clinical work, congresses, routine neuropathology, and publishing scientific papers, my editorial work for acta neuropathologica occupied much of my private time. since we had no computers or internet and i often read articles and proofs during bathing in our home, the journal caused some tension in our private life. many of the clinical and clinicopathological presentations were performed together with members of my team, in particular with wolfgang grisold, one of the best neurologists i ever met, dieter volc, later chief of the parkinson service in a private clinic, and mrs. elisabeth kienzl, student of the technical university vienna and one of my best assistants in the laboratory. with andreas rett, an experienced neuropediatrist, a large number of brain malformations were studied. franz seitelberger, dawna armstrong, an outstanding pediatric neuropathologist from houston, texas, and i were among the first to extensively study the morphology and neurochemistry of rett syndrome. in 1985, i attended workshops in baltimore and bethesda, and was regularly invited to the annual meeting of the british neuropathological society. in addition to neurodegenerative disorders, the morphology and biochemistry of schizophrenia was studied with my cousin eberhart gabriel, director of the vienna psychiatric otto wagner hospital and renowned historian in psychiatry. in 1986, i edited the book “therapy of malignant brain tumours” (springer verlag, vienna), in which international experts reviewed pathology, imaging, neurosurgery, radiation, and chemotherapy of brain neoplasms. between 1986 and 1999, i published chapters about neurodegenerative diseases, brain tumors, and spinal circulation disorders in the book series “neurology in clinic and practice” and, in 1987, about pallidal degeneration in the “handbook of neurology.” in september 1986, elisabeth and i visited stockholm for the 10th international congress of neuropathology, where i presented rett syndrome and changes of subcortical nuclei in ad and pd. we were overwhelmed by this charming city and the hospitality of its citizens. in september 1986 i attended the workshop “histological classification of tumors of the nervous system” in houston, texas, which initiated a new classification of brain neoplasms (fig. 6). in may 1988, i participated in the preparation of the 11th international congress of neuropathology 1990 in kyoto and gave lectures in tokyo, sendai, and niigata, where elisabeth and i were hosted by professor iikuta, head of the institute of brain research in niigata and his wonderful wife, who spoke perfect english. here, we saw the other side of the japanese island (figs. 7 and 8). fig. 6. brain tumor meeting in houston, texas, 1988, with (left to right) lucien rubinstein, kj, lucy rorke-adams and paul kleihues. in march 1989, we had a wonderful austrian neuroscience winter meeting at kitzbühel, and in april 1989 i discussed lewy bodies at a parkinson symposium in chicago. the pathology of parkinson syndrome was published in the handbook of experimental pharmacology, edited by donald calne, a pioneer of pd research. between 1989 and 1991, felicia slowik, a highly experienced neurooncologist in budapest worked with me on hemangiopericytomas, the ultrastructure of malignant lymphomas, and cerebral sarcomas. with werner paulus, the neuropathological basis of different clinical subgroups of pd and their clinical relevance were studied. in 1991, i received the city of vienna prize for medical sciences. the next year, together with e. kienzl and emin sofic, now professor of pharmacology, faculty of sciences, university of sarajevo, selective increase of iron in the sn of pd and changes in the iron-melanin complex in the sn of pd brains with x-ray microanalysis were studied and presented at the meeting of the canadian association of neuropathology, september 1992 in toronto, canada. fig. 7. visit to yashiko shrine hiigata, may 1988 with prof. kreutzberg and prof. ikuta. fig. 8. neuropathology and hospitality in niigate, 1988. more work, more congresses in april 1990, i participated in the who meeting on histological classification of tumors of the cns, organized by my friend paul kleihues, chairman of neuropathology in zürich and later director of the who international agency for research on cancer in lyon, france. werner paulus and i, together with 25 other distinguished brain tumor experts, participated in the meeting of the blue book’s classification of brain tumours. the 2nd edition followed in 1993, the 3rd edition in 1997, and another in 2000, following a meeting in lyon in 1997, where i also participated. in its update, retitled “pathology and genetics of tumours of the nervous system,” edited by p. kleihues and w. cavanee, with werner paulus, malignant lymphomas, histiocytic tumors, and melanotic lesions were reviewed. in august 1990, i presented primary cerebral lymphomas at the international cancer congress in hamburg, and in september 1990, elisabeth and i visited the 12th international congress of neuropathology in kyoto, where we were hosted by prof. yonezawa, who is not only an excellent neuropathologist but also a gourmet and friend of the good life. further highlights were the 5th world congress of biological psychiatry in florence in june 1991, where i presented pathomorphological aspects of schizophrenia, and the 10th international symposium of parkinson's disease in tokyo in october 1991. at the paneuropean congress of neurology in vienna, december 1991, the results of the vienna longitudinal study of dementia were presented, which showed correlations between cognitive impairment and quantitative alzheimer pathology. with christian bancher, the neuropathology of progressive supranuclear palsy (psp) was reviewed in a book edited by irene litvan and yves agid. at this time, the clinical department was fully developed and successful, including a new laboratory for ultrasound examination of the cerebral vasculature. upon the 80th anniversary of lainz hospital, a festschrift was published in 1993 that documented the progress of our department. in april 1993, the neuropathology and classification of psp was discussed in bethesda, md, and i received the eloise troxel memorial grant of the society for progressive supranuclear palsy, baltimore, md. at meetings in tokyo in april 1994, i lectured on new developments in ad and the pathogenesis of pd. a further highlight was the 12th international congress of neuropathology in toronto in september 1994, where our group presented new data about cell death detected by dna fragmentation and phospholipids in the alzheimer brain. in 1994, together in a working group of hans lassmann, pathogenetic problems of multiple sclerosis were studied. that same year, with werner paulus, advances in the pathology of primary cns lymphomas was also studied. a number of neurodegenerative disorders, like psp, multiple system atrophy (msa), and corticobasal degeneration (cbd) were studied in cooperation with an international group headed by irene litvan, now at university of california, san diego, usa. in 1995, christian bancher and i described the neurofibrillary predominant form of senile dementia, a rare subtype with low apoe ε4 genotype, which recently was renamed primary age-related tauopathy (part) [9]. in 1995, the pathology of aids encephalopathy and, together with herbert's group, the neuropathology and diagnostic criteria of cjd and other spongiform prion diseases were studied. in october 1995, i attended the workshop on dementia with lewy bodies (dlb) in newcastle/tyne, a dementing disorder hardly known at that time, and the guidelines for its diagnosis were published in the journal “neurology” in 1996. organizations and committees through the years, i was elected to various national and international scientific organizations and committees. i served as vice president of the international society of neuropathology between 1981 and 1984; was chairman of the eu working group from 1996 to 1999; secretary of the european society of psychopharmacology from 1994 to 2000; member of the fsn study group of neuropathology from 1987 to 2000; vice president of the austrian alzheimer society from 1997 to 2002; president of the viennese association of psychiatry and neurology in 1986/87; and president of the austrian society of neurology and psychiatry from 1990-1992. from 1994 to 2000, i was a member of a committee reporting on neuroscience in austria, on behalf of the austrian ministry of sciences. based on these data, a national program for neuroscientific research for austria was prepared in 1966, coordinated by the lbi of clinical neurobiology as a basis for austria's entry into the european union. with increasing age, i became member and honorary member of national and international scientific societies, including the american, british, german, hungarian, and austrian societies of neuropathology, the german societies of neurology and pathology, the austrian neuroscience association, honorary member of the austrian and german parkinson societies, the scientific commission of the german hirnliga, among many others. during these years, i reviewed articles for multiple national and international journals and became editor or member of the editorial board of many of them. the great adventure: first austrian neurologist in central china in march 1996, a delegation from the austrian society of neurology was invited by the chinese ministry of health to visit medical institutions and hospitals in china. this included 6 persons: hans and jutta lassmann, alexander meng, christian bancher, barbara zeman, and myself. the tour was organized and guided by mrs. zhou quin, program officer of the department of international cooperation of the chinese ministry of health (fig. 9). we visited china from march 31 to april 14, 1996. in beijing, we were informed about the health organization in the people’s republic of china. during the weekend, we visited the great wall, the ming graves, and the imperial palace. we then toured the neurological clinics of beijing hospital and of capital medical college, the two largest hospitals in the city, where hans and i gave lectures about multiple sclerosis (ms) and pd. we flew to chengdu, the capital of sichuan, the largest province of china, where we visited the medical college for traditional chinese medicine, which consisted of traditional and westernized parts, the first specializing in acupuncture and other traditional methods. fig. 9. first delegation of austrian neurologists in central china, with hans lassmann, kj, zhou quin, jutta lassmann, barbara zeman, christian bancher (left to right). we visited the largest herbal pharmacy of china and were invited to an herb dinner. on april 6, a sino-austrian symposium on neurosciences was organized in which lectures about ms, pd, and other topics were presented. by invitation of the minister of health of sichuan, we visited the min river and the buddhist grove sanctuaries in baodingshan and beishan, which impressed us very much. after a short visit to chongqing, a city with many respiratory diseases and lung cancers, we traveled by ship down the jangtze river to wuhan and visited the largest hospital of hubei province, which had modern technical equipment and many german-trained professors. the journey on the jangtse river, with the three gorges, was one of the most impressive adventures in our life. we experienced china's culture, landscape, and people, and passed the largest hydroelectric power station in china spanning the yangtse river, which at that time was in construction and considered one of the most impressive technical projects. for reasons of time, a visit to shanghai was not possible, and we returned to vienna. in conclusion, all of us were deeply impressed by the landscape, the historical relics, culture, health institutions, life in the countryside, and, in particular, by the friendly hospitality of our chinese colleagues. difficult times and new challenges in 1996, the ludwig boltzmann society decided to dismiss heinrich gross, who had been involved in the scandal about the euthanasia of handicapped children in the vienna psychiatric institution “am spiegelgrund” during nazi times, and i was asked to take over his lbi for the research of malformations of the cns, located in the psychiatric hospital baumgartner höhe, which was now combined with the lbi of clinical neurobiology. while the formalin-fixed brains of these children were stored in the cellar of the institute of pathology of this hospital, blocks of those brains had been secretly stored somewhere in the premises of these former lbis and i had the difficult task to find these materials from the archives so that they may be buried in a special memorial, which took place in 2002. this required much time to locate the specific material, to which i never had access before, while other specimens had been stored in the archives of the ni (see [5]). this affair was discussed in the popular media and had unforeseen political consequences, which hurt me, but i will not go into details. in 1996, together with norbert rösler, a postdoc from germany, the validity of csf biomarkers (total tau and amyloid-β-42) in the diagnosis of ad was studied. christian bancher and i visited an alzheimer congress in osaka, japan, and reported about the structural basis of dementia in ad and pd. the next year was of great importance for the department and myself, since i had reached the age of retirement from active service. however, my contract was prolonged for an extra year, during which we opened a stroke unit with 4 beds for surveillance and intensive treatment of patients with acute stroke. furthermore, christian bancher got the lectureship (dozentur) in neurology and neuropathology. in the years before, two other associates, rembert vollmer (neurology and neurophysiology) and wolfgang grisold (neurology) had got their dozentur. on september 30, 1997, i retired from my position as director of the department of neurology at lainz hospital, but remained chief the lbi of clinical neurobiology, which was transferred to a building in the psychiatrical otto wagner hospital. together with my lab assistant, mrs. rappelsberger, i continued neuropathological routine work examining brains from a large psychiatric and geriatric hospital. unfortunately, the archives of all the in-patients during the last 22 years, including the neuropathological protocols and brain material, was not available any more. my successor was dozent manfred schmidbauer, an excellent neurologist interested in neuropathology, book author, and painter. after the fusion of lainz hospital with the nearby neurological hospital rosenhügel, schmidbauer had to succeed the retired gernot schnabert as chief of both neurological departments. in addition, he continued routine neuropathology with focus on peripheral nerve biopsies. he retired in 2016 and became professor of neurodegenerative diseases at the sigmund freud private university in vienna. he was succeeded by christoph baumgartner, a highly-qualified epileptologist. however, this marked the end of neuropathology, which i had initiated and continued for 22 years. but i overcame this blow and continued scientific work. in addition to a small private praxis, i worked as a court-certified expert and still write second opinion expertises about difficult neurological problems. in october 1998, i presented data about movement disorders, malignant lymphomas, lewy body disease, and tangle dementia in new york, washington, bethesda, and montreal. at the 6th conference on ad and related disorders, in amsterdam in july 1998, i described vascular dementia and its relation with ad and received the alzheimer obelisk (fig. 10). in 1999 and 2000, the foci of my interests were pd and related disorders, in particular msa, as well as mechanisms of neuronal cell death, including apoptosis. with hallinah baran, an experienced neurochemist, later at the veterinarian university vienna, we studied kynurenine metabolism in ad, and with christine stadelmann, then assistant in lassmann's institute and now director of the institute of neuropathology, university göttingen, we studied cell death mechanisms in neurodegenerative diseases. at a traditional medicine symposium in vienna, i discussed the basis and impact of acupuncture in neurology. the same year, i was invited as guest professor of neuropathology at the medical university innsbruck, where i gave block lectures and seminars for students and colleagues. since the innsbruck university had no neuropathology at that time, i examined a number of autopsy cases of rare movement disorders, such as msa and psp. the symposium was hosted by prof. werner poewe, chairman of neurology, and gregor wenning, later chief of the division of clinical neurobiology, with whom a long-lasting and fruitful cooperation and deep friendship began. in 2000, i received the burda award of the austrian parkinson society. in the same year, i was invited as guest professor at the medical university of graz, where i lectured about many subjects of clinical and theoretical neuropathology in the clinic of neurology under chairmen erwin ott and later hans-peter hartung, now chairman of neurology in düsseldorf, germany. it was a wonderful time in the beautiful city of graz and its surroundings, which i enjoyed very much. it was a leisure living compared to vienna. in 2000, we described cell death mechanisms in pd and, in 2001, aids-related pathologies before and after antiretroviral therapy. since 2002, i helped the pathological bacteriological institute of the otto wagner hospital in the diagnostic workup of postmortem brains, mainly from elderly demented and non-demented individuals. i performed brain cuttings once a week, and instructed colleagues how to dissect brains, which were examined in the lbi. my trainee and friend johannes attems, then assistant at the local institute of pathology, examined the cases and we discussed them together between 2000 and 2008, thus giving him an excellent basis for his future career and a fruitful cooperation resulting in a number of shared publications. since 2009, johannes is professor of neuropathology and head of the brain bank at newcastle university in england and, since recently, editor-in-chief of acta neuropathological as successor of werner paulus. in 2002, i received the prestigious award for meritorious contribution to neuropathology by the american association of neuropathology, together with s. horoupian, professor emeritus at stanford university in the usa and fusahiro iikuta, director of the brain research center at niigata university, japan. the laudation of my award was written by robert d. terry, one of the most important neuropathologists of the 20th century and co-founder of us alzheimer research (addendum). we met on several occasions, including a visit to his home in california approximately one year before his death in may 2017. fig. 10. presentation of the alzheimer obelisk with dick swab (left) and henry wisniewski (center) at the 6th internat. conference on ad, amsterdam, july 1998. end of an institution and a new beginning in 2002, the lbi of clinical neurobiology was closed despite it being one of the most active scientific institutions outside the university with more than 1,400 publications until 2000. since that year, we received no budget and i had to find other sponsors. the equipment was transferred to the new institute of clinical neurobiology, which was run by the society for the support of research in experimental neurology in vienna, sponsored by donations and previous clinical trials. since 1999, my assistant has been dr. erich mitter-ferstl, phd, who studied at the zurich technical university and is an excellent computer specialist, without whom i hardly would have succeeded in this latest chapter of my career. time went on with both continuing examination of brain material at the wagner-jauregg hospital, evaluating old material, and continuing research. in 2003, i wrote a chapter about the neuropathology of pd in the book “neurodegeneration” (isn neuropath press, basel) edited by the international society of neuropathology and again for the 2nd edition, edited by d.w. dickson and r.o. weller in 2011. in 2007, a review of lewy body disorders appeared in abel lajtha's book “degenerative diseases of the nervous system” (springer, vienna) and since 2011, i continue to write the chapter on neuropathology of movement disorders in youman’s neurological surgery (elsevier saunders, philadelphia, editor w.r. winn), 6th edition, of which the 8th edition is currently in press. in 2014, i contributed the chapters on neuropathology and pathogenesis for the book “multiple system atrophy” (springer, wien) edited by g. wenning and a.fanciulli. lastly, with johannes attems, the chapter on neuropathology for the “textbook of old age psychiatry,” (oxford univ. press, oxford, uk) the 2nd edition of which is now in press. we had a co-operation with the vienna transdanube aging (vita) study, a long-term inter-institutional clinical, neuropsychological, genetic, neuroimaging, and neuropathological assessment of a large group of individuals aged 75 years in the eastern part of vienna, which was performed by the lbi of aging under the direction of hans tragl. for this group, with ildiko wichart, who worked at the university of veterinary medicine, vienna, aβ-42 protein in plasma was studied and correlated as valid marker with many clinical and neuropsychological data, which resulted in a large number of publications and congress reports between 2005 and 2010. unfortunately, the vita study ended for financial reasons, although it had been the first population-based long-term study about risks and development of cognitive changes in the oldest of old people. in 2003, at the 3rd german parkinson congress in dresden, heiko braak, an outstanding neuroscientist, who, among others, introduced the staging of pd and ad pathologies, and i received the lundbeck award for parkinson research (fig. 11). in the same year, i received the karl maria jakob medal of the german association of neuropathology in hamburg. the co-operation with johannes attems studying olfactory involvement and multimorbidity of the aging brain and with gregor wenning’s team on lewy body pathology was successfully continued. furthermore, with mrs. e. kienzl and a research group of the neurological clinic of the technical university of dresden (chief heinz reichmann), the alzheimer associated (alzas) gene was studied as a possible marker for ad. in 2005, i received the istván környey tarscosag award on the occasion of the 100th birthday celebration of this eminent hungarian neurologist, for whom i had the honor to present the laudation. in the same year, i contributed the chapter on synucleinopathies for the polish mossakowski memorial book of neuropathology (wydawnictwo czelej sp. z o.o., lublin, edited by p.d. liberski and w. papierz). since 2006, i lecture on the neuropathology of stroke and vascular dementia at the danube university in krems. the year 2006 was particularly busy. on the occasion of the 100th anniversary of aloys alzheimer's description of the disease, several books appeared. in one edited by george perry, the clinicopathological results of a large series of demented elderly person were presented, and i published a book about the hallmarks of 100 years of scientific research on ad presenting the most important publications between 1938 and 1999 in german (translated by myself). in a book edited by h. herholtz et al., i reviewed the early diagnosis of dementias from the neuropathological point of view based on 1,500 consecutive autopsy cases in comparison to the clinical diagnoses, where i emphasized the impact of multi-morbidity. in october 2007, i lectured about parkinson dementia in south korea, vascular dementia in budapest, and biological ad markers in wroclaw, poland. in 2008, together with lassmann's group, the relations between ms and ad from the neuroimmunological point of view were examined, and, at a parkinson symposium in tokyo, the formation and role of lewy bodies were presented. between 2005 and 2008, the kurt jellinger prize for outstanding scientific writing in neuropathology was sponsored by acta neuropathologica, for which i had served as executive editor for 29 years. the last recipient was goran simic (2008) from the brain research institute in zagreb, who had been guest scientist in my lbi a few years before. in 2008, i found myself among the 20 highly-cited scientists in austria in the isi webb of knowledge. since johannes attems went to newcastle upontyne in great britain, i stopped lab work and concentrated on specific neuroscience problems, reviewing manuscripts, and writing invited review papers. fig. 11. with heiko braak receiving the lundbeck award for parkinson research at the german parkinson congress 2003 in dresden. in 2011, i received the g. cotzias award of the movement study group of the spanish society of neuropathology in barcelona. in honor of my 80th birthday, four of my former scholars and now renowned neuroscientists, hans lassmann, christian bancher, johannes attems and werner paulus, dedicated a “festschrift” of acta neuropathologica [10], for which i am very grateful to them. on the same occasion, my friends and former associates organized a festivity, which all of us enjoyed very much. together with johannes attems and a. thomas, the correlations between cortical and subcortical tau pathologies and their relations with braak tau stages were reviewed, in addition to the pathological correlates of behavioral disorders in dementias with m.f. casanova and s.e. starkstein, and the problem of vascular depression with an international group. the role of α-synuclein in pd was presented in the book “emerging drugs and targets for parkinson's disease,” (the royal society of chemistry, cambridge, uk) edited by a. martinez and c. gil in 2013. invited in february 2013 by g. logroscino, an us-trained italian neurologist and neuroepidemiologist, i gave the lectio magistralis about the role of neuropathology in neurodegenerative diseases at the university of bari, italy. in june 2013, i discussed the role of α-synuclein as the gateway to neurodegeneration and demonstrated brain cutting to the members of the division of clinical neurobiology (head gregor wenning), with whom we celebrated our cooperation since 1993, which still persists. in march 2014, elisabeth and i were invited to a series of lectures at the department of neurology at seoul national university, where we were hosted by prof. beomseok jeon, a wonderful neuroscientist, who had overcome serious injuries in an alpine accident with utmost energy. we were overwhelmed by the megacity of seoul that had been completely destroyed during the civil war, and were deeply impressed by the lifestyle and culture of the korean people. we never had experienced such a warm-hearted hospitality before. after this tour, i was invited for the position of professor of neuropathology at seoul, but i had to decline for age reasons. i like small and intimate meetings, such as those by the society for amelioration of the quality of life, organized by stavros baloyannis, chairman of neurology at thessaloniki medical university, and sponsored by the orthodox church. these meetings occurred in marseille 2008, thessaloniki 2009, delphi 2010, and catania, sicily, in 2009, where franz, mrs. gerstenbrand and i climbed the aetna mountain. other favorite meetings were the “neurology spring” in the austrian waldviertel, organized by christian bancher, now chief of neurology at horn, and the world congresses on controversies in neurology (cony), organized by amos korczyn, professor emeritus in tel aviv, where problems in neurology are discussed by international experts. in may 2015, i presented the pathogenesis of msa at the α-synuclein symposium in innsbruck then once more at the congress on vascular dementia in ljubljana in october 2015. postmortem assessment in vascular dementia, a consensus report on vascular depression, and the relations of diabetes mellitus and dementia were published in collaboration with international experts. together with amos korczyn, an opinion paper discussing whether dementia with lewy bodies and parkinson dementia are the same disease was published in 2018. fig. 12. workshop on neuropathological diagnosis of ad at the ad/pd confernece in lisbon, march 2019, with johannes attems, irina alafuzoff, kj, melissa murray, dietmar thal (left to right). together with johannes attems, dietmar thal, and irina alafuzoff, i helped lead a workshop on neuropathological diagnosis of ad, which was organized by johannes at the aaic congresses in copenhagen 2012, washington 2015, and london 2017; and again at the ad/pd congress in nice 2015 and vienna 2017; and finally, with johannes attems, irina alafuzoff, dietmar thal, and melissa murray at the ad/pd conference in lisbon, march 2019 (fig. 12). my last congressional activities were the annual meeting of the austrian parkinson society in vienna in speaking about parkinson dementia and dementia with lewy bodies, and a biological definition of the alzheimer spectrum at a course about cns proteinopathies in milan, italy in november 29, 2019. now, at the age of 89, i still find it impossible to abandon my interest in neuropathology, and i try to continue scientific work as long and as well as i can. in the last year, i discussed whether braak staging is valid for all types of pd, and, together with gregor wenning's group, protective therapies in msa. after two extensive reviews about the neuropathology and pathogenesis of extrapyramidal disorders, the neuropathology of cognitive impairment in msa and an update of msa were published. two reviews of the pathobiological definition of alzheimer continuum, including the neuropathology of its pathobiological subtypes, are in press. life beyond neurology/neuropathology although i “was married to neuropathology,” together with elisabeth, we had an intensive and happy private life, as busy as it has been. before our wedding in august 1960, we rented a flat that took years to get completely refurnished. elisabeth was a student, and i a poorly-paid university assistant. in summer 1958, she was an au pair with a family in london, while i was paramedic in a summer camp for students in italy. in 1959, we spent a splendid summer vacation on the island of maly losinj at the croatian coast, where i had helped to reestablish the school. in 1960, we spent our honeymoon in lignano and venice, and the next year we went to the greek island of mykonos, where we were among the very few tourists on this beautiful island, which later became a hotspot of elegant tourism. elisabeth finished her studies in english with a thesis about the medieval towneley plays, which i typed. later, she became administrator in the institute of immunology of vienna medical faculty, run by my friend prof. martha eibl. afterwards, elisabeth joined me in literature research and as an assistant in my private medical practice, where she was loved by all my patients. in springtime, we traveled to southern countries, in particular toscana, umbria, the marches, romne, gubbio, urbino, arezzo, parma, sicily, corsica, burgundy, bretagne, and many other interesting regions, where we enjoyed the lifestyles, local gastronomy, cultures, and traditions. in summer we had regular vacations on many greek islands, but also in sicily, mallorca, and sardinia. in summer 1986, we spent our vacation in a beach house of my friend nenad grčević, an excellent croatian neuropathologist, in umag, a small town on the istrian peninsula, when the “prague spring” was suppressed by the soviet army. in 1996, after a peaceful stay at syros, we escaped from the island, afraid of a possible war between greece and turkey, and had to wait in athens for a flight home. later, many summers were spent on the beautiful island of chios, which is next to the turkish border, in a small hotel run by a friendly greek family, but during the last years we could not go there because of the refugee crisis and lack of direct flights. at christmas time and new year, we used to spend some weeks in paris, which we loved for its culture and way of life. unfortunately, during this past year we could not go to our beloved hotel de la bretonnerie in the old marais district because of the yellow jacket riots and the novel coronavirus pandemic. during the rest of the year, we regularly attend the vienna opera and concerts. for years, we have had subscriptions to the vienna philharmonic orchestra and many other chamber music presentations. in previous times, we regularly visited the salzburg festivals, where we met friends, many of them being members of the vienna philharmonic orchestra. during leisure time, i regularly read historical books and try to inform myself about austrian and international history. we like to walk in the idyllic landscape and, in order to exercise our brains, elisabeth and i take regular italian lessons with a nice italian interpreter. in 1969, my late father-in-law built a small house in maiersdorf, a small village at the foot of the hohe wand, a mountain of 1,200 meters, about 65 km south of vienna, which we visited nearly every weekend in summertime and around christmas. since march 13, 2020, and with few interruptions, we have lived in this house during the covid-19 lockdown, and it is here, in the tranquil landscape and beautiful springtime, that i wrote the major part of this report. once the lockdown ends, we hope that “normal life” will resume, although this virus is still an enormous and expanding danger for the whole world. however, we don't agree with the slogan in a humorist vienna journal in july 1918: “the spanish influenza finishes us all,” and, despite all warnings, we are looking optimistically to the future. we just celebrated the 60th anniversary of our wedding not on a greek island but in our rural home. acknowledgment the author thanks mr. erich mitter-ferstl phd, for his excellent secretarial and editorial work, without which this paper could not have been prepared. references 1. ludwin sk (2018) in search of knowledge and joy: my life as a neuropathologist. j neuropathol exp neurol 77:162–175 2. jellinger k, seitelberger f (1958) [acute fatal demyelinizing encephalitis after repeated injections of dry brain cells]. klin wochenschr 36:437-441 3. jellinger ka (2007) alzheimer's disease. in: gilman s (ed) neurobiology of disease. elsevier academic press amsterdam, pp 69-82 4. höftberger r, leisser m, bauer j, lassmann h (2015) autoimmune encephalitis in humans: how closely does it reflect multiple sclerosis ? acta neuropathol commun 3:80 5. budka h (2020) neuropathology through the ages – personal reflections. free neuropathol 1:15. https://doi.org/10.17879/freeneuropathology-12020-12817 6. lampert pw, gajdusek dc, gibbs cj, jr. (1972) subacute spongiform virus encephalopathies. scrapie, kuru and creutzfeldt-jakob disease: a review. am j pathol 68:626-652 7. bernheimer h, birkmayer w, hornykiewicz o, jellinger k, seitelberger f (1973) brain dopamine and the syndromes of parkinson and huntington. clinical, morphological and neurochemical correlations. j neurol sci 20:415-455 8. riederer p, rausch wd, birkmayer w, jellinger k, seemann d (1978) cns modulation of adrenal tyrosine hydroxylase in parkinson's disease and metabolic encephalopathies. j neural transm suppl:121-131 9. crary jf, trojanowski jq, schneider ja, abisambra jf, abner el, alafuzoff i, arnold se, attems j, beach tg, bigio eh, cairns nj, dickson dw, gearing m, grinberg lt, hof pr, hyman bt, jellinger k, jicha ga, kovacs gg, knopman ds, kofler j, kukull wa, mackenzie ir, masliah e, mckee a, montine tj, murray me, neltner jh, santa-maria i, seeley ww, serrano-pozo a, shelanski ml, stein t, takao m, thal dr, toledo jb, troncoso jc, vonsattel jp, white cl, 3rd, wisniewski t, woltjer rl, yamada m, nelson pt (2014) primary age-related tauopathy (part): a common pathology associated with human aging. acta neuropathol 128:755-766 10. lassmann h, bancher c, attems j, paulus w (2011) in honor of the 80th birthday of kurt jellinger: a living legend in neuropathology. acta neuropathol 121:565-568 copyright: © 2020 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. neurodevelopmental disorders: 2021 update feel free to add comments by clicking these icons on the sidebar free neuropathology 2:6 (2021) review neurodevelopmental disorders: 2021 update alfonsa zamora-moratalla1, maría martínez de lagrán1, mara dierssen1,2,3 1 centre for genomic regulation (crg), the barcelona institute of science and technology, dr. aiguader 88, barcelona 08003, spain 2 universitat pompeu fabra (upf), barcelona, spain 3 centro de investigación biomédica en red de enfermedades raras (ciberer), spain corresponding author: mara dierssen · systems biology program · crg-center for genomic regulation · c/ dr. aiguader, 88 · prbb building · 08003 barcelona · spain mara.dierssen@crg.eu submitted: 23 february 2021 accepted: 12 march 2021 copyedited by: cathryn cadwell published: 24 march 2021 https://doi.org/10.17879/freeneuropathology-2021-3268 keywords: autism spectrum disorder, asd, neurodegenerative disorders, next generation sequencing, microbiome, preterm birth abstract one of the current challenges in the field of neurodevelopmental disorders (ndds) is still to determine their underlying aetiology and risk factors. ndds comprise a diverse group of disorders primarily related to neurodevelopmental dysfunction including autism spectrum disorder (asd), developmental delay, intellectual disability (id), and attention-deficit/hyperactivity disorder (adhd) that may present with a certain degree of cognitive dysfunction and high prevalence of neuropsychiatric outcomes. last year, advances in human genomics have begun to shed light on the genetic architecture of these disorders and large-scale sequencing studies are starting to reveal mechanisms that range from unique genomic dna methylation patterns (i.e. “episignatures”) to highly polygenic conditions. in addition, the contribution of de novo somatic mutations to neurodevelopmental diseases is being recognized. however, progressing from genetic findings to underlying neuropathological mechanisms has proved challenging, due to the increased resolution of the molecular and genetic assays. advancement in modelling tools is likely to improve our understanding of the origin of neurodevelopmental disorders and provide insight into their developmental mechanisms. also, combined in vivo editing of multiple genes and single-cell rna-sequencing (scrna-seq) are bringing us into a new era of understanding the molecular neuropathology of ndds. abbreviations adhd attention-deficit/hyperactivity disorder, art assisted reproductive technology, asd -autism spectrum disorder, atac-seq assay for transposase-accessible chromatin using sequencing, cas9 crispr-associated protein 9, cb cingulum bundle, chip-seq chromatin immunoprecipitation sequencing, cnvs copy-number variants, crispr clustered regularly interspaced short palindromic repeats, degs differentially expressed genes, dlpfc -dorsolateral prefrontal cortex, eeg electroencephalographic, e/i excitation and inhibition, encode encyclopedia of dna elements, ept extremely preterm, ewas epigenome-wide association studies, flhs floating-harbor syndrome, geo gene expression omnibus, gf germ-free, go gene ontology, gw gestational week, hd huntington disease, hnpcs human neural progenitor cells, htt huntingtin, id intellectual disability, ipscs induced pluripotent stem cells, iq intelligence quotient, lgd likely gene-disruptive, lincrna long intergenic noncoding rna, mipscs miniature inhibitory postsynaptic currents, mhtt mutant htt, mri magnetic resonance imaging, ndds neurodevelopmental disorders, nfl neurofilament light protein, nlgs neuroligins, npcs neural progenitor cells, nrt nucleus reticularis thalami, nrxs neurexins, pmds phelan-mcdermid syndrome, pscs pluripotent stem cells, prehd premanifest hd gene carriers, prdd-seq parallel rna and dna analysis after deep sequencing, ptms post-translational modifications, rem rapid eye movement, rna-seq rna-sequencing, scrna-seq single-cell rna-seq, rsts1 rubinstein-taybi syndrome 1, sipscs spontaneous inhibitory postsynaptic currents, spf specific pathogen free, swds spike-wave discharges, vz ventricular zone, wes whole-exome sequencing, wgs whole-genome sequencing introduction one of the current challenges in the field of neurodevelopmental disorders (ndds) is still to determine their underlying aetiology and risk factors. ndds comprise a diverse group of disorders primarily related to neurodevelopmental dysfunction including autism spectrum disorder (asd), developmental delay, intellectual disability (id), and attention-deficit/hyperactivity disorder (adhd) that may present with a certain degree of cognitive dysfunction and high prevalence of neuropsychiatric outcomes. last year, advances in human genomics have begun to shed light on the genetic architecture of these disorders and large-scale sequencing studies are starting to reveal mechanisms that range from unique genomic dna methylation patterns (i.e. “epi-signatures”) to highly polygenic conditions. in addition, the contribution of de novo somatic mutations to neurodevelopmental diseases is being recognized. however, progressing from genetic findings to underlying neuropathological mechanisms has proved challenging, due to the increased resolution of the molecular and genetic assays. advancement in modelling tools is likely to improve our understanding of the origin of neurodevelopmental disorders and provide insight into their developmental mechanisms. also, combined in vivo editing of multiple genes and single-cell rna-sequencing (scrna-seq) are bringing us into a new era of understanding the molecular neuropathology of ndds. 1. progress in the use of big data for the understanding the neuropathology of neurodevelopmental disorders most nnds have a major inherited component which compromises correct brain development; however, the evaluation of the genetic causes of ndds remains challenging because of genetic and phenotypic heterogeneity. one well-known example is asd, which has been associated with mutations in a wide range of genes, but relatively few genes or loci are identified in sufficient cases to prove statistical significance at the genome-wide level. such rare genetic developmental diseases are a challenge for diagnosis, as patients with the same genetic defect present with varying degrees of symptoms and phenotypes (haghshenas et al., 2020), that can be due to molecular interactions between their associated genes, as in the case of floating-harbor syndrome (flhs) and rubinstein-taybi syndrome 1 (rsts1). in the last years, the fast pace of development of next-generation sequencing technologies, such as gene panels, whole-exome sequencing (wes), and whole-genome sequencing (wgs) technologies have enhanced our ability to diagnose the ndds. of these, wes achieves a diagnostic rate of 30–53% for ndds and wgs improves the diagnostic rates even more (42-62%). the use of high-throughput allows broader range of variant detection, including noncoding and regulatory regions, and the discovery of novel disease-associated genes, has shed light on the genetic, genomic and epigenomic key players of ndds and will also help in the diagnosis and stratification of the population with those disorders. changes in gene expression are also widely studied to characterize various human diseases and successfully used to predict molecular and cellular processes in complex neurodevelopmental diseases. high-throughput expression profiling has become routine, and as a consequence, a vast amount of public rna-sequencing (rna-seq) datasets has been generated and is available from online repositories, such as gene expression omnibus (geo; barrett et al., 2013). up to the second quarter of 2019, geo hosted more than 112,000 data series comprising more than 3,000,000 samples. this massive amount of biological data brings great opportunity for generating prominent biological hypotheses and is widely used to shed light into neuropathological mechanisms. however, these large datasets were produced for diverse purposes, are sometimes difficult to interpret, and are not friendly to large-scale data integration. therefore, increasing sophistication in the statistical methods and well-organized resources are required to enable efficient and extensive integrated analysis. one interesting example is the work of rahman et al., who performed a meta-analysis using two publicly available rna-seq studies from asd cerebral cortex (rahman et al., 2020). they performed integrative rna-seq gene expression profiling in cortex to identify transcriptional gene signatures altered in 15 asd subjects compared to 15 controls. the study revealed core signatures of differentially expressed genes (degs) associated with asd, including already known markers of asd and novel hub genes. the authors detected 235 unique degs, not identified by the individual studies, supporting the increased statistical power of the meta-analysis approach (rau et al., 2014; walker et al., 2008). seven of these degs (pak1, dnah17, dock8, dapp1, pcdhac2, erbin, and slc7a7), were previously reported to be deferentially expressed in asd. gene ontology (go) and pathways analysis was then used as a tool for identifying molecular pathways enriched by the degs. in their meta-analysis, rahman et al. showed altered osteoclast differentiation, tnf signaling pathway, and complement and coagulation cascade pathways in asd, revealing new previously unidentified genes. moreover, topological analysis of protein–protein interaction of the asd brain cortex revealed proteomics hub gene signatures. however, although meta-analysis is a powerful tool, it is also controversial due to the heterogeneity in terms of platform (different microarray and sequencing techniques), and sources (from peripheral blood to induced pluripotent stem cells [ipscs] or other tissues). jensen et al. (jensen et al., 2020), in an interesting study, added yet another level of complexity: the existence of comorbidities between ndds. this is an important step, given the high degree of co-occurrence of autism with id that may blur the de novo variants and copy-number variants (cnvs) identified in autistic individuals. the study analyzed 2290 individuals from the simons simplex collection for de novo likely gene-disruptive (lgd) variants and cnvs, and determined their relevance regarding intelligence quotient (iq) and social responsiveness scale measures. by analyzing pathogenic de novo genetic variants in individuals with autism who had either id or normal cognitive function, they determined whether genes associated with autism also contribute towards id comorbidity. their study showed that pathogenic de novo variants disrupting autism-associated genes contribute towards autism and id comorbidity so that gene discoveries in autism are biased towards genes that also contribute towards comorbid id (jensen et al., 2020). in contrast, individuals with high-functioning autism are less probable to carry de novo lgd variants in candidate autism genes and tend to present with disruption of genes with less functional relevance towards neurodevelopment. these results highlight the relevance of dissecting phenotypic heterogeneity in family-based sequencing studies of complex diseases. another recent example is the identification of a non-syndromic asd subtype characterized by dyslipidemia using massive multimodal data triangulation from wes, neurodevelopmental expression patterns, electronic health records and healthcare claims (luo et al., 2020). 2. dna episignatures genes associated with asd are enriched for pathways affecting neuronal homeostasis and embryonic development. however, no single genetic variant has been found that accounts for more than 1% of disease liability. this may be in part due to the fact that environmental factors are also known to contribute to asd risk, especially during the prenatal period. epigenetic mechanisms including dna methylation, histone post-translational modifications (ptms), noncoding rna, and chromatin architecture have been proposed to account for the sex bias, gene-environment interactions, and developmental origins of asd aetiology (ciernia & lasalle, 2016). epigenome-wide association studies (ewas) have identified asd-associated locus-specific differential methylation of genes involved in synaptic transmission or microglia, and disease-associated epigenetic signatures (nardone et al., 2014). ewas in post-mortem brains have identified one of the most novel and promising areas of research of ndds, the identification of dna methylation called "episignatures". those are defined as the cumulative dna methylation patterns occurring at multiple cpg dinucleotides across the genome. in the last years, an expanding number of genetic syndromes have been shown to have unique genomic dna methylation patterns. the first clinical genome-wide dna methylation assay, “episign,” used genome-wide dna methylation analysis for the screening of 14 syndromes known to harbor episignatures. this first study showed that dna methylation patterns are stable and specific to certain syndromes, and occur consistently across all of the individuals affected with the same syndrome (aref-eshghi et al., 2018). this has been confirmed recently by the identification of 34 disease-specific episignatures mapping onto 42 genetic syndromes, thus increasing the number of conditions that can effectively be diagnosed through dna methylation testing (aref-eshghi et al., 2020). the authors examined emerging patterns of overlap, and similarities and hierarchical relationships across episignatures. aref-eshghi et al. implemented a uniform approach for mapping dna methylation signatures in numerous syndromes to enable unbiased comparisons. this allowed identification of their key features as they are related to genetic heterogeneity, dosage effect, unaffected carrier status, and incomplete penetrance. through mass screening of a large cohort of subjects with developmental delays and congenital anomalies, they demonstrate the utility of this tool in resolving ambiguous clinical cases and identification of previously undiagnosed cases. an interesting study has taken a slightly different approach. mordaunt et al. using whole-genome bisulfite sequencing identified a distinct dna methylation signature over regulatory regions and genes relevant to early fetal neurodevelopment in the cord blood from newborns later diagnosed with asd (mordaunt et al., 2020). the development of the forebrain, including the assembly of the expanded human cerebral cortex, linked to the distinctively human features affected in ndds, is a lengthy process that involves diversification and expansion of neural progenitors, generation and positioning of layer-specific glutamatergic neurons, cellular migration of gabaergic neurons, and formation and maturation of glial cells. disruption of these cellular events by either genetic or environmental factors can lead to neurodevelopmental disease, including asd and id. these complex cellular processes require highly synchronized regulatory activity underlying these events, which, if perturbed, can cause disease. the authors had previously used whole-genome bisulfite sequencing in placenta samples and identified differential methylation of genes associated with asd (zhu et al., 2019). now, they obtained umbilical cord blood samples from asd and typically developing subjects from two high-familial risk prospective cohorts (i.e., cohorts following child’s early development of younger siblings of a child already diagnosed with asd through) in order to identify an epigenomic signature of asd at birth. their findings suggest that epigenetic dysregulation in asd may originate during early prenatal development in a sex-specific manner and converge on brain-relevant genes to disrupt neurodevelopment. although the study has some limitations as the selection of high-familial risk cohorts and the limitation in statistical power, it opens a new framework for the prognosis and diagnosis of ndds. 3. chromatin dynamics in neurodevelopment as mentioned above, epigenetic gene regulation plays a crucial role in controlling developmental transitions and cell differentiation, and is widely hypothesized to partly mediate risk for ndds such as asd or schizophrenia. therefore, tracking epigenetic changes in specific forebrain cell lineages over long time periods, has the potential to unravel the molecular programs that underlie cell specification in the human cerebral cortex and, by temporally mapping disease risk onto these changes, to identify cell types and periods of increased disease susceptibility. in the last years, chromatin accessibility has emerged as an accurate proxy for the cellular regulatory potential. chromatin encodes epigenetic information in the form of post-translational histone modifications and accessibility to dna binding factors (allis & jenuwein, 2016). chromatin regulation is affected by genetic alterations causative of ndds (de rubeis et al., 2014; pinto et al., 2014; lasalle, 2013), suggesting the existence of two classes of ndds. the first class is produced by mutations in chromatin regulators, as occurs in rett syndrome (schmidt et al., 2020). importantly, those may target several convergent molecular axes, with genes either belonging to the same class (e.g. lysine demethylases) or operating in the same molecular pathway (e.g. polycomb-mediated chromatin regulation). the second class comprises those ndds caused by environmentally-induced epigenetic dysfunction. interestingly, accessible chromatin regions exhibit a high heritability enrichment, indicating that sequence conservation can further refine functional risk genetic variants for disorders with a strong neurodevelopmental component, such as schizophrenia (bryois et al., 2018). efforts to define the transcriptomic and epigenomic landscapes of the developing human forebrain included multilevel analyses with characterization of spatiotemporal gene expression in the cortex (pollen et al., 2015; nowakowski et al., 2018), the molecular signature of cortical progenitors (johnson et al., 2015; pollen et al., 2015), and epigenetics of early brain development (amiri et al., 2018). now, gorkin and partners of the encyclopaedia of dna elements (encode) project, have launched an atlas of chromatin development (gorkin et al., 2020), a genomic resource profiling epigenomics of mammalian development. they initially used a diverse panel of mouse tissues at 8 developmental stages from 10.5 days after conception until birth, including transcriptomes, methylomes and chromatin states to systematically examine the state and accessibility of chromatin in the developing mouse fetus. to map chromatin states, the authors performed chromatin immunoprecipitation with sequencing (chip–seq) for a set of eight histone modifications that can distinguish between functional elements and activity levels. methods like chromatin immunoprecipitation and reduced representation bisulfite sequencing allow the investigation of epigenetic modifications on a genome-wide scale. however, one of the potential limitations of these methods is that you need to already have an idea about what epigenetic mechanisms are at play. thus, the authors complemented this technique with assay for transposase-accessible chromatin using sequencing (atac–seq; buenrostro et al., 2013), optimized for use on frozen tissues, to assay chromatin accessibility. atac-seq identifies accessible dna regions by probing open chromatin with hyperactive mutant tn5 transposase (picelli et al., 2014) that inserts sequencing adapters into open regions of the genome. the authors systematically mapped chromatin state and accessibility across 72 distinct tissue-stages of mouse development, and carried out integrative analyses incorporating additional epigenomic and transcriptomic data sets from the same tissue-stages. importantly, their analysis allowed them to integrate chromatin state annotations, infer the identities of dynamic enhancers and key transcriptional regulators, and characterize the relationship between chromatin state and accessibility during developmental gene regulation. as such, they could identify target genes and demonstrate tissue-specific enrichments of variants associated with disease in humans. approximately 1–4% of the genome differed in chromatin state between tissues at the same stage, and 0.03–3% differed between adjacent stages of the same tissue. this resource will help to map genetic risk for disease and to shed light into gene-regulatory dynamics at previously inaccessible stages of human forebrain development, including signatures of neuropsychiatric disorders. data from this and all phases of encode are publicly available through the encode portal (https://www.encodeproject.org). although encouraging, data on mouse development do not completely mimic human forebrain development, which is, to a large extent, inaccessible for cellular-level study. the lack of availability of primary brain tissue samples and the limitations of conventional in vitro cellular models have precluded a detailed mechanistic understanding of corticogenesis in disease states. using long-term three-dimensional (3d) directed differentiation of human pluripotent stem cells (pscs) into dorsal and ventral forebrain domains as well as primary brain tissue samples, a recently published work (trevino et al., 2020) found that organoids intrinsically undergo chromatin state transitions that are closely related to human forebrain development. trevino et al., also applied atac-seq in combination with rna-seq to map the epigenetic and gene expression signatures of neuronal and glial cell lineages over 20 months in vitro. the authors identified epigenetic alterations putatively driven by specific transcription factors and discovered a dynamic period of chromatin remodeling during human cortical neurogenesis identifying key transcription factors that may coordinate over time to drive these changes. this approach may bring new insights into gene-regulatory dynamics at previously inaccessible stages of human forebrain development, including signatures of neuropsychiatric disorders. 4. sequencing perturbed cortex development the growing number of genetic disruptions identified in human genetic studies far exceeds the ability to study their functions, or perform rigorous genotype-phenotype correlations, which may vary substantially across different cell types and states. until now, genetic screens have systematically analyzed individual gene function in mammalian cells or in vivo in knockout models, analyzing each perturbation separately. also, screens have been performed in a pooled format, measuring, for example, cell autonomous phenotypes, such as growth, drug resistance, or marker expression. both screening methods are time and labor intensive and do not allow the study of genetic interactions. comprehensive analysis of genetic interactions has been performed in yeast between pairs of genes (costanzo et al., 2016). in mammals, only small sets of pre-selected pairs have been assessed for cell viability (bassik et al., 2013) or morphology (laufer et al., 2013), but very few studies have examined higher order interactions or coupled those with a high content scalable readout. the newest addition to the genomic arsenal is single-cell clustered regularly interspaced short palindromic repeats (crispr) screening techniques, independently termed perturb-seq, crisp-seq, or crop-seq, that combine pooled crispr screening with scrna-seq to allow functional crispr screening in single-cells. crispr are dna sequences found in the genomes of prokaryotic organisms such as bacteria and archaea, which are used to detect and destroy dna from similar bacteriophages during subsequent infections. cas9 (or "crispr-associated protein 9") is an enzyme that uses crispr as a guide to recognize and cleave specific strands of dna. cas9 enzymes together with crispr sequences form the basis of the crispr-cas9 technology, used for gene editing. crispr targeting in vivo, especially in mammals, can be difficult and time consuming when attempting to determine the effects of more than a single gene. however, such studies may be required to identify pathological gene variants with effects in specific cells along a developmental trajectory. pool gene targeting followed by single-cell rna-sequencing of perturbed cells in the brain is a powerful methodology to reveal traits of individual cells in heterogeneous populations such as those in brain tissue (dixit et al., 2016). perturbations with single-cell sequencing readouts with increased throughput and enhanced resolution offer the possibility to explore the dynamics of transcription factors in development and genes whose expressions are differential between cell states or across the different areas of a tissue. perturb-seq was developed in 2016 (dixit et al., 2016), involving crispr/cas9 to perform multi-locus gene perturbation with massive parallel scrna-seq. as cellular behaviors typically depend on coordinated expression of many genes and translated proteins, unbiased sequencing methods can extract genome-wide profiles from single cells without prior knowledge. jin et al. (2020) have now used perturb-seq to explore the effects of in vivo genetic disruptions of risk genes of asd or ndds across diverse cells in the developing mouse cortex combined with single-cell transcriptome sequencing. they evaluated 35 asd de novo loss-of-function risk genes in multiple mouse embryos, using crispr-cas9 to introduce frameshift mutations in pools of these risk genes. this allowed them to investigate how diverse mutations affect cell types and states in the developing organism. this method identified networks of gene expression in neuronal and glial cells that suggest new functions in asd-related genes. using weighted gene correlation network analysis, they identified 14 covarying gene modules representing transcriptional programs in different types of cortical cells that affect common biological processes across multiple cell types and/or represent cell type–specific features. perturbations in nine of those asd/ndd genes had significant effects across cortical projection neurons, cortical inhibitory neurons, astrocytes, and oligodendrocytes. one example is ankyrin, which interacts with ion channels in excitatory neurons and stabilizes gabaergic synapses. ank2 mutants show misregulation of intracellular calcium homeostasis and calcium channel expression in excitatory neurons, and ectopic connectivity, but now perturb-seq data could identify additional roles of ank2 in those interneurons co-expressing the ndnf gene. oligodendrocytes and astrocytes were also affected by multiple risk gene perturbations. for example, chd8 modulates oligodendrocyte differentiation and maturation, by directly interacting with oligodendrocyte maturation genes. as highlighted in a comment on this work (treutlein & camp, 2020) in vivo perturb-seq can reveal neuronal and glial effects of sets of asd/ndd risk genes associated with autism. 5. study of lineage diversification in the developing neocortex as shown above, asd susceptibility genes are strongly interconnected and many act as genetic regulators of neurodevelopment of the cerebral cortex. however, the core underlying neuropathologies cannot be fully elucidated without understanding the cellular architecture of the human cortex, underlying its susceptibility to disease. the cerebral cortex is a complex structure formed by a wide repertoire of neural cells shaping its unique configuration. in only two areas of the adult mouse neocortex, single-cell transcriptomics has already identified at least 55 excitatory and 60 inhibitory neuron types (ecker et al., 2017; tasic et al., 2016) and a highly diverse set of excitatory and inhibitory neuron types that are mostly sparse, with excitatory types being less layer-restricted than expected in the middle temporal gyrus of human cortex (hodge et al., 2019). such tremendous cellular complexity of mature neurons is especially intriguing, given that those come from a limited number of progenitors. this apparent paradox leads to one of the most crucial questions in neurodevelopment: how the myriad of neuronal varieties located in the neocortex can arise from a reduced number of progenitor types. to solve this question, researchers are using next-generation single cell functional genomic techniques, which enable comprehensive and unbiased characterization of the progenitor populations from the early fetal stage to neuronal maturation. to elucidate the lineage relationships among different cell types during brain development, last year huang and colleagues (huang et al., 2020) developed a novel method in which parallel rna and dna analysis after deep sequencing (prdd-seq) allows simultaneous reconstruction of neuronal cell type, cell lineage, and sequential neuronal formation in post-mortem human cerebral cortex. this innovative approach revealed some conserved human cell lineage patterns, including that inhibitory and excitatory neurons diverge early in humans, and that excitatory neurons form following a similar “inside-out” order as seen in animal models. their work shows that at least some human neural progenitor cells (hnpcs) demonstrate restricted cell type output and that excitatory and inhibitory neurons are generated from distinct progenitor regions, supporting a model initially established in mice. the authors estimate that the number of progenitor cells that generate the excitatory neurons within a cortical column is ~10. by contrast, inhibitory neurons show complex, subtype-specific patterns of neurogenesis, and not all of them are conserved relative to mouse. using single-cell rna-sequencing and in vivo validation, li et al. (2020) have now revealed previously unrecognized neural stem and progenitor cell diversity within the fetal mouse and human neocortex, including multiple types of radial glia and intermediate progenitors. this novel cellular diversity catalogue they describe is characterized by mixed transcriptional profiles and not so much by morphological classes, which may indicate unique functional or state-dependent transcriptional profiles. in addition, most of the previously unrevealed types of basal progenitors have a human counterpart, indicating again the inter-species conservation of neurodevelopmental processes. li et al. postulate that transcriptional priming, a phenomenon whereby mrna for proteins that will be expressed in progeny is present (but not translated) in the parent cell, underlies the diversification of a subset of ventricular radial glial cells in developing mouse and human brain (li et al., 2020). transcriptional priming of radial glial cells would generate specific types of basal progenitors and neurons, being thus a driver of precursor and lineage diversity. however, those studies still elude the temporal scale in which progenitor populations at the early foetal stage go through neuronal maturation. another recent study has also systematically profiled single-cell transcriptome of the four cortical lobes and the pons. however, in this study they included the temporal dimension by analyzing transcriptional landscapes from human embryo to mid gestation, covering more than 5 months of critical developmental stages (fan et al., 2020). this strategy allowed accurate temporal and spatial resolution of developmental processes. as expected, the authors observed marked distinctions between cerebral cortex and the pons, in both molecular regulations and developmental patterns. the pons, an evolutionarily ancient structure, develops earlier showing abundant interneurons at early embryonic stages whereas in the cerebral cortex interneurons start to appear beginning at the early mid-fetal stage, suggesting a delayed development of neurons in the cerebral cortex. besides, molecular and electrophysiological results pointed out an asynchronous cell development in different regions of the cortex, with maturation occurring earlier in the rostral regions than in the caudal ones, and regional differences on the lateral side of the developing cortex appeared more conspicuous during the neuron maturation stage in the frontal lobe. the authors emphasize the important role of long intergenic noncoding rna (lincrna) in regional and cell type maintenance. these temporal differences in neuronal maturation could be essential for proper neural network construction and be beyond the establishment of certain neurodevelopmental disorders. one limitation of this study, however, is that post-mortem samples are collected at different gestational ages only allow a raw estimation of the temporal evolution of cell lineages but not a longitudinal tracing of the progenitors. a tool that tracks single-cell lineages and their phenotypes longitudinally would reveal whether heterogeneity extends beyond molecular identity. this is exactly what el nachef et al. (el-nachef et al., 2020) have now developed: a novel tool to track in vitro dynamic behaviors at the single-cell level based on the rainbow mouse models. they developed a stable cre-inducible rainbow reporter hpscline that provides up to 18 unique membrane-targeted fluorescent barcodes. because dna recombination is permanent the color barcode of a cell is inherited by daughter cells during cell division, enabling determination of the parental origin of cellular progeny and tracking them longitudinally by live-cell imaging. using this technique, the authors have tracked neural progenitor cells (npcs) over a month of cortical neuron differentiation into 3d cultures. npcs in the monolayer culture had self-organized into radially aligned cells forming a ventricular zone (vz) in these aggregates. the finding that monolayer cultures generate 3d cortical structures suggests that the differentiation protocol robustly recapitulates in vivo signaling where the planar neural plate gives rise to the neural tube and its derivatives through morphogenesis. in addition, longitudinal live imaging enabled determination of the timing of key neurogenesis events, such as vz formation and neurite outgrowth. these results strengthen the use of organoids derived from human pscs for the study of neurodevelopmental processes. understanding cellular mechanisms that lead to dysregulated npc expansion might help the understanding of neurodevelopmental disorders characterized by microcephaly or megacephaly. 6. microbiome: the hidden player behind neurodevelopmental disorders all the works presented above have revealed a remarkably complex molecular neuropathology involving a myriad of genetic architectures and regulatory elements, including unexpected peripheral players, such as the microbiota, in ndds. the human intestine harbors trillions of microbial cells which form a symbiotic relationship with the host and play a vital role in both health and disease (huttenhower et al., 2012). the importance of microbiota to human health is suggested by the observation that dysbiotic shifts in microbial communities have been associated with a number of human diseases, including obesity, inflammatory bowel disorders, or gastrointestinal cancer (schulz et al., 2014; flintoft, 2012; morgan et al., 2012; qin et al., 2012). recent findings also suggest that gut dysbiosis can be involved in the development of a variety of neuropsychiatric disorders, such as asd, depression and schizophrenia (grochowska et al., 2018). however, the high interindividual diversity due to genetics, age, diet, and health condition poses severe limitation to the study of asd gut microbiome (lloyd-price, 2016), the results being highly dependent on the cohort studied. the composition of intestinal microbiome affects health from the prenatal period throughout adulthood, and should be thought of as an organ system with important effects on child development (ronan et al., 2021). in fact, gut microbiota plays a major role in the bidirectional communication between the gastrointestinal tract and the central nervous system and there is increasing evidence of an important role of the gut microbiota in brain development and behavior (hoban et al., 2016; vuong et al., 2017; warner, 2019). to date, most of our understanding of the interaction between the microbiota and the brain has come from rodent models, in which the gut microbiota is linked to brain signaling mechanisms and affective phenotypes, such as anxiety or depression-like behavior. animal studies have clearly demonstrated effects of the gut microbiota on brain gene expression profiles and neurochemical metabolism impacting behavior and performance. interestingly, transfer of fecal gut microbiota from humans with depression into rodents appears to induce animal behaviors that are hypothesized to indicate depression-like states (kelly et al., 2016). germ-free (gf) animals also show clear behavioral differences compared to conventionally raised mice, or mice raised free of specific disease-causing organisms (specific pathogen free; spf) (bäckhed et al., 2007), including social interaction disturbances similar to those of asd; stress-related and anxiety-related behaviors; learning and memory deficits; and impaired motor control (vuong et al., 2017). some of the biochemical changes resulting from germ-free status are irreversible, even after colonization of the animals with normal gut microbiota later in life. other abnormalities, such as anxiety-related behavior, can be ameliorated after reconstitution of the gut microbiota (clarke et al., 2013). dysbiosis has been associated with pediatric diseases, including autism, adhd, asthma, and allergies. however, although several studies of shotgun-based metagenome analysis have been reported for asd gut microbiome (laue et al, 2020; wang et al, 2019), the high interindividual diversity, influenced by a wide range of factors such as genetics, age, diet, and health conditions (lloyd-price et al., 2016) impedes the correct identification of disease-associated microbiome features with stochastic false positives or negatives and the findings are highly dependent on the samples collected (surana & kasper, 2017). to overcome this problem, a team of researchers in china proposed a new analytical strategy, a quasi-paired cohort analysis that successfully allowed them to identify a new gut microbe deficiency in asd (zhang et al., 2020) children. in their paper, zhang et al. applied this novel strategy to a metagenomic study of the asd microbiome. they collected stool samples from 39 children diagnosed with asd, and 40 ageand sex-matched neurotypical children of similar metabolic backgrounds, referring to the profile of metabolic pathways inferred from the metagenomic data. this approach allowed them to transform the original cohort into a paired cohort, thus controlling for individual diversity and increasing statistical power. each of the stool samples were subjected to metagenomic sequencing and the team focused most specifically on 18 microbial species that have previously been linked to asd. using the quasi-paired cohort analytical strategy, the authors identified significant deficiencies in detoxifying enzymes and pathways, strongly correlated with biomarkers of mitochondrial dysfunction in asd children. then they applied diagnostic models based on these detoxifying enzymes, and could accurately distinguish asd individuals from controls of their cohort. in fact, the dysfunction score inferred from the model was proportionally increased with the clinical rating scores of asd. these results were further proofed in an independent cohort of 65 asd children. these results suggest a gut microbiome impact on detoxification in asd, and a potential role of impaired intestinal microbial detoxification in toxin accumulation and mitochondrial dysfunction, a core component of asd pathogenesis (castora, 2019). this is a very interesting finding as toxin exposure is one of the main etiological factors of asd (von ehrenstein et al., 2019). the impaired detoxification capability of the intestinal microbiome would reveal a new mechanism explaining this increased vulnerability to toxicant exposure of patients with asd. moreover, microbial detoxification capabilities could thus be a new therapeutic target for patients with asd. emerging evidence suggests that the microbiota’s influence on the central nervous system seems likely to be established as traits early in life, and potentially lead to later adverse mental health outcomes (walker et al., 2008). in fact, the gut microbiome development evolves throughout the lifespan from a composition that is simpler and more unstable during development to a highly diverse and stable composition in the adult (fouhy et al., 2012). even transient changes in gut microbial composition, induced by various intrinsic and extrinsic factors, may drive enduring shifts in the microbiota composition when the gut microbiota has not fully matured or is generally unstable, such as during early life. in fact, the vulnerability of microbiota composition during adolescence may explain why this period shows increasing incidence of psychiatric illnesses, including anxiety and mood disorders, psychosis, eating disorders, personality disorders and substance abuse (borre et al., 2014). the pathophysiology of these disorders has been commonly interpreted as arising from aberrations of maturational changes that normally occur in the adolescent brain. now, a recent study (lach et al., 2020) by lach et al. has revealed that even transient microbiota depletion has long-lasting effects on microbiota composition and leads to increased anxiety-like behavior. the study was performed in mice exposed to antibiotic treatment for 3 weeks to achieve microbiota depletion during the adolescent period or in the adulthood. this transient microbiota depletion had long-lasting effects on microbiota composition but only when antibiotics were administered in adolescent mice, in which it was associated with a significantly increased anxiety-like behavior. to further understand what pathways of communication between the gut and the brain are responsible for such changes at this time period, the authors analyzed gene expression in the amygdala and again they showed that it was more severely affected in mice treated with antibiotics during adolescence. this highlights the vulnerability of the gut microbiota during the adolescent period, influencing gene expression and behavior even in adulthood. this is especially important in the actual covid-19 crisis (fore et al., 2020; headey et al., 2020), which poses severe risks to the nutritional status of young children and adolescents, especially in lowand middle-income countries, as diet is a potentially modifiable determinant of the functionality of gut microbiome throughout life (ghosh et al., 2020; jebeile et al., 2019; liu et al., 2019; sonnenburg & sonnenburg, 2019). it is thus important not to neglect the later consequences of this crisis, forcing millions of families to rely on nutrient-poor alternatives that may not only lead to later obesity and diabetes, but also to a mental health pandemic (pan et al., 2021). 7. neuroligin in neurodevelopmental disorders alterations in synapse formation and function are a hallmark of neurodevelopmental and neuropsychiatric disorders. synapses are formed by a complex process that is controlled by synaptic organizer molecules, including trans-synaptic adhesion molecules and secreted factors (ferrer-ferrer & dityatev, 2018; yuzaki, 2018). a key component of these organizational protein complexes are the synaptic adhesion proteins called neuroligins (nlgs), a family of post-synaptic cell-adhesion proteins which act trans-synaptically with neurexins (nrxs) another group of pre-synaptic cell-adhesion molecules. the nlgs and nrxs were one of the first pairs of synaptic adhesion molecules to be characterized, and their mutation was quickly shown to be associated with autism, and later with schizophrenia (jamain et al., 2003; südhof, 2008; sun et al., 2011). synaptic organizational protein complexes play central roles both in orchestrating synapse formation and in defining the functional properties of synaptic transmission that together shape the flow of information through neuronal networks. however, although a myriad of evidence supports the involvement of genetic alterations in neuroligin genes, including point mutations, missense mutations and internal deletions in patients with asd, their precise pathogenetic mechanism in ndds still remains elusive. there are five members of the nlg family in humans and other primates: nlg1, 2, 3, 4x, and 4y. mutations in nrx1, nlg3 and nlg4 genes are linked with asd and other neurodevelopmental disorders (parente et al., 2017; etherton et al., 2011; sun et al., 2011; südhof, 2008; dean & dresbach, 2006; lisé & el-husseini, 2006). several nlgn2 variants have been identified in individuals with schizophrenia, autism, and anxiety disorder (parente et al., 2017). one longstanding hypothesis in the field is that asd arises from a disruption of the neuronal network activity due to perturbation of the synaptic excitation and inhibition (e/i) balance. the nlgs family regulates the balance between excitation and inhibition activity in the brain circuit by being able to modify the excitatory (nlgn4) / inhibitory (nlgn2) balance. in particular, the nlg2 subtype is highly expressed at inhibitory postsynaptic structures, and it is required to establish appropriate inhibitory synapse function during development, but also appears to play a role in synapse maintenance in adulthood (liang et al., 2015; troyano-rodriguez et al., 2019). as such, nlg2 is a potent regulator of the e/i balance: knockdown of nlg2 reduces gaba receptors and gaba-mediated synaptic transmission, resulting in increased neuronal excitability (liang et al., 2015) while overexpression of nlg2 in transgenic mice leads to motor discoordination, social impairment and abnormal electroencephalographic (eeg) activities, characteristics shared by asd and epilepsy (hines et al., 2008). this is interesting since high prevalence of epileptiform activity emerges as a common pathophysiological hallmark of autism (subota et al., 2017; horváth et al., 2016; plioplys et al., 2007). nlg2 might be thus a common player explaining the comorbidity, as asd and epilepsy frequently coexist in the same individual, suggesting a common neurodevelopmental basis for these disorders. however, even though genome-wide association studies recently allowed for the identification of a substantial number of genes involved in asd and epilepsy, there was no direct evidence to support the specific involvement of nlg2 in epileptic seizures. now, in a recent paper, cao et al. (cao et al., 2020) demonstrate that loss of nlg2 produces seizures and behavioral deficits that are associated with gabaergic transmission in thalamic neurons. they systematically performed cortical eeg recordings in freely moving nlg1, nlg2, and nlg3 knockout mice and their wild-type littermates in awake, rapid eye movement (rem) sleep, and non-rem sleep states, simultaneously monitoring their motor activity by electromyographic recordings. interestingly, only nlg2 knockout mice exhibited abnormal 5-8hz spontaneous spike-wave discharges (swds), an activity pattern that was correlated with behavioral arrest episodes characteristic of absence seizures. supporting their hypothesis that those arrest episodes were reflecting absence, these were ameliorated by the anti-absence seizure drug ethosuximide. this might result from the markedly reduced gabaergic and glycinergic inhibitory synaptic transmission, as assessed in the form of miniature or spontaneous inhibitory postsynaptic currents (mipscs or sipscs) in nlg2 knockout mice. in fact, restoring gabaergic transmission by optogenetic activation in a projection from the thalamic reticular nucleus to the ventrobasal thalamus also partially normalized these phenotypes, indicating that they may result from loss of feedforward inhibition in the circuit, the only region, together with the central amygdala, reported to date to have near-normal inhibitory synaptic transmission in the absence of nlg2 (babaev et al., 2016). the same rescuing effect was achieved by postsynaptic nlg2 expression in the thalamic neurons and optogenetic activation of the nucleus reticularis thalami (nrt) thalamic pathway was able to partially rescue gabaergic transmission, swds, and behavior arrests in nlg2 knockout mice. previous results have indicated that the imbalance in the excitation/inhibition tone in the thalamocortical circuitry is linked with the swds, absence seizures and other forms of epileptic behaviors (fogerson & huguenard, 2016; maheshwari & noebels, 2014; paz et al., 2011). the results of cao and colleagues now provide strong evidence that nlgn2-mediated gabaergic transmission is a powerful regulator of thalamocortical network activities related to absence seizures and may provide a molecular mechanism underlying high rates of comorbidity of asds with increased epileptic seizures, providing a link between asd phenotype and absence seizures through a nlg2-mediated inhibition at the nrt-thalamic synapse. 8. are neurodegeneration diseases programmed from development? since some years ago, accumulating evidence is revealing similarities and frequent complex interactions between neurodevelopmental and neurodegenerative disorders. neurodevelopmental conditions can manifest as lifelong weaknesses in cognitive functions and a number of works suggest that certain neurodevelopmental disorders may entail greater risk for specific neurodegenerative disorders. an interesting example is huntington disease (hd) a neurodegenerative disease characterized by dyskinesia, progressive involuntary movements, behavioral and psychiatric disturbances, and dementia. these symptoms are accompanied by widespread neurodegeneration that likely starts in the striatum (nopoulos, 2016) but also involves fronto-striatal circuitry malfunction. it is prevalent in adult individuals, with a mean age at onset of ~45 years (langbehn et al., 2010) and initiates with motor disabilities, such as involuntary movements. hd is caused by mutation of the huntingtin (htt) gene, as a result of cag trinucleotide repeat expansion with repeat length ranging from 10 to 35 in the normal population. repeat lengths between 36 and 39 cause hd at reduced penetrance, and when expanded to 40 or more repeats, mutant htt (mhtt) causes hd at full penetrance. despite symptoms of hd manifesting in adulthood, the aberrant htt protein is present much earlier in persons carrying the disease-causing mutation. in fact, htt is involved in brain development, axonal transport, synaptic function and cell survival and mhtt impairs npc division, neuronal migration and maturation (saudou & humbert, 2016). mutation of htt in early life is enough to induce hd features in adult mice and shows that there is a developmental component in the disease (arteaga-bracho et al., 2016; molero et al., 2016) and hd mice present in fact thinner cortices (godin et al., 2010). there are a number of lines of evidence that show cognitive function starts to decline and functional deficits appear around 15 years before clinical onset (lee et al., 2012; paulsen et al., 2008; tabrizi et al., 2020). an interesting approach was taken by the kids-hd study, which includes a unique cohort of children and adolescents who are at risk for adult-onset hd (lee et al., 2017). a recent study on this cohort using neuroimaging data of children and adolescent carriers of mhtt showed that developmental trajectories in the striatum and globus pallidus were markedly different between gene-expanded and gene-non-expanded individuals (van der plas et al., 2019), a pattern that was exaggerated with cag expansion length > 50. the striking difference in developmental patterns suggests that pathogenesis of hd begins with abnormal brain development, where children who carry the gene expansion exhibit different trajectories of brain growth than those who did not inherit the expansion. now, scahill et al. (2020) have showed that clinical biomarkers of hd neurodegeneration such as cerebrospinal fluid neurofilament light protein (nfl) are already significantly elevated as early as 24 years from predicted onset of clinical symptoms in a cohort of premanifest hd gene carriers (prehd) young adults, in which motor, cognitive, and psychiatric function are preserved,. a curious finding of tereshchenko et al. (tereshchenko et al. 2020) also indicates that measures of growth are abnormal in child and adolescent carriers of mhtt decades before hd onset. specifically, gene expanded males were taller than gene non-expanded males in adolescents of similar weight. furthermore, barnat et al. (barnat et al. 2020) and difiglia (difiglia 2020), have gone even earlier, and studied human brain cortex of hd mutation carrier fetuses and healthy controls at gestational week 13 (gw13) and of a hd knock-in mouse model at embryonic day 13.5 (e13.5), which correspond to gw13. in this phase of development, progenitor cells in the vz are extending processes towards the apical and basal surfaces of the neuroepithelial wall. the authors found, both in human fetuses and in mouse embryos, abnormalities in the developing cortex, including mislocalization of mhtt and junctional complex proteins, defects in npc polarity and differentiation, abnormal ciliogenesis, and changes in mitosis and cell cycle progression. in normal development, npcs in the vz reach out to both the apical and basal surfaces of the neuroepithelial wall, and their cellular nuclei shuttle back and forth as the cell cycle progresses. with the aberrant mhtt, epithelial junctions are disrupted, epithelial polarity is disturbed, and the cell cycle favors premature neuronal differentiation. these data are in good agreement with previous experimental data that htt regulates cellular adhesion, polarity, and epithelial organization (godin et al., 2010). in summary, alterations in neurodegenerative genes or proteins during developmental stages might program neurodegeneration disorders in adulthood. however, given their neurodevelopmental effects, it is unknown why these disorders do not emerge until adult stages. 9. shank-3: the missing link the wide variety of ndds that present with motor symptoms is well established. in recent years, there has been an increased interest in the motor deficits of asd in order to find a target to diagnose the disease as early as possible in young children. however, the landscape of motor deficits is quite broad and may involve brain, peripheral nervous system or neuromuscular junction. asd patients may present with motor-related abnormalities such as delayed motor development (ozonoff et al., 2008), impairment of gross and fine motor functions (provost et al, 2007) and deficits in basic motor control (jansiewicz et al., 2006). some patients may have trouble with coordinating movements between the left and right side of the body, low muscle tone or balance. one possible candidate to explain part of these symptoms is shank3. haploinsufficiency of the shank3 gene represents one of the most common single-gene mutations in asd and has been associated with neural circuit alterations in several brain areas (holder and quach, 2016; leblond et al., 2014; betancur et al., 2013). shank3 is a structural protein accumulated in postsynaptic densities, which contributes to excitatory synapse function and plays various roles in development (wang et al., 2014). moreover, the shank3 gene is the main contributing factor in phelan-mcdermid syndrome (pmds) that is generally characterized by neonatal skeletal muscle hypotonia despite normal growth, severely delayed speech, moderate to profound developmental delay, and mild dysmorphic features (sarasua et al., 2014; kolevzon et al., 2014; soorya et al., 2013). hypotonia is critical for diagnosis because it is one of the earliest clinical hallmarks of pmds. indeed lutz et al. (lutz et al., 2020) now reported the first experimental evidence that directly links the mutation of shank3 with peripheral muscle atrophy. they investigated the role of shank3 on motor unit development using a combination of patient-derived human ipscs, shank3δ11 (−/−) mice, and pmds muscle biopsies from patients. the authors show that hypotonia in shank3 deficiency might be caused by dysfunction in all elements of the voluntary motor system: motoneurons, neuromuscular junctions and striated muscle fibers. these findings provide a new perspective on the function of shank3, beyond the central nervous system, and suggests a future treatment strategy for shank3-associated asd. furthermore, recent evidence shows that somatosensory neurons in shank3-mutated mice are dysfunctional and contribute to tactile phenotypes (orefice et al., 2019). therefore, impaired proprioception and altered somatosensory feedback to the central nervous system of patients with pmds and asd may worsen symptoms and induce a delay in motor development. abnormal functional communication between brain areas has recently arisen as a feasible biomarker for asds (emerson et al., 2017; lewis et al., 2013). interestingly, zhou et al. (2019) showed evidence of atypical behavior and altered brain circuits in shank3-mutant macaques. the authors used crispr-cas9-mediated gene-editing technology to genetically engineer non-human primate models (macacafascicularis and their offspring), which might better approximate the behavioral and neural phenotypes of asds than do rodent models. magnetic resonance imaging (mri) in shank3 mutants revealed altered local and global connectivity patterns among brain areas that were indicative of circuit abnormalities. this was paralleled by social and learning impairments, sleep disturbances, repetitive behaviours and motor deficits. the works of lutz et al. (2020) and zhou et al. (2019), together, make it clear that shank3 protein is affecting the physiological functioning of the motor system from the cerebral cortex to the nerve endings of the body's extremities. in addition to these findings, using multiparametric mri analysis by diffusion tensor imaging and volumetry, it has recently been shown that the lack of shank3 induces mainly changes in white matter (jesse et al., 2019). in shank3-deficient patients with pmds the authors found a significant pattern of white matter alterations in the long association fiber tracts. white matter alterations were also observed in heterozygous isoform-specific shank3 knockout mice, with a minimal change in gray matter. the delayed achievement of motor milestones seen in pmds may be associated with white matter changes in the sensorimotor, cortico-striatal and cortico-spinal tracts. these abnormalities during development could affect myelin formation and connections between different brain areas, altering the flow of information in brain networks, and leading to the motor disturbances and asd associated behaviors observed in shank3 mutant mice (jamarillo et al., 2020). interestingly, early genetic restoration of shank3 improves the behavioral deficits (figure 1). asd shows a very complex pathology associated with shank3 mutations that induce alterations in functional and structural motor network organization within the central and periphery nervous system. further investigation using different animal models will be needed to move these findings from basic research to the clinic and ultimately in order to find new therapeutic strategies at early ages. figure 1: possible mechanisms associated with shank3 mutation. mutant shank3 induces deficits in the central and peripheral nervous system. early genetic restoration of shank3 is able to prevent the beginning of the asd-like behavioral deficits. possible mechanisms associated with shank3 rescue are depicted. 10. the effects of being too early prematurity, gestational age, or weight at birth have been identified as a risk factor for ndds, including those distinguished by autistic features. brain injury is more severe in extremely preterm (ept) newborns (those born with less than 25-30 weeks gestational age). their survival has increased remarkably in the last decades, making even more obvious the consequences of underdevelopment. data from 70’s indicated that survival was less than 20%, while recent data in the united kingdom reports that 35% of the babies born at 22 weeks, 38% born at 23 weeks, and 60% born at 24 weeks, survive (mactier et al., 2020). among the babies who survive, the risk of complications or ongoing disability increases with decreased gestational age. among the possible complications are anxiety, asd, cerebral palsy, epilepsy and a significant number of cases of cognitive impairment is detected during childhood. however, the majority of survivors do not have a severe, life-limiting disability (marret et al., 2013). thus, to what extend ept children have long-term deficits at different neurological levels needs to be explored. last year, erdei et al. reported that ept children had higher rates of cognitive impairment relative to their term-born peers, with odds rate of any impairment ranging from 3.7 at the age of two, to 5.0 at age 6 (erdei et al., 2020) however, most children born ept experience mild (27.2%) rather than severe (10.7%) cognitive impairment at 12 years. also, a recent study from the epicure team that followed a cohort of 5,391 infants born ept in the united kingdom and ireland from 1995, found that 60% of young adults (19-year-old) who were ept are impaired in at least one neuropsychological area, often cognition and visuomotor abilities (o’reilly et al., 2020). the cohort continues to present lower scores compared to term-born peers in general cognitive functioning (iq), visuomotor skills, as well as in prospective memory and aspects of language and executive functions. interestingly, the damage seems progressive, as the proportion of ept participants with intellectual impairment (iq <70) increased by 6.7% between 11 and 19 years of age. for a small proportion of ept individuals, intellectual impairment may only become apparent later in adolescence or adulthood when cognitive demands become more complex. the heterogeneity in the onset and severity of the neuropsychological and cognitive outcomes among the ept individuals and how to predict neurodevelopment impairments is one of the unsolved issues in the field. from the neurobiological point of view, the ept prenatal period concurs with the final stages of neurogenesis in the human telencephalon, neuronal migration, differentiation and maturation, and the very early stages of cortical myelination, conferring to the brain high susceptibility. therefore, interruption of the normal fetal environment during the development process is expected to have an impact in the brain conformation and connectivity. many neuropathological hallmarks of brain injury are found in preterm born infants (burkitt et al., 2019; yum et al., 2019; strunk et al., 2014) including neuroinflammation, oligodendrocyte maturation arrest and hypomyelination, axonopathy, reduced fractional anisotropy and cortical volume as determined by mri, and many of these differences persist into adolescence and adulthood. the neuroimaging studies revealed alterations in white and grey matter microstructure, impaired cortical folding and disturbances to regional brain growth (hinojosa-rodríguez et al., 2017). a recent study comparing brain connectivity using resting state functional mri in very premature infants and age-matched healthy fetuses with structurally normal brain mris, revealed stronger connectivity in sensory input and stress-related areas in premature infants suggesting that extra-uterine environment exposure alters the development of select neural networks even in the absence of structural brain injury (de asis-cruz et al., 2020). brain structures whose maturation is highly dependent on external stimuli during the postnatal period are more susceptible to being affected due to the housing conditions, poverty of maternal care and associated pathologies along with the ept condition. this is the case of the thalamocortical pathway whose maturation is subjected to critical developmental windows during the neonatal period (kostoví & judaš, 2002). the thalamus is a relevant structure as it acts as a hub for integration of cortical activity which regulates cortical power across a range of frequencies (malekmohammadi et al., 2015; fitzgerald et al., 2013). recently, the relation between thalamic myelination level and its neurophysiological functioning at rest in preterm infants has been explored (nunes et al., 2020). this study revealed that the thalamic volume reduction is associated with atypical frequency-specific neurophysiological power in preterm birth. moreover, thalamic intensity is associated with negative neurocognitive outcomes. neurocognitive difficulties were associated with increased frontal and occipital delta, and occipital theta, as well as decreased power mostly involving frontal and occipital beta activity. these data together suggest that environmental exposure in preterm newborns might play a crucial role in the outcome of neurophysiological outcomes, being relevant to the development of standardized protocols of medical care during the earlier postnatal life of ept individuals. other studies are focusing more on the analysis of the spatial microstructure alterations and not so much on long-range connections, with the aim of predicting the outcomes based on the heterogeneity among ept individuals. one latest example is the imaging study of the cingulum bundle (cb), an anterior-to-posterior white matter tract connecting prefrontal areas with the insula, amygdala and hippocampal and parahippocampal cortices that has been associated with complex attention processes (i.e., shifting and divided attention [schulte et al., 2008]). the maturation of the cb can be compromised in ept since myelination begins postnatally and continues until the first 2 years of life. brenner et al. have shown in a longitudinal study in preterm infants at 5 years of age that white matter microstructure is altered specifically in the right anterior cb and correlates with the emergence of psychiatric symptoms (brenner et al., 2021). this is interesting as dysfunction of anterior cb has been associated with internalizing disorders, autism, and attention-deficit/hyperactivity disorder. based on the existing knowledge of the precise spatial and temporal mapping of cortical gene expression during the human fetal period, ball et al. (ball et al., 2020) have linked molecular mechanisms using gene expression patterns to non-invasive measures of cortical development by mri in early life (ball et al., 2020). the authors demonstrated that non-invasive imaging of the cortical structure in the neonatal brain is sensitive to differential spatiotemporal patterns of gene expression during gestation. specifically, regional variation in cortical morphometry and microstructure reflects differences in developmental maturity and tissue composition across cortical areas. in addition, they found that interruption to this developmental programming by preterm birth is associated with significant cortical alterations that appear to reflect the selective vulnerability of developing cortical glial populations. nowadays, mainly due to late maternal age, assisted reproductive technology (art) is widely used as a solution for specific infertility problems. however, although the association between the use of art and preterm birth and associated neurodevelopmental risks has been explored in several studies (liu et al., 2017) the result is still inconclusive and has mainly been explored for asd. to analyze the existent of risk of neurodevelopmental disorders among children who are born from this technology, djuwantono and colleagues (djuwantono et al., 2020) published a meta-analysis aimed at quantifying the relative risks of cerebral palsy, intellectual disability, asd, and behavioral problems in children born from different art methods. their study indicates that children conceived by art are at higher risk of acquiring cerebral palsy. cerebral palsy has been associated with preterm birth and low body weight, features also common with art. remarkably, the intracytoplasmic sperm injection technique increased risks of intellectual disability and asd (catford et al., 2018). this technique is mainly recommended in cases of sperm immobility and poor-quality oocytes, associated with higher rates of dna fragmentation as are also advanced maternal age, unexplained infertility, or cryopreservation (inaba et al., 2016). this might be related to neurodevelopmental disorders, as some studies correlate infantile autism with elevated degree of dna damage (porokhovnik et al., 2016). sperm dna damage might be associated with reduced numbers and proportion of trophectoderm cells, promote embryo arrest and induce the activation of apoptotic machinery, all associated with higher prevalence of neurodevelopmental disorders (sedó et al., 2017). colophon the combination of new sequencing techniques and more efficient gene editing tools, with machine learning and deep learning techniques is revealing unexpected complexities in the cellular and molecular neuropathology of ndds. also, revisiting older concepts with new perspectives is changing the perspectives of how development may influence brain function in the long term. we are certainly entering in a new and exciting era of the genomic neuropathology, that combined with classical techniques will help in understanding the rules behind pathogenesis of disturbed brain development. acknowledgements the lab of md is supported by the secretaria d’universitats i recerca del departament d’economia i coneixement de la generalitat de catalunya (grups consolidats 2017 sgr 926). we also acknowledge the support of the agencia estatal de investigación (pid2019-110755rb-i00/aei / 10.13039/501100011033), the european union's horizon 2020 research and innovation programme under grant agreement no 848077, jerôme lejeune foundation, nih (grant number: 1r01eb 028159-01), marató tv3 (#2016/20-30), jpnd heroes project. we acknowledge support of the spanish ministry of science and innovation to the embl partnership, the centro de excelencia severo ochoa and the cerca programme / generalitat de catalunya. the ciber of rare diseases (ciberer) is an initiative of the isciii. references allis, c. d., & jenuwein, t. 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(2019). placental dna methylation levels at cyp2e1 and irs2 are associated with child outcome in a prospective autism study. human molecular genetics, 28(16), 2659–2674. https://doi.org/10.1093/hmg/ddz084 copyright: © 2021 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. reflections on an unconventional neuropathology career feel free to add comments by clicking these icons on the sidebar free neuropathology 1:18 (2020) reflections reflections on an unconventional neuropathology career margaret miriam esiri nuffield department of clinical neurosciences, oxford university, john radcliffe hospital, oxford, ox3 9du, uk corresponding author: margaret miriam esiri · neuropathology department · level 1, west wing · john radcliffe hospital · oxford ox3 9du · united kingdom margaret.esiri@ndcn.ox.ac.uk submitted: 06 july 2020 accepted: 07 july 2020 copyedited by: deanna fang published: 13 july 2020 https://doi.org/10.17879/freeneuropathology-2020-2903 the author´s cv has been attached as electronic supplementary material: cv supplementary material keywords: neuropathology, oxford university, personal reflections i have no idea if this short account will be of any interest to others but in writing it i have a strong sense of good fortune for having had the career i have had as a neuropathologist. my interest in joining the medical profession emerged in my teens and i found myself in 1960, at the age of 18, commencing a degree in physiology at st hugh’s college, oxford university, as the first stage towards attaining this ambition. this 3-year course was to be followed by three years of clinical training and a supervised year of hospital practice before full registration as a medical doctor. women made up about 10% of the medical students at oxford at that time and, in due course, we were distributed in a wide range of medical specialties. the early stages my professional development was complicated by meeting and marrying a fellow medical student from nigeria and embarking on bringing up a family while still a clinical student. this influenced my choice of specialty because it was almost impossible to specialise part-time and front line clinical specialties demanded very long hours of training which would have been incompatible with bringing up a family. therefore, after completing my clinical studentship, i took up a scholarship from the uk medical research council to have training in research methods as the next stage of my education. the training was undertaken under the supervision of professor graham weddell in the department of anatomy. my brief was to spend a year learning the relatively new technique of electron microscopy and applying it to a study of diseased muscle in mice which had been infected with the leprosy bacillus, mycobacterium leprae, after being immunosuppressed by whole body irradiation. graham weddell was an elderly, avuncular character with a kind but rather vague air. he was an expert in the structure of peripheral nerves, a site that the leprosy bacillus was known to damage. by reducing the immune response to the bacillus by subjecting the experimentally infected mice to irradiation, it was thought that light might be cast on how nerves were damaged in the naturally occurring human disease. this was a completely new field of endeavour for me. i knew nothing about electron microscopy, nor leprosy, nor muscle. my first task was to learn how to prepare the tissues, sent to us from a collaborating laboratory in london, for light and electron microscopy. there were other people in the laboratory who were using the electron microscope for other purposes and they could share their expertise with a newcomer such as myself. it took some time to become familiar with the hugely magnified features of the cells being examined with the electron microscope – it was another world that was being displayed before one’s eyes. i went to the books and scientific papers to get help in deciphering the images i was seeing. i had never felt so much need to follow every letter of the message in papers and books about these ultrastructural features. my attention was much more riveted on what i was reading than it had been when i was reading papers for my tutorials as an undergraduate. now the details were needed to help me make sense of what i could see. some of the muscle samples that i studied had been prepared for examination using a light microscope. this was to enable me to get more of a ‘bird’s eye’ view of what i was studying under the electron microscope. i was frustrated to find that for quite a long time i could not find any leprosy bacilli in my electron microscope preparations despite the fact that i could clearly see clusters of them with the light microscope. this happened because the tissue samples used for electron microscopy were tiny compared with those viewed with the light microscope and it was easy to miss such minute structures as bacilli. i began to wonder if i would ever find any bacilli at the ultrastructural level, but then came a red letter day when a whole cluster of them, appearing large and black, were suddenly there in front of me in a new sample of tissue (fig. 1). i fell on them, photographing them with abandon to preserve a record of their structure and their localization in the muscle fibres. fig. 1. mycobacterium leprae in muscle   colony (c) of mycobacterium leprae in a foot-pad striated-muscle fibre. the bacillary membranes can be clearly distinguishedinthose sectioned transversely. the organisms are lying freely in a sarcoplasmic matrix. myofibrils (mf) and mitochondria (m) are also present. thymectomised-irradiated mouse given 107 myco. leprae i.v. 11 mth previously. ua, lc, em x36,000. published with permission from esiri mm et al. 1972; 106: 73-80. for the electron microscopy work i clearly needed to be present in the anatomy department but for the light microscopy i was given the loan of a microscope to take home so that some of my work could be carried on there at times that were the most convenient to me. it was typical of graham weddell that he went out of his way to enable my work to fit in with my home life. from time to time we would have a discussion about the work so that he could satisfy himself that it was progressing satisfactorily but most of the time he just left me to get on with it. in due course i wrote a short thesis and we prepared a paper describing our findings which was submitted to a scientific journal. i was aware as i carried out my practical work in the anatomy department that professor weddell had regular visits from someone from the local teaching hospital, the radcliffe infirmary, who wished to familiarise himself with the electron microscopy of muscle and nerve. he wished to be able to extend to the ultrastructural level the examination he made of muscle and nerve biopsies from patients that were taken for diagnostic purposes. his name was dr trevor hughes (fig. 2) and he worked at the neuropathology department of the radcliffe infirmary. trevor told me that he was hoping to obtain funds to support a junior research officer post in his department that would be devoted to muscle disease research. would i be interested in applying? i explained that i needed to obtain my full general medical council registration by occupying a year of preregistration house officer posts, but we agreed to keep in touch. fig. 2. dr j trevor hughes while i was carrying out this year of leprosy research, i had kept my hand in at dealing with patients by helping out once a week in an endocrinology clinic run by one of the consultants, dr derek hockaday, whose clinical firm i attended for my second medical attachment as a student. derek was aware of my difficulty in obtaining pre-registration house jobs while responsible for a young family. it was necessary to obtain the full gmc registration to enable me to progress to any subsequent clinical specialty. derek was sympathetic to my plight. he had a wife who worked part-time as a paediatric neurologist and they had 3 young children. he eventually offered me a supernumerary pre-registration house job that would not pay me but would give me the experience necessary to secure full registration. this dealt with the 6 months’ medical experience. to complete the additional 6 months’ surgical experience that i needed, i managed to gain approval to work as a house officer in obstetrics, a specialty that did not have routine pre-registration jobs. the advantage of taking this route was that it entailed a 1-in-3 on call rota at night, whereas in surgery this rota would have been 1-in-2. it was also critical that i had the offer of such an obstetric post from the head of the obstetric service, professor john stallworthy, whereas none of the surgeons was willing to offer me a post. thus, i had secured the possibility of getting my full medical registration, albeit with an unconventional set of posts. i also needed to secure the home situation and here we were very lucky to find a very good young nanny, jenny, who was willing to live in so that i could do my nights on call. i enjoyed this year of clinical work but i knew it would be impossible for me to carry on with this type of work in the future. at the end of the year, i therefore applied for, and obtained, the junior research officer post that trevor hughes had secured. the post was for 3 years and it would enable me to work for a dm research degree while allowing me at the same time to see what i thought about a career in neuropathology. i recognised that this was a very small specialty with only about 20 departments in the uk and those confined to major medical centres. i had always kept at the back of my mind the possibility that i might end up working in nigeria. if i trained in neuropathology i would need to re-train if i were going to nigeria. but since our young family had appeared, nigeria had been experiencing a bloody civil war, ruling it out for settling in for the time being. the prospect of three further years living and working in oxford helped us to decide that it would be best if the children were to be brought up with their roots in the uk. we did not think it would be a good idea to move the children to and fro between nigeria and the uk even if the civil war had ended. so, in 1970, i started as a junior research officer in neuropathology. i needed to decide on a topic to research on muscle disease. perusal of the textbooks about human muscle disease seemed to suggest that although there was a great variety of rare diseases, the two categories of disease that were more common were inflammatory myopathies and muscular dystrophies. i chose to study inflammatory myopathies because i had become interested in the role of the immune system in disease during my experience of studying leprosy. there were also not then the tools readily available to carry out genetic studies of muscular dystrophy that are now very effectively in play. inflammatory myopathies can occur at any age but are most common in late middle age. to make a definitive diagnosis, a small sample of muscle – a biopsy – needs to be removed and studied under the microscope to see if inflammation is present. the cause of the inflammation is not certain but it was thought likely that it was due to an immune reaction to some component of muscle, triggered by division and activation of subsets of lymphocytes. i decided to investigate blood lymphocytes taken from people with recently diagnosed inflammatory myopathies and compare their reactions in culture to a small concentration of homogenised muscle with the reactions of healthy people (myself and my colleagues). to do this i needed to find an immunology laboratory that could accommodate me and my lymphocyte cultures. there was such a laboratory in the nuffield department of medicine. it was headed by dr ian mclellan. although the laboratory was already full of researchers, he very generously allowed me to join them. i carried out my human lymphocyte studies there and also studied rats in which i attempted to produce a similar condition of inflammatory myopathy by injecting them with muscle homogenate combined with an adjuvant. while the lymphocytes were being cultured, i also examined the muscle biopsies from the same patients with light and electron microscopy. although there would be no foreign organisms expected to be present in the muscle if this inflammatory myopathy is an autoimmune disease, it still seemed worthwhile to check for any invading organisms, such as viruses, which might be detectable with an electron microscope. to start with, i used the electron microscope in the anatomy department that i was already familiar with but the radcliffe infirmary soon acquired one of its own in the pathology department where it was particularly useful in diagnosing the nature of kidney disease from studying renal biopsies. i was able to use this electron microscope soon after it was installed and it turned out to be very useful for diagnosing some of the rare muscle diseases. just as i had initiated these studies and satisfied myself they were running well, i was overtaken by an unexpected event – a massive fire in march 1971 that destroyed the whole of the neuropathology department and the adjacent hospital library at the radcliffe infirmary. luckily no one was injured. the fire occurred at night and i came in the next morning to find the department, including all my records, razed to the ground. the hospital authorities had to scurry round searching for somewhere to put us. the professor of surgery generously offered an office and there was some recently vacated animal house accommodation, rather dark and damp, into which we could move. having scavenged a few chairs and a desk or two from sympathetic colleagues, we sat ourselves in the animal house to draw breath when who should appear but a former neuropathologist who had moved a few years earlier to london, dr sabina strich. she had heard the news of the fire on the radio and promptly come to visit and commiserate and to provide us with a new electric kettle, mugs, coffee and chocolate wholemeal biscuits. it was such a welcome gesture! it raised all our spirits and made us determined to make the best of the situation. fortunately, my lymphocyte studies could continue in the immunology laboratory which was in a different part of the hospital, even though the records of the first experiments were lost. there were also negatives from the electron microscope studies that could be retrieved and reprinted. some of the brain specimens from earlier years were still retained in a hut in the grounds of another hospital and those that were most instructive for teaching purposes were retrieved and sections re-cut for microscopy. some second hand equipment such as microscopes and microtomes, needed to cut thin sections, were rapidly acquired and the elements of a re-formed department thus assembled. eventually the whole destroyed floor of the hospital was re-built, though this was not to happen for several years. the three years of the junior research officer post passed quickly with my energies divided between the laboratories and the home. we now had a new nanny, jane, who stayed with us until all the children were at full time school. the combination of research and pathology was much more compatible with family commitments. my husband was pursuing a surgical training and holding a succession of short term posts all over the country as was the usual practice in those days. he returned home whenever he was able to. the lymphocyte studies yielded modest evidence of lymphocyte sensitivity to muscle in inflammatory myopathies. it was not a dramatic effect and could have been the result, rather than the cause, of the condition. it was difficult to provoke any more than a slight inflammatory reaction in the muscle of the sensitised rats. in the electron microscopic studies, there was only one biopsy that contained what might have been virus-like particles. thus, the fruits of my studies were far from conclusive. i believe it remains the case today, more than 40 years after these studies were performed, that inflammatory myopathies are really little better understood. however, the thesis i wrote describing these studies earned me a dm degree and i published a couple of papers summarising the findings. i had found satisfaction from this work but now felt increasingly curious about the central nervous system and its diseases that i had started to become familiar with. however, before i was certain that neuropathology should become my chosen specialty, i needed to become familiar with other branches of pathology as part of my training. what did these two short periods of research teach me? first they reinforced my admiration for science. i particularly admired the way science was prepared to embrace the contributions of people wherever they came from and whatever their background provided they worked with integrity and had innovative ideas. i admired the way science relied on reproducibility of findings to give them credence and not on the advertising ability of their perpetrators to give validity to their findings. i came to appreciate the importance of karl popper’s dictum that scientists should seek to refute their ideas, not confirm them. scientific ‘facts’ are always provisional and can be revised if subsequent experiments are incompatible with them. i learnt to recognise the importance of quantitation in recording results of experiments and the need, in studying disease, to include control samples as well as samples from the disease under investigation. it was important for the investigator, whenever possible, to be unaware of which samples were from controls and which from disease so that there was no chance of bias creeping into the observations being made. it seemed to me that if careful quantitative studies were performed they could almost always yield useful information even if they showed that the ideas that prompted the experiments turned out to be incorrect. in short, science satisfied my urge to ask questions and seek answers to them and having commenced my efforts in that direction i was going to continue if i could. the next stage to become a neuropathologist in the 1970s i needed first to gain experience of other specialties of clinical pathology. i became a trainee registrar with a post that rotated through haematology, bacteriology, virology and histopathology. the longest time (2 years) was spent in histopathology (also known as cellular pathology). there were exams to take for membership of the royal college of pathologists once the practical experience had been obtained. it was essential to gain this qualification before applying for a post as consultant in the nhs. the exam had a heavy component of histopathology, hence the longer time spent in that specialty. i didn’t find the haematology or bacteriology very interesting. the numbers of samples to be analysed in each of these departments were so overwhelming that much of the work had been automated. many samples would typically be sent to each of these departments during the course of each patient’s hospital admission and, in addition, many samples were sent in each day from local general practitioners. the departments were primarily concerned with delivering a diagnostic service and there was relatively little that i, as a newcomer, could contribute. they did not undertake active research. histopathology was of greater interest, being more directly relevant to neuropathology. i learnt how to perform post mortem examinations (in the process giving myself the only nightmares i’ve ever had, in which the corpse came to life while i was busy and i attempted to replace the larynx so that it could speak!) and to report on surgical specimens of tissue. because of the large variety of specimens and of the conditions they exhibited, it took a fair time to be able to make correct judgements on the nature of the disease, often cancer. samples were examined under the microscope, first by myself and then by a consultant who was able to share his or her experience and pass on to me the critical visual clues that yielded the essential evidence about the nature of the disease process. the head of the histopathology department was dr robb-smith, a slightly shy and enigmatic character whom i had encountered as a student at post mortem demonstrations. he was interested in attracting women to his specialty and had secured a trainee post that could be held part-time. this enabled me to have two free afternoons each week. i found the children were all occupied at these times so i used this time to write up my dm thesis, a task i had been putting off while i dealt with more immediate concerns at home and at work. one of the great advantages for me of working in the histopathology department was that i not only gained essential diagnostic experience but i was also able to participate in research. there was a research officer there, dr ian burns, who was experimenting with the use of antibodies to detect specific proteins in tissue sections. if antibodies could be produced towards a purified protein of interest and tagged with a marker substance such as a coloured dye or a fluorescent dye, this could be used to detect specific proteins in tissue sections with much greater specificity than could be provided with the traditional tinctorial stains. this was the principle behind the work that ian burns was carrying out and which has now become the hugely successful branch of pathology, immunohistology or immunocytochemistry. when ian was working in the early 1970s, he was concentrating on the use of antibodies to detect immunoglobulins, themselves antibody proteins produced by plasma cells. i was able to make use of ian’s reagents to study plasma cells containing different types of immunoglobulins in sections of central nervous system tissue affected by different diseases. this was research i undertook when i had moved back from the histopathology department to be a registrar in the neuropathology department. my experience in more general pathology had not seduced me into getting more interested in that field. rather, i thought there were important things i had learnt there, particularly this new subject of immunohistology, that i could usefully apply in neuropathology. back in the neuropathology department, i was also gaining experience of diagnostic aspects of the specialty by examining biopsies removed from brain tumours by the neurosurgeons as well as other biopsies such as those from muscle and peripheral nerves. i was also performing post mortem examinations on cases of neurological disease and brain trauma. this diagnostic work i found still left time for a limited amount of research in which i applied the antibody techniques i had been lucky enough to learn about from ian burns to nervous system diseases. this was an example of the wonderful way that collaboration can be developed in science and i was to become a great advocate of such collaboration, prizing it far above competition which, in some fields or departments of medical science sadly seemed to predominate. i was lucky enough to obtain the modest funds needed to enable me to employ a technician to assist with the application of immunohistology in neuropathological material. i started by studying the lesions or areas damaged in the central nervous system in multiple sclerosis, most generally considered as an autoimmune disease1. this was a condition that was of interest to the other consultant in the neuropathology department, dr david oppenheimer (fig. 3), who had written a dm thesis on it. he had seen many examples of this condition in post mortem studies and the tissues from some of these cases were still available in the department despite the fire that had destroyed much material in 1971. one of the beauties of the immunohistological technique was that it could be used on tissue that had been preserved in the department after being fixed in formalin and then embedded in paraffin wax to enable thin sections to be cut. although some proteins that may be of interest in tissues are destroyed by such treatment, meaning that fresh frozen sections are needed to study them, there are many others that survive this treatment. the advantage of being able to use formalin-fixed, paraffin-embedded sections is that this tissue shows better preservation of its structure than can usually be seen in frozen sections. fig. 3. dr david oppenheimer i followed up the work on multiple sclerosis with studies of diseases that did have a foreign organism in the damaged tissues, starting with poliomyelitis of which we had cases dating from an epidemic of polio in the uk in the late 1950s. later i extended the work to a study of cases of the rare complication of measles, subacute sclerosing panencephalitis. i wanted to know if these diseases showed a similar distribution of plasma cells containing immunoglobulins as i saw in multiple sclerosis. although this turned out to be the case, the difficulty in interpreting the significance of the finding was that the arrangement of plasma cells might be just the same in autoimmune disease as in one with a foreign organism. while these studies were proceeding, more antibodies for use in immunohistology were being produced, including some directed to viruses. i was to make use of these for a later study when i was a research fellow. the neuropathology department when i joined it was a small one. there were 2 consultants, drs hughes and oppenheimer, a registrar (myself), 3 technicians and a laboratory assistant. trevor hughes had a particular interest in muscle disease and spinal cord disease. he had moved into neuropathology from histopathology. david oppenheimer had a less conventional background. he had started his academic career with degrees in philosophy, politics and economics and also music. during the second world war, he had been a conscientious objector and worked with an ambulance team in london rescuing injured people in the blitz. this experience awakened his interest in medicine and he read for his medical degree immediately after the war. initially he worked on neuroanatomy and it was from there that he moved to neuropathology. he was initiated into diagnostic work by his predecessor, sabina strich, over a matter of a few months. he recognised that his lack of experience in histopathology and other pathology specialties was a handicap but his knowledge of neuroanatomy was most valuable. it made him particularly interested in the many important diseases of the nervous system in which there is a neuroanatomical basis for the way the disease is expressed or, in some cases, seems to spread. i had been introduced to the neuropathology department by trevor hughes but as time went on i became more interested in the conditions that interested david oppenheimer, starting with multiple sclerosis. one of the first opportunities that arose from the diagnostic work that i carried out was a chance to study a rare case of herpes zoster (shingles) in an elderly woman who happened to die just after the eruption developed. it wasn’t the herpes zoster that killed her but an advanced state of multiple myeloma which had rendered her susceptible to developing herpes zoster. my patient had herpes zoster affecting the region supplied by part of the trigeminal nerve. herpes zoster is caused by the same virus that causes chickenpox, varicella-zoster virus. the theory had been developed that in the natural history of infection with this virus, chickenpox is the first manifestation of infection. as the virus is overcome by the immune response that enables the person with chickenpox to recover, some virus survives and lies latent in the nerve cells of one or more sensory nerves. circulating antibody which persists after recovery from chickenpox makes any reactivation of the virus uncommon until later in life when the level of antibody wanes or when the person has their immunity compromised by disease. when the virus reactivates in the sensory nerve cells of a particular nerve, it travels along the nerve to the skin where it causes the eruption of herpes zoster. although this theory was widely accepted, the reactivation of the virus in nerve cells had never been demonstrated. it was this reactivation that i was able to show by electron microscopy which revealed the virus particles in the nerve cells of the trigeminal ganglion (fig. 4). a colleague in the virology department, dr albert tomlinson, carried out immunofluorescence using an antibody to the varicella-zoster virus on frozen sections of the ganglion and the nerve and was able to show the virus at both sites. fig. 4. herpes zoster viral particles in the cytoplasm (c) of a trigeminal ganglion neuron.   from esiri mm, tomlinson ah j neurol sci 1982; 15(1); 35—48. the varicella-zoster virus that dr tomlinson was able to show in frozen sections of the nerve and ganglion of our case could not be seen when we tried to detect the virus in formalin fixed tissue – the viral protein was an example of an antigen that could not survive formalin fixation. this observation made me think that there could be more we could learn from our post mortem and surgical specimens if we thought carefully about whether we should preserve a portion of tissue by snap freezing it in liquid nitrogen and then storing it in a -70◦c freezer. snap freezing in liquid nitrogen preserved the tissue structure better than slowly freezing tissue in a freezer but only if the tissue sample was small. the opening of our minds to the potential importance of deep freezing some tissue was to prove important in years to come, not only to enable immunohistology to be performed for detection of antigens that were sensitive to formalin but also to enable biochemical and genetic studies to be carried out. it led to oxford being early in the field of brain banking for research (for a history of brain banking in the uk see 2). the highly trained technicians that we worked with in neuropathology played an absolutely essential role in all that we did. unlike in histopathology, where most tissue sample sections were subjected to a single stain, in neuropathology it was common to have consecutive thin sections from the same block of tissue stained with several different stains, some of which were quite difficult to perform. each technician had their special expertise in carrying out a selection of these stains. some were histochemical stains that enabled enzymes to be displayed in frozen sections. these histochemical stains were mainly used on muscle biopsies. each of the tinctorial stains picked out a particular feature of the nervous system to highlight. nowadays the need for these special tinctorial stains is much reduced because immunohistology can convey the same information using antibodies that are more specific and more straightforward to apply. some of the reagents used in the tinctorial reactions are quite toxic and it is far preferable that technicians can now avoid them. my closest colleagues were those in the neuropathology department. trevor and david took it in turns, several months at a time, to supervise my diagnostic work and i had the close assistance of the technicians in the preparation of the sections i needed to do this work. i liaised closely with the neurosurgeons and neurologists over the results. at times there was another trainee doing a project in the department or a visitor making use of the material we had available for them to study. i remember particularly a very pleasant, recently retired, canadian professor of anatomy, richard saunders, who came to spend a few winters in oxford with his wife to escape the worst of the winter in nova scotia. they had a son who was an oxford rhodes scholar. richard had an intimate knowledge of the structure of the hippocampus which he did his best to impart to me. it turned out to be useful knowledge when i became interested in alzheimer’s disease. i made regular visits to local district general hospitals where i provided a diagnostic service on neurological post mortem cases. i discovered that histopathologists in these hospitals had to deal with quite a lot of neurological cases as the hospitals had consultant neurologists working in them. this visiting service yielded many interesting cases from which i brought back to our department selected samples for expert processing. i would then send reports on these cases back to the histopathologists. once or twice a year there were meetings of the uk neuropathology community. trainee and consultant neuropathologists could apply to join the british neuropathological society, which organised these meetings, after they had delivered one sponsored paper or poster at one of the meetings. some overseas neuropathologists also joined the society. my first paper to the society, sponsored by trevor, described the work i had done on experimental leprosy infection in the anatomy department. it was interesting to get to know neuropathologists working in other centres and to compare notes on our respective research projects. i found them to be a very congenial group. collaboration with some of them became an important asset in devising some research projects for which more cases of a particular disease might be needed than were available in one centre. it was also helpful at times to be able to send a puzzling diagnostic specimen to someone in another centre to obtain a second opinion on the diagnosis. towards the end of my time as a registrar i made the acquaintance of a recently appointed consultant in geriatric medicine, gordon wilcock. gordon had come to enquire if there might be someone interested in collaborating with him to study the neuropathology of dementia, particularly alzheimer’s disease. he was himself interested in learning some neuropathology and he had many patients under his care in the geriatric medicine wards on whom, if they agreed, he had carried out a simple cognitive test to determine whether or not they had dementia. a considerable number of them had dementia but there were others who did not, including quite a number who had had strokes. if any of these patients subsequently died, and their next-of-kin was willing for a post mortem to be performed, we were able to see what abnormalities there were in the brain that contributed to making them demented. it was known that in alzheimer’s disease there were two distinct microscopic abnormalities: the presence of plaques and tangles. gordon and i wanted to see if we could relate either plaques or tangles (or both) to the presence of dementia in his cognitively tested patients. we had the impression that the distribution of tangles in alzheimer’s disease affected parts of the brain that were becoming known to be anatomically linked together. we took to discussing the neuroanatomy of alzheimer’s disease on friday afternoons with experts on these cortico-cortical and cortico-hippocampal connections, drs tom powell and his former dphil student, carl pearson. tom had spent his career on meticulous studies of the cerebral cortex and was greatly pleased to find that some of his work might have relevance to an understanding of such an important disease as alzheimer’s disease. from these discussions came a study of the detailed distribution of plaques and tangles in the different layers of the cerebral cortex3. the findings fitted well with what was known of the manner in which one part of the cortex was connected to others and to the hippocampus. those regions most closely linked to the hippocampus had many tangles while those parts of the cortex which were most remote from the hippocampus had the fewest tangles. this work was my introduction to trying to understand alzheimer’s disease from a neuropathological perspective. another influence in fuelling my new interest in dementia was a publication by a neurochemist at london’s institute of neurology, dr david bowen. in the mid-1970s, he published a paper showing that in post mortem brain specimens that had been frozen at -70◦c immediately after removal from the body there were many enzymes whose activity could be reliably measured. he furthermore showed that in alzheimer’s disease the enzyme, choline acetyl transferase, required for the synthesis of acetyl choline, was deficient. a similar finding was reported from two other laboratories at about the same time. this paper was a real eye-opener for me. i had previously assumed that after death all enzyme activity would cease. gordon and i had discussions with david about providing him with frozen brain samples from the patients gordon had tested for cognition on the wards and we were soon adopting the policy of deep freezing one side of the brain for david’s studies and carrying out our microscopic studies on the other side. this led to a realisation that it is the burden of neurofibrillary tangles that is held within the brain that determines the extent of dementia in alzheimer’s disease, much more so than the amyloid load4. i have watched with dismay the dominance of amyloid in thinking about alzheimer’s disease and how to prevent it that has lasted for decades. hopefully, now that may change5. not long after we had set up this programme to study dementia, gordon was offered a professorship in care of the elderly at bristol university and left oxford but there was an interested consultant in geriatric psychiatry at the large littlemore mental hospital near oxford, dr john robinson, who was able to play a similar role in supplying cases of dementia, though he was not in a position, as gordon had been, to supply cases that did not have dementia. this collaboration with clinicians in our research on dementia was the first time i had had the opportunity to work directly with doctors looking after patients to devise a research strategy. neuropathologists are too remote from direct patient care to be in a position to recruit patients to a study themselves. some neuropathologists developed interests in brain tumours that they could follow using the biopsy specimens they received. others developed experimental models of diseases that could be studied alongside human post mortem material. but i think the most valuable contribution that clinical neuropathologists can make is to work closely with their clinical colleagues to recruit patients into clinic-pathological studies of human disease. by the mid-1970s, i had acquired enough experience to take the royal college of pathologists examination in histopathology slanted to neuropathology. the written examinations were taken in london and for the practical exam i was instructed to go to glasgow. i remember casting my eye hastily over my notes on the long train journey to glasgow and thinking that to be taking examinations at the age of 35 years was too old! fortunately, i passed this last one and was then in a position to think about the next step in my career. for the sake of our children who were happily settled in schools in oxford, i wanted to remain there but there was only one academic post in neuropathology and that would be occupied by david oppenheimer for another few years. one of my colleagues in the histopathology department made a most helpful move at this point. he wrote to the medical research council on my behalf and asked if there might be a prospect of getting support from this source to fund research for a few years in oxford. an encouraging reply to this enquiry led to me applying for a senior clinical fellowship from the medical research council. becoming a senior clinical fellow i put together an application for this senior clinical fellowship to study a disease that had long been of interest to me – herpes simplex encephalitis. this is a rare condition but one that had a considerable mortality at that time and which i had the chance to study post mortem. there were several enigmatic aspects to this disease which intrigued me. it seemed to affect people of a wide range of ages in whom it developed quite unexpectedly. yet it was caused by a virus that infected some 90% of people in some of whom it caused recurrent cold sores. like its cousin, varicella-zoster virus, which i had already encountered, it had the capacity to lie latent in sensory ganglia (for this virus, usually the trigeminal ganglion, because it usually causes a facial infection) from which it could reactivate under a range of conditions such as fever, exposure to strong sunlight or the common cold. what was the process by which it could occasionally cause a devastating encephalitis? i knew i would be able to study the distribution of the virus in the brain using immunohistology and an anti-herpes simplex antibody, and i had tissue from enough cases, which had survived for varying periods of time after disease onset, to enable me to try to piece together the pathological process. armed with this intention i was lucky enough to get the fellowship for 5 years at consultant level salary plus some part-time technical help and costs of consumables. this period of full-time research reinforced my passion for investigating brain diseases of which there seemed so many without an effective treatment, in part a consequence of inadequate understanding of the disease process – hardly surprising given the complexity of the nervous system and the difficulty of accessing brain tissue during the course of many of these diseases. at the time, brain imaging was still rudimentary compared with how it is today and the post mortem examination, for those diseases that were fatal, gave the best lead to increase understanding. herpes simplex encephalitis has a very intriguing distribution of damage in the brain with an emphasis on the temporal lobes but with the damage usually asymmetrically distributed6. the focus of damage overlaps with that in alzheimer’s disease with the hippocampus and sites connected to it badly affected. from my visits to the anatomy department to discuss alzheimer’s disease with tom powell and carl pearson, i knew these areas had close connections with the olfactory system so i studied the olfactory bulbs in these cases of herpes simplex encephalitis because it seemed possible that the virus, with its propensity to travel along axons, might reach the brain along this route. i could indeed detect the virus there but the olfactory bulbs were not as damaged as sites in the amygdala and piriform cortex to which the olfactory tracts projected. thus it seemed possible that the virus reached the olfactory bulbs by moving centrifugally, not centripetally, perhaps from a site of latency within the brain, perhaps the hippocampus. the asymmetry of damage to the temporal lobes, which was more marked in those dying early in the course of the disease, might be explicable if it reactivated from inside one hippocampus and then travelled to the contralateral one via anatomical connections between the two. to take this work further i teamed up again with albert tomlinson, the virologist who had helped me examine the case of herpes zoster. we developed a mouse model of herpes simplex encephalitis and showed that inoculation of the face led to the virus ascending to the brain via both the trigeminal and olfactory routes and the development of both brain stem and temporal lobe inflammation. others have pursued the possibility that latent infection of the brain with herpes simplex virus may predispose to the development of alzheimer’s disease but the opportunity to further investigate the human acute encephalitis was lost on account of the fortunate development of an effective treatment which reduced the fatality of the disease and resulted in many people affected recovering at least partially. a change of direction? while i was intent on this research, my children had grown into their teens and my husband was exploring the possibility of returning to nigeria to provide medical care there. this posed a dilemma for me. should i abandon my neuropathological career and re-train in tropical medicine or could i carry on with my neuropathology in oxford part-time while taking periods of unpaid leave to join my husband in nigeria during school terms when our two younger children were at boarding school? the other alternative to pursue neuropathology in nigeria, in the very remote region in which my husband decided to set up a medical practice would have been impossible. i was able to negotiate to carry on part-time in oxford for the last part of my research project, working full-time while in oxford and visiting nigeria between times. strange though such an arrangement might seem, it actually worked well and, by the time the research was completed, david oppenheimer had retired, giving me the expectation that i might apply for his (now vacant) post. that expectation was thrown into confusion by the government of the day deciding to impose drastic cuts to university financial support. as a result, all re-filling of posts was frozen and many posts were lost. i remained in a form of professional limbo with short-term medical research council support lasting 3 months at a time until a new professor, john newsom-davis, was appointed in the clinical neurology department on the retirement of the previous postholder. john offered to attempt to reinstate the lectureship in neuropathology which had previously been held in the cellular pathology department, but only if it could be held in the clinical neurology department. the post was rescued in this way and i was appointed to it on a part-time basis that allowed me to continue with my visits to nigeria. my husband’s choice of where to establish his clinic was partly determined by his wish to live in a part of the country that is decidedly cooler than the rest. he hated the tropical heat of most of the country and he knew i would too. his clinic was established in a town, gembu, on the mambilla plateau, some 1,500 metres high and not far from the cameroon border which lies to the east of nigeria. the countryside is made up of grass-covered hills, interspersed with deep, fertile, valleys and the climate is really perfect, with temperate, warm, sunny weather and abundant rainfall. the roads were rudimentary in the extreme and getting to the place took some 12-15 hours of uncomfortable land rover motoring after a domestic air flight to the nearest place with an airport, yola. over the years, a tarred road of sorts was created that mounted the steep escarpment to the plateau but it was soon full of potholes that were hardly ever repaired. spending 2 months at a time in gembu in the spring and autumn of each year became my routine for about 20 years from the early 1980s. because i couldn’t continue with neuropathological research while i was away, i decided to turn my attention to writing about the diseases i was interested in. my aim was to inform those coming after me about what i had learnt so far. my first book was written with john booss, a neurologist from yale, who came to oxford on a sabbatical to study multiple sclerosis. together we wrote a book on viral encephalitis7. to write my parts, i took piles of references with me to read and digest in gembu, mainly in the afternoons and evenings after i had helped in the mornings at my husband’s clinic. my offerings there were decidedly amateurish but one felt that in such a place, where medical care barely existed, one was justified in doing one’s best. once i had gained a little confidence from seeing people recover or, at worst, return the next day for a re-think about the diagnosis, i thoroughly enjoyed this experience. with no diversions such as newspapers or television to occupy the time later in the day i was able to think and write without interruption, although, in the evenings, any activity had to be carried out by the dim illumination offered by a ‘tilly’ light, fuelled by kerosene. to follow up after publication of viral encephalitis, i approached david oppenheimer, who had recently retired, to see if he would be willing to write a book on diagnostic neuropathology with me. i felt greatly honoured to have had much tuition from david during my training. he was a truly inspiring person and i felt it would be good if other people could benefit from his teaching even if it had to be in the form of a textbook. i knew he wrote superbly so could convey his knowledge in that way. he thought about it and agreed to write a ‘pamphlet’ in which we would divide up the topics evenly between us. he had kept the photographic slides that he had collected over his career so was able to make use of these and entered into the writing of the ‘pamphlet’ with enthusiasm. i continued to learn from him as we wrote and it was a complete pleasure, at the end of a working day when i was in oxford, to visit him and his wife on my way home and to share the new text he had written during the day. i think he also enjoyed being able to extend his teaching of neuropathology in this way. the result was a textbook of nearly 400 pages that was published as diagnostic neuropathology8. the third book that i initiated was one on the neuropathology of dementia9, which was co-edited with my colleague james morris, who had stepped into the post that trevor hughes had vacated when he retired. that all these three books went in to 2nd editions pleased me as it suggested they had found useful homes among those in the neuroscience community. by this time, a major longitudinal study of ageing and dementia, the oxford project to investigate memory and dementia (optima,) had been started by david smith, professor of pharmacology in oxford, and we were benefitting from the opportunity to study the brains of dementia sufferers and healthy elderly controls who had been followed up in a detailed way during life and had consented to donating their brain for research after their death. this study grew slowly, as such longitudinal studies do, but we eventually collected over 500 brains that helped to increase understanding of dementia, and still continue to do so. forging ahead once i had been appointed to an academic position in neuropathology in the mid-1980s, i started to supervise graduate students who wished to study the brain and its diseases. i always insisted that students should have two co-supervisors because of the periods i spent in nigeria and out of contact with the department. two months was the maximum that i considered possible to remove myself from all the activities that go on in a department that has diagnostic, teaching and research interests. sometimes i would return to find a pile of problems to solve but usually everything had continued smoothly and i had the pleasure of hearing about the new findings that had emerged while i was away. because of these periods of leave i kept my other absences from the department to a minimum which meant attending very few conferences and meetings – a sacrifice that i was willing to make in return for the privilege of being able to continue with my professional work in my rather unusual personal circumstances. the department was expanding at this time (the late 1980s) with the arrival in oxford of professor tim crow, a psychiatrist with a deep interest in biological aspects of psychiatric disease, particularly schizophrenia. we were able to expand into a new small laboratory and recruit an experienced technical manager and a small group of research students to work on psychiatric disease. we were also joined by a research group created by a local academic psychiatrist, dr paul harrison. i was never in any doubt that study of the brain was needed as much to understand these diseases as it was for dementia and ageing and for neurological diseases. in the neuropathology department, i had wonderful collaborations with psychiatrists as well as neurologists and neurosurgeons. it seemed very odd to me that there was little direct communication between clinical neurologists and psychiatrists and little shared training for the two disciplines but this may be changing now. there were also superb opportunities in oxford to collaborate and learn from neuroscientists in the basic science departments from which we benefitted enormously. a great boost to our research on schizophrenia came from the generous provision of a substantial group of brains donated for research in belfast, northern ireland. professor ingrid allen, the senior neuropathologist in belfast, and her colleague in psychiatry, dr steven cooper, were instrumental in creating this initiative with tim crow. tim had a fascinating hypothesis about schizophrenia: that it was somehow related to subtle differences in cerebral asymmetry, that differs normally between men and women, but which he thought might be disturbed in those with schizophrenia. this led to our studies of the brain in schizophrenia being carried out having regard to sex and cerebral hemisphere side. we also studied the corpus callosum and other tracts that connect the two hemispheres. we found tantalising findings on this basis that seemed to indicate, among other things, that schizophrenic males have cerebral hemispheres more akin to normal females than occurs normally in males and vice versa for female schizophrenics. we should not have found these differences if we had studied schizophrenia without distinguishing between the sex of subjects whose brains we studied. although it has become fashionable in psychology to downplay differences in brain structure between males and females i think these differences are important and may have significant roles in psychiatric disease that we are not yet able to understand. while these attempts to understand psychiatric disease were launched, we were also continuing to investigate dementia and ageing with the optima resource. a graduate student, zsuzsa nagy, who remained for a while in the department as a postgraduate research scientist, discovered that neurons expressed antigens indicating they had re-entered the cell division cycle in elderly subjects. this discovery, which has been repeatedly confirmed by others, led to studies of lymphocytes and the suggestion that properties of these cells in culture might have diagnostic value for alzheimer’s disease. although this work has been followed up, we are still without a simple method of diagnosing this disease, particularly in its earliest phase, when symptoms are absent or minimal, but at the best time for any intervention to be made to slow or stop its development. my interest in multiple sclerosis was re-ignited at about this time by two developments. the first was provoked by a paper that came out in the lancet linking episodes of clinical expression at the start and in relapse in multiple sclerosis to episodes of previous sinusitis. the author, frederick gay, was a general practitioner and he made his observations using general practitioner records. he thought it possible that bacterial antigens derived from microorganisms in the paranasal sinuses might reach the cns compartment in some individuals and lead to an immune response that created the demyelinating, inflammatory lesions typical of this condition. we worked together on this idea but, given its unfashionable nature at a time when most interest lay in viruses having a role in the disease (if, indeed, any foreign antigens were involved), funding to make progress was hard to acquire. the idea, however still has great attractions, in my opinion, and it is still being actively pursued. the second development that brought back my active interest in multiple sclerosis was applying immunohistology yet again but this time using an antibody to amyloid precursor protein as a marker of damaged axons. although axons have long been known to be damaged in ms, the extent of that damage, its timing and its role in progression of the disease were little appreciated largely because there had been no sensitive method of assessing its extent. being able to detect damaged axons with great sensitivity, which detection of amyloid precursor protein provided, changed this situation and when we applied the technique to multiple sclerosis lesions of differing age i was greatly surprised by the result. i had expected that we should see damaged axons in chronic lesions which are common in progressive disease but it was actually in the most recent lesions that axon damage was most prolific10 (fig. 5). this was despite the fact that recent lesions are more common at earlier phases of the disease when recovery from relapses is common. initially it seemed hard to understand how damage to axons, which we assumed was irreversible, was compatible with a history of relapse followed often by remission. this pattern of illness seemed more compatible with pathology that was reversible, likely demyelination and the inflammation that accompanied it. but we soon realised that because there is much redundancy in the cns, progression of damage would only occur when a threshold of axon loss and other forms of neurodegeneration was reached, leaving room for recovery between episodes of inflammation before that point was arrived at. fig. 5. graphs to illustrate the profile of axonal injury (app stippled areas) and the number of macrophages (black areas) in a typical acute multiple sclerosis lesion (a), active chronic multiple sclerosis lesion (b) and chronic multiple sclerosis lesion (c).   from ref. 10 this reassessment of multiple sclerosis and, in particular, its neurodegenerative aspects, made me acutely aware of how progress in understanding disease depends on new techniques being applied to enable new knowledge to be acquired. the spectacular developments in neuroimaging over the past few decades have enabled much progress to be made in understanding cns diseases and this will continue. nevertheless, there is still a place for pathological studies because these are still needed to penetrate to the cellular and molecular levels of change that accompany disease. without knowledge at these levels rational treatment or prevention cannot be achieved. the new frontier relating to knowledge that has grown up in the last two decades is about genetics. here there is such a wealth of new information that new ways of managing data are needed but i remain convinced that it is the protein and other structural aspects of cells that will provide the key answers to what happens to the state of the body in disease and, therefore, what can be done to remedy it. fig. 6. margaret m esiri (2000) the future along with new technical developments enabling fresh knowledge to be acquired about disease, it is vital that there are new ideas. these arise from human imagination and ingenuity. ideas can come to a researcher from all sorts of experiences and at any stage of their career but, in the long run, a steady supply of new research students with novel ideas is essential. that is why i have been so fortunate in having a career in an outstanding university in which tutors take extraordinary efforts to select the best students some of whom can then become the generators, along with newcomers from elsewhere, of the next wave of insights. they start off without the baggage of long indoctrination with the previous and current dogmas and bring creativity to a research team. students that i have supervised have repaid my confidence in them in spades and i feel their hands are fully capable of taking forward the fields that came to fascinate me. the person i have kept in closest touch with is gabriele deluca, now a consultant neurologist at oxford with a lively research group, who brings energy, creativity and flare to his research and teaching, making him an inspiration for yet another generation of young medical students and scientists. one thing writing a memoir brings home is the manner in which attitudes and knowledge reach you and which you, in turn, pass on to others. there is a social angle to all this, with an amplifying effect of teaching and example, that, at best, is inspiring and uplifting. at the age of 78, i still find it impossible to abandon my interests in neuropathology and i am incredibly lucky to have a department that still welcomes me into its fold. fig. 7. left: margaret m esiri, right: a composite painting depicting the three buildings in which i worked as a neuropathologist: radcliffe infirmary (left), st hugh’s college (centre), john radcliffe hospital, west wing (right). references 1 esiri mm. multiple sclerosis: a quantitative and qualitative study of immunoglobulin-containing cells in the central nervous system. neuropathol appl neurobiol 1980; 6: 9-21. 2 wellcome trust witness seminar on the development of brain banks in the uk 1970-2010. 3 pearson rc et al. anatomical correlates of the distribution of the pathological changes in the neocortex in alzheimer’s disease. proc natl acad sci 1985; 82: 4531-4. 4 wilcock g, esiri mm. plaques, tangles and dementia: a quantitative study. j neurol sci 1982; 56: 343-56. 5 esiri mm. target tau, not amyloid, to prevent and treat alzheimer’s disease. obm geriatrics 2020; 4, issue 1 doi 10.21926/obm.geriatr2001103 6 esiri mm. herpes simplex encephalitis. an immunohistological study of the distribution of viral antigen within the brain. j neurol sci 1982; 54: 209-26. 7 booss j, esiri mm viral encephalitis: pathology, diagnosis and management. blackwell scientific publications, 1986. 8 esiri mm, oppenheimer dr. diagnostic neuropathology: a practical manual. blackwell scientific publications, 1989. 9 esiri mm, morris jh (eds) the neuropathology of dementia. cambridge university press, 1997. 10 ferguson b et al. axonal damage in acute multiple sclerosis lesions. brain 1997; 120: 393-9. copyright: © 2020 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. sars-cov-2 vaccination induced cerebral venous sinus thrombosis: do megakaryocytes, platelets and lipid mediators make up the orchestra? feel free to add comments by clicking these icons on the sidebar free neuropathology 2:18 (2021) opinion piece sars-cov-2 vaccination induced cerebral venous sinus thrombosis: do megakaryocytes, platelets and lipid mediators make up the orchestra? kate chander chiang1, ravi raghavan2, ajay gupta1,3 1 kare biosciences, orange, ca 92869, usa 2 department of pathology, loma linda university medical center, loma linda, ca 92354, usa 3 department of medicine, university of california irvine (uci) school of medicine, orange, ca 92868, usa corresponding author: ajay gupta · division of nephrology, hypertension and kidney transplantation · university of california irvine (uci) school of medicine · orange, ca 92868 · usa ajayg1@hs.uci.edu submitted: 6 june 2021 accepted: 1 july 2021 copyedited by: biswarathan ramani published: 7 july 2021 https://doi.org/10.17879/freeneuropathology-2021-3395 keywords: sars-cov-2, covid-19, vaccine, thrombosis, cerebral venous sinus thrombosis, aspirin, thromboxane, cox-2, adenoviral vector, megakaryocyte abstract the covid-19 vaccines comprised of adenoviral vectors encoding the spike (s) glycoprotein of sars-cov-2 are highly effective but associated with rare thrombotic complications. the adenovirus vector infects epithelial cells expressing the coxsackievirus and adenovirus receptor (car). the s glycoprotein expressed locally stimulates neutralizing antibody and cellular immune responses. these vaccines have been associated with thromboembolic events including cerebral venous sinus thrombosis (cvst). s glycoprotein stimulates the expression of cyclooxygenase-2 (cox-2) and leads to massive generation of thromboxane a2 in covid-19. megakaryocytes express car and we postulate that s glycoprotein stimulated generation of thromboxane a2 leads to megakaryocyte activation, biogenesis of activated platelets and thereby increased thrombogenicity. cerebral vein sinuses express podoplanin, a natural ligand for clec2 receptors on platelets. platelets traversing through the cerebral vein sinuses could be further activated by thromboxane a2-dependent podoplanin-clec2 signaling, leading to cvst. a prothrombotic hormonal milieu, and increased generation of thromboxane a2 and platelet activation in healthy females compared to males is consistent with increased risk for cvst observed in women. we propose that antiplatelet agents targeting thromboxane a2 receptor signaling such as low-dose aspirin merit consideration for chemoprophylaxis when administering the adenovirus based covid-19 vaccines to young adults at risk of thrombosis provided there are no contraindications. abbreviations txa2 thromboxane a2, dic disseminated intravascular coagulopathy, cox cyclooxygenase, ttp thrombotic thrombocytopenic purpura, cvst cerebral venous sinus thrombosis, clec c-type lectin-like receptor tlr, toll-like receptor, car coxsackievirus and adenovirus receptor, covid-19 coronavirus disease 2019, sars-cov-2 severe acute respiratory syndrome coronavirus 2, pf4 platelet factor 4 introduction covid-19 disease is caused by a novel positive-strand rna coronavirus (sars-cov-2), which belongs to the coronaviridae family, along with the severe acute respiratory syndrome (sars) and the middle east respiratory syndrome (mers) coronaviruses.1 the genome of these viruses encodes several non-structural and structural proteins, including spike, envelope, membrane, and nucleocapsid proteins.2 the majority of the vaccines for covid-19 that employ administration of viral antigens or viral gene sequences aim to induce neutralizing antibodies against the viral spike protein (s), preventing uptake through the ace2 receptor, and thereby blocking infection.3 the janssen covid-19 vaccine (johnson & johnson) is comprised of a recombinant, replicationincompetent ad26 vector, encoding a stabilized variant of the sars-cov-2 spike protein. the chadox1 ncov-19 vaccine (azd1222, vaxzevria®) was developed at oxford university and consists of a replication-deficient chimpanzee adenoviral vector chadox1, encoding the s protein.4 in us phase iii trials, vaxzevria has been demonstrated to have 79% efficacy at preventing symptomatic covid-19, and 100% efficacy against severe or critical disease and hospitalization, with comparable efficacy across ethnicity, gender and age.5 however, vaxzevria has been associated with thrombotic and embolic events including disseminated intravascular coagulation (dic) and cerebral venous sinus thrombosis (cvst), occurring within 14 days after vaccination, mostly in people under 55 years of age, the majority of whom have been women.6 data from europe suggests that the event rate for thromboembolic events may be about 10 per million vaccinated. antibodies to platelet factor 4 (pf4)/heparin complexes have been recently reported in a few patients.7 however, the significance of this finding remains to be established. as of april 12, 2021, about 6.8 million doses of the janssen vaccine have been administered in the u.s.8 cdc and fda are reviewing data involving six reported u.s. cases of cvst in combination with thrombocytopenia.8 all six cases occurred among women between the ages of 18 and 48, and symptoms occurred 6 to 13 days after vaccination.8 sars-cov-2 is known to cause thromboinflammation leading to thrombotic microangiopathy, pulmonary thrombosis, pedal acro-ischemia (“covid-toes”), arterial clots, strokes, cardiomyopathy, coronary and systemic vasculitis, deep venous thrombosis, pulmonary embolism, and microvascular thrombosis in renal, cardiac and brain vascularture.9-14 cerebral venous sinus thrombosis (cvst) has also been reported in covid-19 patients.15 amongst 34,331 hospitalized covid-19 patients, cvst was diagnosed in 28.16 in a multicenter, multinational, cross sectional, retrospective study of 8 patients diagnosed with cvst and covid-19, seven were women with an average age of 63 years.17 in a combined series of 41 patients with covid-19 and cvst, the average age was about 50 years (sd, 16.5 years).17 the pathobiology of thrombotic events associated with the astrazeneca vaccine should be viewed in the context of mechanisms underlying thromboinflammation that complicates sars-cov-2 infection and covid-19 disease. a. role of cox-2 and thromboxane a2 in thromboinflammation complicating adenovirus-based covid-19 vaccine encoding the spike protein of sars-cov-2 thromboinflammation in covid-19 seems to be primarily caused by endothelial, platelet and neutrophil activation, platelet-neutrophil aggregates and release of neutrophil extracellular traps (nets).13,18 platelet activation in covid-19 is fueled by a lipid storm characterized by massive increases in thromboxane a2 (txa2) levels in the blood and bronchoalveolar lavage fluid.19,20 cyclooxygenase (cox) enzymes catalyze the first step in the biosynthesis of thromboxane a2 from arachidonic acid, and cox-2 expression is induced by the spike protein of coronaviruses.21 we postulate that an aberrant increase in thromboxane a2 generation induced by the spike protein expression from adenovirus vaccines leads to thromboinflammation and cvst. the support for the above proposed mechanism comes from the following observations. first, when mice of different age groups were infected with sars-cov virus, the generation of txa2 was markedly increased in younger mice compared to middle aged mice.22 furthermore, in children with asymptomatic or mildly symptomatic sars-cov-2 infection, microvascular thrombosis and thrombotic microangiopathy occur early in infection.19 these observations are consistent with the higher risk for thrombosis in adults under 60 years of age, compared with the older age group.6,7 second, platelets from female mice are much more reactive than from male mice.23 furthermore, txa2 generation, txa2-platelet interaction and platelet activation is increased in women compared to men.24,25 these observations are consistent with disproportionately increased risk of thrombosis in women following astrazeneca and janssen covid-19 vaccines. the adenoviral vector chadox1, containing the ncov-19 spike protein gene, infects host cells through the coxsackievirus and adenovirus receptor (car).26 car-dependent cell entry of the viral vector allows insertion of the sars-cov-2 spike protein gene and expression of spike protein by host cells (figure 1). car is primarily expressed on epithelial tight junctions including on enterocytes.27,28 car expression has also been reported in platelets,29 and, since platelets are anucleate cells, car expression by megakaryocytes can be inferred. therefore, astrazeneca and janssen vaccines would be expected to induce expression of spike protein in megakaryocytes, platelets (figure 1) and enterocytes. we postulate that the expression of car receptors by basolateral membrane on enterocytes allows uptake of adenovirus vector,28 leading to spike protein-induced cox-2 expression and generation of thromboxane a2. delivery of thromboxane a2 into the splanchnic circulation could activate platelets locally, leading to splanchnic vein thrombosis associated with adenovirus vaccines.30 figure 1. astrazeneca or janssen covid-19 vaccine-induced thromboinflammation and cerebral venous sinus thrombosis (cvst) proposed mechanisms: adenovirus carrier delivers sars-cov-2 dna encoding the spike (s) protein to the lung megakaryocytes via the coxsackie-adenovirus receptor (car). spike protein induces cox-2 expression in megakaryocytes leading to megakaryocyte activation, biogenesis of activated platelets that express cox-2 and generate thromboxane a2 (txa2). cerebral venous sinuses express podoplanin, a natural ligand for clec2 receptors on platelets. platelets traversing through the cerebral venous sinuses would be further activated by thromboxane a2-dependent podoplanin-clec2 signaling, leading to release of extracellular vesicles, thereby promoting clec5aand tlr2-mediated neutrophil activation, thromboinflammation and cvst. young age and female gender are associated with increased txa2 generation and platelet activation, respectively, and hence increased risk of thromboembolic complications following sars-cov-2 vaccination with adenovirus-based vaccines. spike protein of coronaviruses is known to induce cox-2 gene expression.21,31 cox-2 expression is induced during normal human megakaryopoiesis and characterizes newly formed platelets.32 while in healthy controls <10% of circulating platelets express cox-2, in patients with high platelet generation, up to 60% of platelets express cox-2.32 generation of txa2 by platelets is markedly suppressed by cox-2 inhibition in patients with increased megakaryopoiesis versus healthy subjects.32 therefore, we postulate that expression of spike protein induces cox-2 expression and generation of thromboxane a2 by megakaryocytes. txa2 promotes biogenesis of activated platelets expressing cox-2. platelet txa2 generation leads to platelet activation and aggregation, and thereby thromboinflammation (figure 1). extravascular spaces of the lungs comprise populations of mature and immature megakaryocytes that originate from the bone marrow, such that lungs are a major site of platelet biogenesis, accounting for approximately 50% of total platelet production or about 10 million platelets per hour.33 more than 1 million extravascular megakaryocytes have been observed in each lung of transplant mice.33 following intramuscular injection of the astrazeneca and janssen vaccines, the adenovirus vector will traverse the veins and lymphatics to be delivered to the pulmonary circulation, thereby exposing lung megakaryocytes in the first pass. interestingly, under thrombocytopenic conditions, haematopoietic progenitors migrate out of the lung to repopulate the bone marrow and completely reconstitute blood platelet count.33 since thrombocytopenia is a common feature of cvst associated with adenovirus sars-cov-2 vaccines, migration of megakaryocytes from the lungs into the systemic circulation including the cerebral circulation could further predispose to cvst. in this regard, it is relevant that megakaryocytes have been found in the cerebral circulation in patients deceased from severe covid-19.34 the prothrombotic mechanisms postulated above could be operative alone or in conjunction with other underlying factors that have been proposed including the presence of anti-platelet factor 4 (pf4) antibodies as reported by greinacher and others.7 notably, amongst 31 cases of cvst in close temporal relationship with a covid-19 vaccination, anti-pf4 antibodies were positive in 22 cases and negative in 9.35 therefore, cvst following covid-19 vaccination appears to be a multifactorial disease. further studies using cultured megakaryocytes and animal models are needed to define the underlying mechanisms. even though the risk of cvst is increased about 10-fold by the adenovirus based covid-19 vaccines,36 it remains unclear why cvst remains a rare event. to further define the importance of these causative factors, biomarkers such as plasma thromboxane b2, anti-pf4 antibodies and anti-phospholipid antibodies should be measured in the thrombosis-free vaccinated population. such studies may help define the role of anti-pf4 antibodies as causative versus an epiphenomenon. interestingly, almost all patients with thrombosis and thrombocytopenia were found to have anti-pf4 antibodies.35 on the other hand, majority of patients with thrombosis without thrombocytopenia lacked anti-pf4 antibodies.35 the postulated mechanisms may be especially relevant to thrombotic events occurring in the absence of thrombocytopenia or anti-pf4 antibodies. b. predilection of cerebral venous sinuses for thrombosis following vaccination recent studies have demonstrated that arterial, venous and sinusoidal endothelial cells in the brain uniquely express markers of the lymphatic endothelium including podoplanin.37 podoplanin (d2-40) serves as a ligand for clec2 receptors on platelets.38 thromboxane a2-dependent clec2 signaling leads to platelet activation (figure 1), while a txa2 receptor antagonist nearly abolished clec2 signaling and platelet activation.38 txa2-dependent clec2 signaling promotes release of exosomes and microvesicles from platelets, leading to activation of clec5a and tlr2 receptors respectively on neutrophils, neutrophil activation and release of neutrophil extracellular traps (nets) (figure 1).39 neutrophil activation, more than platelet activation, is associated with thrombotic complications in covid-19.13,18,40 as proposed above, the expression of podoplanin by cerebral venous sinuses may be responsible for the predilection of brain vascular bed to thromboinflammation and cvst as a complication of covid-19 vaccines. the valves at the lymphovenous junction and the podoplanin-clec2 lymphovenous hemostasis act as “fail-safe” mechanisms to prevent movement of blood into the lymphatic system, thereby preventing coagulation in the lymphatics despite expression of podoplanin in the lymphatic endothelium.41 c. chemoprophylaxis with antiplatelet agents in animal models of endotoxin-mediated endothelial injury and thromboinflammation, antagonism of thromboxane a2 signaling prevents ards, reduces myocardial damage and increases survival.42-44 considering the key role played by platelets in thromboinflammation, we propose consideration of anti-platelet agents, either aspirin or txa2 receptor antagonists, as chemoprophylactic agents when the astrazeneca vaccine is administered to adults between 18 and 60 years of age.45 high bleeding risk because of another medical condition or medication would be contraindications to use of antiplatelet agents.45 medical conditions that increase bleeding risk include previous gastrointestinal bleeding, peptic ulcer disease, blood clotting problems, and kidney disease.45 medications that increase bleeding risk include nonsteroidal anti-inflammatory drugs, steroids, and other anticoagulants or anti-platelet agents.45 aspirin appears to be safe in covid-19. in a retrospective observational study in hospitalized patients with covid-19, low-dose aspirin was found to be effective in reducing morbidity and mortality and was not associated with any safety issues including major bleeding.46 therefore, aspirin is likely to be safe as an adjunct to covid-19 vaccines even in the event of a subsequent infection with sars-cov-2 virus. can aspirin influence the host immune response to the covid-19 vaccines? this issue merits further investigation. when healthy adults > 65 years of age were given influenza vaccine and randomized to receive 300 mg aspirin or placebo on days 1, 2, 3, 5 and 7, the aspirin group showed 4-fold or greater rise in influenza specific antibodies.47 the risk-benefit analysis, based on above information, suggests that a one to three week course of low-dose aspirin merits consideration in order to prevent the thromboembolic events associated with the astrazeneca vaccine. summary thromboembolic disease, cvst and thrombocytopenia have been reported in association with astrazeneca and janssen covid-19 vaccines, especially in younger women. many countries have halted use of these vaccines for adults under 30 to 60 years of age. european and north american countries generally have access to mrna vaccines. however, in many asian and african countries the choices are limited to adenovirus-based covid-19 vaccines. the governments in such countries are forging ahead with vaccinating all adults, including those under 60 years of age, with vaxzevria, covishield (the version of vaxzevria manufactured by the serum institute of india) or the janssen vaccines. this has led to grave concern and anxiety amongst the citizens and medical professionals. considering the profound global public health implications of limiting the use of these vaccines, it is critical to understand the pathobiology of vaccination induced thrombotic events in order to guide strategies aimed at prevention. in this regard, studies are urgently needed to examine lipid mediators and the thromboxane a2 platelet axis following vaccination with these vaccines compared with mrna vaccines. the risk-benefit analysis based on information presented here suggests that chemoprophylaxis using a short course of low-dose aspirin in adults under 60 years of age and lacking any contraindications to aspirin may be justified in conjunction with adenovirus-based covid-19 vaccines in order to prevent thromboembolic events and enhance safety. acknowledgment we thank prof. andrzej breborowicz, md, phd, department of pathophysiology, poznan university of medical sciences, poland for a careful review and critique of this work. funding no funding was required. conflict of interest ag and kcc have filed a patent for use of ramatroban as an anti-thrombotic and immune modulator in sars-cov-2 infection. the patents have been licensed to kare biosciences. kcc is an employee of kare biosciences. author contributions ag and kcc conceptualized, created the framework and drafted the original version. all authors reviewed and edited the final version. references 1. ortiz-prado e, simbaña-rivera k, gómez-barreno l, et al. clinical, molecular, and epidemiological characterization of the sars-cov-2 virus and the coronavirus disease 2019 (covid-19), a comprehensive literature review. diagnostic microbiology and infectious disease. 2020;98(1):115094. 2. du l, he y, zhou y, liu s, zheng b-j, jiang s. the spike protein of sars-cov — a target for vaccine and therapeutic development. nature reviews microbiology. 2009;7(3):226-236. 3. kyriakidis nc, lópez-cortés a, 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an immunohistochemical study of lymphatic elements in the human brain. proceedings of the national academy of sciences. 2021;118(3):e2002574118. 38. badolia r, inamdar v, manne bk, dangelmaier c, eble ja, kunapuli sp. g(q) pathway regulates proximal c-type lectin-like receptor-2 (clec-2) signaling in platelets. journal of biological chemistry. 2017;292(35):14516-14531. 39. sung p-s, huang t-f, hsieh s-l. extracellular vesicles from clec2-activated platelets enhance dengue virus-induced lethality via clec5a/tlr2. nature communications. 2019;10(1). 40. ng h, havervall s, rosell a, et al. circulating markers of neutrophil extracellular traps are of prognostic value in patients with covid-19. arteriosclerosis, thrombosis, and vascular biology. 2021;41(2):988-994. 41. welsh jd, kahn ml, sweet dt. lymphovenous hemostasis and the role of platelets in regulating lymphatic flow and lymphatic vessel maturation. blood. 2016;128(9):1169-1173. 42. carey ma, bradbury ja, seubert jm, langenbach r, zeldin dc, germolec dr. contrasting effects of cyclooxygenase-1 (cox-1) and cox-2 deficiency on the host response to influenza a viral infection. the journal of immunology. 2005;175(10):6878-6884. 43. kuhl pg, bolds jm, loyd je, snapper jr, fitzgerald ga. thromboxane receptor-mediated bronchial and hemodynamic responses in ovine endotoxemia. american journal of physiology. 1988;254(2 pt 2):r310-319. 44. altavilla d, canale p, squadrito f, et al. protective effects of bay u 3405, a thromboxane a2 receptor antagonist, in endotoxin shock. pharmacological research. 1994;30(2):137-151. 45. peters at, mutharasan rk. aspirin for prevention of cardiovascular disease. jama. 2020;323(7):676. 46. chow jh, khanna ak, kethireddy s, et al. aspirin use is associated with decreased mechanical ventilation, intensive care unit admission, and in-hospital mortality in hospitalized patients with coronavirus disease 2019. anesthesia & analgesia. 2021;132(4). 47. saleh e, moody ma, walter eb. effect of antipyretic analgesics on immune responses to vaccination. human vaccines & immunotherapeutics. 2016;12(9):2391-2402. copyright: © 2021 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. neurooncology: 2022 update feel free to add comments by clicking these icons on the sidebar free neuropathology 3:4 (2022) review neurooncology: 2022 update pieter wesseling1,2, jacob s. rozowsky2 1 department of pathology, amsterdam university medical centers/vumc, brain tumor center amsterdam, de boelelaan 1117, 1081hv amsterdam, the netherlands 2 laboratory for childhood cancer pathology, princess máxima center for pediatric oncology, heidelberglaan 25, 3584 cs utrecht, the netherlands corresponding author: pieter wesseling · department of pathology · amsterdam university medical centers/vumc · de boelelaan 1117 . 1081 hv amsterdam · the netherlands p.wesseling@amsterdamumc.nl submitted: 14 february 2022 accepted: 23 february 2022 copyedited by: henry robbert published: 24 february 2022 https://doi.org/10.17879/freeneuropathology-2022-3804 keywords: brain tumor, neuropathology, molecular diagnostics, glioblastoma, medulloblastoma abstract this ‘neurooncology: 2022 update’ presents topics that were selected by the authors as top ten discoveries published in 2021 in the broader field of neurooncological pathology. this time, the spectrum of topics includes: papers with a direct impact on daily diagnostic practice of cns tumors in general and with information on how to improve grading of meningiomas; studies shedding new light on the oncogenesis of gliomas (in particular ‘optic gliomas’ and h3-mutant gliomas); several ‘multi-omic’ investigations unraveling the intra-tumoral heterogeneity of especially glioblastomas further; a study indicating the potential of ‘repurposing’ prozac® for the treatment of glioblastomas; liquid biopsy using csf for assessment of residual medulloblastoma. in the last part of this review some other papers are mentioned that didn’t make it to this (quite subjective) top ten list. introduction for the third year in a row, the first author of this paper was invited to contribute a review on the top ten discoveries in neurooncology published in the previous year. rather than checking over 10,000 papers on tumors of the central nervous system (cns) published in 2021 (as was done for the previous review), he now restricted himself to screening the higher-ranked neurooncology and neuropathology journals and the top-journals in oncology and science with a broader scope. furthermore, he teamed up with the last author, a young fulbright scholar from the usa that he supervises for a period of 9 months in the netherlands. the last author mainly used a twitter®-based approach for identifying promising candidates for our list of top ten discoveries in 2021. interestingly, there was quite some overlap between the results of these different search strategies (but more research is needed for sorting out if twitter® is a reliable source of information in this respect, also because it appears that in other areas of life the information shared by tweets can cause chaos rather than order). the final list of topics selected by the authors as very interesting and/or very important for the broader field of neurooncological pathology this time looks like this: who 2021 classification of cns tumors 1 glioma driver mutations in the normal human brain 2 neuronal activity-dependent initiation of nf1-mutant ‘optic gliomas’ 3 oncogenic role of h3 mutations in histogenesis context 4,5 epigenomic insights into glioma cell differentiation and plasticity 6,7 proteogenomic and metabolomic characterization of glioblastomas 8 genomic and immunologic heterogeneity of gliomas and brain metastases 9 therapeutic interference with glioblastoma cell membranes using prozac® 10 liquid biopsy assessment of residual medulloblastoma 11 improved ‘histomolecular’ grading of meningiomas 12-14 of note, all the papers just mentioned became officially available in 2021, but the printed version may have lagged somewhat behind. also, and like in previous years, one has to realize that there is of course quite a subjective component in the selection process that resulted in this list. for example, the strong interest of the last author in computational biology aspects of cns tumors may well have played a role in the selection of topics 5 and 6 especially. to make up to some degree for this rather subjectively composed bouquet of papers, in the discussion section of this review some papers/topics are mentioned that didn’t make it to this top ten list but that certainly are interesting as well. hopefully, this review thereby again helps to appreciate the wealth of (sometimes mind-boggling) information provided by the papers that were used as building blocks for the present manuscript. topic 1: who 2021 classification of cns tumors 1 publication of a new who cns tumor classification may in itself not be perceived as a major scientific achievement. however, quite some of the changes implemented in the fifth edition of the who classification of tumors of the central nervous system (who cns5 classification) are the result of very good science, followed by (hopefully) ‘smart translation’ of the findings into a new classification, which definitely has a major impact on clinical neurooncology. since november 2021, the who cns5 classification is available online via https://tumourclassification.iarc.who.int, and the printed ‘blue book’ version can now be purchased as well (figure 1). the review by louis dn et al. published in neuro-oncology provides a very informative summary of major general changes in the who cns5 classification and of specific changes in the different taxonomic categories 1. figure 1. covers of the who cns tumor blue books throughout the years.in these blue books, the actual who cns tumor classification is the list of tumors that are recognized as distinct tumor types (see e.g. table 1 for the 5th edition); the rest of the information in these books concerns explanation of the pathological (nowadays histological and molecular) characteristics of these tumors, and the most salient information on their clinical and radiological context. molecular characteristics were for the first time introduced as defining criteria in the revised 4th edition, especially for diffuse gliomas in adults and for some embryonal tumors. in the 5th edition, molecular features are introduced as essential diagnostic criteria for many more tumors. in line with recommendations of the cimpact-now consortium 15, salient changes in this new classification are the separation of (groups of) pediatric-type low-grade and pediatric-type high-grade diffuse gliomas from adult-type diffuse gliomas, further refinement of the classification of ependymal tumors, and the addition of a few newly recognized tumors in the category of cns embryonal tumors. table 1 provides an overview of these changes in the who cns5 classification (with # and * indicating the newly introduced and the provisionally accepted tumor types, respectively). another important change is that grading of neoplasms is performed in the who cns5 classification within (rather than across) tumor types, with arabic (instead of roman) numerals now used for the different grades. thus, in the who cns5 classification there is only one entry for e.g. astrocytoma, idh-mutant, and one for meningioma (rather than separate entries for e.g. anaplastic astrocytoma, idh-mutant, or for atypical meningioma). also, glioblastoma, idh-mutant should now be diagnosed as astrocytoma, idh-mutant, cns who grade 4. table 1. who classification of primary cns tumors, 5th edition (2021).major changes in the who cns5 classification are the separation of (low-grade and high-grade) pediatric-type diffuse gliomas from adult-type diffuse gliomas; the recognition of several new ependymal, embryonal and other tumor types; assigning of cns who grades within tumor types; coining the most malignant idh-mutant diffuse astrocytic tumor as astrocytoma, idh-mutant, cns who grade 4; and changing the name of diffuse midline glioma (dmg), h3 k27m-mutant into dmg, h3k27-altered. see summary of louis dn et al. 1 or, even better, the (digital version of the) ‘blue book’ for much more information of these changes. in red & capitals & italics: overarching categories of tumors; in bold and italics: groups of tumor types, and under these groups (with a few exceptions) the individual tumor types; # = newly defined tumor type compared to the 2016 edition; * = provisional tumor type; @ = tumor with revised nomenclature or revised placement. click here to view a large version of this table. furthermore, the presence of tert promoter mutation, egfr amplification and/or the combination of gain of complete chromosome 7 and loss of complete chromosome 10 can now be used for diagnosing an adult-type, histologically lower grade, idh-wildtype diffuse glioma as glioblastoma, idh-wildtype (cns who grade 4), and the presence of homozygous cdkn2a/b loss to diagnose a histologically lower grade, idh-mutant diffuse astrocytoma as cns who grade 4. for yet other tumors the name or their placement in the classification was changed as well (@ in table 1). for example, h3k27m-mutant after diffuse midline glioma (dmg) was changed into h3k27-altered because there are h3-wildtype dmgs especially in children that do show loss of nuclear h3k27me3 staining and with a similar prognosis as dmgs, h3k27m-mutant. also, the name rela fusion-positive for a subset of supratentorial ependymoma was changed into zfta fusion-positive, as zfta (‘zinc finger translocation associated’, the new name for c11orf95) appears to be the more frequent fusion partner in these tumors (most frequently showing fusion with rela). compared to the revised fourth edition, the who cns5 tumor classification certainly is an improvement. however, as discussed in somewhat more detail in the last part of this review, this new classification brings several challenges as well, e.g., related to the (lack of) availability of molecular diagnostic tools, finding the optimal therapeutic management for newly defined tumor types, and the fact that a more refined taxonomy of cns tumors makes it more difficult to perform studies on a large number of patients. topic 2: glioma driver mutations in the normal human brain 2 cancer is a disease characterized by the accumulation of genomic aberrations that confer a proliferative advantage. also, somatic mutations and copy number variations have been described to accrue with age in (seemingly) normal tissues 16. however, relatively little is known about this phenomenon in the normal brain. ganz j and maury ea et al. (with lee ea and walsh ca as corresponding authors) published a paper in cancer discovery 2 in which they used targeted gene sequencing on cerebral gray and white matter samples of 110 individuals to investigate the presence of oncogenic variants in normal brain tissue. the 121-gene panel was selected for genes implicated in different diseases and previously characterized oncogenic drivers. the authors hypothesized that somatic mutations would accumulate in brain tissue over time and (as neurons have a low proliferation capacity) would be enriched in cerebral white matter. the authors identified 35 variants present in normal brain tissue: 12 of the variants were in known proto-oncogenes or tumor suppressor genes, and 6 were known driver mutations of gliomas (idh1, ptpn11, pten, nf1, apc, mtor). the authors validated the variant allele frequencies (vafs) from their gene panel with ultra-deep ion torrent sequencing. the most frequently found mutation was idh1 r132h. in one patient, two adjacent white matter samples had vastly different variant allele frequencies (vafs) of idh1 r132h (5% vs 0.9%). because a 5% vaf is high for a localized oncogenic mutation, the authors suggest that in this tissue, the mutation should be considered as a clonal event that conferred cells with a proliferative advantage. using single-nucleus rnaseq, they found that the idh1 r132h mutation was enriched in glial cells. interestingly (and thus in contrast to what has previously been described for other organs), in this study the accumulation of oncogenic mutations in brain tissue did not positively correlate with age. in fact, all of the patients with identified somatic variants were under 30. furthermore, in this study oncogenic mutations were found to be present in 5.4% of the non-diseased human brains, while the incidence of primary brain tumors in the population is much lower. these results thus raise some intriguing next questions, such as: what are the mechanisms for postnatal elimination of the mutant clones? what is the ‘tipping point’ that eventually causes brain tumor formation? maybe the acquisition of a second hit (such as tp53 and/or atrx mutation for idh-mutant astrocytomas, and 1p/19q codeletion for idh-mutant oligodendrogliomas) 17? and do maybe these findings help to explain the occurrence of ‘dual genotype’ oligoastrocytomas with histologically distinct astrocytic and oligodendroglial areas 18? topic 3: neuronal activity-dependent initiation of nf1-mutant ‘optic gliomas’ 3 over the past few years, we have begun to appreciate the unique role of neurons for glioma growth. for example, two papers that were already summarized in topic 7 of the ‘top ten discoveries in 2019’ review in this journal demonstrated the presence of bona fide neuron-glioma synapses in high-grade gliomas, with electrochemical-induced depolarization of the glioma membrane promoting proliferation of the tumor cells 19,20. these discoveries have boosted the interdisciplinary field of ‘cancer neuroscience’ 21. therapies that target the neuron-glioma interface, especially through neuroligin-3 signaling (nlgn3), are now under investigation as a possible treatment for these malignant gliomas 22,23. in 2021, pan y et al. (with monje m and gutmann dh as corresponding authors) published a study in nature on the impact of neuronal activity in the optic nerve on the formation and growth of ‘optic gliomas’ in mice with nf1 mutation 3. the authors started with an authenticated genetically engineered mouse model which mimics the neurofibromatosis type 1 (nf1) childhood predisposition syndrome: a germline nf1 mutation (nf1+/-) combined with an acquired somatic nf1 mutation in neural progenitor cells (nf1-/-). when these mice are 9 weeks of age, gliomas form along the optic nerve, resembling what is seen in children with nf1 (and in the vocabulary of the who cns tumor classification then generally concern pilocytic astrocytomas). by stimulating the retinal ganglion cells, the authors found an increase in optic nerve volume and proliferation rate, proving that optic nerve activity can increase optic glioma growth. next, the authors investigated the effect of visual experience on the initiation and growth of these optic gliomas. rearing mice in the dark from 6 weeks of age (before optic glioma formation) prevented the formation of tumors. additionally, compared with mice raised in a regular light cycle, dark-rearing mice rescued retinal ganglion cell death and prevented optic nerve damage. pan y et al. further characterized the role of nlgn3, a synaptic adhesion molecule, in these models and found that it operates under a similar mechanism as in high-grade gliomas: adam10 sheddase cleaves and releases nlgn3 into in the tumor microenvironment, which stimulates proliferation of optic glioma cells. when the authors introduced a brain penetrant inhibitor of adam10 in their model, no tumor formed in the optic nerve. these insights raise many interesting follow up questions. for example, would limiting light exposure—e.g., with light-blocking glasses—and/or interference with nlgn3 during certain developmental periods in children help to prevent optic glioma formation? hopefully, these findings will sooner or later provide new opportunities for the therapeutic management of children with nf1. topic 4: oncogenic role of h3 mutations in histogenesis context 4,5 mutations affecting histone h3 are a defining feature in a particular subset of high-grade diffuse gliomas in children and suggest an epigenetic driver of cancer. the two more common mutations, h3.3 p.k28m (k27m) and h3.3 p.g35r/v (g34r/v), are mutually exclusive and typically lead to tumor formation in different cns locations (h3 k27m in midline structures, and h3 g34r/v in the cerebral hemisphere). two recent studies aimed to discern the oncogenic mechanisms of these h3.3 mutations using stem cell models of brain development and gliomagenesis. haag d et al. (with wernig m and pfister sm as senior authors) published a study in cancer cell in which they investigated the effects of the h3 k27m mutation, which co-occurs in diffuse midline gliomas (dmgs) with a mutation in tp53 in 77% of cases 4. previous single cell rnaseq studies of dmgs, h3 k27m-mutant revealed that these tumors may originate from an oligodendrocyte progenitor cell (opc) 24. haag et al. inserted a k27m mutation in neural stem cell (nsc) and opc lines, which increased proliferation in these cell lines. interestingly, only nsc lines with k27m and knock-out of tp53 formed malignant tumors in mouse xenografts that resembled the pathology of dmgs. as this finding is inconsistent with prior reports of the cell-of-origin, the authors hypothesized that the k27m mutation could cause nsc to adopt an opc-like transcriptional profile. to this end, the authors used chromatin immunoprecipitation sequencing (chip-seq) to describe the interactions between mutant h3.3 and dna. they found h3.3 enrichment in bivalent chromatin domains which maintains the expression genes necessary for pluripotency and stem cell identity. this can explain how k27m-mutant nscs can mimic opc transcriptional programs and could drive these tumors. bressan rb et al. (with pollard sm as lead contact) described the oncogenic effects of the g34r mutation in nsc lines derived from the forebrain and hindbrain in a paper in cell stem cell 5. forebrain nsc lines with only the g34r mutation had low tumorigenic capacities. however, combination of knockout of tp53 and amplification of pdgfra in these cells resulted in increased proliferation and the formation of malignant tumors when xenografted in mice. the authors then sought to describe the epigenetic effects of the h3 g34r mutation. unlike the k27m mutation, which causes wide-spread epigenetic and transcriptional changes, the authors found that the g34r mutation reduces binding with zmynd11, a tumor suppressor that alters the elongation and splicing of highly expressed genes. in the context of brain tumors, this results in higher expression of genes that regulate forebrain development and locks nscs into a proliferative and non-differentiated state. together, these papers very nicely show how pre-clinical stem cell modeling can untangle the mechanisms of cancer initiation in general, and of the oncogenesis of h3.3 mutant tumors in particular. topic 5: epigenomic insights into glioma cell differentiation and plasticity 6,7 single-cell (sc) rnaseq studies of diffuse gliomas have revealed unprecedented insights into their intra-tumoral heterogeneity, wherein glioma cells from the same tumor exist in different transcriptionally defined cell states 25-27. many of these cell states mirror the neurodevelopmental hierarchy, and it has been suggested that tumor cells can transition between more or less differentiated states. such a plasticity may well contribute to resistance to therapy. as gene regulation and dna methylation (dname) can largely control these cell states, back-to-back papers published in nature genetics by johnson kc and anderson kj et al. (with verhaak rgw as senior author) 6 and chaligne r, gaiti f, silverbush d, and schiffman js et al. (supervised by suvá ml and landau da) 7 measured dname and rna expression at the single-cell level of idh-wildtype glioblastomas in adults (idh-wt gbms) and in idh-mutant (idh-mt) diffuse gliomas. using scdname, both articles report on how glioma cells from the same patient exist in different methylation states, while cells from different patients can exist in the same states. these studies re-affirm that diffuse gliomas, especially gbms, have high intra-tumoral heterogeneity, with malignant cells adopting different epigenotypes. obviously, the bulk methylation assays that are frequently used to classify (glial) brain tumors fail to account for this level of intra-tumoral heterogeneity that is uncovered by such single-cell analyses. both articles took different approaches to describe the epigenomic landscape that gives rise to the differentiation hierarchies in gliomas. chaligne et al. report that, compared to idh-mt gliomas, polycomb complexes in idh-wt gbms are more hypermethylated in stem-like cells than in differentiated cells. this suggests a role for dname in maintaining a stem-like phenotype of gbm cells, thereby contributing to their treatment resistance and progression. however, this was not seen in idh-mt gliomas, suggesting a different role of dname in maintaining their stem-like cell states. furthermore, the authors constructed lineage trees using scdname and found that cells within each clade had the same chromosomal aberrations. this highlights how sub-clonal mutations are related with the epigenotype of glioma cells. interestingly, when the authors inferred glioma cell state (npc, opc, ac, mes) based on scrnaseq for idh-wt gbms, they found that cells within the same lineage adopted different transcriptional states. however, the reverse was found for idh-mt glioma, where closely related cells obtained similar cell states. altogether, these results confirm that idh-wt gbms have higher cell plasticity than idh-mt glioma, and dname contributes to the glioma stem-like identity. johnson et al. also investigated the contribution of ‘dname disorder’ (i.e., aberrant methylation which allows cells to adapt to diverse methylation states) for maintaining the stem-like identity in gliomas. dname disorder could allow for the glioma cells to overcome environmental stressors and contribute to resistance to treatment. this hypothesis was tested in vitro by exposing two glioma cell lines to hypoxic conditions and irradiation. the authors found that under such environmental stressors, dname disorder increases with time, potentially allowing the cells to adopt and retain their stem-like identities. topic 6: proteogenomic and metabolomic characterization of glioblastoma 8 wang l-b et al. (with the clinical proteomic tumor analysis consortium) published a study which integrated whole exome/whole genome sequencing, bulk and single-nucleus rna sequencing (rnaseq), dna methylation, proteome, phospho-proteome, acetylome, lipidome and metabolome datasets for 100 treatment-naïve glioblastomas. landmark studies previously already comprehensively characterized the genome and transcriptome of glioblastoma (see e.g. 28). a deep ‘multi-omic’ approach, like wang l-b et al. undertook, provides very interesting further insights into functional and potentially targetable differences between glioblastoma subtypes. based on their findings, especially with regard to gene expression, and protein and phosphoprotein abundances, wang l-b et al. defined three ‘multi-omic subtypes’ which closely resemble tcga (the cancer genome atlas) glioma subtypes: the proneural-like subtype was enriched for neurotransmission and synaptic vesicle gene-sets; the mesenchymal-like subtype was enriched for immune system activation, phagocytosis, and glycolysis; finally, the classical-like subtype was enriched for chromatin modification, dna repair, and mrna splicing. the authors also defined immune-based subtypes based on gene-set enrichment analysis of rnaseq data. subtypes ranged from low-enrichment of all immune cells (especially classical and proneural-like glioblastomas) to enrichment of lymphocytes, microglia, and macrophages (mesenchymal-like glioblastomas). the authors integrated these observations with single nucleus rnaseq of 18 samples spanning all multi-omic and immune subtypes, and characterized expression programs found in the neoplastic, stromal, and immune cells. interestingly, this rnaseq analysis revealed upregulation of epithelial-mesenchymal transition related genes in both glioma and immune cells of mesenchymal-like glioblastomas. these differences could be observed on the protein-level as well. last but not least, the authors also noticed subtype-specific enrichment of lipids and metabolites. for example, they noticed idh mutation status-associated differences in glycolysis-related metabolites. not surprisingly, idh-mutant gliomas showed increased levels of 2-hg, as well as decreased glutamate and serine. phosphoproteomic data highlighted therapeutic opportunities for treating glioblastoma, with plcg1 and ptpn11 as signaling hubs for receptor tyrosine kinases. importantly, all the data published by wang et al. is open access and can thus be used as a resource for future studies of glioblastomas. indeed, such a rich dataset adds depth to the previous knowledge from genomic and transcriptomic investigations of glioblastomas and can hopefully pave the way for more effective, personalized treatments for these tumors. topic 7: genomic and immunologic heterogeneity of gliomas and brain metastases 9 immunotherapies for primary and secondary brain tumors are a ‘hot topic’ in the field of neurooncology (see for example also topic 9 of the ‘top ten discoveries of the year 2019’ review in this journal). in 2021, schaettler mo and richters mm et al. (with griffith m and dunn gp as supervising authors) published a study in cancer discovery investigating intra-tumoral heterogeneity of somatic variants, neo-antigens, and infiltrating t-cells in gliomas as well as in brain metastases. for this study, the authors collected 93 spatially separated tumor samples from 30 patients. these samples were subjected to whole exome, rna and t-cell receptor sequencing (wes, rnaseq, tcr-seq). interestingly, while brain metastases had considerably more somatic variants per tumor than gliomas, these variants were mostly clonal, so spanning all tumor samples of a patient. when analyzing the sub-clonal variants of gliomas, the authors noted that sequencing only one region of the tumor missed 40% of variants that were obtained when sequencing three regions. additionally, they found that 9/16 gliomas in this study showed characteristics of multiple transcriptional subtypes (classical, mesenchymal, neural and/or proneural). integrating wes and rnaseq data allowed the authors to predict hla class i and ii neo-antigens expressed in the tumors. they found that the neo-antigens in brain metastases were more often clonal. this could have implications for the development of personalized immunotherapies, as clonal neoantigens would be better targets. using rnaseq data, the authors also described the immune composition of the tumors. brain metastases had robust infiltration of monocyte-derived macrophages, while gliomas were more enriched for microglia gene signatures. finally, the results of tcr-seq analysis revealed that spatially distinct samples of brain metastases had more similar t-cell clones than those of gliomas. altogether, these findings highlight the considerable intra-tumoral heterogeneity in somatic variants, transcriptome, and neo-antigens of gliomas. this can be expected to pose significant challenges for developing effective immunotherapies. comparatively, brain metastases are more homogenous tumors, which could partially explain the clinical benefits of immune checkpoint inhibition when treating patients with those latter tumors. topic 8: therapeutic interference with glioblastoma cell membranes using prozac® 10 so far, little progress has been made with improving the prognosis for patients with idh-wildtype glioblastomas (idh-wt gbms). gbms are known to have an altered lipid composition, with molecular alterations being linked to differential plasma membrane remodeling 29. for example, gbms are frequently driven by the amplification of egfr on extra-chromosomal dna (ecdna). furthermore, variants in the extracellular egfr domain (especially egfrviii) cause constitutive activation 30,31. these signaling molecules are organized into lipid rafts on the plasma membrane. in a study of bi j et al. published in cell reports (with mischel ps as lead contact), the authors identified that gbms are dependent on sphingomyelin phosphodiesterase 1 (smpd1), an enzyme that converts sphingomyelin to ceramide and alters the composition of the plasma membrane. in the tcga (the cancer genome atlas) cohort of gbm samples, increased expression of smpd1 was associated with shorter overall survival, suggesting that targeting this pathway could be a therapeutic strategy for gbm. interestingly, fluoxetine (sold under the brand name prozac®; a brain-penetrant, fda-approved selective serotonin reuptake inhibitor (ssri) used to treat multiple psychiatric disorders) inhibits smpd1 activity. in vitro experiments of gbm patient-derived cell lines treated with fluoxetine was found to result in an increase in lysosomal stress and dose-dependent cell death. also, temozolomide showed synergistic effects with fluoxetine. these results were confirmed in in vivo studies of gbm orthotopic xenografts, wherein 6 of 8 mice receiving both temozolomide and fluoxetine showed no tumor recurrence after 5 months of treatment. over-expression of smpd1 in vitro and in vivo reversed the effects of fluoxetine. next, the authors examined electronic medical records from 180 million americans who were diagnosed with gbm between 2003 and 2017. the patients with gbm who, for psychiatric reasons, were also treated with fluoxetine (prozac®) during the disease course of their brain tumor had significantly longer overall survival. this result was not found for gbm patients treated with other ssris. in conclusion, the results of the study of bi et al. indicate that fluoxetine/prozac® can maybe be ‘re-purposed’ and integrated with standard of care of patients diagnosed with gbm in order to help improve their survival. obviously, controlled clinical trials are necessary to substantiate this idea further. also, it would be interesting to learn more about other compounds that might be good candidates for therapeutic interference with (the composition of) gbm cell membranes. topic 9: liquid biopsy assessment of residual medulloblastoma 11 a current problem in the management of children with medulloblastoma is assessing residual disease during treatment and predicting which patients relapse locally or through dissemination to the leptomeninges (which, unfortunately, frequently leads to death) 32. currently, mri and cytology assessment of the cerebrospinal fluid (csf) are used to detect relapse. however, these diagnostic modalities are generally only useful once the tumor has progressed substantially. medulloblastomas typically have few driver mutations and are characterized by high levels of chromosomal instability (copy number variants). in a study from 2020, escudero et al. found that csf-derived cell-free dna (cfdna) recapitulated molecular characteristics of the initial medulloblastomas, including the oncogenic drivers 33. cfdna from the csf could also be used to classify medulloblastomas into the four molecular sub-groups. following up on these findings, liu api, smith ks, and kumar r et al. (with gajjar a, robinson gw and northcott pa as senior authors) published a paper in cancer cell evaluating the utility of cfdna to assess measurable residual disease (mrd) for guiding the therapeutic management of medulloblastoma patients 11. they performed low-coverage whole genome sequencing on cfdna from 476 csf samples, representing 123 patients, and derived cnvs as a biomarker for mrd. of the 105 csf samples collected at baseline, the authors detected cnvs in 64% of the samples. cnvs were not detected in cfdna from csf of patients without oncological diseases that were used as control. using multivariate regression, they found that detection of baseline samples was associated with tumor location and subgroup, wherein detection was lowest for tumors centered within cerebellar hemispheres or vermis, and for the shh subgroup. additionally, the detection of mrd in samples post-radiotherapy, mid-chemotherapy, or at the end of therapy was associated with worse progression free survival (pfs), while baseline cnv detection was not prognostic for pfs. the authors did find a high correlation between cnvs detected at baseline in the csf and in the primary tumor, confirming the results from escudero et al. comparing serial samples of cfdna, 75% of patients had a more ‘unstable’ genome with a loss of chromosome 10q at recurrence. mrd was detected for all patients with persistent disease recurrence, demonstrating the high accuracy for this assay. of note, of the patients with medulloblastoma recurrence, mrd was detected by csf cfdna analysis at least 3 months before appearing on mri or based on cytology analysis. this study demonstrates that detection of cnvs from csf-derived cfdna may allow for a more sensitive evaluation of disease progression in children with medulloblastoma than the currently available tools. the authors therefore advocate for incorporating such cfdna analysis of liquid biopsies to inform management of these aggressive cancers. topic 10: improved (histo)molecular grading of meningiomas 12-14 until recently, the diagnosis who grade ii meningiomas was based on the presence of increased mitotic activity, brain invasion, presence/absence of three or more of the following features: high cellularity, small cells with a high nucleus: cytoplasm ratio, prominent nucleoli, patternless/sheet-like growth, and foci of necrosis. furthermore, particular histological phenotypes were used to assign a who grade ii (clear cell, chordoid) or who grade iii (rhabdoid, papillary) to meningiomas, while very high mitotic activity and/or overtly malignant cytomorphology were considered as sufficient for the diagnosis of malignant/anaplastic meningioma (who grade iii) as well. however, the prognostic meaning of this histology-based grading system was suboptimal, and it became increasingly clear that molecular data can be used to refine grading of these tumors. indeed, in the who cns5 classification, presence of tert promoter mutation and/or homozygous cdkn2a and/or cdkn2b deletion are now listed as criteria for meningioma, cns who grade 3. furthermore, in this new classification, the rhabdoid and papillary phenotypes are no longer considered as cns who grade 3 based on their histological phenotype alone 1,34. nassiri f et al. (with aldape k and zadeh g as supervising authors) published a paper in nature providing a wealth of matched multidimensional bulk and single-cell molecular and clinical data on a large cohort of meningiomas, enriched for the higher-grade tumors according to who 2016 criteria 12. unsupervised sample-wise clustering of gene-level somatic copy-number alterations (cnas), dna methylome, and transcriptome data in isolation revealed six stable subgroups for each datatype with clinically relevant and significant differences in outcome. additional (second-order) clustering revealed four stable molecular groups (mg1–mg4) without a clear one-to-one relationship between molecular group and who grade. classification by molecular groups was independently associated with recurrence-free survival as assessed by multivariable cox regression, even after accounting for known prognostic clinical factors. mg3 and mg4 tumors (carrying the most unfavorable outcomes) were found to be high-aneuploidy tumors with losses in chromosomes 22q, 1p, 6q, 14, and 18. in addition, mg4 meningiomas showed a gain of chromosome 1q and a loss of chromosome 10. such findings have the potential to supersede existing molecular and clinically used classifications and grading schemes. indeed, two studies published in 2021 show that prognostication for patients with meningioma can be improved by taking particular copy number variations (cnvs) into account (figure 2). driver j et al. (corresponding authors bi wl and santagata s) published a paper in neuro-oncology evaluating whether the use of chromosomal copy-number data provided more accurate prediction of time to recurrence for patients with meningioma than the traditional who grades 14. their discovery cohort consisted of 527 patients diagnosed with meningioma, and two independent cohorts of 172 meningioma patients were used for validation of the findings. based on mitotic count and the presence/absence of the loss of (arms of) particular chromosomes (1p, 3p, 4, 6, 10, 14q, 18, 19), and of homozygous deletion of cdkn2a the authors developed a scheme with three integrated grades (1-3). this grading approach was found to more accurately identify meningioma patients at risk for recurrence than the traditional who grading system. figure 2. assessment of chromosomal copy number aberrations for improved grading of meningiomas.both in the study by driver j et al. 14 and by maas sln et al. 13 the presence/absence of particular chromosomal losses is used for assessment of prognosis of meningiomas. in the ‘driver approach’, this is combined with information on cdkn2a/b status and number of mitoses, while in the ‘maas approach’ the grade as traditionally assigned based on histological features and the suggested methylation family using the meningioma classifier (benign – intermediate – malignant) are taken into account as well. importantly, next to methylation array-based grading system for meningiomas, the study of maas et al. presents some alternatives for a more stepwise approach for grading of meningiomas, taking into account that particular histological phenotypes (angiomatous, psammomatous, secretory) are strongly associated with cns who grade 1 behavior, while presence of tert promoter mutation and/or cdkn2a/b loss indicate high-risk tumors. maas sln, stichel d, hielscher t, sievers p et al. (supervisors von deimling a and sahm f) published a study in the journal clinical oncology in which dna methylation profiling and copy-number information were generated for 3,031 meningiomas of 2,868 patients and mutation data for 858 samples 13. both cnvand methylation family-based subgrouping independently resulted in increased prediction accuracy of risk of recurrence compared with who grading. prediction power for outcome was assessed in a retrospective cohort of 514 patients, and validated on a retrospective cohort of 184 as well as on a prospective cohort of 287 cases. combining different risk stratification approaches into an integrated molecular-morphologic score resulted in substantial further increase in accuracy. again (like in the study of driver et al.), the integrated scores were found to separate tumors more precisely for risk of progression, especially so at the diagnostically challenging interface of cns who grade 1 and grade 2 tumors. discussion in 2021 again an amazing amount of information with relevance for neurooncological pathology has been published. hopefully, this review helps readers keep up with what’s new in this respect. like who tumor classifications in general, and as already stated in the section on topic 1, the who cns5 classification represents work in progress with room for further improvements. obviously, this new classification brings further challenges as well. for example, for more cns tumors it is now impossible to reach a state-of-the-art ‘histomolecular’ diagnosis in case molecular tools for assessment of essential diagnostic characteristics (or immunohistochemistry for reliable surrogate markers, see below) are not available. in those situations, adding nos (not otherwise specified) to the histology-based diagnosis is the way to go 35. furthermore, designing the optimal therapeutic management for newly defined tumor types is challenging. and while a more precise classification facilitates enrollment of more homogeneous populations of patients in clinical studies, the higher granularity of cns tumor taxonomy makes it more difficult to perform studies on a large number of patients for particular tumor types. still, one would like to think that patients suffering from a cns tumor are better served by a more precise diagnosis because this allows for a better estimation of prognosis and, hopefully sooner than later, for a more tailored and effective therapeutic approach. immunohistochemistry for surrogate markers can indeed provide a way for making a bona fide, ‘histomolecular’ who cns5 diagnosis. some examples of stains that are already often used in clinical practice are immunohistochemistry for mutant proteins (idh1 r132h, h3 k27m, h3 g34v/r, braf v600e), for loss of staining for normal proteins in the tumor cell nuclei (atrx, ini1, brg1, h3k27me3), and for abnormal location or intensity of a protein in the tumor cell nuclei (e.g. stat6, p53). the information provided by the studies of driver et al. and of maas et al. (see topic 10) shows that the care for patients with meningioma can be improved by assessment of particular molecular characteristics for the grading of these tumors 13,14. at the same time, there may be an opportunity for immunohistochemistry here as well: for example, loss of h3k27me3 staining of tumor cell nuclei in meningioma has been reported as an additional tool for identification of meningiomas with higher risk of recurrence 36,37. furthermore, in the study of nassiri f et al. (see topic 10), particular proteins were found to be highly enriched in the different molecular groups (mg1: s100b; mg2: scgn; mg3: acadl; mg4: mcm2) 12. further validation of such immunohistochemical approaches and comparison with the results as presented by driver et al. and by maas et al. is needed. also, acknowledging that the molecular underpinnings in meningiomas diagnosed in kids are distinct from those in adults 38, pediatric meningiomas may require an adapted grading system. while cns tumors can now be much more precisely characterized than a few decades ago, the translation of this increased knowledge into more effective treatments is seriously lagging behind. quite some of the topics discussed in this review concern studies that further elucidate the pathobiology of particular cns tumors. hopefully, such knowledge can be exploited for more effective therapies as well, e.g., by targeting of epigenetic regulation of neural or oligodendrocyte precursor states/cells (topic 4), by remodeling the epigenome of glioma cells in order to alter the differentiation hierarchy of gliomas (topic 5), by exploiting the new information obtained by multi-omic investigations for improved targeting of the pathways involved in glioblastomas (topic 6), and/or by improving strategies for selection of patients for targeted (immune)therapies based on immunophenotyping studies and information on the spatial distribution of genomic alterations of gliomas/glioblastomas (topic 7). in a study of > 10,000 cancer patients across 20 different cancer types, transcriptomic analysis allowed for the identification of four distinct tumor-microenvironment subtypes which correlated with patient response to immunotherapy. by integrating transcriptomic and genomic data, a global tumor portrait can be designed, describing the tumor framework, mutational load, immune composition, anti-tumor immunity, and immunosuppressive escape mechanisms, guiding therapeutic decision-making 39. the finding that visual experience and neuronal activity is required for the formation and growth of optic gliomas may provide new targets for therapeutic interference for these tumors (topic 3), and maybe some ‘old drugs’ can indeed be re-purposed in order to improve the outcome for patients with glioblastoma, e.g. because they interfere with the cell membranes of the tumor cells (topic 8). interestingly, a recent (‘seed & soil’) study on the metastatic potential of 500 human cancer cell lines spanning 21 types of solid tumor, breast cancer cells capable of metastasizing to the brain were found to have an altered lipid metabolism, perturbation of which indeed resulting in curbed development of brain metastasis 40. as discussed under topic 9, a liquid biopsy approach using detection of cnvs in csf-derived cfdna may allow for improved recognition of disease progression in children with medulloblastoma. however, in a very recent study of 258 pediatric brain tumors ‘across all histopathologies’, cnvs were detected in only 20% of csf-derived cfdna, and the fact that no genomic aberrations could be detected in liquid biopsies from patients with low-grade gliomas may indicate that its utility is more promising for patients with aggressive brain tumors 41. papers published in 2021 that the authors considered as (very) interesting, but that didn’t make it to the top ten list include: publications presenting potential new tumor types (e.g., supratentorial neuroepithelial tumors, plagl1 fusion-positive 42 and histologically polyphenotypic neuroepithelial tumors, patz1 fusion-positive 43); the report of detailed molecular analysis of (plus clinical variables in) a series of 191 medulloblastomas in adults 44; a study reporting that germline variants in the e-cadherin gene cdh1 are (in addition to non-cns tumors, especially of the stomach and breast) associated with increased risk of neuroepithelial tumors/oligodendrogliomas, idh-mutant, 1p/19q-codeleted 45; papers reporting the accuracy of different molecular tests for assessment 1p/19q status 46, and for mgmt promoter methylation status 47,48. also, for those with a keen interest in neuroimmunooncology it may be good to know that dural sinuses appear to act as a ‘neuroimmune interface’ where brain antigens are surveyed, and that may show ageand disease-related dysfunction 50, and that bone marrow niches in the skull and vertebral column act as myeloid cell reservoirs for the meninges and cns parenchyma 51. acknowledging that for multiple decades histological slides formed the basis for the diagnosis of (cns) tumors, one can argue that for a long time clinical (neuro)pathologists have acted as masters of ‘thin slicing’ (i.e., making quick inferences about characteristics of a tumor using a limited amount of tissue). nowadays, however, for the pathological diagnosis of a rapidly increasing number of cns tumors assessment of particular molecular characteristics is required as well. and in the near future, spatial and/or multi-omics analysis of these tumors may also be required. one can expect that by that time, diagnostic support tools will become available, such as tools for artificial intelligence (ai)-guided analysis of digitized, histological slides 52, and visualization tools for integration of the multi-omic profiling data and for guidance of therapeutic decision-making 39 (figure 3). while such a transition from thin slicing towards augmented reality indicates that the future of neurooncological pathology is bright, it wouldn’t mean too much unless these developments indeed can help to drastically improve the prognosis of patients that suffer from a cns tumor. figure 3. neurooncological pathology: from thin slicing to augmented realityas histological slides formed the basis for the diagnosis of (cns) tumors for multiple decades, one can argue that for a long time clinical (neuro)pathologists have acted as masters of ‘thin slicing’. this term was initially used especially in psychology and philosophy for a situation in which one makes very quick inferences about the state, characteristics or details of an individual or situation with minimal amounts of information. in the context of tumor pathology, it could literally mean the ability to make quick inferences about characteristics of a tumor using very thin slices of tissue. nowadays, however, for an increasing number of cns tumors, molecular characterization is necessary as well. in the near future, spatial and/or multi-omics analysis of cns tumors may be required for an optimal assessment of the diagnosis (including prognosis) and for prediction of the best therapeutic management. one can expect that by that time, diagnostic (ai-guided and visual) support tools have become available that guide the neurooncological pathologist from a more bounded rationality into an augmented reality. meanwhile, it is important to realize that the judgement of an experienced (neuro)pathologist based on thin-slicing/histological slides 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promoter methylation testing to predict overall survival in people with glioblastoma treated with temozolomide: a comprehensive meta-analysis based on a cochrane systematic review. neuro oncol 23:1457-1469, 2021 carlson jc, cantu gutierrez m, lozzi b, et al: identification of diverse tumor endothelial cell populations in malignant glioma. neuro oncol 23:932-944, 2021 rustenhoven j, drieu a, mamuladze t, et al: functional characterization of the dural sinuses as a neuroimmune interface. cell 184:1000-1016 e27, 2021 cugurra a, mamuladze t, rustenhoven j, et al: skull and vertebral bone marrow are myeloid cell reservoirs for the meninges and cns parenchyma. science 373, 2021 jin l, shi f, chun q, et al: artificial intelligence neuropathologist for glioma classification using deep learning on hematoxylin and eosin stained slide images and molecular markers. neuro oncol 23:44-52, 2021 copyright: © 2022 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. the ’accidental neuropathologist' – on 40 years in neuropathology feel free to add comments by clicking these icons on the sidebar free neuropathology 1:24 (2020) reflections the ’accidental neuropathologist' – on 40 years in neuropathology harry v. vinters pathology & laboratory medicine & neurology, david geffen school of medicine at ucla, los angeles, ca, usa corresponding author: professor harry v. vinters · laboratory medicine & pathology · university of alberta · faculty of medicine & dentistry · edmonton, alberta · canada hvinters@mednet.ucla.edu submitted: 14 august 2020 accepted: 16 august 2020 copyedited by: christian thomas published: 25 august 2020 https://doi.org/10.17879/freeneuropathology-2020-2956 additional resources and electronic supplementary material: supplementary material keywords: neuropathology, ucla, personal reflections this article is dedicated to the memory of my brother raymond john vinters (1952-2020). beginnings in canada my family were latvians who, by the end of world war ii, had become refugees from the soviet union. for the repressive communist regime that controlled the ussr, my family (on both sides) were or would soon become ‘criminals’ who owned land and were involved in commerce. they saw no future under a communist regime, and the fate of relatives who remained in latvia after world war ii confirmed the wisdom of their decision and choice – some were exiled to siberia for many years, others simply had their property confiscated. my family – grandparents, their two children (one my father) and assorted household staff who joined them – fled the country as the red army moved in to ‘liberate’ its people and begin a brutal and merciless tyranny that would last until the early 1990s. during that happy time in the early 1990s the ussr thankfully collapsed under the weight of grotesque corruption and reliable, predictable, frequently comical soviet incompetence. members of my family lived in various refugee camps in europe between 1944 and 1949, at which time they were sponsored by distant relatives to relocate to canada; some of their lasting and most intimate friendships were made in the camps during those postwar years. my parents had wed in lübeck, germany. it was their (and my) good fortune that our family ended up on the northwest shore of lake superior, in the small city of port arthur (now integrated into a larger municipality, thunder bay), where i was born. if one envisions lake superior as having the shape of a wolf’s head, thunder bay is about where its eyes would be located. this beautiful small city is situated on rolling hills overlooking the lake, and is home to one of the harshest (winter) climates of any metropolitan area in canada. it provided me with an outstanding primary and high school education at the hands of, and with the mentorship of, dedicated and devoted teachers who inspired a love of learning and a first-rate work ethic, of which they themselves were emblematic. very lucky break for me! winters were long and brutal in port arthur. i well remember sitting in my high school classes, looking out onto the harbor – port arthur was a port city thanks to the st. lawrence seaway (opened in the late 1950s, queen elizabeth ii herself came to our fair city for the official opening), which allowed ocean-going vessels to come into the great lakes. the harbor was still frozen in april and may, and i longed for warmer days – never dreaming then that i would spend most of my working career in a climate that can safely be described as endless summer. i was also fortunate to have support and encouragement from a wonderful father [figure 1] who, as a single parent, raised me and my brother after our mother left in the early 1960s to begin a new life in the usa and my parents divorced. he died in 1980 but never a day goes by that i don’t think – with much love and gratitude – of him and his dedication to his sons and family, his generosity, support and kindness. our loving and resourceful family included grandparents, aunts and uncles, eventually cousins, all trying to find their way in the brave new world into which they had been thrust by world war ii and its aftermath and sequelae. as with many immigrant families trying to establish life in a foreign land, barely speaking english and with limited finances, extended family members (usually from three generations) occupied small apartments and houses, then eventually spread their wings and left to make lives of their own in their new homes, when they could finally afford them. my favorite home in those early days was a ramshackle house – little more than a cottage – just outside the port arthur city limits, with two tiny bedrooms and one miniscule bathroom (bath, no shower), situated on the banks of a small meandering river and on a gorgeous piece of land. the property was also home to several massive pine trees and came to be known (by the latvian expatriate community) as eglaines (loosely translated, the home of tall pine trees). seven of us called this home for many years. it had an old coal furnace in which fuel needed to be replenished during the icy winter nights, a task shared by my father and grandfather. my grandparents, who had been ‘saimnieks and saimniece’ (the master and mistress of their household) and oversaw a large farm in latvia, lovingly cultivated their gardens of flowers, berries and vegetables on the two acres that eglaines occupied. i often wondered what it would be like to start one’s life over, at the age of 49 or 50, in a foreign land and culture, as my resilient grandparents had; the task was easier – though no less challenging! – for my father and mother, who later became a medical technologist. my father and grandfather worked in the grain elevators that surrounded the harbor (the grain coming from canada’s prairie provinces and destined to be loaded onto seaway-bound ships) until my dad became a real estate agent, then subsequently a real estate broker. my paternal grandmother worked as a domestic. while i loved port arthur (that affection continues to this day) i yearned to pursue higher education in a larger center, and a variety of scholarships allowed me to do so at the university of toronto (u of t), another lucky twist of fate. being a toronto resident introduced me to the joys (and challenges…) of big city metropolitan life, and i have enjoyed urban living ever since. i adore the buzz of large cities, almost without exception. idyllic country life, a dream for some, is not for me. fig 1: my father gunnar (gunars) vinters, 1925-1980 during the summer between my graduation from high school and the beginning of university (1968) i had the marvelous opportunity to work in the laboratory of a family friend (and my aunt’s godmother), irene grodums, who after immigrating to canada had completed her ph.d. and become a virologist at the university of saskatchewan in saskatoon. prof. grodums instilled in me a respect for the importance of careful observation in tissue studies, whether done by light or electron microscopy (she worked on coxsackie virus effects on the heart) and respect for experimental animals used in biological research. in the 1970s medical school was increasingly regarded as a place of graduate studies, following upon an undergraduate experience. my first degree was in biology and most of my courses given in the botany department (these days, plant sciences). u of t undergraduate instruction was loosely based in one of the constituent colleges, and my academic (and residential) home was university college. i thrived on living on an historic campus situated in the middle of a world class city, one with a great sense of history. one of my first summer research jobs was in the botany department, studying the kinetics of water movement into and out of giant algae with profs. jack dainty and mel tyree (both accomplished biophysicists); this resulted in my first publication [1]. while in medical school at the university of toronto (1972-76) i developed an abiding affection for the neurosciences, though i realized i would never have the skill set, insights, patience or scientific acumen to be a solid basic neuroscientist. our inspiring professors included the legendary neuropharmacologists phil seeman and oleh hornykiewicz; dr. seeman (probably one of the best lecturers i have ever encountered) had previously discovered how anesthetics and tranquilizers interact with cell membranes [2] and subsequently contributed to the molecular understanding of complex genes that control dopamine receptor function, while dr. hornykiewicz was the discoverer of the dopamine deficiency [3] that leads to parkinson disease. the other option was to choose a clinical neuroscience in which i would have both clinical responsibilities and an opportunity to do research and teaching. i decided to pursue postgraduate training in neurology, having eliminated neurosurgery as a career path – i lacked the required stamina and manual dexterity, both (in addition to excellent clinical judgement) requirements of a first-rate neurosurgeon. fortunately, throughout my career i have had the opportunity to work with some of the very best in the latter field, in numerous productive collaborations and diagnostic challenges (see below). how neuropathology chose me neuropathology was a largely invisible specialty while i was in medical school. admittedly, in the early 1970s it was itself a fledgling discipline in the clinical neuroscience firmament, certainly in canada. we received wonderful lectures from neuropathologists – including dr. barry rewcastle – but were never really apprised of how a neuropathologist spends her/his time and contributes to the diagnosis and understanding of neurologic disease. indeed, we hardly knew that such individuals existed or how one went about becoming a neuropathologist, this despite several significant neuropathologic discoveries having been made at u of t’s banting institute on college street, then home of the research components of the pathology department. (frederick banting, the discoverer of insulin, was the first and is still one of the few canadians to be awarded the nobel prize in medicine or physiology). the initial report of progressive supranuclear palsy [4] was co-authored by the great jerzy olszewski, though he had unfortunately passed on by the time i was in medical school. i spent my internship year at the university of alberta hospital in edmonton, in retrospect a wise choice because it introduced me to the beauty of the rugged canadian west (stunning prairie skies and amazing geography) and facilitated my meeting people who remain steadfast friends to this day. of course that year also provided a terrific opportunity to learn important fundamentals of medicine from energetic and knowledgeable instructors. the university of alberta hospital would, many years later, become a place where i would do an extended locum. i began training in neurology in london, ontario in 1977 in the department of clinical neurological sciences (cns) at the university of western ontario / uwo (subsequently renamed western university). this department represented a logical fusion of talent from neurosurgery, neurology, neuroradiology, and neuropathology. its leadership included two legends of 20th century neurosurgery (charles g. drake) and neurology (henry j.m. barnett). london was also a major center for stroke research; the clinical specimens we were shown in surgical pathology and at autopsy conferences inspired an appreciation and respect for the importance of cerebrovascular disease in neurology. the weekly 3-hour city-wide clinical neuroscience rounds included presentations from all four of the sub-disciplines, including a clinicopathologic conference weekly or every two weeks. i was coming to an important, somewhat uneasy career juncture: neurology felt like perhaps not quite the optimal choice of specialization for me. however… an inspirational figure during that first year of neurology training came to be dr. john c.e. kaufmann, chief of neuropathology at the (recently built) university hospital on the main uwo campus in the north end of the city. dr. kaufmann (or jcek, as his trainees knew him, figure 2) had been recruited from south africa to be the first neuropathologist at uwo, and one of only a handful in canada at the time. more than being a marvelous diagnostician, jcek was the essence of a ‘gentleman and scholar’, someone who took responsibility for the wellbeing and mental and physical health of his trainees. i daresay most of us lucky to be in that multi-year cohort try to emulate jcek in our own careers. he and his wife suzanne kaufmann loved art (they had an inspiring collection which included an andy warhol), entertaining on a grand scale, fine (exquisite) cuisine usually prepared in their own kitchen, good and witty conversation and travel. the presentations jcek oversaw at the weekly clinical neuroscience rounds were marvels of clarity and insights into neurologic disease. throughout that year of training in neurology, i came to the conclusion that neuropathology might be a more appropriate career path for me. fortunately, dr. barnett was very passionate about the vital importance of neuropathology in neurodiagnostics. when i requested the possibility of doing a full year of neuropathology training (then configured as part of my neurology residency) dr. barnett was favorable to the idea, as was jcek. i embarked on the road to my career as a neuropathologist with some trepidation, but a passion for the study of the anatomic basis of neurologic diseases using a multitude of special stains, which in those days (late 1970s) were, together with electron microscopy, the ‘gold standard’ for neuropathologic investigations. oh yes, things have changed! fig 2: dr. john c.e. kaufmann, chief of neuropathology, university hospital, london, ontario, canada my other mentors in london were dr. melvyn j. ball, who was already a prominent alzheimer researcher, and dr. joseph gilbert [figure 3], who had trained in neurology and neuropathology in cleveland, though practiced the latter. neuropathology in canada is configured as a specialty rather than a ‘subspecialty’ requiring fellowship training. jcek and joe gilbert were in charge of np training at the two major uwo teaching hospitals, university and victoria, and possessed complementary, mutually reinforcing didactic skills. jcek was an experienced, meticulous and insightful neurohistologist. joe gilbert was a master of providing clinicopathologic insights based upon careful correlation of neuropathologic findings with clinical signs and symptoms (structural brain imaging in the late 1970s was still in its infancy). the experience of working with them both and learning from them was exhilarating, humbling and yes – a little daunting. fig 3: dr. joseph j. gilbert, neuropathology mentor, london, ontario joe gilbert also suggested to me a translational research project that would determine and define my research interest for the coming decades: diseases of the cerebral microvasculature. as a stroke referral center, uwo was encountering many patients with lobar intracerebral hemorrhages caused by, or at least associated with, cerebral amyloid angiopathy (caa). joe suggested that we collect our cases of (putative) caa-related brain hemorrhage and write a descriptive paper on this association; a companion study would assess the topography and severity of caa in relation to brain aging. in the late 1970s and early 1980s (well before the discovery of beta-amyloid protein in 1984, see below) caa was evaluated by the use of congo red staining and polarization microscopy (alternatively using thioflavin stains and fluorescence microscopy). the ‘topography/severity of caa with age’ study was performed simply by staining a large number of brain sections, from uniformly sampled autopsy brains, with congo red and evaluating caa in these. the two resultant manuscripts went through several drafts, were eventually submitted to the journal stroke as ‘back-to-back papers’, and published in 1983 [5,6]. it is gratifying that the findings we described using fairly primitive (by today’s standards) technology have been replicated using beta-amyloid immunohistochemistry in later studies; it is perhaps especially satisfying that the papers are still being cited 37 years after their publication (combined total citations of 500+ per the web of science). examining the pathogenesis and consequences of caa and other microangiopathies in the brain would constitute a major theme of my subsequent research endeavours. my np residency was spent feasting on the instruction and support of my mentors and the remarkable and stimulating milieu created by the clinical neurologists, neuroradiologists and neurosurgeons at uwo. this showed and highlighted the importance of collaborative activities in understanding complex neurologic diseases. luminaries in neurology and neurosurgery (fred plum, raymond adams among them) were frequent visitors to this small but ‘neurologically important’ city half way between toronto and detroit. one of my interests was in the effects on cerebral arteriovenous malformations (avms) of agents used in novel embolization therapies that were just being developed, many of them at uwo. two skilled endovascular specialists (drs. fernando vinuela and allen fox) were pioneering new agents and approaches to non-surgical therapies for vascular brain lesions, and continued to do so throughout their careers. when these lesions (usually avms) were subsequently resected, they showed unique and sometimes dramatic reactive changes caused by the embolotherapy agents (about which more later…). many of my research interests have been driven by clinical or clinicopathologic questions. here were two large and important ones: why (and how) do embolotherapy agents work through their interactions with vessel walls in vascular malformations? what might be some neuropathologic clues to why they sometimes fail? they were also questions that could be effectively addressed by simple observation (using routine stains) of previously embolized lesions when they made their way to the surgical neuropathology laboratory. there was discussion of my returning on faculty to uwo, but only after i had spent some time developing basic research skills pertinent to the study of cerebrovascular disease. the university would provide or facilitate funding for my research endeavours, but i had the freedom to choose a laboratory in which i might develop appropriate experimental techniques and skills that i could repatriate to uwo. at that time, some of the most exciting work on cerebral microvascular biology (especially the blood-brain barrier/ bbb) was being done using cell biologic techniques in the laboratories of drs. pasquale (pat) cancilla [figure 4] and michael n. hart [figure 5] at the university of iowa (u of i) [7,8]. it was arranged that i would spend two years working with them, after a 6-month period refining my diagnostic skills in pediatric neuropathology with dr. margaret norman in vancouver; marg became and remained a good friend for the rest of her life. fig 4: dr. pasquale (pat) and enid cancilla, undated photograph trying my hand at (basic) research i arrived in iowa city (in which the university of iowa is located) in january 1982, in the midst of a nasty midwestern blizzard. during a lonely weekend in a slightly seedy motel in coralville (a suburb of iowa city) i frequently thought: ‘what on earth have i done?’ during my first day in the new laboratory environment at the u of i, pat cancilla asked if i could go with him for a cup of coffee. then came the bombshell: he had just accepted the chair of pathology position at university of california los angeles (ucla). he suggested that, since i had independent funding that was not tied to a specific institution, i might either stay at the u of i and work with michael hart, or move with him to ucla. as i looked outside at the heavy snowfall and felt the subzero temperature en route back to the lab, i had essentially made my decision, and moved with dr. cancilla to ucla that summer. during the six months i resided and worked there, however, i developed an affection for iowa city, sometimes described as the ‘athens of the midwest’—famous for its world class medical center (including brilliant translational neuroscience and behavioral neurology) and the iowa writers workshop. my colleagues there included drs. jonathan fratkin and david beck, the latter an outstanding neurosurgeon (then still a resident) bursting with excellent research ideas that might optimize the use of cultured cells in understanding bbb physiology and abnormalities [9]. fig 5: dr. michael noel hart ucla, destined to become my academic home for most of the remainder of my career (though that decision was still in the future), was research heaven for a young scientist. it was fun helping to set up pat cancilla’s laboratory (our senior technician at the time was jim bready) with top-of-the-line equipment and facilities that were the perks of being a newly recruited chair and entering the academic milieu of a major research university. our ‘lab warming’ party was a smash hit throughout the medical center. pioneering studies of cerebral microvessels (especially the bbb) were ongoing in the laboratories of two ‘bills’ – william pardridge and william oldendorf. dr. oldendorf had previously (1975) won the lasker award, and probably deserved the nobel, for suggesting the principles that led to computerized tomographic (ct) scanning, but was also an innovative and creative investigator looking at the physiology of the bbb, especially using isotope tracer techniques. he was also bill pardridge’s mentor. pat cancilla’s lab became part of an informal consortium of investigators (also including drs. judith a. berliner, an experienced cell biologist, and eain cornford at the west la va medical center) interested in cerebral microvasculature (especially its endothelium) and optimal ways to study it. our laboratory was mainly intrigued by the potential use of tissue culture approaches and had been able to isolate and culture endothelial and smooth muscle cells from brain microvessels, initially from mouse brains but eventually (some years later) even those derived from human corticectomies for intractable epilepsy. i was also smitten by the city of angels, los angeles. it represented a far different world from the one in which i grew up. l.a. was (and is…) a city with a near perfect climate (summer and winter), far cleaner air than is widely believed, stunning geography with canyons, hills and flatlands, a brilliant seaside location on santa monica bay, and remarkable ethnic and cultural diversity. it reminds itself almost daily that it is the ‘world capital of the entertainment industry’, which is probably true, and ‘the creative center of the planet’ – the latter a claim that london (england), amsterdam, tokyo, paris and a few other cities might dispute. i remember reading that the german novelist gunther grass had once described berlin as representing a city that ‘faced the realities of the 20th century’ (this may be an erroneous recollection…), and thinking that would probably apply quite accurately to l.a. in the late 20th and early 21st centuries. though i at first lived near the ucla campus, i found myself spending many weekends at venice beach, where i have now had a home for the past 30+ years. (rationale: if you live near the pacific ocean, why not at one of the most famous beaches in the world?) as i was nearing the end of my postdoctoral fellowship, i was working in the lab on a saturday morning. pat cancilla called me into his office (immediately adjacent to the laboratory) and rather directly asked if i might be interested in a ucla faculty position – one had just come open in neuropathology because of the retirement of dr. jann brown. we discussed the possibility briefly. this was the second time in my career that a casual chat with pat cancilla would alter the course of my career. return to london, ontario and uwo (briefly) because of visa issues and funding, i was committed to return to london, ontario, but my faculty job there was a ‘soft money’ position, unlike the tenure track post for which i was being considered at ucla. i had formally applied for the ucla faculty position while still a postdoc there, interviewed for it, and eventually (a few weeks after returning to london, ontario) was offered and decided to accept the job. it was a decision made over an agonizing weekend during which i weighed the many pros and cons – the university of western ontario would have made a wonderful academic home for me, and london a welcoming and very livable city (i had already bought a home there). john kaufmann and joe gilbert were amazing mentors and colleagues. yet i had enjoyed my postdoc at ucla so much… that i felt i had to ‘give it [the faculty position] a try’. having heard that american academic medicine could be rather brutal and fiercely competitive (though i had seen little evidence of this during my postdoc), i began to wonder whether i had the stomach for this. i rationalized the decision by thinking i would remain at ucla for 2-3 years, then return to london, ontario (assuming a position was still open there…). at the appointed time, i considered this move back, and decided to remain at ucla, where i am (part time, semi-retired) today. my 18 months in london (pending receipt of the green card / permanent resident status that would allow me to take up life in the usa) were delightful and rewarding both professionally and personally – so much so that i began having some doubts as to whether i had made the correct decision in accepting the ucla job. (timing is everything. were i making this same career-changing deliberation in the current unwelcoming usa immigration climate, i would almost certainly have stayed in london!) i managed to set up a small tissue culture laboratory at university hospital – thanks to the fact that a fully equipped laboratory with appropriate equipment had just been made available by a departing or retiring faculty member. we were able to successfully culture endothelial and smooth muscle cells from human cerebral microvessels [10]. i continued to have an interest in ‘embolized avms’. as a result of the neurosurgical skills and expertise of drs. charles drake and sidney (‘skip’) peerless, brilliant endovascular therapy experts vinuela and fox (see above) and their respective teams, london and uwo attracted complex avm cases from around the world that might be candidates for this unique therapy. if one is going to do translational research, it helps to have a unique collection of abundant clinical and pathologic specimens to study – and our neuropathology laboratory had just such an array of treated avms, an amazing experiment of nature, though one admittedly manipulated by physicians. with the help of jcek and a very industrious undergraduate student (mark lundie) i set about a careful examination of avms that had been treated by embolization with isobutyl cyanoacrylate, one of the widely used embolotherapy agents at that time. (it is an agent that is injected through a catheter in liquid form, but polymerizes at various rates depending upon how much contrast medium is in the mixture. with this agent, timing of the polymerization process was of paramount importance – if it polymerized too quickly, the catheter could become stuck to the intima of the avm ‘feeding’ vessel; if too slowly, it would end up passing through the avm and into a treated patient’s lungs.) we had vascular malformations with a wide range of post-embolization intervals that allowed for inferences about how this polymer was interacting with abnormal vessel walls in avms. we put together a significant (and rather unique) data set, which i brought with me to ucla when i returned there in july, 1985, hoping i would have the time to analyze it in detail and prepare a manuscript. fig 6: dr. michael a. farrell, friend, colleague and ‘partner in crime’ i also had the good fortune to meet, during that 1 ½ years back at uwo, dr. michael farrell [figure 6], a newly arrived neuropathology trainee from dublin, ireland. michael became a great friend and colleague during that time and after he returned to a consultant post in dublin. over the years, we have written many papers (see below) and co-authored a textbook, he has often visited l.a. (even doing a mini-sabbatical in my laboratory during the early 1990s), and i have gone to dublin dozens of times; it is on my list of favorite cities around the globe. great and generous friend. through michael i eventually came to know the small but vibrant irish neuropathology community, including drs. katie keohane and francesca brett. on faculty at ucla how does one summarize 35 years of academic and diagnostic work at one institution? there are many things i love about ucla: the physical beauty and location of the campus in westwood (the ucla medical center, now ronald reagan ucla medical center, is at the southern edge of the main campus), the collegial and collaborative nature of the faculty, the wealth of expertise in all aspects of basic and clinical science, especially neuroscience, talented and intelligent students and trainees, and the cultural and ethnic diversity of both students and faculty who are committed to scholarly work. yes, there are things not to like (but one endures them…): the numbing, frequently obstructive bureaucracy, the ‘bend over backwards’ approach to being politically correct are among them. i returned to l.a. and the ucla family the first week of july, 1985. i used the ‘down time’ in setting up a small research laboratory, to finesse two projects. one was collating the embolized avm data into a coherent story. as i wrote the manuscript and put the results into perspective, i thought the material was sufficiently novel that it might be worthy of submission to the prestigious new england journal of medicine, with the thought that ‘nothing ventured, nothing gained’. as i put the manuscript in the mail (yes, what we now call snail mail...) i was already reformatting the paper for a lower level, less prestigious journal because of the certainty of rejection. then a few days later i received a surprising phone call from the nejm telling me the manuscript had been accepted, with minimal revisions; they were calling me to verify that there was no overlap with a review article on cyanoacrylates we had recently written – i assured them there was none [11]. the nejm paper was published in 1986 [12] and represents one of my two appearances as an author of a paper in this high-profile venue, the second coming almost exactly 20 years later as a co-authored contribution on a totally different topic [13]. my other project was to write a review article on (what else?) caa – i suggested this to henry barnett, then executive editor of stroke, and he liked the idea. that paper eventually appeared (after a few revisions) in 1987 [14] and is still frequently quoted today. in the meantime, i resumed my tissue culture studies but decided to embark on other scientific adventures. while i was impressed by the simple questions that could be addressed (and answered) using in vitro methods, i was (like many investigators) concerned about how these phenomena could be understood in the context of complex multi-cellular tissues and organs, especially in the brain. being a junior faculty member at ucla was the academic equivalent of being a child at a candy store. colleagues were always approaching one about working on any variety of projects, some utilizing human cns specimens, others exploiting interesting animal models, transgenic and knockout mice, etc. neuropathologists were and are in the unique position of understanding the structure of the nervous system (normal and abnormal), and how best to study it. i re-established a close collaboration with william pardridge (see above), an endocrinologist in the dept. of medicine who was interested in the biochemistry (and eventually molecular biology) of transport mechanisms through the bbb, but was now also looking for a way to study abnormalities (diseases?) of small blood vessels. we had many discussions about cerebral amyloid angiopathy (caa), which of course affects arterioles and capillaries and, together with arteriolosclerosis, is one of the two most common microvasculopathies found in the brain. in 1984, glenner and wong [15] had isolated the ‘a4’ protein from amyloid-laden meningeal vessels of an alzheimer patient. bill pardridge was amazingly skilled at making antibodies and proceeded to make a polyclonal antibody to ‘a4’, using the partial sequence of the peptide provided by glenner and wong in their paper. it worked magnificently in immunohistochemical protocols to demonstrate both senile plaques and caa, resulting in several publications [16,17]. bill pardridge was also the best person in the school of medicine to review the draft of a grant proposal; i am eternally grateful for his merciless critique of one of my first independent national institutes of health (nih) proposals, which through his intervention helped me transform an unfocused and improbable set of studies into a fundable application. my chief of neuropathology was m. anthony verity, and we divided the service and diagnostic commitments equally – each of us covered 6 months of the year. my interests were obviously in cerebrovascular disease and dementia, tony’s in neurotoxicology and neuromuscular diseases. when one of our outstanding neurologists, dr. jeffrey cummings, decided to submit an nih (national institute on aging) grant that, if successful, would fund an alzheimer disease center (adc) at ucla in the late 1980s/early 1990s, i seemed the logical choice to head the neuropathology core and brain bank. we were funded on the first submission and i was thrilled to formally become part of the alzheimer research enterprise at ucla, even though my main academic focus (caa and related microvascular diseases) were at the periphery of most debates on ad pathogenesis. this situation has changed drastically in recent years. it is also worth noting that glenner and wong had isolated a4 from amyloid-laden meningeal microvessels (‘a novel cerebrovascular amyloid protein’) rather than senile plaques (sps); as is now well known, the anti-a4 antibody indeed labels sps as well as caa, but not neurofibrillary tangles, despite initial claims that it might. over the years until his departure from ucla, jeff cummings was an amazing leader: a superb diagnostician and organizer who supported basic research efforts and consistently brought out the strengths in investigators and his staff. and oh yes, he was a masterful fund raiser, successful in obtaining private donations that were crucial to supporting the adc when nih funding lapsed or became thin. he eventually secured an endowment that led to re-naming of the adc as the mary easton alzheimer center. i had obtained nih and other funding for my own projects within 3-4 years of arriving at ucla, but have always felt most at home scientifically in the environment of a collaborative center or program project grant; pieces of these have constituted most of my research efforts and funding support while on faculty at ucla. of course, in the early to mid-1980s other events were taking center stage in the world of medicine: the disease that was eventually named acquired immune deficiency syndrome (aids) presented in a handful of gay men, initially studied in new york city and l.a. (please also see herbert budka’s thorough and excellent description of the beginnings of this epidemic in his reflections piece published earlier this year.) as pathologists, we were witnessing the evolution of a feared illness that, at the time and for many years to come, had no effective treatment and was almost uniformly fatal. by the mid-1980s it was known to be caused by the retrovirus hiv-1 (initially named htlv-iii). in that same time frame, thanks to the work of many investigators including george shaw, beatrice hahn, clayton wiley, leroy sharer and others, it became appreciated that hiv frequently infected the brain in both children and adults, causing a syndrome initially described as aids dementia complex (in adults) though later re-named several times. but i’m getting ahead of myself… pathologists have now (and always have had) a key role in providing morphologic evidence for unique disease mechanisms – some of them based simply upon careful observation of novel but convincing histopathologic changes within tissues. consider, as one example outside the nervous system, the astute observation by robin warren (pathologist) and barry marshall (gastroenterologist) of ‘unidentified curved bacilli in the stomach of patients with gastritis and peptic ulceration’. this description of helicobacter pylori, initially published in lancet (in 1984) eventually led to their being awarded the nobel prize in 2005 because the observation led to a complete re-examination of the rationale for ways to treat peptic ulcer disease, a major paradigm shift. moving back into the cns, neuropathologists have made countless key observations (often in human biopsy or autopsy specimens) pertinent to the pathogenesis of neurodegenerative disease, intractable epilepsy, stroke, developmental disorders, tumors, and the list goes on. with the beginning of the aids epidemic, here was another opportunity to see a new infectious disease evolving before our eyes. this might be (often is…) called ‘opportunistic research’, with the slight implication that it is not as challenging or important as ‘basic research’. however, when unique opportunities to carefully study a human disease present themselves, one would be foolish not to take advantage. at ucla center for the health sciences (chs), one of the bright young residents from canada, karl anders [figure 7], suggested we review the neuropathologic features of our large and growing autopsy collection of brains from aids patients. karl and i, both recent arrivals from canada, bonded as soon as i arrived at ucla. we had both grown up in two cities located about 400 miles apart (karl in winnipeg), were raised in the households of immigrant families, and we both liked looking at slides – a lot of slides. fig 7: dr. karl h. anders, pathologist and frequent collaborator on projects, currently in calgary, alberta, canada we also engaged in this neuropathologic enterprise the collaboration of dr. uwamie tomiyasu [figure 8], an amazingly insightful, organized, helpful and collegial neuropathologist at the west l.a. va medical center (who over the years became a close friend), and involved a promising young ucla medical student wayne guerra in the project. karl anders had been the first author on the report of our initial series of aids autopsy cases (less than 30) written while i was still a research fellow at ucla. we had submitted this detailed report to clinical neuropathology. the manuscript had been almost immediately accepted for publication in late 1983 or early 1984. then, to our amazement and shock, clinical neuropathology ‘sat on’ this very topical manuscript for over 2 years before publishing it – in the meantime printing (arguably) less impactful and timely studies that had clearly been accepted after our paper [18]. by 1986 we had compiled data on almost 90 aids autopsy cases, and submitted this updated report to the american journal of pathology, where it was accepted with minimal revisions, and published a few short months after the much delayed clinical neuropathology paper [19]. karl and i wondered whether someone reading both papers (published in 1986) would question how we accumulated 60 new aids cases in a few short months, which of course we had not. the neuro-aids work took some interesting twists and turns, largely as the result of collaborations with some of my formidably talented colleagues in virology and neurology interested in viral neuropathogenesis. i co-authored two papers together with david ho and his research group while he was briefly on staff at cedars sinai medical center in los angeles [20,21]; these are the only two publications i have written with a physician-scientist who would eventually become time magazine’s man/person of the year in 1996. (i very much enjoyed basking in the reflected glow of david’s richly deserved fame.) i was also approached by a brilliant and productive retrovirologist, dr. irvin s.y. chen, who had trained with one of the discoverers of reverse transcriptase (howard temin at the university of wisconsin) before becoming a faculty member at ucla at about the same time as me. irvin was (and remains) keenly interested in the molecular pathogenesis of interactions between hiv and the brain. his laboratory used autopsy brain and csf specimens from neuro-aids subjects to characterize a neurotropic strain of hiv, and exploited pcr to quantify hiv viral load in the cns of affected patients [22,23]. it seemed an ideal collaboration between a talented basic virologist and someone (hvv) interested in morphologic brain changes induced by hiv. at the same time, my laboratory remained fascinated by the interesting opportunistic infections (ois) – especially other viral agents, that can cause puzzling varieties of devastating neurologic diseases in immunosuppressed aids patients. cytomegalovirus was one of the viral ois capable of producing variable cns disease in aids, including (at its worst extreme) severe encephalitis, ventriculitis and myeloradiculitis, yet minimal abnormalities in others. contributors to these studies included karl anders, a postdoctoral fellow from germany, dr. regina von einsiedel and marcia cornford, who was one of our first trainees in neuropathology and is still a faculty member at harbor-ucla medical center in torrance, california [24,25]. i became the pathologist of record for the ucla site of the multi-center aids cohort study (macs, pi dr. roger detels), one of the longest funded nih projects in history. karl anders and i co-authored a monograph on the neuropathologic features of aids [26]. fig 8: dr. uwamie tomiyasu, (dec 2004), former neuropathologist, west l.a. va medical center and ucla faculty member, friend by the midto late 1980s and into the 1990s there were other interesting developments in ucla’s active epilepsy surgery programs. it already had a longstanding research effort on temporal lobectomy for intractable temporal lobe seizures, under the capable and inspiring direction of jerome (‘pete’) engel (neurologist) and paul crandall (neurosurgeon). pete was, and continues to be, one of the great advocates for bringing the possibility of curative temporal lobe surgery or ‘neocorticectomy’ to epilepsy patients throughout the world. paul was a meticulous neurosurgeon who provided us with anatomically intact (including the hippocampus) temporal lobes. the arrival at ucla of pediatric neurosurgeon dr. warwick peacock from south africa in the mid-1980s ushered in a new era of pediatric epilepsy surgery. using high resolution neuroimaging (including structural and metabolic/positron emission tomographic imaging) modalities linked with electrocorticography, warwick and the pediatric epilepsy group (under the visionary leadership of drs. donald shields and raman sankar) developed the concept that generalized epilepsies in infants and children (e.g. leading to infantile spasms) might have a focal origin – a ‘zone of cortical abnormality (zca)’ within the cerebral cortex – and that removing such a focus might effectively treat the seizures. at the time, this was considered a radical, almost heretical – possibly even dangerous! – approach, but our clinicians were certain they had convincing evidence for the zca and proceeded with corticectomies for intractable infantile and pediatric epilepsy, sometimes removing a significant portion of one cerebral hemisphere in an infant. the challenge for neuropathologists was to identify what the underlying structural abnormality (abnormalities) causing the seizures might be. again, we were handed the opportunity to study unique specimens that might explain important clinical signs and symptoms. by the late 1980s and early 1990s we had accumulated a significant experience with these specimens. fortuitously, my friend and colleague dr. michael farrell came over from dublin in 1991 to do a mini-sabbatical at ucla, and set about collating the data on our collection of pediatric epilepsy surgery cases. the most common neuropathologic change (especially among the youngest symptomatic patients) was a malformation of cortical development that had first been described by david taylor and colleagues in the 1970s. it had a striking resemblance to the tubers found within the brains of patients with tuberous sclerosis complex (tsc) even though affected individuals showed none of the visceral or cutaneous stigmata of this neurocutaneous disorder [27]. ironically, ‘tuberectomy’ has now become an accepted treatment for some tsc patients with intractable seizures. a much less common finding among corticectomies was an inflammatory disorder first described in 1958, rasmussen encephalitis (re). dr. farrell was first author on our initial ‘pediatric corticectomy’ paper [28] and we continued to collaborate on other projects, including one aimed at elucidating the detailed neuropathologic changes of re [29]. paul mischel, then a neuropathology fellow and now a leading neuro-oncology researcher at uc san diego, was a lead author on a paper in which we proposed a ‘grading’ scheme for the (as yet poorly understood) condition of cortical dysplasia (cd) [30] – a scheme that served its purpose in the mid-1990s and has been superseded by others, as it should be. the team of clinicians and basic researchers who collaborated in the pediatric epilepsy surgery program was a pleasure to work with. this was team science at its finest – everyone in the group respected (and utilized) the unique skills of all other basic and clinical neuroscientists, and the resulting multi-disciplinary studies benefited from this synergy. experts in linguistics, neuropsychology, neuroradiology, neurophysiology and psychiatry interacted productively with a simple neuropathologist. our goal was not only to understand what structural changes in the brain may have contributed to the catastrophic seizure disorder, but how corticectomy might impact a child’s behavior and language development. dr. gary mathern, a ucla-trained neurosurgeon and protégé of paul crandall and warwick peacock, eventually became the surgeon-of-record for most of the pediatric epilepsy cases. dr. mathern also has the ‘soul of an investigator’, is possessed of enviable organizational skills and leadership qualities, and fostered new and exciting directions in which the ucla pediatric epilepsy surgery program might move, places where it might thrive. dr. carlos cepeda, michael levine, (the late) robin fisher, veronique andre and others became integral to the study of electrophysiological and morphological properties of the epileptogenic tissue originating from infants and children with intractable seizures [31-33]. dr. noriko salamon in neuroradiology contributed her powerful diagnostic insights and observations [34,35]. in 2006, and based upon our morpho-anatomical and electrophysiologic studies, we proposed the ‘dysmature cerebral developmental hypothesis’ as a possible etiology for cd [32]. at the same time, we continued to explore the possible relationship of sporadic cd to tsc tubers. the genes that are mutated to cause tsc (first tsc2, then tsc1) had been discovered in the 1990s; we made probes to the relevant gene transcripts and eventually antibodies to the gene products (tuberin and hamartin), using these to study gene expression in normal brain, tubers of tsc and sporadic cortical dysplasia [36-39]. this led to a valuable collaboration with leading tsc researcher dr. david kwiatkowski and his associates in boston – which included sophisticated molecular genetic analysis of many of our corticectomies and even allowed us to examine a novel animal model of tsc, one of the first to be developed by his group [40,41]. i became ucla’s chief of neuropathology in 1993, when tony verity decided to take advantage of the university’s ‘voluntary early retirement incentive plan, verip’, though thankfully he remained active as an emeritus faculty for many years, sharing his expertise in neuromuscular diagnosis (dr. negar khanlou became his protégé and successor) and his passion for cricket. tony was also an excellent artist and i am proud to have one of his paintings on the wall of my living room in venice. i served in the chief of neuropathology post until i myself took ‘semi-retirement’ in 2016, and had the pleasure to mentor junior colleagues who went on to highly successful careers: cynthia welsh, dennis chute, jeff twiss, paul mischel among them. dr. william yong, my current chief, was a medical student who i tutored in the 1980s, later a neuropathology fellow in our program, and has been a marvelous colleague – alas, recently departed for a position at university of california irvine medical center. for several years, i was honored to hold the sarkaria chair in diagnostic medicine. in the mid-1990s i was asked by university of southern california (usc) neurologist dr. helena chui to become part of a california-wide study she was planning, examining the clinical, neuroimaging and neuropathologic substrates of individuals with documented (or at risk for) cerebrovascular disease. the overarching (and perhaps overly ambitious) theme and goal of the program project grant was to highlight the neurobehavioral, imaging and neuropathologic features of ischemic vascular dementia (ivd), on the assumption that such an entity exists. helena was an inspiring and superbly organized leader, and an insightful judge of character, the consummate solver of problems. nih program project grants (ppgs), including the one that funded this study, involve the interactions of widely differing personalities and the principal investigator of such an endeavor (i.e. helena chui) needs to manage those people and their disagreements as well as their productive interactions. our group involved neuropsychologists, epidemiologists, neurologists, and experts in neuroimaging: bruce reed, dan mungas, mike weiner, bill jagust, ling zheng, wendy mack and many others. my fellow investigators in the neuropathology core included william ellis (uc davis), ewa borys (now in chicago) and chris zarow (then a talented neuroanatomist at usc). our studies, over the long duration of the ivd-ppg, showed that ‘pure’ ivd was a comparatively rare phenomenon, but that risk factors for cerebrovascular disease probably contribute to accelerated brain aging, and even some components of alzheimer disease (ad), a view that is gaining traction in the neuroscientific community and a subject that has generated much research interest [42-44]. two papers from the ivd group focused on the ‘neuropathologic substrates’ of cognitive impairment resulting from ischemic brain lesions [45,46], though crucial neuropathologic data were integrated into many other publications. for several years, i have had an enjoyable collaboration with dr. wolff kirsch, a neurosurgeon at loma linda university medical center (approximately 100 miles east of l.a.) who, rather than quietly retire after a stellar career in neurosurgery, decided to become a researcher with an interest in, among other things, brain aging and cerebral amyloid angiopathy (see table 1). dr. kirsch is devoted to the idea of eventually preventing or treating this microangiopathy and has enlisted materials from the easton center brain bank in studies with that goal. very entrepreneurial and creative neuroscientist and much fun to work with in joint projects. as the above summary indicates, much of my research has been collaborative and translational. grant reviewing agencies have, i am quite certain, considered it ‘scattershot’ and unfocused. ‘can vinters not focus on a single topic?’ – the answer is clearly ‘no’. i’ve enjoyed all of the disparate research endeavours in which i have been involved. they have included interactions with investigators possessed of unique talents and abilities, providing the possibility of forming irresistible, tantalizing scientific alliances. table 1 summarizes the key individuals with whom i have worked and published most intensely over the years; it is by no means a complete list. my advice to people entering neuropathology would be to choose your collaborators carefully, wisely. consider individuals who can bring projects to completion in a timely fashion and make effective use of a neuropathologist’s unique skill set. have i myself been successful in following that mandate for timely completion of projects? well… not always… but often enough. work with people who will criticize your work and writing, sometimes brutally. i’ve been fortunate to co-author a manuscript with a pharmacologist, dr. louis ignarro, destined to win the 1998 nobel prize in medicine/physiology for his discovery of nitric oxide [47]. (disclosure: dr. ignarro did not win his nobel on the basis of our study!) my favorite projects and papers have been those that translated basic science observations to address and inform important clinicopathologic issues – ones that might eventually be used to help patients avoid or recover from devastating neurologic illnesses. in 2009, dr. michael sofroniew in ucla’s dept. of neurobiology approached me with the idea to write a review of basic astrocyte biology (his expertise), and how astrocytic dysfunction might mediate neurologic disease (my interest) [48]. it resulted in my most cited publication and led to further productive collaborations that continue now – studies of astrocyte derangement in certain seizure conditions. i benefit daily from my colleagues (drs. william yong, shino magaki, negar khanlou), fellows (currently drs. zesheng chen and josh byers) and technically super-skilled histotechnologists and staff research associates (e.g. kazu williams, current laboratory manager of the easton center brain bank, nelly vehabedian, jennifer yi, cecilia choi). part of the group is shown in figures 9 and 10, masked and ‘social distancing’ (at least in figure 10) as appropriate for the pandemic. when one of the technical staff contributes significantly to a research project and the resultant publication, i include her/him as a co-author on the manuscript. table 2 lists key senior research technicians and lab managers who have each co-authored several publications, many of which have been cited 100+ times (per the web of science). i take pride that many of these skilled technicians have a publication record that surpasses (in both quantity and quality) those of some junior faculty. sadly, one of the individuals on that list (justine pomakian) passed earlier this year after a long battle with leukemia. another (spencer tung) will graduate this year as a physician. a huge bonus or perk of academic life is the possibility to teach, train and influence others – including medical students, basic science colleagues, graduate students, postdoctoral fellows, residents and clinical fellows. i am especially proud of the many neuropathology fellows who have graduated from the ucla training program and have gone out to initiate clinical and/or research programs of their own. our goal as teachers and mentors should be to foster the creation of individuals who are better than we are – more creative, more energetic, better and more insightful diagnosticians. i flatter myself to think that overseas visitors who spent significant time at ucla neuropathology – drs. sung hye park from korea, remco natte from the netherlands, orestes solis from the philippines, hajime miyata from japan, regina von einsiedel from germany, many others – brought back to their home countries skills and knowledge gained in los angeles in ucla’s laboratory of neuropathology. fig 9: team neuropathology at ucla, 2020 (briefly suspending social distancing rules during the covid 19 epidemic) the joy of scientific meetings, sabatticals, visiting professorships and academic visits academics can be fun, terrific fun! most of us enjoy scientific meetings – well, used to enjoy them before they devolved into sitting in front of a computer screen, asking ‘should i share my screen? unmute yourself… can everyone hear me? interesting living room décor, or is it an artificial background? oh no, black screen again’, etc. i have a nagging fear that the trainees of today will come to think: this really isn’t so bad! perhaps it is acceptable as a temporary substitute, but of course there is nothing to compare with the give and take of in person conferences, the body language and passion that are evident in heated or even subdued scientific debates, the stimulating discussions – and yes, gossip! – that occur over cocktails and meals. i recall an american association of neuropathologists meeting (in the early days of my career) during which two senior prominent academics who both worked on neurodegenerative diseases (names withheld to protect the guilty… though alas, both are now deceased…) almost came to blows in front of a poster focusing on the pathogenesis and significance of senile plaques. a little embarrassing, but also somewhat gratifying that each should care so completely and enthusiastically about the significance of these tiny (though controversial) brain lesions – whose importance and relevance to alzheimer’s disease are still debated today. fig 10: team neuropathology at ucla, practicing social distancing what would academic life be without sabbaticals? i spent a marvelous year in the early 1990s with dr. clayton a. wiley at uc san diego, learning the basics of molecular biology and diagnostics as applied to human brain specimens, especially those originating from corticectomies for intractable seizures [49]. i had the opportunity to work with an outstanding technician, rebecca wang (who sadly died of metastatic melanoma a few years later) and dr. cris achim, who remains a friend and collaborator while now on faculty at ucsd. in 1993, i was kindly and generously funded by the daad (german academic exchange service) to work for several weeks with heiko and eva braak in frankfurt – learning how elegant neuroanatomic methods that they had perfected could provide insights into alzheimer’s disease. i’ve had the opportunity to speak and study in countries i would probably not have visited, save for an invitation to lecture or work there – usually at the expense of the host nation/city/academic institution, for which generosity i am sincerely grateful. i’ve even given a lecture in riga, latvia (2015) and was interviewed in latvian (of course i speak the language, in fact english is my second language) on their national radio network. in 1997 (for 6 months) and later in 2004 (for a shorter time interval), i had the chance to do research and teaching as a visiting professor (‘hoogleraar’) at the leiden university medical center, located in an historic city that is home to an equally ancient and revered university – what might be described as a college town in the usa. leiden is situated between amsterdam and the hauge (closer to the latter) in the netherlands and is a short bicycle ride to katwijk on the north sea coast. i shared an office, and many stimulating discussions (about neuropathology, politics, the dutch royal family and other non-scientific matters) with dr. marion maat-schieman [figure 11]. i had the privilege to study, in great detail and with marion’s assistance and much input from dr. remco natte (now a successful pathologist in holland), the fascinating and unique dutch form of hereditary cerebral amyloid angiopathy that results from a distinctive app codon 693 mutation. this causes perhaps the purest form of vascular dementia resulting from microvascular disease, at least beta-amyloid caa [50]. remco later spent several productive months performing complex tissue culture experiments (using vascular-derived smooth muscle cells) in my lab at ucla. in 1998, burroughs wellcome generously provided me with funds to work for a month with dr. michael farrell at the beaumont hospital in dublin. for several years in the early 2010s, i had the privilege to travel to manila in the philippines to lecture at the university of santo tomas and several national and international meetings held in that fascinating country. manila itself is full of paradoxes – great wealth and grinding poverty among them, sometimes existing in close physical proximity – and is one of the most welcoming and hospitable nations in the world. fig 11: dr. marion maat-schieman, retired neuropathologist, leiden university medical center, the netherlands i am proud to be both canadian and american, hold two passports and even have a home in vancouver, canada – which i hope to see again some day after the covid19-related canadian/usa travel restrictions are lifted or at least decreased. for several summers from 2013-2017, i spent a 1-2 week micro-sabbatical in vancouver, working with dr. ian mackenzie and his associates at the vancouver hospital, refining my diagnostic skills in the area of frontotemporal dementia and lobar degenerations, enjoying this almost impossibly beautiful city, and ian’s amazing knowledge of this complex group of disorders. one of the great tragic consequences of covid 19 is the impact it has had on international travel. joining in the life of the university ucla medical center (for many years now the ronald reagan ucla medical center/ rrumc) is located at the southern part of the larger ucla campus. unlike many metropolitan medical centers, it is thus closely linked not only to the non-medical scientific departments with which we interact (including most notably chemistry and biochemistry, biomedical engineering) but also the arts and humanities. while i have been at ucla, two faculty members in the department of chemistry/biochemistry (donald cram and paul boyer) have been awarded nobel prizes; excellent resource to have when one is contemplating experiments with a biochemical component. since arriving at ucla as a junior faculty member, i’ve taken advantage of this proximity, having served for several years on the campus-wide graduate council and, more recently, the university’s council on academic personnel (cap). cap is a committee of 12-15 senior faculty members representing all (most) segments of ucla, who review the dossiers of other ucla academic faculty for initial appointment, major academic promotions (e.g. to the rank of distinguished professor) or appointment to an endowed chair. serving on cap provided a remarkable overview of the diversity of talent and skills at the university. it also gave an interesting perspective into the heterogeneous activities that qualify as creative accomplishment in ucla departments such as near eastern languages and culture, english, philosophy and digital media, to name four among many. life does not end with retirement – nor should it… and closing comments i formally retired from my full time equivalent (fte) in july 2016, and have been on ‘recall’ for clinical and academic work since then, continuing to the present time. for some unknown reason, ucla allows recall faculty to work 43% of time (??!!), though i probably spend more time than that in my office and lab. in 2017, i was honored by the british neuropathological society with the alfred meyer medal and lectureship, received and delivered in london; the british surely are the masters at doing this sort of festive event, and i was suitably ‘chuffed’ and grateful for the honor. that same year i received the award for meritorious contributions to neuropathology, from the american association of neuropathologists. agreed – awards are not that important, but they are a lovely symbol of regard from one’s colleagues. over many years, i have established the gunnar vinters bursary at lakehead university in my home town of thunder bay. last year, i also endowed the elise and arnold vinters scholarship, in memory of my grandparents, at the northern ontario medical school in the same university. i enjoy building on the endowment for each academic award, annually and encouraging others to contribute. since 2018 i have been doing locum work at the university of alberta, in a unit known as alberta precision laboratories (apl). my wonderful colleagues there are drs. laura schmitt, frank van landeghem and sumit das [figure 12]. the work is currently on hiatus (since february, 2020), pending resumption of travel between canada and the usa, but i hope that happens soon. i recently co-authored my first paper with colleagues at apl [51]. it is a form of coming full circle, since i interned at the old university of alberta hospital in 1976-77. fig 12: my neuropathology colleagues at alberta precision laboratories, edmonton, alberta, 2020 (l to r drs. laura schmitt, frank van landeghem, sumit das) i love travel, including (and especially) air travel… but as of this writing have not been on an airplane or near an airport for 5+ months. however, there is much still to do at ucla, projects that – so i hope – benefit from my input, complex cases on which i can shed some light. in my spare time, i revel in the delights of venice beach, california, where there is – shall we say – never a dull moment. it is a beachside region of los angeles founded in the early 1900s by abbot kinney, a tobacco heir who was so impressed with venice, italy, that he thought it would be fun to replicate it in southern california. i have great friends there, throughout l.a. and in canada and five accomplished godchildren whose antics keep life interesting. in my spare time, which seems sometimes not to be terribly abundant, i write short stories and oped pieces, none of which is destined for publication, though i’ve published a few ‘back in the day’. now it is time to end this reflection, which seems to have expanded to the length of a novella! when i was in medical school, i never dreamed i would spend most of my adult life in southern california. as i heard or read somewhere, ‘life is what happens to you while you are making plans…’ – how true. i appreciate free neuropathology giving me the opportunity to write this piece. acknowledgements kazu williams assisted with preparation of figures. dr. shino magaki and kazu williams assisted with critical reading of the manuscript. references 1. vinters h, dainty j, tyree mt. cell-wall elastic properties of chara corallina. canadian journal of botany. 1977; 55: 1933-1939 2. seeman p. membrane actions of anesthetics and tranquilizers. pharmacological reviews. 1972; 24: 583-655 3. hornykiewicz o. dopamine (3-hydroxytyramine) and brain function. pharmacological reviews. 1966; 18: 925-964 4. steele jc, richardson jc, olszewski j. progressive supranuclear palsy. a heterogeneous degeneration involving the brain stem, basal ganglia and cerebellum with vertical gaze and pseudobulbar palsy, nuchal dystonia and dementia. archives of neurology. 1964; 10: 333-359 5. gilbert jj, vinters hv. cerebral amyloid angiopathy—incidence and complications in the aging brain. 1. cerebral hemorrhage. stroke. 1983; 14: 915-923 6. vinters hv, gilbert gg. cerebral amyloid angiopathy—incidence and complications in the aging brain. 2. the distribution of amyloid vascular changes. stroke. 1983; 14: 924-934 7. debault le, cancilla pa. gamma-glutamyl transpeptidase in isolated brain endothelial cells: induction by glial cells in vitro. science. 1980; 207:653-655 8. debault le, cancilla pa. induction of gamma-glutamyl transpeptidase in isolated cerebral endothelial cells. advances in experimental medicine biology. 1980; 131: 79-88 9. beck dw, vinters hv, hart mn, cancilla pa. glial cells influence polarity of the blood-brain barrier. journal of neuropathology & experimental neurology. 1984; 43: 219-214 10. vinters hv, reave s, costello p, girvin jp, moore sa. isolation and culture of cells derived from human cerebral microvessels. cell tissue research. 1987; 249: 657-667 11. vinters hv, galil ka, lundie mj, kaufmann jce. the histotoxicity of cyanoacrylates. a selective review. neuroradiology. 1985; 27: 279-291 12. vinters hv, lundie mj, kaufmann jce. long-term pathological follow-up of cerebral arteriovenous malformations treated by embolization with bucrylate. new england journal of medicine. 1986; 314: 477-483 13. small gw, kepe v, ercoli lm, prabha s, bookheimer sy, miller kj, lavretsky h, burggren ac, cole gm, vinters hv, thompson pm, huang s-c, satyamurthy n, phelps me, barrio jr. pet of brain amyloid and tau in mild cognitive impairment. new england journal of medicine 2006; 355: 2652-2663 14. vinters hv. cerebral amyloid angiopathy. a critical review. stroke 1987; 18: 311-324 15. glenner gg, wong cw. alzheimer’s disease: initial report of the purification and characterization of a novel cerebrovascular amyloid protein. biochemical and biophysical research communications 1984; 120: 885-890 16. vinters hv, pardridge wm, yang j. immunohistochemical study of cerebral amyloid angiopathy: use of an antiserum to a synthetic 28-amino-acid peptide fragment of the alzheimer’s disease amyloid precursor. human pathology 1988; 19: 214-222 17. vinters hv, nishimura gs, secor dl, pardridge wm. immunoreactive a4 and gamma-trace peptide colocalization in amyloidotic arteriolar lesions in brains of patients with alzheimer’s disease. american journal of pathology 1990; 137: 233-240 18. anders k, steinsapir kd, iverson dj, glasgow bj, layfield lj, brown wj, cancilla pa, verity ma, vinters hv. neuropathologic findings in the acquired immunodeficiency syndrome (aids). clinical neuropathology 1986; 5: 1-20 19. anders kh, guerra wf, tomiyasu u, verity ma, vinters hv. the neuropathology of aids. ucla experience and review. american journal of pathology 1986; 124: 537-558 20. li xl, moudgil t, vinters hv, ho dd. cd4-independent, productive infection of a neuronal cell line by human immunodeficiency virus type 1. journal of virology 1990; 64: 1383-1387 21. ho dd, bredesen de, vinters hv, daar es. the acquired immunodeficiency syndrome (aids) dementia complex. annals of internal medicine 1989; 111: 400-410 22. koyanagi y, miles s, mitsuyasu rt, merrill je, vinters hv, chen isy. dual infection by of the central nervous system by aids viruses with distinct cellular tropisms. science 1987; 236: 819-822 23. pang s, koyanagi y, miles s, wiley c, vinters hv, chen isy. high levels of unintegrated hiv-1 dna in brain tissue of aids dementia patients. nature 1990; 343: 85-89 24. vinters hv, kwok mk, ho hw, anders kh, tomiyasu u, wolfson wl, robert f. cytomegalovirus in the nervous system of patients with acquired immune deficiency syndrome. brain 1989; 112 (pt 1): 245-268 25. von einsiedel rw, fife td, aksamit aj, cornford me, secor dl, tomiyasu u, itabashi hh, vinters hv. progressive multifocal leukoencephalopathy in aids. a clinicopathological study and review of the literature. journal of neurology 1993; 240: 391-406 26. vinters hv, anders kh. neuropathology of aids. boca raton, florida: crc press, 1990; 229 pp 27. taylor dc, falconer ma, bruton cj, corsellis ja. focal dysplasia of the cerebral cortex in epilepsy. journal of neurology, neurosurgery and psychiatry 1971; 34: 369-387 28. farrell ma, derosa mj, curran jg, secor dl, cornford me, comair yg, peacock wj, shields wd, vinters hv. neuropathologic findings in cortical resections (including hemispherectomies) for the treatment of intractable childhood epilepsy. acta neuropathologica 1992; 83: 246-259 29. farrell ma, droogan o, secor dl, poukens v, quinn b, vinters hv. chronic encephalitis associated with epilepsy. immunohistochemical and ultrastructural studies. acta neuropathologica 1995; 89: 313-321 30. mischel ps, nguyen lp, vinters hv. cerebral cortical dysplasia associated with pediatric epilepsy. review of neuropathologic features and proposal for a grading system. journal of neuropathology and experimental neurology 1995; 54:137-153 31. cepeda c, hurst rs, flores-hernandez j, hernandez-echeagaray e, klapstein gj, boylan mk, calvert cr, jocoy el, et al. morphological and electrophysiological characterization of abnormal cell types in pediatric cortical dysplasia. journal of neuroscience research 2003; 72: 472-486 32. cepeda c, andre vm, levine ms, salamon n, miyata h, vinters hv, mathern gw. epileptogenesis in pediatric cortical dysplasia: the dysmature cerebral developmental hypothesis. epilepsy & behavior 2006; 219-235 33. cepeda c, chen jy, wu jy, fisher rs, vinters hv, mathern gw, levine ms. pacemaker gaba synaptic activity may contribute to network synchronization in pediatric cortical dysplasia. neurobiology of disease 2014; 62: 208-217 34. chandra ps, salamon n, nguyen st, chang jw, huynh mn, cepeda c, leite jp, neder l, koh s, vinters hv, et al. infantile spasm-associated microencephaly in tuberous sclerosis complex and cortical dysplasia. neurology 2007; 438-445 35. salamon n, andres m, chute dj, nguyen st, chang jw, huynh mn, chandra ps, andre vm, cepeda c, levine ms, et al. contralateral hemimicrencephaly and clinical-pathological correlations in children with hemimegalencephaly. brain 2006; 129: 352-365 (part 2) 36. menchine m, emelin jk, mischel ps, haag ta, norman mg, pepkowitz sh, welsh ct, townsend jt, vinters hv. tissue and cell-type specific expression of the tuberous sclerosis gene, tsc2, in human tissues. modern pathology 1996; 1071-1080 37. vinters hv, park sh, johnson mw, mischel ps, catania m, kerfoot c. c. cortical dysplasia, genetic abnormalities and neurocutaneous syndromes. developmental neuroscience 1999; 21 (3-5): 248-259 38. johnson mw, miyata h, vinters hv. ezrin and moesin expression within the developing human cerebrum and tuberous sclerosis-associated cortical tubers. acta neuropathologica 2002; 104: 188-196 39. johnson mw, emelin jk, park sh, vinters hv. co-localization of tsc1 and tsc2 gene products in tubers of patients with tuberous sclerosis. brain pathology 1999; 9: 45-54 40. qin w, chan ja, vinters hv, mathern gw, franz dn, taillon be, bouffard p, kwiatkowski dj. analysis of tsc cortical tubers by deep sequencing of tsc1, tsc2 and kras demonstrates that small second-hit mutations in these genes are rare events. brain pathology 2010; 20: 1096-1105 41. goto j, talos dm, klein p, qin w, chekaluk yi, anderl s, malinowska ia, di nardo a, et al. regulable neural progenitor-specific tsc1 loss yields giant cells with organellar dysfunction in a model of tuberous sclerosis complex. proceedings of the national academy of sciences of the usa 2011; 108: e1070-e1079 42. reed br, mungas dm, kramer jh, ellis w, vinters hv, zarow c, jagust wj, chui hc. profiles of neuropsychological impairment in autopsy-defined alzheimer’s disease and cerebrovascular disease. brain 2007; 130 (pt 3): 731-739 43. jagust wj, zheng l, harvey dj, mack wj, vinters hv, weiner mw, ellis wg, zarow c, mungas d, reed br, et al. neuropathological basis of magnetic resonance images in aging and dementia. annals of neurology 2008; 63: 72-80 44. chui hc, zarow c, mack wj, ellis wg, zheng l, jagust wj, mungas d, reed br, kramer jh, decarli cc, et al. cognitive impact of subcortical vascular and alzheimer’s disease pathology. annals of neurology 2006; 60: 677-687 45. vinters hv, ellis wg, zarow c, zaias bw, jagust wj, mack wj, chui hc. neuropathologic substrates of ischemic vascular dementia. journal of neuropathology and experimental neurology 2000; 59: 931-945 46. vinters hv, zarow c, borys e, whitman jd, tung s, ellis wg, zheng l, chui hc. review: vascular dementia: clinicopathologic and genetic considerations. neuropathology and applied neurobiology 2018; 44: 247-266 47. mitrovic b, ignarro lj, vinters hv, akers ma, schmid j, uittenbogaart c, merrill je. nitric oxide induces necrotic but not apoptotic cell death in oligodendrocytes. neuroscience 1995; 65: 531-539 48. sofroniew mv, vinters hv. astrocytes: biology and pathology. acta neuropathologica 2010; 119: 7-35 49. vinters hv, wang r, wiley ca. herpesviruses in chronic encephalitis associated with intractable childhood epilepsy. human pathology 1993; 24: 871-879 50. vinters hv, natte r, maat-schieman mlc, van duinen sg, hegeman-kleinn i, welling-graafland c, haan j, roos rac. secondary microvascular degeneration in amyloid angiopathy of patients with hereditary cerebral hemorrhage with amyloidosis, dutch type (hchwa-d). acta neuropathologica 1998; 95: 235-244 51. chan j, magaki s, zhang xr, chin c, greenspan s, linetsky m, kattar m, vinters hv. intravascular carcinomatosis of the brain: a report of two cases. brain tumor pathology 2020; 37: 118-125 copyright: © 2020 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. clustering of activated microglia occurs before the formation of dystrophic neurites in the evolution of aβ plaques in alzheimer’s disease. feel free to add comments by clicking these icons on the sidebar free neuropathology 1:20 (2020) original paper clustering of activated microglia occurs before the formation of dystrophic neurites in the evolution of aβ plaques in alzheimer’s disease. patrick jarmo paasila 2, danielle suzanne davies 1, greg trevor sutherland 2, claire goldsbury 1 1 discipline of anatomy and histology, school of medical sciences, faculty of medicine and health, the university of sydney, nsw 2006, australia 2 discipline of pathology, school of medical sciences, faculty of medicine and health, the university of sydney, nsw 2006, australia corresponding author: dr claire goldsbury phd · brain and mind centre · 94 mallett street · camperdown, nsw 2050 · australia · tel +61 2 9351 0878 claire.goldsbury@sydney.edu.au submitted: 16 june 2020 accepted: 29 july 2020 copyedited by: jeffrey nirschl published: 04 august 2020 https://doi.org/10.17879/freeneuropathology-2020-2845 keywords: alzheimer’s disease, inferior temporal cortex, microglia, post-mortem human brain tissue, primary motor cortex abstract alzheimer’s disease (ad) is a late-onset disease that has proved difficult to model. microglia are implicated in ad, but reports vary on precisely when and how in the sequence of pathological changes they become involved. here, post-mortem human tissue from two differentially affected regions of the ad brain and from non-demented individuals with a high load of ad-type pathology (high pathology controls) was used to model the disease time course in order to determine how microglial activation relates temporally to the deposition of hallmark amyloid-β (aβ) and hyperphosphorylated microtubule associated protein tau pathology. immunofluorescence against the pan-microglial marker, ionised calcium-binding adapter molecule 1 (iba1), aβ and tau, was performed in the primary motor cortex (pmc), a region relatively spared of ad pathological changes, and compared to the severely affected inferior temporal cortex (itc) in the same cases. unlike the itc, the pmc in the ad cases was spared of any degenerative changes in cortical thickness and the density of betz cells and total neurons. the clustering of activated microglia was greatest in the pmc of ad cases and high pathology controls compared to the itc. this suggests microglial activation is most prominent in the early phases of ad pathophysiology. nascent tau inclusions were found in neuritic plaques in the pmc but were more numerous in the itc of the same case. this shows that tau positive neuritic plaques begin early in ad which is likely of pathogenic importance, however major tau deposition follows the accumulation of aβ and clustering of activated microglia. importantly, findings presented here demonstrate that different states of microglial activation, corresponding to regional accumulations of aβ and tau, are present simultaneously in the same individual; an important factor for consideration if targeting these cells for therapeutic intervention. introduction alzheimer’s disease (ad) is neuropathologically characterised by inclusions of microtubule-associated protein tau (tau) and extracellular deposits of β-amyloid (aβ). intraneuronal tau pathology includes neurofibrillary tangles (nfts) in the cell soma and neuropil threads (nts), which occur mostly in the dendritic compartment, but also in the axonal domain though to a lesser extent (1, 2). nfts and nts are both comprised of paired helical filaments and straight filaments of polymerised hyperphosphorylated tau protein (3, 4). the extent of tau pathology follows a predictable spatiotemporal progression through functionally integrated brain regions (5, 6) and there is an extensive body of literature that demonstrates an inverse correlation between the accumulation of nfts and cognitive status (7) such that the spread and regional level of nfts reflects the severity of dementia with time (8). contrastingly, aβ plaques follow a seemingly more haphazard regional pattern of accumulation throughout the neocortex, indeed plateauing relatively early in the disease time course (9, 10), and therefore correlate poorly with disease status until substantial argyrophilic neuritic tau pathology is also present (5, 7, 11). whilst aβ load may be an unreliable indicator of disease severity, it is generally accepted that the extent of its spread, in combination with tau pathology, is useful for staging purposes (9). aβ plaques can be classified with immunostaining into at least three morphologically distinct categories: diffuse, fibrillar, or dense-cored. diffuse plaques have long been proposed to be a structural precursor of other plaque forms, but whether these categories represent separate entities with independent mechanisms of development or are temporally linked is still unclear. neuritic plaques (nps) are those aβ plaques that also feature dystrophic neurites (dns) with silver staining. dns can occur in all these morphological subtypes of aβ plaques, though more commonly in dense-cored and fibrillar plaques than morphologically diffuse plaques (12). most dns are tau-positive and morphologically similar to nts which are elongated in shape, but may also be globular, and possibly represent swollen presynaptic (axonal) terminals (13). nps that retain sparse dns but show minimal aβ staining have previously been described and were termed ‘remnant plaques’ which were proposed to result from glial phagocytosis of insoluble aβ (14). there remains significant debate as to the sequence of the neuropathological changes that precede the onset of ad symptomatology. the amyloid cascade hypothesis posits aβ, and in particular the soluble, oligomeric, non-fibrillar fraction, as the initiating factor (15-20). a competing view is offered by others who contend that the sequence of pathological events begins with neurofibrillary pathology (21, 22) which precedes the formation of insoluble aβ pathology (23). indeed, there is evidence that tau pathology occurs prior to aβ pathology as it is more common in the brains of non-demented individuals (21) but this might represent a non-ad scenario described as primary age-related tauopathy (part) (24). notwithstanding the order of events, both hypotheses suggest that an activated glial response is an integral component of the pathogenesis of ad. the study of microglia morphology in post-mortem human brain tissue presents a simple method with which to gauge the involvement of microglia in instances of changed physiological conditions or to the development of a disease. microglia with a ramified morphology, characterised by thin, evenly distributed, highly branched processes with a small, spherical soma, represent the healthy cell population (25-27). activated microglia are characterised by reduced morphological complexity including hypertrophy of the soma and processes and may also display the formation of distal phagosomes (23, 25, 28). dystrophic microglia display features consistent with cellular senescence (29), including a loss of processes, tortuosity of remaining processes, and discontinuous iba1-immunolabelling (30-32). lastly, clusters of activated microglia in ad have been noted previously in post-mortem human brain tissue and represent the direct interaction between microglia and aβ and tau pathology (10, 32, 33). by investigating brain regions differentially affected by ad-type pathology in non-demented and demented individuals it may be possible to model the sequence of pathological changes in the disease, in particular the activated microglial response. previously we demonstrated an increased density of activated microglia in the inferior temporal cortex (itc) of non-demented controls with similar levels of ad-type pathology (‘high pathology controls’ – hpcs) as clinically and neuropathologically-confirmed ad cases (26). this suggested that the microglial response occurs in the preclinical phases of ad but we wished to confirm this finding in a belatedly affected region of the ad brain, the primary motor cortex (pmc), compared to an earlier and more severely affected region, the itc, of the same cases. further, we wished to determine the sequence of aβ, tau, and microglia pathological changes that occur in the cortex. the findings here demonstrate that nascent nts and dns in the pmc begin early in the pathogenesis of ad, but follow the clustering of activated microglia. by contrast, looking at the severely affected itc in the same cases, findings suggest that microglial clustering at plaques dissipates once tau and aβ pathology is long established and there is also a substantial loss of iba1-immunoreactivity (26, 34). although an early toxic microglial gain of function cannot be ruled out, these findings appear most consistent with a scenario where microglial activation is neuroprotective early in the pathogenesis of ad. we suggest that this is followed by a gradual exhaustion of microglial function that contributes to cognitive deterioration in the ad brain. table 1. cohort characteristics methods this study was approved by the university of sydney’s human research ethics committee (hrec#2015/477). all tissue samples for this study, as well as demographic and clinical information, were supplied by the new south wales brain tissue resource centre (nswbtrc) and the sydney brain bank (sbb), collectively the new south wales brain banks (nswbb), following approval from their scientific advisory committee. methods for case ascertainment and tissue preparation by nswbb have been previously published (35). the demographic and clinicopathological characteristics of the cohort (table 1) and data from the itc have been previously published (26). immunofluorescence immunofluorescence staining procedures were performed on free-floating 45 μm fixed sections derived from the caudal aspect of the superomedial area of the pmc of controls (n = 10), hpcs (n = 5), and pathologically confirmed ad cases (n = 8) as previously described (26). double-labelled sections using antibodies against aβ, total tau (ttau), and the pan-microglial marker ionised calcium-binding adapter molecule 1 (iba1), were used for the quantification of aβ and tau loads and microglial morphological subtypes. for aβ and iba1 quantification, heat-induced epitope retrieval was performed using a sodium citrate solution (ph 8.5) at 60°c overnight, followed by a 12-minute formic acid (90%) incubation at room temperature. blocking was performed in 10% normal goat serum (gibco #16210072), and primary (mouse aβ, 1:1000, biolegend 803002; rabbit iba1, 1:1000, wako 019-19741) and secondary (1:200; thermo scientific: alexa fluor (af) 488 goat anti-mouse, #a11001; af 568 goat anti-rabbit, #a11011) antibody incubations were performed at 4°c with gentle agitation. nuclear counterstaining was performed in the last 40 minutes of the secondary antibody incubation by the addition of hoechst 33342 dye (1 µg/ml; thermo scientific 62249). sections were mounted using prolong diamond antifade (invitrogen p36961). double-immunolabelling of ttau (rabbit; 1:500; dako k9ja/a0024) and iba1 (mouse; 1:50; millipore mabn92) was carried out as previously described (34). ttau immunostaining of nfts, nts, and dns has previously been demonstrated to give comparable immunostaining to standard phosphotau antibodies (12e8 and at8) (36). briefly, heat-retrieval was performed with sodium citrate (ph 6.0) for 10 minutes at 100°c, before permeabilising, blocking with bsa, and incubating in primary antibodies for three hours at room temperature or overnight at 4°c, and finally incubating in secondary antibodies (1:200; invitrogen: af555 goat anti-mouse, a214424; af647 goat anti-rabbit, a21244). hoechst 33342 was added to counterstain nuclei and sections were mounted in prolong gold antifade. image acquisition and analysis aβ and iba1 double-labelled sections were imaged using a zeiss lsm 800 confocal microscope using the ‘tile scan’ function at the advanced microscopy facility, bosch institute, the university of sydney. a previously validated modified disector sampling approach that utilises one section per individual was used here for the analysis of the aβ and iba1 immunostained sections (37). briefly, a total of three cortical strips per section from areas where the pial surface and the grey-white boundary were strictly in parallel were acquired for the quantification of microglia, aβ plaques and aβ-positive pixels. these cortical strips were constructed of serial images 500 μm in width, 6 μm in z-depth with three z-slices (z-step = 3 μm), spanning all of the cortical laminae of the pmc using a 20×/0.8 numerical aperture (na) objective. clusters of microglia and individual microglia, which were categorised as having either a ramified, activated, or dystrophic morphology as previously described (26), were enumerated in image analysis software (fiji; nih). the terminology used to describe the different populations of morphologically diverse populations of microglia was informed by previous investigations (25, 28, 30). microglia with thin, highly branched processes, and a spherical nucleus were categorised as ‘ramified’. ‘activated’ microglia (previously termed ‘deramified’ (34)) included those cells that displayed hypertrophy of the soma or processes with retraction of secondary or tertiary processes. ‘dystrophic microglia’ were identified on the basis of a loss of processes with the remaining processes displaying significant tortuosities or discontinuous iba1-immunolabelling with or without blebbing or punctate iba1-labelling (previously dystrophic microglia were subcategorised as either ‘punctate’ or ‘discontinuous’ to reflect these observations (34)). a ‘cluster of microglia’ was counted if three or more soma occurred within, or were touching the margins of, a 20 μm2 virtual graticule subregion. larger clusters were counted as one cluster if the graticule subregion could be moved and still incorporate at least three somata. clusters were counted whilst visualising only the 568 nm (iba1-positive) channel to distinguish these from individual microglia and to minimise potential false-positive counts in the presence of either aβor ttau-immunostaining. the total number of microglia counted per section averaged 519 with a coefficient of error (ce) of ≤0.2 for all counts of morphological subtypes, with the exception of microglial clusters (ce ≤0.3) which were relatively rare and displayed significant variance between cases. quantification of ttau was carried out using an olympus vs120 slide scanner at the sydney microscopy and microanalysis, brain and mind centre, the university of sydney. whole section dic and fluorescence overviews of a single section from each case were generated using a 10× objective and were used to systematically map out four representative 500 μm2 regions of interest (= 1mm2 per section) within the mid-cortical laminae (iii-v) for manual counts of nfts and for the quantification of ttau-positive pixels. individual images were captured using a 40×/0.9 na objective and were comprised of seven z-slices with a depth of 6 μm (z-step = 1 μm). image analysis was performed in fiji using manually thresholded images. positive pixel counts were generated for aβ and ttau staining and expressed as a percentage of total pixels (% aβ and % ttau respectively). high resolution imaging of all immunolabelled sections were carried out using a nikon a1r or zeiss lsm 710 confocal microscope (sydney microscopy and microanalysis, charles perkins centre and brain and mind centre, the university of sydney). features were imaged with either a 40×/0.95 na or 100×/1.4 na objective and shown as maximum intensity projections. nissl staining nissl stains of free-floating 45 μm thick formalin-fixed sections were performed for the measurement of cortical thickness and neuronal counts. sections were incubated in 0.1% cresyl violet acetate (0.02% glacial acetic acid; added immediately before use) for 15 minutes at 60°c. differentiation was achieved by sequentially washing sections for three minutes in 70% and 95% ethanol. the final level of staining was adjusted by briefly dipping sections in 100% ethanol and confirmed by light microscopy before clearing in xylene for ten minutes and mounting in dpx. cortical thickness measurements and neuronal counts were performed on three cortical strips from one section per individual using an eyepiece graticule on an olympus bx50 microscope using a 20×/0.75 na objective. a height measure of 45 μm was used in determining density estimates to eliminate artefactual tissue shrinkage during staining; with the microtome accuracy to cut precise sections having been previously validated (37). ces using the modified disector technique outlined above were <0.1 for cortical thickness and <0.15 for the density of betz cells and total neurons. statistical analyses the normality of data was tested using the shapiro-wilk test. equality of variances was tested using brown-forsythe test. group differences were investigated by either welch’s analysis of variance or kruskal-wallis test for non-gaussian distributions, with either games-howell or dunn’s test, respectively, for pairwise comparisons. regional differences to the previously reported itc (26) were investigated by either welch’s t test or wilcoxon rank-sum test for non-gaussian distributions. the pearson correlation coefficient (r) and coefficient of determination (r2), or spearman rho (ρ) for non-gaussian distributions, were calculated for univariate correlations to investigate relationships between ad-type pathology, microglial morphologies, and apoe ε4 status. stepwise regression models which included age, sex, brain ph, post-mortem interval, and fixation period were performed to exclude effects of potential confounders. a p-value <0.05 was considered statistically significant. all statistical analyses were performed using jmp pro 14 (sas institute inc). graphs were produced using microsoft excel. results the alzheimer’s disease primary motor cortex exhibits mild tau and β-amyloid deposition but no evidence of neurodegeneration this study involved 23 autopsy cases that had previously been clinicopathologically characterised as ‘probable ad’ or controls based on abc score and clinical dementia rating (table 1) (39). ad cases (n=8) included individuals with an intermediate–high likelihood of ad dementia following routine neuropathological diagnostic testing (38) and who presented with typical ad dementia prior to death. ‘high pathology controls’ (hpc) (n=5) were grouped as such on the basis of no cognitive impairment but satisfied a diagnosis of intermediate ad likelihood on post-mortem examination. controls (n=10) included individuals with a range of abc scores (a0–3; b0–2; c0–3), though only satisfying a ‘not’ or ‘low’ outcome after diagnostic testing. there was no cortical atrophy (fig. 1a) or neuronal loss (fig. 1b), including the prominent layer vb betz cells (fig. 1c), in the pmc of ad cases or hpcs compared with controls. following a positive pixel analysis, grey matter aβ areal fraction (% aβ) was higher in the pmc of ad cases compared to controls (p = 0.0005), but not hpcs (p = 0.07). the density of total aβ plaques, fibrillar, and dense-cored plaques was also significantly higher in ad cases compared to controls (totalp = 0.003; fibrillarp = 0.002; dense-coredp = 0.004) but not hpcs (totalp = 0.1; fibrillarp = 0.06; dense-coredp = 0.2). total tau areal fraction (% ttau) and the density of nfts was higher in the pmc of ad cases compared to controls (%ttaup = 0.001; nftp = 0.001) and hpcs (%ttaup = 0.007; nftp = 0.01) (table 2). amongst the ad cases, the pmc had significantly reduced % aβ (p = 0.006), total aβ plaques (0.001), and fibrillar plaques (p = 0.0002), but not dense-cored plaques (p = 0.2) compared to the itc. the % ttau (p = 0.02) and the density of nfts (p = 0.02) were also significantly reduced in the pmc compared to the itc of ad cases. amongst controls the % ttau was significantly reduced in the pmc compared to the itc (p = 0.04). there were no other regional differences in terms of pathological load in controls, including amongst hpcs (table 3). in the itc, all of the control, hpc, and ad cases had neuritic tau pathology at varied levels. aβ pathology in the itc occurred in 4/10 controls, and all hpcs and ad cases. the clustering of activated microglia in the itc was apparent in 6/10 controls (including three with only tau pathology), and all of the hpcs and ad cases except one (m12) (itc case data is shown in fig. 1d). in the pmc, neuritic tau pathology was present in 7/10 controls, 4/5 hpcs, and all ad cases, while aβ deposition in the pmc occurred in 6/10 controls, 4/5 hpcs, and all ad cases. microglial clustering in the pmc was observed in 5/10 controls, and all of the hpcs and ad cases, with the exception of one case (m12) (pmc case data is shown in fig. 1e). in controls, the % ttau correlated with age in the pmc (r2 = 0.44, p = 0.04) but not in the itc, which was near significant (r2 = 0.39, p = 0.05). figure 1 characteristics of the pmc and regional neuropathological comparisons. a–c the pmc is spared of ad-related neurodegenerative changes as measured by cortical thickness (a), total neuronal density (b), and the density of layer vb betz cells (inset demonstrates a pyramidal betz cell with multiple asymmetrically distributed perisomatic neurites, a prominent nucleolus, and a dark dense deposit of cytoplasmic lipofuscin) (c). d–e a cohort wide comparison of the percentage of aβ and ttau immunolabelling and microglial clustering in the itc (d) and pmc (e) demonstrates age-related tau build-up in a majority of control brains in both regions and an early build-up of aβ in hpcs (that were scored as a2-3, b2, c0-3 on diagnostic slides) with a concomitant microglial clustering response that is more prevalent in the pmc compared to the itc and which appears to dissipate with severe ad pathology in the itc. scale bar = 50 μm (c) table 2. summary of neuronal and neuropathological data of the primary motor cortexa amean ± standard deviation. banova results; see text for p-values of pairwise comparisons. cinclusive of betz cells. ddensity of betz cells in layer v. table 3. regional neuropathological comparisonsa amean ± standard deviation. clusters of activated microglia are more commonly found in the pmc than the itc of hpcs and ad cases the density of individual activated (fig. 2a–e), ramified (fig. 2f), dystrophic (fig. 2g–h), or total microglia did not differ between any of the three groups in the pmc (table 4). microglial clusters (fig. 2i–j), although a small proportion of total microglia, were significantly more common in hpcs (fig. 2k) and ad (fig. 2l) cases compared to controls in the pmc ( hpc p = 0.01; ad p = 0.04). clusters in the pmc were also higher than in the itc of hpcs (p = 0.02) and ad cases (p = 0.03) (fig. 3a), whilst the density of total (p = 0.04) and ramified microglia (p = 0.002) were reduced in the itc compared to the pmc of ad cases. immunofluorescent double-labelling for aβ and iba1 in the pmc showed that the clusters of activated microglia, occurred preferentially within the boundaries of fibrillar aβ plaques, with a significant correlation in a combined analysis of control and ad brains (spearman ρ = 0.54, p = 0.006; fig. 3a). the percentage of aβ plaques associated with a cluster of activated microglia in the pmc ranged from a mean of 41% in controls and 43% in ad cases to 60% in hpcs, with one control (m15), two hpcs (m14 and m18), and one ad case (m13) having a higher density of microglial clusters than aβ plaques. figure 2 response of microglia to ad pathology. a–e a spectrum of microglial activation can be identified by a series of morphological changes including an enrichment of iba1 labelling of the soma and primary processes (a), hypertrophy of the primary processes (b), retraction of tertiary processes ± the formation of morphological features consistent with phagosomes (arrow heads) (c), further retraction of secondary processes (d), until amoeboid in shape (e). f–h healthy ramified microglia have a small, spherical soma and thin, evenly distributed processes (f), contrasting with dystrophic microglia that have either deramified and tortuous processes (g) or pseudo-fragmentation of processes when marked with iba1 (h). i–j microglia that form a cluster within the boundary of an aβ plaque may be either dystrophic or have reached a phase of early (i) or late/amoeboid (j) activation. k–l mosaics of iba1 staining demonstrating the size and distribution of microglial clusters (arrows), defined as three or more somata occurring within, or touch the boundaries of, a 20 μm2 virtual graticule subregion, in the pmc of an hpc (k; m23) and ad case (l; m13). scale bar in j = 40 μm (a–j); in l = 100 μm (k–l) figure 3 characteristics of microglial clustering in the pmc. a the density of microglial clusters was significantly greater in the pmc of hpcs and ad cases compared to controls. b microglial clusters were more frequently associated with fibrillar neuritic plaques in a combined group analysis; spearman ρ = 0.54, p = 0.006. †significantly reduced compared to pmc of hpcs; ‡significantly reduced compared to pmc of ad cases. *†‡p<0.05. table 4. quantification of the morphological subtypes of microglia in the primary motor cortexa acells/mm2; mean ± standard deviation. banova results; see text for p-values of pairwise comparisons of microglial clusters. a graded extent of neuritic tau pathology and clustering of microglia occurs within nascent aβ plaques, with the persistence of dystrophic neurites and the loss of iba1-immunoreactivity and aβ-immunoreactivity occurring in established neuritic plaques all of the aβ plaques examined in the itc, and a majority in the pmc, contained dns. however, the extent of the accumulated neuritic tau pathology within each plaque was lower in the pmc (fig. 4a) than the itc of the same case (fig. 4b). both aβ deposits and globular dns were seen perivascularly (fig. 4c), consistent with previous observations (39). the density of dns was greatest in the itc of ad cases (fig. 4c), which also had the highest density of remnant plaques characterised by accumulations of dns associated with weak or absent aβ-immunoreactivity (fig. 4d). in the itc a diffuse lattice of elongated nts occurred throughout the neuropil and independently of aβ plaques (fig. 4e). dns appeared radially projecting from aβ plaques (fig. 4e) and showed either elongated or globular morphology (fig. 5a). hoechst dye marked cell nuclei around the periphery of aβ plaques and also partly stained the fibrillar deposits of aβ (fig. 5a). hoechst has previously been reported to stain aβ plaques in transgenic mice (40). immunofluorescent double-labelling showed no colocalisation of aβ and ttau (fig. 5b). figure 4 immunofluorescent double-labelling for aβ and ttau. a–c the majority of aβ plaques examined here were associated tau-positive dystrophic neurites, with a clear gradation visible between the pmc (a) and itc (b) in controls (m01 pictured in a and b) and ad cases (itc of m09 pictured in c). ad cases had the most extensive build-up of dns in fibrillar and dense-cored plaques, as well as perivascular (capillary) aβ deposits (c). d remnant plaques (arrows) are characterised by absent or weak aβ staining and dense accumulations of tau pathology and were much more common in the itc than the pmc (also seen in b). e severely affected regions of the ad brain, such as the itc, showed a diffuse network of elongated nts throughout the parenchyma as well radially projecting dns. scale bar = 20 μm (a–c), 60 μm (d), 40 μm (e) figure 5 high power image of an aβ plaque in ad. a image panel showing a fibrillar neuritic plaque in an ad case (m03) with globular and threadlike dns distributed throughout the plaque, which is also surrounded by a network of nts (dotted box in merged image demonstrates roi shown in b). hoechst staining labelled cell nuclei around the periphery of the plaque as well as the fibrillar aβ component inside the plaque. b colocalisation study showed no coincidence aβ and ttau staining in any of the sections examined here (dotted line represents a 6.5 μm length along which pixel intensities have been compared in this exemplar roi). scale bar = 20 μm (a) microglia cell processes exhibiting evidence of phagocytic activity are interspersed around the periphery and core of aβ plaques microglia that occurred in proximity to diffuse, fibrillar, and cored plaques commonly displayed structures morphologically consistent with phagosomes on distal processes with enriched iba1 immunolabelling that closely associated with the aβ element in the periphery and core of plaques (fig. 6a; fig. 7a). as previously reported for the itc, superior frontal gyrus, and primary visual cortex, the overall density of dystrophic microglia was inversely correlated with brain ph in the pmc (r2 = 0.3, p = 0.01) (26). however, it was also noted that individual plaques with dense dns, that were associated with weak or absent aβ staining which were more abundant in the itc, were associated with dystrophic microglia (fig. 6b) rather than a cluster of activated microglia which more commonly occurred where the extent of dns was not yet fully developed (fig. 6c–d). confocal views showed co-localisation of microglial cell processes with aβ in ad (fig. 7a–b), but no co-localisation with tau pathology (fig. 7c–d). figure 6 responses of microglia to ad neuropathology. a the itc of a control case (m01) demonstrating activated microglia with morphological features consistent with the formation of phagosomes (arrows) responding to peripheral and core elements of an aβ plaque. b in the itc of ad cases (m17 pictured), dystrophic microglia were more commonly associated with plaques that contained dense accumulations of dystrophic neurites, however the overall density of dystrophic microglia was inversely correlated with brain ph and not with ad. c–d conversely, plaques with a lower density of dystrophic neurites were more commonly associated with a cluster of activated microglia (m01 pictured) in both the pmc (c) and itc (d). scale bar = 20 μm (a–d) figure 7 exemplars from an investigation into the potential internalisation of aβ and tau pathology by microglia. a activated microglia in an aβ plaque in the itc of a control case with morphological features consistent with the formation of phagosomes (arrows) (m01; dotted box represents region of interest in b). b coincidence of aβ and iba1 pixel intensities along a 7.8 μm length (dotted line) suggests potential internalisation of aβ by microglia. c activated microglia in close proximity to tau-positive dns in the itc of an ad case (m17; dotted box represents region of interest in d). d there was no evidence of the internalisation of tau pathology by microglia in any of the sections investigated here; exemplar shows aβ and ttau staining intensities over a 7.2 μm length (dotted line). scale bar 20 μm (a, c) discussion ad is a uniquely human disease with a long prodrome and has proved difficult to model. the combination of using different regions of post-mortem brain tissue from individuals with or without dementia and with variable amounts of ad-type pathology may allow the pathological sequence of events to be elucidated. for example, the level of disease severity could be ordered from lowest to highest as follows: pmc-controls < itc-controls < pmc-hpc < itc-hpc < pmc-ad < itc-ad. in particular, regions such as the itc in hpcs and the pmc in ad cases could harbour the pre-symptomatic targets required to therapeutically delay or prevent ad. prior to using this model to understand the role of microglia in ad, a quantitative neuropathological analysis of the pmc was carried out to ensure that it met expectations for being a relatively unaffected region of the ad brain. as expected, the pmc of ad cases had a significantly higher % aβ, aβ plaque count, % ttau, and nft density compared controls. there were no nfts observed in the pmc of controls and hpcs, which also had very similar levels of % ttau (which correlated with increasing age), but differed in their level of % aβ. hpcs were defined according to standard neuropathological diagnostic criteria – having an intermediate abc score (38). incidentally, hpcs and ad cases (intermediate–high abc scores) were similar in their aβ load but differed in their tau levels in both regions. in contrast to the primary visual cortex previously investigated (26), the pmc in ad did have significantly reduced levels of aβ and overall tau pathology, including nfts as well evidenced elsewhere (41-46), compared to the itc of the same cases. overall there was no evidence of neurodegeneration in the pmc of ad cases, unlike the itc, with cortical thickness, number of total neurons, and giant layer vb pyramidal betz cells remaining unchanged as expected (47, 48). examination of the microglial morphologies in the pmc using the previously validated modified disector sampling approach across all cortical laminae yielded no significant differences between controls, hpcs, and ad cases. however, group differences could be seen locally around ad-type pathology within the cortex with an increase in clustering of activated microglia in the pmc of hpcs compared to controls, and in the pmc compared to the itc of ad cases. moreover, a higher percentage of plaques contained clusters of microglia in hpcs than in ad cases and also a portion of microglia clusters that were not spatially associated with aβ. the presence of a strong microglial clustering response in the pmc of ad cases and in the hpcs aligns with pet imaging studies demonstrating early activation of microglia in preclinical ad cases (49). although the presence of clusters unrelated to aβ pathology may be a non-specific observation, it is interesting to note that a previous animal study using a 5×fad model also reported the presence of microgliosis prior to the formation of insoluble aβ plaques (50), with another mouse model also indicating microglial activation in relation to synaptic dysfunction prior to aβ deposition (51). here it is suggested that clusters of activated microglia in the pmc represent a neuroprotective response correlating with the deposition of aβ. we have demonstrated clusters of activated microglia that display phagocytic capabilities in the mildly affected pmc before the development of extensive tau pathology. it is conceivable that once the phagocytic potential of microglia is overwhelmed, a transition to a more neurotoxic proinflammatory phenotype occurs and that this represents a pivotal moment preceding tau-related neurofibrillary degeneration. studies in mouse models of aβ overexpression suggest proinflammatory microglia, which may be induced by the binding of oligomeric and fibrillar aβ species to nlr family pyrin domain containing 3, receptor for advanced glycation endproducts, scavenger receptors, and toll-like receptors, among others (52-58), are associated with poorer cognitive and survival outcomes, have impaired phagocytic capabilities (59), and are capable of secreting an expansive complement of neurotoxic compounds including reactive oxygen species, nitric oxide, peroxynitrite, tumour necrosis factor α, interleukin 1 β, and prostaglandin-e2 (60). however aβ-independent mechanisms of microglia activation or exhaustion in human ad cannot be excluded and require further research considering the so far limited efficacy of the pharmacological clearance of aβ in clinical trials (61, 62). activated microglia tended to be associated with fibrillar nps, and higher resolution confocal photomicrographs showed evidence of aβ internalisation by microglia in both regions. the latter may explain the remnant plaques observed here (that contain weak or absent aβ-immunolabelling; in which higher levels of aβ were associated with activated microglia, but dystrophic microglia where aβ-immunoreactivity was very minimal or absent, particularly where dystrophic neurites were extensive) and described elsewhere (14) and is potentially relevant to the proposed dynamic equilibrium between soluble aβ oligomers and insoluble fibrils (63). however, it will be important to confirm the internalisation of aβ by microglia with super resolution techniques such as direct stochastic optical reconstruction microscopy (dstorm). in contrast, microglia did not specifically cluster around any of the three forms of tau pathology, nfts, nts and dns, nor did they appear to internalise tau in co-localisation studies, although processes of microglia were coincidentally found adjacent to dns. the microglial clustering response dissipated over the modelled disease course with advanced stages, represented by the itc of ad cases, being characterised by reduced iba1 immunoreactivity, as reported elsewhere (31, 64). this suggests a process of microglial incapacitation in the context of increased tau load, a concept which is supported by a growing body of literature (65, 66). from these results it is hypothesised that the activation of microglia coincides with cortical aβ deposition. neuritic inclusions of tau in the cortex are evident early in the disease process, represented here by the pmc of ad cases, but mainly develop after the deposition aβ and the activation of microglia. this sequence of pathological changes is ostensibly consistent with the amyloid cascade hypothesis for ad pathogenesis (19) given the presence of elevated aβ in the pmc of hpcs, however it should be stressed that the levels of aβ and tau pathology were present at similar levels in the pmc of confirmed ad cases. therefore it could be argued that insoluble aβ and tau deposits begin forming concurrently in the cerebral cortex of ad brains. this would be consistent with those arguing in favour of the pathogenetic importance of tau deposition (22, 23, 67, 68) and the possibility that it in fact acts as a causative factor behind ad-related microglial activation (69). certainly, animal models suggest that microglial activation augments tau pathology and specifically tau phosphorylation (70). this scenario would then be consistent with our observation that microglial activation wanes with increased tau deposition and with the idea that ageing impairs the housekeeping functions of microglia (71). finally, even in the presence of extensive tau pathology, the increased presence of remnant plaques in the itc suggests that microglia retain the ability to clear aβ peptides. understanding the functional significance of these dynamic spatiotemporal changes in microglial activity along the time course of ad pathophysiology will be critical before new treatments targeting these cells can be imagined. given that microglia of different brain regions display different activation states simultaneously depending on the graded extent of ad-type pathology present, the implementation of either antior pro-inflammatory microglia-based therapies would presumably be beneficial in one brain region but detrimental in another. in future work the genetic characterisation of subjects investigated here may also provide further insight into how genotype affects individual susceptibility to differential microglial function, represented by the highly variable clustering response of microglia in hpcs and ad cases in particular. overall, findings from the post-mortem model used here suggest that the clustering of activated microglia occurs concomitantly with the formation of aβ plaques, and that tau-related neuritic degeneration follows these changes along with a loss of clustering. acknowledgements the authors would like to thank the donors and their families for their kind gift. brain tissue was received from the nsw brain tissue resource centre and sydney brain bank. these brain banks are supported by the nhmrc of australia, the university of new south wales, neuroscience research australia, and the national institute of alcohol abuse and alcoholism (nih (niaaa) r24aa012725). the authors also acknowledge the facilities used at the bosch institute and microscopy australia at the australian centre for microscopy & microanalysis both at the university of sydney. data availability statement the data that support the findings of this 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synovial sarcoma of the central nervous system feel free to add comments by clicking these icons on the sidebar free neuropathology 3:11 (2022) letter a potential diagnostic pitfall: primary synovial sarcoma of the central nervous system arnault tauziède-espariat1,2, nicolas macagno3, daniel pissaloux3, dominique figarella-branger4,5, romain appay4,5, dorian bochaton6, sanaa tazi7, paul kauv8, lauren hasty1, alice métais1,2, fabrice chrétien1, pascale varlet1,2 1 department of neuropathology, ghu paris psychiatry and neuroscience, sainte-anne hospital, paris, france 2 inserm, ima-brain, institute of psychiatry and neurosciences of paris, université de paris, umr s1266, paris, france 3 department of biopathology, léon bérard cancer center, lyon, france 4 department of pathology, toulouse university hospital, toulouse, france 5 inserm u1037, cancer research center of toulouse (crct), toulouse, france 6 institut curie hospital, laboratory of somatic genetics, paris, france 7 department of neurosurgery, henri mondor hospital, créteil, france 8 department of radiology, henri mondor hospital, créteil, france corresponding author: arnault tauziède-espariat · department of neuropathology · ghu paris psychiatry and neuroscience, sainte-anne hospital · 1 rue cabanis, 75014 paris · france a.tauziede-espariat@ghu-paris.fr submitted: 14 march 2022 accepted: 25 april 2022 copyedited by: cinthya agüero published: 26 april 2022 https://doi.org/10.17879/freeneuropathology-2022-3811 keywords: ss18, ssx, synovial sarcoma, bcor introduction synovial sarcoma (ss) is an aggressive soft tissue sarcoma that occurs primarily in the juxta-articular extremital regions of young adults, followed by the trunk and the head and neck. they are universally defined by a fusion of ss18 with one of the ssx genes (predominantly ssx1). primary ss of the central nervous system (cns) have been rarely reported in the literature (n=22 molecularly confirmed cases) and have not yet been added to the mesenchymal non-meningothelial chapter of the world health organization (who) classification of cns tumors. here, we report one case of ss18-rearranged cns-ss, with a review of the literature to determine its clinical, radiological, and histopathological features and its main differential diagnoses. case presentation a 62-year-old woman presented with an intraventricular hemorrhage (figure 1 a-b). the patient had a history of a hemorrhagic lesion diagnosed as a central neurocytoma and was treated by surgery 3 years earlier. the tumor exhibited histologically dense, solid, hemorrhagic, and basophilic cell proliferation (figure 1c), composed of monotonous spindle cells, with round to oval nuclei lacking any pleomorphism or mitotic activity. the cytoplasm was scant or more abundant and vacuolated (figure 1d). there was no collagenous stroma, myxoid changes, “hemangiopericytoma-like” vasculature, whorls, psammoma bodies, neurocytic rosettes, papillae, rhabdoid, or glandular formations. necrosis and microvascular proliferation were absent, and the ki67 labeling index was low (1%) (figure 1e). tumor cells were immunonegative for ae1/ae3, ck7, ck20, ck18, sstr2a, ema, cd34, stat6, cd45, cd99, nut, olig2, gfap, sox10, ps100, hmb45, chromogranin a, synaptophysin, neun, neurofilament, desmin, and smooth muscle actin. the expression of h3k27me3 and brg1 was retained. ini1 was maintained with a diminished expression in comparison to the endothelium (figure 1f). the tumor expressed vimentin, cd56, bcl2, tle1, and bcor (but with less intense staining compared to our control, a molecularly confirmed cns tumor with internal tandem duplication of bcor) (figure 1g-j). histopathological characteristics were like those of the previous surgical sample. the initial tumor was diagnosed as a neurocytoma due to its intraventricular location, its expression of cd56 and because of its absence of any sign of malignancy (particularly no mitosis). initial and recent whole-body imaging did not show other tumor locations. digital droplet pcr failed to reveal an internal tandem duplication of bcor, and whole-exome rna sequencing [1] demonstrated a ss18::ssx1 gene fusion (figure 1k). an additional immunostaining targeting the chimeric protein ss18::ssx was secondarily performed and revealed a strong and diffuse expression in the primary tumor and its recurrence (figure 1k). using dna-methylation analysis, the tumor was not classifiable by the heidelberg brain tumor classifier (v12.5) and the sarcoma classifier (v12.2). a final diagnosis of primary cns-ss was suggested. forty months after the initial diagnosis, the patient was alive without a tumor residue. figure 1. radiological, histological, and molecular features of the recurrent tumor. (a) axial ct scan showing a voluminous left intraventricular mass with hemorrhage. (b) axial ct scan after contrast injection showing a heterogeneous enhancement of the mass. (c) densely cellular proliferation with hemorrhagic changes (hps, magnification x100). (d) monotonous basophilic spindle cells with round to oval nuclei and vacuolated cytoplasm (hps, magnification x400). (e) low ki67 labeling index (magnification x400). (f) retained expression of ini1 with low intensity (magnification x400). (g) diffuse but moderate immunoexpression of bcor (magnification x400). (h) diffuse immunoexpression of vimentin (magnification x400). (i) diffuse immunoexpression of cd56 (magnification x400). (j) diffuse immunoexpression of bcl2 (magnification x400). (k) whole exome rna-seq analysis highlights a fusion between ss18 (pink) and ssx1 (blue) genes, respectively located on chr.18 and chrx. immunopositivity for the chimeric protein between ss18 and ssx with retained protein domain of ss18 detected by immunohistochemistry (insert magnification x400). black scale bars represent 50 μm for figures d-j; and 250 μm for figure c. hps: hematoxylin phloxin saffron. clicking the figure will lead you to a virtual slide (h&e). discussion and conclusion molecularly confirmed examples of primary cns-ss are very rarely reported in the literature (n=22 cases, table s1) [2–8]. like their soft tissue counterpart, they affect young adults (median age 21 years with ranges from 1 to 81) or children (32% of cases) [2,3] with a male predominance (sex ratio male to female: 1.75) [2–8]. they are mainly supratentorial (19/22, 86% of cases) [2–8] and 9% of them are intraventricular as was the case for our patient [2]. clinical symptoms depend on the location of the tumor, but interestingly, 4/22 cases (18%) are revealed by an intracerebral hematoma [2,5,8]. radiologically, they present as a solid (13/22 cases) or cystic mass (8/22 cases) that is well defined from the parenchyma (11/16 cases) [2–4,8]. some of them present a dural attachment (4/22 cases) [4–7]. histopathologically, ss is classically divided into three subtypes within the soft tissue: biphasic (composed of a mixture of spindle cells and epithelial component), monophasic (only composed of spindle cells), and poorly differentiated (composed of round or spindle cells with a high mitotic index and pronounced atypia). rare ss examples can exhibit a round cell morphology [9–11]. in the cns, all these variants have been reported, the monophasic subtype (as our case) being the most frequent (10/21 cases compared to 9/21 biphasic and 2/21 poorly differentiated cases) [2,4–7]. recently, a highly specific and sensitive antibody targeting the ss18::ssx fusion protein has been developed independently of the ss18 fusion gene partner among ssx genes [12], which can serve as a surrogate to molecular testing [12,13]. like their soft tissue counterparts, ss frequently and strongly express bcl2, vimentin, and cd99, which are not specific. the classical phenotype includes a variable, usually focal aberrant epithelial phenotype, diffuse and intense nuclear expression of tle1 [14], and reduced expression of ini1 [15]. the glial markers, sstr2a, cd34, and stat6, are not expressed, which allows one to rule out differential diagnoses (gliosarcoma, meningioma, and solitary fibrous tumor). however, the occasional expression of bcor may be a confusing finding [16], but additional molecular analyses did not confirm the presence of bcor alterations and found a ss18::ssx1 fusion, the most frequent fusion transcript described in soft tissue ss. in the cns, no detailed fusion has been reported, except in one case (ss18::ssx2) because only fish analyses for ss18 have been performed [2–8]. like many fusion-driven neoplasms, the cellular origin of ss remains unknown. a mesenchymal hypothesis was suggested with respect to its preferential location in soft tissue. because meningeal involvement has been reported in a part of cns cases, it is reasonable to suggest a possible mesenchymal dural origin. recently, dna-methylation-based classification of cns tumors has been shown to be a robust tool for molecular tumor classification, most likely because individual tumor types maintain an epigenetic ‘memory’ of their distinct cell of origin [17]. in v12.2 of the sarcoma classifier, there is a methylation class of synovial sarcoma [18]. however, due to the low cellularity in our hemorrhagic sample, the dna methylation profiling did not properly cluster and did not allow us to classify this tumor. the prognosis of soft tissue ss depends on the fnclcc grade [19] and genomic index [20]. cns-ss is associated with a unfavorable outcome, high rates of recurrence (18/21 cases, 86%), and death (16/21 cases, 76%) with a median overall survival of 14 months [2–5,7,8]. our case represents an exception with the longest overall survival ever described without adjuvant treatment (40 months), possibly due to the low histological grade of the tumor. in conclusion, cns ss with a ss18::ssx1 fusion represents a rare mesenchymal non-meningothelial tumor, not yet included in the who classification of cns tumors. although its diagnosis remains challenging, different diagnostic tools (immunohistochemistry, fish, rna sequencing) exist to identify ss. further studies are needed to elucidate its cellular origin in the cns. declarations ethics approval this study was approved by the ghu paris psychiatry neurosciences, sainte-anne hospital’s local ethic committee. consent for publication the patient signed informed consent forms. competing interests the authors declare that they have no conflicts of interest directly related to the topic of this article. funding the authors declare that they have not received any funding. authors’ contributions ate, pk and st compiled the mri and clinical records; ate, am, fc and pv conducted the neuropathological examinations; nc, dp, dfb, ra and db conducted the molecular studies; ate, lh, and pv drafted the manuscript. all authors reviewed the manuscript. acknowledgements we would like to thank the laboratory technicians at the ghu paris neuro sainte-anne for their assistance. references macagno n, pissaloux d, de la fouchardière a, karanian m, lantuejoul s, galateau salle f, et al. wholistic approach: transcriptomic analysis and beyond using archival material for molecular diagnosis. genes chromosomes cancer. 2022; 29:1–12. zhang g, xiao b, huang h, zhang y, zhang x, zhang j, et al. intracranial synovial sarcoma: a clinical, radiological and pathological study of 16 cases. eur j surg oncol j. 2019; 45:2379–85. xiao g, pan b, tian x, li y, li b, li z. synovial sarcoma in cerebellum: a case report and literature review. brain tumor 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otherwise stated. multiple system atrophy a clinicopathological update feel free to add comments by clicking these icons on the sidebar free neuropathology 1:17 (2020) review multiple system atrophy a clinicopathological update kurt a. jellinger institute of clinical neurobiology, vienna, austria corresponding author: kurt a. jellinger · institute of clinical neurobiology · alberichgasse 5/13, a-1150 vienna, austria kurt.jellinger@univie.ac.at submitted: 25 may 2020 accepted: 24 june 2020 copyedited by: nicole schwab published: 3 july 2020 https://doi.org/10.17879/freeneuropathology-2020-2813 keywords: multiple system atrophy, α-synuclein, glio-neuronal degeneration, animal models, etiopathogenesis, prion-like seeding, biomarkers, experimental therapeutics abstract multiple system atrophy (msa) is a fatal, adult-onset neurodegenerative disorder of uncertain etiology, clinically characterized by various combinations of levo-dopa-unresponsive parkinsonism, and cerebellar, motor, and autonomic dysfunctions. msa is an α-synucleinopathy with specific glioneuronal degeneration involving striatonigral, olivopontocerebellar, autonomic and peripheral nervous systems. the pathologic hallmark of this unique proteinopathy is the deposition of aberrant α-synuclein (αsyn) in both glia (mainly oligodendroglia) and neurons forming pathological inclusions that cause cell dysfunction and demise. the major variants are striatonigral degeneration (msa with predominant parkinsonism / msa-p) and olivopontocerebellar atrophy (msa with prominent cerebellar ataxia / msa-c). however, the clinical and pathological features of msa are broader than previously considered. studies in various mouse models and human patients have helped to better understand the molecular mechanisms that underlie the progression of the disease. the pathogenesis of msa is characterized by propagation of disease-specific strains of αsyn from neurons to oligodendroglia and cell-to-cell spreading in a "prion-like" manner, oxidative stress, proteasomal and mitochondrial dysfunctions, myelin dysregulation, neuroinflammation, decreased neurotrophic factors, and energy failure. the combination of these mechanisms results in neurodegeneration with widespread demyelination and a multisystem involvement that is specific for msa. clinical diagnostic accuracy and differential diagnosis of msa have improved by using combined biomarkers. cognitive impairment, which has been a non-supporting feature of msa, is not uncommon, while severe dementia is rare. despite several pharmacological approaches in msa models, no effective disease-modifying therapeutic strategies are currently available, although many clinical trials targeting disease modification, including immunotherapy and combined approaches, are under way. multidisciplinary research to elucidate the genetic and molecular background of the noxious processes as the basis for development of an effective treatment of the hitherto incurable disorder are urgently needed. abbreviations αsyn α-synuclein, adnc alzheimer disease neuropathological changes, bg basal ganglia, caa cerebral amyloid angiopathy, ci cognitive im-pairment, cn caudate nucleus, cns central nervous system, csf cerebrospinal fluid, dat dopamine transporter, ftld frontotemporal lobar degeneration gci glial cytoplasmic inclu-sion, gdnf glia-derived neurotrophic factors, gp globus pallidus, gwas genome-wide association study, hpr hyperintensive putaminal rim, lbd lewy body disease, lbs lewy bodies, mbp myelin basic protein, mci mild cognitive impairment, msa multiple system atrophy, msa-c msa with prominent cerebellar ataxia, msa-p msa with predominant parkinsonism, opc olivopontocerebellar, opca olivo¬pontocerebellar atrophy, os oxidative stress, part primary age-related tauopathy, pd parkinson disease, pet positron emission tomography, prpc cellular prion protein, psp progressive supranuclear palsy, sn substantia nigra, snd striatonigral degeneration, tg transgenic, tppp tubulin polymerization-promoting protein, wt wild type introduction multiple system atrophy (msa) is a rare adult-onset and lethal neurodegenerative disorder clinically characterized by rapidly progressing autonomic and motor dysfunctions. the pathological hallmark of msa, a specific form of α-synucleinopathy, is abnormal accumulation of fibrillar α-synuclein (αsyn) in oligodendrocytes as glial cytoplasmic inclusions (gci) [1], which may represent a primary pathologic event [2]. degeneration of many neuronal pathways causes multifaceted clinical phenotypes: a parkinsonian variant (msa-p), associated with striatonigral degeneration (snd), and a cerebellar (msa-c) variant with olivopontocerebellar atrophy (opca) [3]. in addition to combined or "mixed" msa, there are several disease variants [4-6]. the underlying molecular mechanisms are poorly understood, but converging evidence suggests that a "prion-like" spreading of disease-specific αsyn strains is involved in the pathogenic cascade leading to a specific multisystem neurodegeneration in this (oligodendro)glioneuronal proteinopathy [4, 7-12]. the aim of the present review is to describe recent advances in msa neuropathology, clinical diagnosis, neuroimaging, and candidate biomarkers. it further provides an overview of the mechanisms underlying msa pathogenesis and of possible novel therapeutic targets that have emerged from animal studies and preclinical interventional trials [13-16]. epidemiology msa is a rare disease with an estimated incidence of 0.6-0.7/100,000 person-years [17], although studies from russia and northern sweden have reported incidences of 0.1 and 2.4/100,000 person-years, respectively [18, 19]. prevalence estimates range from 1.9 to 4.9/100,000 [20] but may reach up to 7.8 after the age of 40 years [21], and up to 12/100,000 after the age of 70 [22]. msa-p accounts for 70-80% of cases in the western world [23], whereas msa-c is more frequent in asian populations (67-84%) with rather frequent mixed phenotypes [24-27], probably due to genetic and environmental factors [5]. etiology and genetics msa is generally considered a sporadic disease [17], but msa pedigrees with both autosomal dominant and autosomal recessive inheritance patterns have been reported in europe and asia [28-33]. a genome-wide association study (gwas) found an estimated heritability of 2-7% [34], but unlike parkinson disease (pd), no single gene mutations linked to familial forms and no definite environmental risk factors have been identified [35]. screening for pd causal genes (mapt, pdyn, parkin, pink1, and several single nucleotide polymorphisms/snps) did not reveal any association with msa [36-38], while lrrk2 exon variants may contribute to its susceptibility [39]. glucocerebrosidase (gba) variants were associated with autopsy-proven msa [40, 41], particularly with msa-c [42], while others have found no association [43]. furthermore, c9orf72 repeat expansions [44] and snca polymorphisms as risk factors of msa [45, 46] have not been confirmed [47-49]. no significant associations of the apoe locus nor the prion prnp with risk of msa was observed [50, 51], and there is no evidence of autosomal dominant msa or of de novo mutations in this disorder [52]. a british family with snca mutation showed neuropathologic features of both pd and msa [53], but they are distinct from pd patients carrying the h50q or snca duplication [54]. none of the nucleotide polymorphisms (fbxo47, elovl7, edn1, etc.) reached genome-wide significance [34], and polymorphisms of the lingo1 and lingo2 (nogo receptor interacting protein-1 and -2) do not decrease the risk of msa [55]. the possible involvement of the snca, coq2, mapt, gba1, lrrk2 and c9orf72 genes in msa pathogenesis was examined recently [56]. the link between v393a mutations and the coq2 gene, encoding the coenzyme q10 (coq10), and familial or sporadic msa in japanese and other asian populations [44, 57-61] has not been confirmed in other populations [34, 62-64]. thus, coq2 polymorphisms may be region-specific and may not represent common genetic factors for msa. decreased levels of coq10 in cerebellum and plasma of msa patients [65, 66] suggest that its deficiency may contribute to pathogenesis due to decreased electron transport in the mitochondria and increased vulnerability to oxidative stress (os) [67]. rna analyses of msa brain tissue revealed alterations in αand β-immunoglobuline [68], dysregulations of micrornas that regulate gene expression in the pons and cerebellum [69, 70], and disruption of long intervening non-coding rnas (lincrna) in the frontal cortex along with protein coding genes related to iron metabolism and immune response regulation [71, 72]. epidemiological studies suggested that epigenetic factors or environmental toxins may be associated with the risk for msa [73], but there are no convincing data correlating increased risk of msa with exposure to pesticides, solvents, other toxins, or alcohol consumption [74, 75]. pathogenesis although our understanding of msa remains incomplete, evidence from animal models and human post mortem studies indicates that the accumulation of misfolded αsyn, particularly in oligodendrocytes but also in neurons, plays an essential role in the disease process [10, 76-78]. the impact of the neuronal endosomal-lysosomal system in the processing of αsyn in pd is well established, while lysosomes contribute to the pathogenesis of msa, enabling oligodendroglial and neuronal uptakt of αsyn [79]. reduced oligodendrocyte-derived enriched microvesicles (oemvs) could be an important mechanism related to pathological αsyn aggregation in oligodendrocytes [80]. although it has been speculated that primary neuronal pathology leads to secondary oligodendroglial degeneration, as suggested by the widespread occurrence of ncis even in areas lacking gcis [77], the distribution and severity of neurodegeneration reflects subregional gci density and supports the assumption that msa is a primary oligodendrogliopathy [2, 81]. the role of oligodendroglia in introducing the neurodegenerative process was confirmed experimentally in transgenic (tg) mice overexpressing αsyn in oligodendrocytes [10, 13, 82-84]. these and other results highlight the role of endogenous αsyn and p25α in the formation of αsyn assemblies in oligodendrocytes and provide in vivo evidence of the role of oligodendroglial αsyn in the establishment of αsyn pathology in msa [85]. early events are an ectopic appearance of αsyn in oligodendrocytes, loss of the camp-regulated phosphoprotein of 32kda (darpp-32), and calbindin indicating calcium toxicity and disturbance of phosphorylated proteins [86]. recent findings suggest the possibility of endogenous αsyn accumulation in oligodendrocyte precursor cells that contribute to gci formation and perturbation of neuronal/glia support in msa brain [86a]. reduced oemvs could be an important mechanism related to pathological αsyn aggregates in oligodendroglia, inducing dysfunction of the snare protein complex, which regulates membran fusion in eukaryotic cells. the concentrations of oemvs in msa were significantly reduced compared to those in pd [80]. decreased expression of glia-derived neurotrophic factors (gdnf) in msa brains [87] indicates that αsyn aggregation in oligodendrocytes impacts their trophic transport to neurons. oligodendroglial changes are more widespread than αsyn positive gcis, suggesting that oligodendroglial pathology induces degeneration of the oligodendroglia-myelin-axon-neuron complex [2, 26]. the selectivity of the neurodegeneration in msa is determined by the interaction of multiple noxious factors. some of these factors include: ectopic αsyn accumulation in oligodendrocytes and neurons, "prion-like" propagation of disease-specific strains of misfolded αsyn [88], targeting distinct brain regions and cell types [89, 90], impaired protein degradation, proteasomal and mitochondrial dysfunctions [91, 92], alterations of the autophagic pathway [91, 93, 94], perturbed iron homeostasis [95], lipid dysfunction involved in myelin synthesis [96-98], genetic polymorphism [55], microglial activation [97, 99], neuroinflammation [100], proteolytic disturbance, autophagy [101], and microrna dysregulation [102] driving inflammation, disrupting myelin, and contributing αsyn accumulation via the dysregulation of autophagy and prion mechanisms [103]. these and other factors are contributing to os, which is suggested to be a major pathogenic factor in msa and related diseases [104]. these multiple mechanisms interact to result in the system-specific pattern of neurodegeneration in msa (fig. 1). tnfα-dependent neuroinflammation may play a key role in msa pathogenesis, and its relevance has been underlined in various models of the disease [105]. αsyn, which shows specific conformational strains [88, 106] that are primarily generated by neurons, can be toxic once released to the extracellular environment [107] and can spread throughout the brain in a "prion-like" manner [9, 108-111]. extracellular αsyn, interacting with neuronal and non-neuronal cell types, mediates neuroinflammation and cell-to-cell spread [112, 113]. neuron-to-oligodendrocyte transport of misfolded αsyn plays a major role in the pathogenesis of msa [114, 115]. msa and pd show different phosphorylation signatures of αsyn and distinct seed characteristics, indicating that distinct strains underlie these diseases [90, 116, 117]. after propagation in tgm83 tg mice, strain-specific phenotypic differences are maintained after serial transmission, providing evidence that disease heterogeneity among the synucleinopathies is caused by distinct αsyn strains [89]. msa strains show several similarities to pd strains, and less so with dlb strains, but more potently induce motor deficits, nigrostriatal degeneration, αsyn spreading, and inflammation, reflecting the aggressive nature of this disease [118]. fig. 1. pathogenetical features of msa causing neurodegeneration. spontaneous misfolding of αsyn results in formation of abnormally folded dimers and further assembly results in oligomers and amyloid formations. αsyn-rich gcis involving oligodendroglia result in demyelization and neurodegeneration. the red arrow shows the “prion-like” cell-to-cell transfer of misfolded αsyn. courtesy of victoria sidoroff, md, dept. of neurology, medical university of innsbruck, innsbruck, austria recent animal model studies that only partially replicate the human disorder have provided some progress in our understanding of msa pathogenesis [13, 15, 84, 119, 120]. early accumulation of p25α (tppp), a potent stimulator of αsyn aggregation, may decrease myelin basic protein (mbp), favoring both the deposition and fibrillation of αsyn and changing myelin metabolism [121]. relocation of p25α from the myelin sheaths to the oligodendroglial soma, due to mitochondrial dysfunction, and the formation of cytoplasmic p25α inclusions precedes the aggregation of transformed αsyn in oligodendrocytes. endogenous αsyn and p25α induce the formation of pathological αsyn assemblies in oligodendrocytes and provide in vivo evidence of their contribution to the pathogenesis of msa [85]. although large inclusions appear in a later disease states, small, soluble assemblies of αsyn, promoted by p25α, are pathogenic [122]. the source of αsyn in oligodendroglia is unclear, but it contains αsyn mrna expression and αsyn may be secreted by neurons and taken up by oligodendrocytes, which is facilitated by protein cx32 via direct protein-protein interaction in both neurons and oligodendroglia [115]. 21% of proteins found consistently in gcis and lbs are synaptic vesicle-related, suggesting that misfolded αsyn may be targeted via vesicle-mediated transport, and may explain the presence of this neuronal protein within gcis [123]. thus, msa represents a specific form of oligodendroglial proteinopathies [124], while others suggest that it is a primary neuronal disease with secondary accumulation of αsyn in oligodendrocytes [77]. induced pluripotent stem cell (ipsc) studies indicate a pathological phenotype of msa neurons, independently from oligodendrocytes. these data together with findings in animal models suggest that both neurons and oligodendrocytes are affected in msa [91]. the disease is currently viewed as a primary synucleinopathy with specific glio-neuronal degeneration developing via the oligo-myelin-axon-neuron complex [2, 4]. histopathology and molecular pathology the histological core features of msa encompass the following types of different severity: (1)specific αsyn-immunoreactive inclusion pathology with five types of inclusions: gcis within oligodendrocytes (also referred to as papp-lantos bodies [125], the presence of which is mandatory for the post mortem diagnosis of definite msa [1]) and less frequently glial nuclear inclusions, neuronal nuclear inclusions, astroglial cytoplasmic inclusions, and neuronal threads, also composed of αsyn [126]; (2) selective neuronal loss and axonal degeneration involving multiple regions of the nervous system; (3) extensive myelin degeneration with pallor and reduction in mbp with astrogliosis; and (4) widespread microglial activation [127] and neuroinflammation [128, 129], with extensive cd4 and cd8 t-cell infiltration [130]. gcis and the resulting neurodegeneration show a characteristic distribution, involving not only the striatonigral and opc systems, but also cortical regions, autonomic and motor nuclei in the brainstem, spinal cord, preganglionic autonomic nerve structures [131-134], and the peripheral nervous system [135-138], characterizing msa as a multi-system/-organ disorder [2, 77, 139]. phosphorylated αsyn is accumulated in subpial and periventricular astrocytes after long disease duration [140]. however, αsyn-positive astrocytes in subpial and perivascular regions are seen in both msa and lewy body disease (lbd), suggesting that this pathology is not a specific feature of msa [141]. inclusion pathology gcis are argyrophilic, triangular, sickle-/half moon-shaped or oval cytoplasmic aggregations, composed of fibrillar αsyn, ubiquitin and various multifunctional proteins, including 14-3-3 protein, lrrk2, aggressomal proteins, etc. [125] (fig. 2). they form a meshwork of loosely packed filaments or tubules 15-30 nm in diameter with a periodicity of 70-90 nm and straight filaments, both composed of polymerized αsyn granular material and other filaments. the central core contains phosphorylated (ser129) αsyn cryo-em showed that αsyn inclusions from msa are made of two types of filaments, each of which consists of two different protofilaments. each type contains non-proteinaceous molecules at the interface of the two proteofilaments. thus, they differ from those in dlb brain, which suggests that distinct conformations/strains are characteristic for specific synucleinopathies. in addition, αsyn filament extracts from msa tissue differ from those formed in vitro using recombinant proteins, which may have implications for the mechanisms of protein aggregation and neurodegeneration [142]. soluble αsyn in gcis differs from the insoluble form in lewy bodies (lbs) [143]. purification of αsyn containing gcis revealed 11.9% αsyn, 2.8% α-β-crystallin, and 1.7% 14-3-3 protein compared to 8.5%, 2.0% and 1.5% in lbs [144]. in the msa brain, αsyn 140 and 122 isoform levels are increased, whereas αsyn 126 is decreased, in the substantia nigra (sn), striatum, and cerebellum. in early disease states, diffuse αsyn staining in neuronal nuclei and cytoplasm occurs in many gray matter areas, indicating that aggregation of non-fibrillary αsyn occurs early in neurons [26]. recent studies using a proximity ligation assay revealed a wide distribution of αsyn oligomers not only in oligodendrocytes but also in neocortical neurons and purkinje cells, suggesting that αsyn oligomer-mediated toxicity is an early event in msa, inducing neuronal loss in msa [145]. fig. 2. (a–c) glial cytoplamic inclusions in msa: (a) in globus pallidus (gallyas silver impregnation), (b) in pontine basis (α-synuclein) and (c) in frontal white matter, anti-ubiquitin. (d) neuronal cytoplasmic inclusion and neurites in pontine basis (α-synuclein). (a–d) original magnification 34,000. from [126]. on the other side, interactions exist between extracellular αsyn and each of the major central nervous system (cns) cell types. this has thepotential to contribute to secondary disease processes such as neuroinflammation, synaptic dysfunc-tion, and cell-to-cell spread, with vehicles such as microglia and exosomes that mediate spread of αsyn pathology to peripheral brain regions [113]. cathepsin-d, calpain-1 and kallikrein-6 are elevated in the putamen, pontine basis, and cerebellar white matter, indicating that αsyn accumulation is not due to reduced activity of these proteases, but rather that their upregulation is compensatory to increased αsyn [146]. iron levels in basal ganglia (bg) and sn are higher in msa than in pd and controls, indicating perturbed iron homeostasis as a potential pathogenic factor in msa neurodegeneration [95]. quantitative analyses of neuronal death and gci density showed a positive correlation with each other, indicating the pivotal role of gcis in neuronal death [81, 147], and additionally, both lesions increase with disease duration [148-150]. in the sn, severe neuronal loss is accompanied by low gci density, indicating that this and other areas affected in early disease have been burned out [139]. glial nuclear inclusions show a distinct distribution from gcis (fig. 2d), and similarly the density of neuronal cytoplasmic inclusions (ncis) and neuronal nuclear inclusions are unrelated to that of gcis [151]. ncis are more widespread and show a hierarchical pattern related to the duration of disease but are independent of neuronal destruction, suggesting that other factors may induce the subtype-dependent neuronal loss [77]. region-specific astrogliosis is positively correlated with αsyn pathology in msa, in contrast to pd [152], and in general parallels the severity of neurodegeneration [148]. microglial activation in degenerated regions accompanies gci pathology and is most abundant in white matter areas with mild to moderate demyelination [153]. in msa-c, the cerebellar subcortical white matter and cerebellar brainstem projections are the earliest involved, followed by other cns regions. distribution of lesions a grading system for snd was proposed based on semiquantitative assessment of atrophy, neuronal loss, and the presence of gcis [154]: neuronal loss in the sn pars compacta is grade 1; extension to the putamen is grade 2; further involvement of the caudate and globus pallidus (gp) is grade 3. subsequently, the grading system was extended for both snd and opca [155]. of 42 patients, 22 were assigned as msa-p and 20 as msa-c, but none displayed "pure" opca pathology or more severe opca pathology than snd (i.e., opca iii+snd i/ii). these clinicopathological subtypes correlated with initial symptoms and clinical features of both types. post mortem mri changes in the putamen (type 1, mild atrophy and isointensity; type 2, atrophy and diffuse hypointensity with a hyperintensive putaminal rim/hpr; type 3, putaminal atrophy and isoor hypointensity with hpr) reflect various degrees of brain damage [156]. in two large series from the uk and japan, another grading system for msa was proposed [148]: each case of snd and opca was divided into three grades based on semiquantitative assessment of neuronal loss in regions of interest: for snd, the putamen, gp and sn; and for opca the pontine nucleus, cerebellar hemisphere and vermis, inferior olivary nucleus and sn. this classification showed significant clinicopathological correlations. snd phenotypes showed more severe bradykinesia, and the opca phenotype more frequently showed cerebellar signs. no patients showed "pure" snd or "pure" opca. however, there is an increasing overlap of αsyn pathology with increased duration of the disease the extent of αsyn pathology [157]. damage to the striatonigral system is most severe in the dorsolateral caudal putamen and lateral sn, suggesting transsynaptic degeneration of the striatonigral fibers. consistently and severely affected areas are the putamen, cn, sn, pontine and medullary tegmental nuclei, inferior olives, and cerebellar white matter; moderately affected areas are the motor cortex and gp, and mild lesions involve the cingular cortex, hypothalamus, nucleus basalis of meynert, thalamus, subthalamus, and pontine tegmentum [158]. degeneration of the gp and sn leads to dysfunction of these inhibitory nuclei projecting to the motor thalamus, but the sn loss is of dopamine, not gaba (gamma aminobutyric acid), neurons. stereological studies of the bg revealed a substantial loss of neurons in the sn, putamen, and gp, whereas astrocytes were more frequent in the putamen and caudate nucleus (cn). microglia were found in all cns regions with greatest frequency in the, otherwise unaffected, red nucleus. these data support the region-specific pattern of pathological changes in msa [159]. another neuropathological study showed that the striatonigral region was most severely affected in 34% of snd and in 17% in opca cases, while in almost half of them both regions were equally affected [133]. in view of the frequent overlap and mixed forms, the value of grading systems for evaluation of msa is under discussion [139]. there is widespread involvement of the neocortex with significant loss of neurons and increase of astrocytes and microglia in the frontal and parietal areas, but no change in the total number of oligodendrocytes [160]. early degeneration of the bg drives late onset cortical atrophy due to fronto-striatal degeneration [161, 162]. reduced neuronal numbers in the anterior olfactory nucleus and intrabulbar part of the primary olfactory (pyriform) cortex may underlie olfactory dysfunction in msa [163]. limbic tdp-43 pathology is rare in msa, but co-localization with αsyn suggests an interaction between the two molecules [164-167]; tdp-43 positive cases showed significantly older age at death than negative ones, suggesting that tdp-43 pathology in msa is an age-related phenomenon rather than a disease-specific change [141]. demyelination of variable intensity affecting all parts of the nervous system [168] is associated with reduction of mbp by about 50% [96]. gcis and microglial burden are greatest in mild to moderate white matter lesions and decrease with progression of myelin damage that increases with disease duration [169]. the regional vulnerability of the white matter to msa pathology is poorly understood, but recent gwass revealed dysregulation of various methylated loci, including hip1, lman2, mobp, and others, giving the first evidence that dna methylation changes contribute to the molecular processes altered in msa [170]. early msa stages show increased microglia (about 100%) in the white matter [127], without concomitant astrogliosis or oligodendroglial degeneration [171]. both microglial activation and αsyn-containing oligodendrocytes trigger neuroinflammation in the white matter [128]. the loss of tubulin polymerization-promoting protein (tppp)/p25α immunoreactivity correlated significantly with the degree of microglial reaction and loss of mbp density as a marker of tract degeneration [124]. white matter degeneration causes degeneration of neuronal loops, leading to dysfunction of cerebral autoregulation [172]. gliosis in the degenerated areas of the msa brain usually correlates with αsyn pathology and the severity of neurodegeneration [153, 173], which is in contrast to pd [174]. significant increase of monoaminoxidase b (mao-b), a biomarker of astrogliosis, in the degenerated putamen (+83%) was associated with astrogliosis and showed a positive correlation with αsyn accumulation [175]. microglial activation accompanying αsyn pathology and phagocytosing degenerating myelin is prominent in all degenerating regions [176], particularly in white matter input tracts to the extrapyramidal system and cerebellum [177]. stereological studies revealed a significant increase of microglia in the white matter without concomitant astrogliosis and with absence of significant oligodendroglial degeneration [171], suggesting that microglia cells play an important role in the initiation and progression of neurodegeneration in msa [100, 178]. this is supported by tg mouse models indicating an active contribution of microglial activation by triggering neuroinflammatory responses in the msa brain [179]. in msa-c, gcis are most prominent in the cerebellum, pons, and medulla [169]. the cerebellar purkinje cells are more severely affected in the vermis, with atrophy of olivary nucleus, cerebellopontine fibers, and pontine basis, causing interruption of specific cerebellocortical circuits [180]. the motor subnetwork in msa-c is significantly altered in both bg and cerebellar connectivity [181], with hyperintensity of the middle cerebellar peduncle [182]. involvement of autonomic and peripheral nervous systems degeneration of preganglionic autonomic neurons of the brain stem and spinal cord cause multidomain autonomic failures in msa [133, 183, 184]. supraspinal lesions involve cholinergic neurons of the ventrolateral nucleus ambiguous [185, 186], tegmental nuclei [187], ventral periaqueductal dopaminergic neurons [188], medullary and arcuate nucleus, noradrenergic locus ceruleus [134], serotonergic medullary groups, ventrolateral medulla [189], caudal raphe neurons [190, 191], catecholaminergic neurons of rostral ventral medulla, and noradrenergic neurons of the caudal ventrolateral medulla [185, 192]. the medullary serotonergic and catecholaminergic systems are involved in early stages of msa [193]. other involved areas are the dorsal vagal nucleus [185], periaqueductal gray [132], the westphal-edinger nucleus and posterior hypothalamus, the tuberomamillary and suprachiasmatic nuclei [194], and the pontomedullary reticular formation [149]. the density of αsyn pathology did not correlate with neuronal loss, and there was no correlation between the αsyn burden and disease duration in these regions, indicating that the loss of monoaminergic neurons may progress independently from αsyn accumulation [195]. sympathetic preganglionic neurons in the intermediolateral cell columns of the thoracolumbar spinal cord [26, 134, 196] and sympathetic ganglia and schwann cells in autonomic nerves are involved [197]. neuronal loss affects onuf's nucleus in the sacral region [198], with minor loss of upper and lower motor neurons [26] and variable involvement of anterior horn cells [134]. mild degeneration of cardiac sympathetic innervation has been reported in some cases of msa [199, 200], which accounts for a mild to moderate decrease in the number of tyrosine hydroxylase, but not of neurofilament-immunoreactive nerve fibers in the epicardium. however, depletion of cardiac sympathetic nerves is closely related to the presence of αsyn pathology in the sympathetic ganglia of the cns [200, 201]. the peripheral nervous system shows αsyn deposits in sympathetic ganglia, skin nerve fibers [138, 202, 203], and schwann cells [204], but lack of αsyn immunoreactivity in dermal fibers in contrast to pd [203, 205]. filamentous αsyn aggregates involve the cytoplasm of schwann cells in cranial, spinal and autonomic nerves in msa [141, 197, 206]. clinical features the onset of motor symptoms is 56±9 (mean ± sd) years, with both sexes equally affected [207], however 20-75% of msa patients have a prodromal/preclinical phase with non-motor symptoms. this phase includes cardiovascular and other autonomic failures (urogenital and sexual dysfunctions, orthostatic hypotension, and rem sleep behavior disorder (rbd), which occurs in 88% or more [208, 209]), which may precede the motor presentation by months to years [210, 211] and indicates more rapid progression of the disease [212, 213]. average age at disease onset is earlier in msa-c compared to msa-p, the latter leading to more severe disability [214-216]. average duration after clinical diagnosis is 6-10 (mean 9.5) years [12, 23], with few patients surviving more than 15 years [217]. others have reported a 5 year survival of 78% [218] and a 43% death rate during 3 years of follow-up [135]. a pan-american multicenter study reported that 68% of the participants presenting as msa-p showed an age at onset of 61.5 years, and those as msa-c of 57.4 years [219], while a prospective cohort in the usa reported a median survival of 9.8 (95% ci 8.8-10.7) years [220]. early autonomic dysfunctions and severity of orthostatic hypertension have negative impact on both disease progression and survival [221] and more than triples the risk of shorter survival [222, 223], and a meta-analysis identified severe dysautonomia, early combined autonomic and motor failure, and early falls as unfavorable predictors of survival, whereas msa phenotype and sex did not predict survival [224]. parkinsonism with rigidity, slowness of movements, postural instability, gait disability, and a tendency to fall, characterize the motor presentation of msa-p [12]. parkinsonism is rapidly progressing to wheelchair confinement within 5 to 10 years from symptom onset, poorly responsive to l-dopa, and is often associated with atypical features [17]. unilateral parkinsonism occurs in 40% of msa patients [220] and typical tremor in 4-10% [225]. early postural instability and gait difficulties with recurrent falls are also seen in msa [35]. polyminimyoclonus, not included in the current diagnostic criteria of msa, has now been recognized as a specific clinical feature of msa. among motor and non-motor symptoms in early msa, dysarthria was the most prevalent feature (98.4%), followed by sexual dysfunction (95%), rbd (90.2%), constipation (82%), snoring (70.5%), dysphagia (69%), and stridor (42.6%), which was more common in msa-c than in msa-p [226]. a resting tremor is rare, whereas irregular postural and action tremor may occur [227, 228]. cerebellar ataxia, widespread gait, uncoordinated limb movements, action tremor, and spontaneous or gaze invoked nystagmus predominate msa-c [35]. hyperreflexia and a babinski sign occur in 30-50% of patients, while abnormal postures, such as bent spine, antecollis, and hand or foot dystonia are rare [229]. early generalized and rapidly progressive autonomic failure is typical of msa [230] and, in the absence of parkinsonism or cerebellar signs, indicating pure autonomic failure, which converts to msa within a few years in about 28% [231-233]. among non-motor symptoms observed in 75-95% of patients [234], urinary urgency and increased frequency are common in early disease stages [35]. in a subset of msa patients with early urinary retention, the disease may begin in the sacral spinal cord and then spread to other regions [235]. orthostatic hypotension with recurrent syncope, which occurs after the onset of urogenital symptoms, is a hallmark feature of msa; less specific are dizziness and nausea. other symptoms are anhydrosis, gastrointestinal dysfunction with early dysphagia and constipation [225], pupillary autonomic involvement with blurred vision and dry eyes. [236]. dysproportional antecollis and pisa syndrome are common postural deformities in msa [35]. about 50% of patients with msa-p develop cerebellar signs and even a higher proportion of msa-c cases develop parkinsonian features [23, 220]. dystonia, repeated falls, drooling, dysphagia, dysphonia, and pain occur in advanced stages of the disease [237]. laryngeal stridor is rare [210]. respiratory disturbances including diurnal or nocturnal inspiratory stridor and sleep apnea are frequent [238, 239]. diagnostic biomarkers despite numerous studies, to date there are no reliable diagnostic and prognostic biomarkers available. while multimodal imaging of structural and functional brain changes gave insight into the pathophysiology and may evaluate disease progression, recent studies suggest that the combination of neuroimaging and fluid biomarkers may be more successful than using single markers to increase the accuracy of the clinical (differential) diagnosis of msa [240]. fluid and tissue biomarkers studies of αsyn levels in cerebrospinal fluid (csf) and plasma have been shown to not be useful in the discrimination between msa and pd or psp [5, 241, 242]. a recent meta-analysis of available csf data showed that reduction of p-tau, αsyn, aβ-42 and total tau and elevated nfl are indicators for msa [243]. currently, the most promising approach is a combination of csf dj-1, phospho-tau, light chain neurofilament protein (nfl) and aβ-42 that may be helpful in the differential diagnosis between msa and other parkinsonian disorders [5, 240, 243, 244] (fig. 3). other studies have shown increased csf levels of cytokines such as mcp-3, mdc, fractalkine, and mip-1β [246]. phosphorylated αsyn in red blood cells may be a potential diagnostic biomarker for msa [247]. the results of proteomics for biomarker discovery and mrna expression need further elucidation [248]. fig. 3. candidate biomarkers of multiple system atrophy compared to parkinson’s disease and controls. msa: multiple system atrophy; pd: parkinson’s disease; nfl: neurofilament light chain; fh: complement factor h; c3: complement 3; mhpg: 3-methoxy-4-hydroxyphenylethyleneglycol; igf-i: insulin-like growth factor i; uch-l1: ubiquitin carboxy-terminal hydrolase l1; oxdj: oxidized dj-1 protein; mirna: microrna. modified from [245]. molecular and functional imaging a cardiac sympathetic postganglionic denervation distinguishes pd from msa, showing intact innervation. i-123 mibg (metaiodobenzylguanidine) scintigraphy can help differentiate the two diseases with a pooled specificity of 77% (95% ci: 68-84%) [199]. recent meta-analyses suggest that mibg imaging is useful to discriminate pd from msa in moderate to advanced disease stages, but unreliable in early stages [199, 249]. however, interactions with many drugs limit the value of this method [250]. the anteroposterior diameter of the medulla oblongata is a potential imaging marker of parasympathetic dysfunction in msa [251]. in recent years, several brain magnetic resonance imaging (mri) features have been described as helpful in the differential diagnosis of parkinsonian syndromes. they include atrophy of the putamen, pons, cerebellum, and middle cerebellar peduncle, a dilated fourth ventricle, and various signal intensity variations on mri [252]. mri abnormalities including the "hot-cross bun" sign, a cruciform hyperintensity in the pons [253], and the "putaminal rim sign", which marks hyperintensive bordering of the dorsolateral margins of the puta men in t2-weighted mri reflecting degeneration and iron deposition, may differentiate msa-p from pd [254-258]. they are, however, non-specific signs and therefore not included in the recent consensus criteria [3], in contrast to putaminal atrophy which shows 92.3% specificity but low sensitivity (44.4%) [259, 260]. putaminal atrophy together with hypointense putaminal signal changes on iron-sensitive routine sequences seem to be specific for msa-p [252]. others showed significantly increased putaminal diffusivity volumes in the small anterior region of interest in msa-p versus pd [261]. another distinguishing feature is the extensive and widespread volume loss across the entire brain in msa-p [262]. in quantitative mri studies, the bilateral r2* increase in the putamen best separated msa-p from pd [263]. putaminal and infratentorial volume information classified 96.8% of msa cases [260]. diffusion tensor imaging permits differentiation between pd and msa-p, the latter showing higher values of the diffusion coefficient in the inner capsule, corona radiata, and lateral periputaminal white matter [264], while a meta-analysis of putaminal diffusivity measurements showed sensitivity of 90% and specificity of 93% in distinguishing msa-p from pd based on putaminal diffusivity [265]. combined use of diffusion ratios and magnetic susceptibility values/quantitative susceptibility mapping allowed differentiation of msa-p and msa-c from other parkinsonian syndromes with sensitivities and specificities of 81-100% [266]. hyperintensity of the middle cerebellar peduncle and hot cross bun sign should be added into the list of additional neuroimaging features of possible msa-c [182]. several studies assessed the diagnostic potential of multimodal mri [267-270]. in conclusion, the sensitivity of conventional mri findings in msa compared to pd and healthy controls is inconsistent (36-83%), the specificity of mri abnormalities differentiating msa from pd is high (88-100%). automated imaging differentiation in parkinsonism (aid-p) and magnetic resonance parkinsonism index (mrpi) are robust biomarkers for pd and msa [271]. diffusion weighted images, t2* weighted images and proton density weighted images are useful for diagnosis msa-p in early stages [272]. fluorodeoxyglucose-positron emission tomography (fdg-pet) can distinguish msa-p from pd, showing different patterns of decreased glucose metabolism with a positive predictive value of 97% [273, 274]. targeting postsynaptic dopaminergic functions using 123fβcit spect differentiates pd (normal or increased signal) from msa (normal or increased signal) [275]. dopamine transporter (dat) imaging showed more prominent and earlier dat loss in the anterior caudate and ventral putamen in msa than in pd [276], although normal dat imaging does not exclude msa [277]. in autopsy-confirmed cases a greater asymmetry of striatal binding was seen in msa than in pd [278], but it is highly correlated with sn cell loss [279]. 18f-dopa-pet showed more widespread bg dysfunction in msa than in pd without evidence of early compensatory increase in dopa uptake [280]. future studies will be needed to determine the usefulness of tau-pet imaging for the characterization of αsyn filaments and the differential diagnosis of atypical parkisonian disorders. interpretation of tau-pet should be done cautiously, since some msa cases with severe gci pathology may be false-positive [281, 282], even though the affinity of pbb3 is 10 to 50 times less than αsyn [283]. 1-(2-chlorophenyl)-n-methyl-n-(1-methylpropyl)-3-isoquinoline carboxamide (pk11195) for imaging microglia-mediated processes showed elevated tracer binding in many areas of the msa brain, consistent with the known neuropathologic distribution [284]. diagnostic accuracy and differential diagnosis revised consensus guidelines define 3 degrees of certainty of clinical diagnosis of msa: definite, probable and possible [3] (table 1, fig. 4). fig. 4. diagnostic scheme for msa according to the current consensus diagnostic criteria. definite msa requires post mortem evidence of widespread αsyn inclusions with concomitant snd or opca [1]. probable msa is defined as a sporadic, progressive disorder in adults, clinically characterized by severe autonomic failure, urinary dysfunction and poor l-dopa-responsive parkinsonism or cerebellar ataxia. a diagnosis of probable msa is based on clinical features and ancillary diagnostic tests. possible msa can be diagnosed when a sporadic progressive adult-onset disorder with parkinsonism or cerebellar ataxia is accompanied by at least one of the following additional features within 3 years of motor onset: dysphagia, gait ataxia and other cerebellar symptoms (table 1). table 1. diagnostic clinical markers for msa. modified from [240]. msa, multiple system atrophy; msa-c, msa with cerebellar features; msa-p, msa with predominant parkinsonism. "red flag" diagnostic features the presence of "red flag" (warning sign) features highly specific for msa may provide important clues for a correct and early diagnosis. they include orofacial dystonia; inspiratory signs, contractures of hands and feet, jerky myoclonic postural/action tremor, polyminimyoclonus, severe dysphonia and dysarthria, pathological laughter and crying, snoring, disproportional antecollis, camptocormia and/or pisa syndrome, and cold hands and feet [225, 229] (table 2). in addition, severe disability milestones include: frequent falls, use of urinary catheters, wheelchair dependence, unintelligible speech, cognitive impairment, severe dysphagia, and residential care. in a recent clinicopathological study of 203 clinically diagnosed msa patients, a lifetime recorded number of red flags in both msa-p and msa-c was compared to lbd and psp [225]. recognition of patients with early or possible msa may be supported by one or more red flags, and two or more out of six had a specificity of 98.3% and a sensitivity of 84.2% [228, 229], while no differences were found in the frequencies of red flags within 3 years from disease onset between msa and msa look-alikes [225]. recent studies confirmed the validity of an eight-item pilot scale for the assessment of early msa [285]. table 2. clinical features supporting and non-supporting a diagnosis of multiple system atrophy. modified from [240]. due to the heterogeneity of clinical phenotypes and lack of specific biomarkers, it is a challenge to make a correct antemortem diagnosis of msa [286]. the sensitivity of the second consensus criteria was 41% for possible and 18% for probable msa at first clinical visit and 92% and 63% at last clinical visit, respectively [287]. in two recent brain bank studies, among patients diagnosed with msa during life, only 62% and 79% met the pathological criteria [225, 286], while 25% of patients with the diagnosis of "possible" msa had different pathological diagnoses, including pd and psp [225]. the most common misdiagnoses were dlb (13 and 14%, respectively), psp (6 and 11%) and pd (6%). autonomic failure was the leading cause of misdiagnosis in pd and dlb, and cerebellar ataxia that of misdiagnosis in psp [286]. sporadic spinocerebellar ataxia (sca) with autonomic failure can masquerade as msa-c. a study reported that 7% of patients with clinically diagnosed msa had mutations in sca genes [288]. fragile x tremor-ataxia syndrome and x-linked adrenoleukodystrophy can also be misdiagnosed as msa-c [61]. the possible explanations for the suboptimal diagnostic accuracy of the current consensus criteria for msa that saw a positive predictive diagnosis even in later disease stages from 60 to 90% [286, 287] have been recently discussed [289]. atypical msa almost all cases of msa display neuronal loss in both striatonigral and opc structures [24, 148], with only 11 of 42 cases assigned to the category of "pure" snd [155]. however, msa has a wider range of presentations, which expands the list of differential diagnoses. several subtypes of msa do not fit into the current classification [290]. "minimal change" msa is a rare aggressive form with gcis and neurodegeneration almost restricted to the sn, putamen, and locus coeruleus, thus representing "pure" snd [291-294], suggesting that gci formation is an early event and may precede neuronal loss. one patient with "minimal" msa-c showed abundant gcis in pontine nuclei, middle cerebellar peduncle and cerebellar white matter, with ncis and neuronal nuclear inclusions restricted to the pontine basis, cerebellar vermis, and inferior olivary nuclei, which were associated with neuronal loss indicating a link between both lesions in early disease [295]. neurologically normal individuals are rarely found to have gcis at autopsy as coincidental or incidental findings limited to the pons and inferior olivary nuclei with mild neuronal loss restricted to the sn, suggesting that these regions may be afflicted first in msa-p [296, 297]. the presence of gcis may represent an age-related phenomenon not necessarily progressing to overt clinical disease, classifying these cases as "incidental" or "prodromal/preclinical" msa, similar to incidental lbd [298]. young-onset msa with a mean age of 36.4 years shows more l-dopa-induced dyskinesia but less common myoclonus and pyramidal signs compared to late-onset cases. on post mortem analysis, the "minimal change" variant was more common in young-onset msa [299]. the other extreme are "benign" msa cases with prolonged survival up to 15 years in about 2-3% of patients [217, 300]. most of them showed slowly progressing parkinsonism with subsequent rapid deterioration after development of autonomic failure [301]. many of them developed motor fluctuation and l-dopa-induced choreiform dyskinesias [302, 303]. other cases of survival up to 18 years revealed extensive distribution of gcis in the cns [304]. another variant of pathological confirmed msa showed neither parkinsonism nor cerebellar symptoms [305]. an atypical case of frontotemporal lobar degeneration (ftld)-tdp type a with msa phenocopy syndrome showed severe striatal degeneration and cerebellar involvement [306], while four cases with clinical features of ftld, but without autonomic dysfunction, showed frontotemporal atrophy and severe limbic αsyn neuronal pathology with pick body-like, but tau-negative, inclusions. these cases were suggested to represent a novel subtype of ftld associated with αsyn (ftld-αsyn) [307]. rare cases in a family with pathologic hexanucleotide repeat expansions in c9orf72, a gene linked to amyotrophic lateral sclerosis, demonstrated clinical and neuroimaging features indistinguishable from msa [308], and a cerebello-brainstem dominant form of x-linked adrenoleukodystrophy presented as msa [61]. recently, rare cases of msa with transitional or diffuse dlb developing clinical features of pdd or dlb have been reported. those with neuronal loss in sn but not in striatal or opc systems with widespread gcis were considered "minimal change" msa, in which lbd was considered the primary pathology and msa as coincidental. apoe allele frequency was not different between these forms [309]. these and other subtypes should be considered in establishing a correct diagnosis of msa. cognitive impairment in msa unlike other synucleinopathies, msa has not been associated with significant cognitive impairment (ci), which has been considered an exclusion criterion for the diagnosis of msa [3]. however, a recent position statement by the neuropathology task force of the movement disorder society indicated that ci may be an under recognized feature in msa occurring in 17-47% of msa patients, while severe dementia is rare [310]. because ci has been underestimated in msa, not all patients have undergone formal cognitive assessments and, therefore, the frequency could be higher than reported in several studies. the degree of ci in msa patients ranges from mild to moderate decline and affect executive, attentional and visuospatial functions, while memory is less often impaired [197, 310-312]. ci may occur in early stages of msa, but it is generally common in advanced cases [313] and often correlates with disease duration [314]. mild cognitive impairment (mci) has been reported in up to 40% of msa-p patients, mainly characterized by frontal dysfunction [310, 315]. mild or moderate ci has been reported in 14-37% of pathologically proven msa cases [134, 286, 302, 316]. more severe and widespread cognitive dysfunction was seen in msa-p than in msa-c patients [317], probably due to prefrontal impairment [315], whereas others saw no differences in cognitive variables between the two groups [318] or more severe cognitive dysfunctions in msa-c [319]. ci has been regarded as a result of cortical and subcortical structural changes [320], frontal lobe dysfunction [321, 322], cortical dysfunction driven by focal frontostriatal degeneration [162], alterations in the corpus callosum [323], the dorsolateral prefrontal cortex network [324], or neocortical neuronal loss [159], while others have not found any differences in the severity of pathological findings between cases with and without ci [325]. recent studies indicated that nci burden in the hippocampus and parahippocampal gyrus is associated with memory impairment in msa [326]. alzheimer’s disease neuropathological changes (adnc), cerebral amyloid angiopathy (caa), and cerebrovascular lesions did not differ between cases with and without ci [325], whereas others showed a greater burden of ncis in medial temporal regions, the hippocampus or perirhinal regions [77, 197, 316, 326, 327]. adnc has been reported in only 2/35 (7%) autopsy-proven cases of msa [134], whereas two cases of combined msa and ad (braak stages iii and vi) have been reported, in which only a few neurons shared αsyn and tau [328]. a recent retrospective clinicopathological study of 48 msa patients (33 msa-p and 15 msa-c) with a mean age at death of 60.5±7.8 (range 46-82) years, reported mci in 10 cases (20.8%), in which three had associated moderate cortical tau pathology (braak i-ii), and moderate ci in seven patients (14.5%), for which six had associated cortical amyloid plaques and moderate cortical tau pathology (braak ii-iii), one had probable primary age-related tauopathy (part), and one female aged 82 years with severe dementia showed fully developed ad. cortical lewy pathology, observed in four cases, was not associated with clinical ci. 77.1% of the msa cases were free of adnc, compared to 42% in controls, while lewy pathology was higher than in the control groups (8.4%) [329]. in view of the limited data on the molecular basis of ci (and other neuropsychiatric symptoms) in msa, further studies on the pathological basis of ci in msa are needed. msa a prion-like or prion disease? the spread of αsyn pathology from one cell to another and even from one nervous structure to another has been demonstrated in vivo [11, 330-335]. this pattern, resembling prion spreading, has led to the concept of prion-like propagation of αsyn and tau [110]. self-propagation of αsyn oligomers, however, is not sufficient to declare them as prions, because they show "seeding" activity rather than infectivity of αsyn [336]. however, the applicability of the prion hypothesis in α-synucleinopathies and, in particular, msa remains controversial, since injections of brain lysated from msa patients failed to replicate the oligodendroglial αsyn pathology that is typical for msa. while studies in wild type (wt) mice provided insights into the mechanisms of oligodendroglial αsyn aggregations in msa, intracerebral inoculation studies in non-human primates to the best of our knowledge have not been performed yet. there are other challenges to the hypothesis that msa is a prion disease. first, endogenous wt αsyn is insufficient to propagate αsyn pathology; mutant αsyn is needed as a template. the transmission of αsyn "prions" to a second synucleinopathy model and their ability to propagate between two distinct mouse cell lines while retaining strain-specific properties was suggested to provide evidence that msa is a prion disease [337]. however, these and other mouse experiments have not yet explained why in msa αsyn pathology predominantly accumulates in oligodendroglia, as msa-derived αsyn does not appear to have the ability to induce strain-like cell-specific aggregates. this demonstrates that the intrinsic properties of a53t αsyn in the m83 mouse model dominate over any strain features harbored by misfolded αsyn in msa brains [9]. furthermore, gcis have never been identified in wt mouse brains inoculated with msa-derived αsyn [338]. hence, αsyn aggregates ("prionoids") derived from msa patients created a neurodegenerative pattern that is atypical for msa [336]. moreover, αsyn aggregates, the morphological hallmarks of msa, were not detected in msa-inoculated tgm83+/mice [339, 340], and no study has definitely propagated patient-derived seeds from cell-to-cell or mouse-to-mouse, or fully characterized αsyn strains from msa vs. pd [117]. the variety of seeds, animal models, and methodologies currently prevents clear conclusions regarding αsyn-related spreading and toxicity, as well as translation of preclinical findings to human disease [341]. a recent study found no evidence of binding between cellular prion protein (prpc) and αsyn oligomers, while prpc neither binds to αsyn oligomers nor mediates their detrimental effects [342]. however, there may be different species of αsyn oligomers, which have different binding capacity with prpc, and it remains possible that future studies could demonstrate that both prpc-dependent and -independent pathways could play a role in the pathogenesis of synucleinopathies [343]. accordingly, it could be possible that aggregated αsyn is potent in cross-seeding of prion protein misfolding and aggregation in vitro, producing self-propagating states that can lead to prion diseases upon serial passing in wt animals [344]. however, recent studies showed that abnormal misfolded cellular prion protein was able to efficiently propagate in the brain of animals even in the absence of αsyn, suggesting that this protein may not act as a key modulator of prion propagation. thus, αsyn may take part in this process of self-propagation but is not specifically required for sustaining prion conversion and propagation [345]. finally, gene analyses have shown that the homozygous state of positions 129 in the prnp gene is not a risk factor for msa and no variants of the prnp gene were associated with increased risk for msa [50]. review of clinical notes from patients who had died of msa showed no evidence of neurosurgical transmission [346], and studies of couples whose spouses had autopsy-confirmed pd, psp, or msa, did not suggest an increased risk of synucleinopathy development in the other spouses [347, 348]. although there is no evidence of iatrogenic or direct transmission in autopsy-confirmed msa cases, this is no evidence of absence of human transmission or misfolded proteins other than prions and β-amyloid, and further research is necessary before any conclusion can be drawn [349]. in conclusion, it seems reasonable to postulate that even if prion-like spreading in experimental systems may justify the view that the progression of neurodegeneration in msa reflects a cell-to-cell spread of pathological αsyn, this is not sufficient to define msa as classical prion disease [336]. new therapies so far there are no causative or disease-modifying treatments available for msa and symptomatic therapies are limited [35, 350]. the first-line treatment of a hypokinetic-rigid syndrome is dopaminergic treatment with l-dopa, the initial responsiveness to which has been reported in 83% of msa patients [228], but its effect is usually transient, and only 31% showed a response for a period of 3.5 years [23]. l-dopa response was observed in 42-57% of msa-p and in 13-25% of msa-c patients [220]. recent animal studies suggest that l-dopa failure can be induced by restricted lateral striatal lesions combined with dopaminergic denervation [351]. in some patients, motor fluctuations with wearing-off phenomena or off-bound dystonia were observed [352]. l-dopa-induced dyskinesias were reported in 24.7% of definite msa patients [23]. dopamine agonists are not considered a therapeutic option, as they show poor efficacy and may involve severe side effects, particularly the worsening of orthostatic hypotension [353]. for cerebellar symptoms, no efficient drug treatments are available. deep brain stimulation in msa patients showed only transient improvement of motor symptoms, but was rapidly counteracted by the occurrence of disabling symptoms [303]. non-pharmacological treatment options such as physiotherapy and occupational therapy play an important role in improving symptoms and patients' quality of life, and should be integrated into the therapeutic concept [354]. translational and novel therapeutic approaches based on the current knowledge about the pathogenesis of msa and the different findings in animal models, a number of therapeutic strategies have been proposed to target disease progression in msa [5, 15, 16]. based on the ability of αsyn to be transferred from cell to cell and to spread through the brain in a prion-like manner, inhibition of αsyn oligomerization and aggregation may constitute a promising therapeutic strategy for disease modification, and interesting efforts have been made in this direction. these include (1) αsyn inhibition, (2) αsyn degradation enhancement, (3) intervening neuroinflammation, and (4) neuronal loss. numerous randomized, placebo-controlled trials of putative disease-modifying agents have been performed including riluzole, minocyline, lithium, rifampicin, fluoxetine, rasagilin, neuroprotective mesenchymal stem cells, epigallocatechin gallate, intravenous immunoglobulins and others. although most of these treatments were efficient in cellular or animal models of msa, in human patients they showed no clinical effects [16, 341]. among drugs targeting αsyn aggregation, promesa studies on the effect of epigallocatechin gallate, a polyphenol found in green tea which reduces aggregation and toxicity of αsyn oligomers [329], did not modify disease progression [355]. among αsyn degradation enhancing compounds, rapamycin, an autophagy enhancer, showed a reduction of αsyn aggregates in some brain areas [356] in preclinical studies, and is now under clinical trial [357]. another approach concerns the possible involvement of toll-like receptor 4 (tlr4) and its selective antagonist monophosphoryl lipid a (mpla) that reduced gcis and motor deficits in mice [358]. targeting neuroinflammation, the inhibition of myeloperoxidase as well as the reduction of tnfα-dependent reactions are promising disease-modifying targets and are being clinically tested in msa patients [16]. the use of microglia inhibitors, such as minocycline, that rescues dopaminergic neurons in msa mice, and the anti-inflammatory substance fluoxetine, however, fail to change disease progression. an alternative approach was used in msa patients to target neuroinflammation by delivering intravenous immunoglobulin, but the results were inconclusive [359]. the compound fty720-mitoxy, an fda-approved immunosuppressive for multiple sclerosis, reduced parkinsonism by increasing brain-derived neurotrophic factor (bdnf), and protected movement and mitochondria in wt and cnp-αsyn mice [360]. numerous efforts have been undertaken to address neuronal loss, including bone marrow-derived mesenchymal stem cells [361, 362] and the antioxidant target of rapamycin (mtor) receptor [363], however all of these efforts have failed to slow or halt disease progression [16]. several studies have successfully proven the therapeutic potential of anti-αsyn immunotherapy by preventing αsyn spreading [5, 15]. based on the fact that active immunization of mbp mice reduced αsyn accumulation and neurodegeneration [364], two αsyn vaccines (pd03a, pd01a) were evaluated in phase i studies with msa and pd patients and showed good safety and tolerability [365, 366]. active immunization against αsyn and combination with anti-inflammatory treatment may also be promising therapeutic strategies [367, 368]. gene therapy may constitute another feasible approach to diminish os excitotoxicity and subsequent neuronal loss, but none of the used compounds demonstrated effects on disease progression and the underlying neurodegeneration [16]. new strategies targeting αsyn are in progress [16, 280, 369], based on completed or ongoing interventional trials by the msa coalition [12]. therefore, there is a strong need to clarify the pathogenic mechanisms of msa in order to develop new therapeutic strategy options, including combined approaches by targeting different msa-specific pathogenetic effects. conclusions and further outlook current evidence supports the hypothesis that misfolded αsyn contributes to os that induces a cascade of deleterious events, including proteasomal and mitochondrial dysfunctions, neuroinflammation, and energy failure that is associated with deposition of aberrant αsyn in both glia (mainly oligodendroglia) and neurons resulting in neurodegeneration and demyelination. currently, the cascade of events that underlies the pathogenesis of msa is not completely understood. recent studies using animal models that only partially replicate human pathology and the molecular dynamics of the neurodegenerative process have provided progress in our understanding of msa pathogenesis. the disease is viewed as a primary synucleinopathy with specific (oligodendro)glial-neuronal degeneration developing secondarily via the oligo-myelin-axon-neuron complex [2, 4, 370]. strong evidence against a primary neuronal pathology with the formation of gcis, resulting from secondary accumulation of pathological αsyn that may be of neuronal origin [371], is the fact that gcis are the hallmark of msa and not of pd, a disease with similar patterns of αsyn inclusions (lbs) but resulting from different strains of αsyn, differentiating the two disorders [88, 89, 106, 372]. the source of αsyn in msa and the pathogenic cascade leading to "prion-like" spreading of its strains contributing to progression of the disease need further elucidation, and there is no convincing evidence for the suggestion that msa is a prion disease. although disease-modifying treatments are currently not available, better knowledge about the molecular pathogenesis of msa derived from animal models and human post mortem experience have contributed to the development of future therapeutic strategies to target disease progression in msa. acknowledgements the author thanks mr. e. mitter-ferstl, phd, for secretarial and editorial work. funding the study was partially funded by the society for the promotion of research in experimental neurology, vienna, austria. references [1] 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[372] halliday gm (2015) re-evaluating the glio-centric view of multiple system atrophy by highlighting the neuronal involvement, brain 138:2116-9. copyright: © 2020 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. notes on the career of jacqueline mikol feel free to add comments by clicking these icons on the sidebar free neuropathology 2:25 (2021) reflections notes on the career of jacqueline mikol jacqueline mikol honorary emeritus professor, université de paris, france address for correspondence: jacqueline mikol · université de paris · france jacqueline.mikol@wanadoo.fr submitted: 7 september 2021 accepted: 13 september 2021 copyedited by: jeffrey nirschl published: 20 september 2021 https://doi.org/10.17879/freeneuropathology-2021-3532 additional resources and electronic supplementary material: supplementary material keywords: neuropathology, reflections, autobiography, euro-cns i was born in neuilly sur seine in 1936 but my parents lived in rumaucourt, a small village in the north of france (pas de calais). both were from jewish romanian descent, but they met in france. my father was a family physician and my mother, who had interrupted her medical studies before wwii, resumed them after the end of the war when i was at primary school and became a radiologist. during the war, my father was not present at home. he was first a prisoner in germany, then deported to a concentration camp (auschwitz-monowitz/flossenburg). fortunately, he managed to survive and to come back home in 1945 but after this traumatic experience, life would be never easy again. my parents did not want to have any other children. from 1940 to 1945, my mother and i had lived in beaulieu sur dordogne, in corrèze. at the time, as a little girl, i did not realize that our life was in danger and that i was very protected. this gave me some degree of naivety and the feeling, during my first decades, not to be enough prepared to the ups and downs of private and professional life. in 1956 i got married, then divorced and remarried to françois mikol (1961) who was “interne”, in the department of neurology of professor raymond garcin at salpêtrière hospital, when i was “externe”. i had with françois three children, two boys (1962, 1964) and a girl (1970) and later eight grandchildren. my children often criticized me to spend so much time at work. i had to wait for years, when one of them began to do research, to be told that when you begin an experience, you never know exactly when you are going to finish it. my husband, who became a neurologist, was nominated the head of the department of neurology of the rothschild foundation in paris. this gave me the opportunity, after 1980, to frequently collaborate with him till his retirement. i had been very admirative of my father who resumed his practice, very soon, after coming back from deportation. at the time, a physician took care of medicine treatments, fractures, childbirths, day and night. i was also impressed by the book of balzac, the country physician. so, i decided to study medicine. i began my medical studies in 1954, in paris medicine university, after one year of basic sciences referred to as “physics, chemistry and biology”. i succeeded to become “externe” (1956), “interne” of paris hospitals (1960) then “chef de clinique” in the clinic of neurological diseases in salpêtrière hospital (1966-1969). simultaneously, i was trained in pathology (medicine university: 1963-1966) and histology (science university: 1966-1967), as i had decided to become a neuropathologist. although it seems obvious by now, my training was to investigate the relationships between clinical data and histology to determine the physiopathology of the neurological diseases. having a dual clinical and biological training was then feasible, which unfortunately, is not the case anymore in france. so, i was qualified as a “neuropsychiatre” (1968) and had a research degree in human biology (grade neuropathology-1972). i began my practical training when i was in the laboratory of the department of professor garcin, writing reports on infarcts, then called softenings, and tumors. claude vital and philippe evrard and later jean de recondo were my classmates, under the supervision of professor jean lapresle. i defended my thesis in 1966 on binswanger’s encephalopathy and related forms, directed by professor garcin and professor lapresle. from 1968 to 1983, i became a researcher at inserm (institut national de la santé et de la recherche médicale). the unit was successively directed by doctor jean gruner and professor michel fardeau. during this period, i was responsible for the laboratory of the “clinique neurologique”, at salpêtrière hospital, professor boudin having replaced pr. garcin, to perform day-life practice of neuropathology (1969-1976). afterwards, i moved to groupe hospitalier lariboisière-fernand widal-saint-lazare to set up a neuropathology unit (central nervous system-nerve-muscle), in the department of pathology, at the request of dean raymond houdart, neurosurgeon, professor bernard pepin neurologist and professor jean roujeau, pathologist, my former teacher in faculty. i was alone, in charge, to ensure neuropathology analysis for a long time until 1988 when doctor marc polivka joined me. i was nominated professor of pathology in 1985. this was not an easy journey, as a biologist and a woman. during this period, i particularly studied wilson’s disease, for which professor b. pepin was a clinical expert and also prion diseases which was part of a national project (professor j. j. hauw project leader). i coordinated and participated in the writing of a monograph dedicated to csf. professor annie galian had succeeded to professor roujeau. she was specialized in gastro-enterology diseases. she was the one who encouraged me to study the gut nervous system, which must be included in the training of neuropathology. after her early retirement, i became head of the department, in october 1995. the first part of my career was mostly focused on science and the second part included some management as well. during my residencies, two events have impacted my career. michel fardeau who was responsible of muscle and nerve biopsies in the department of professor garcin went to the united states for a scientific mission. i was required to learn and to practice the surgical technique till he returns. this triggered my interest in muscle pathology. reports on ocular myopathies, carnitine deficiency, inclusion myositis, both with andrew engel, mitochondrial myopathies… will be published years later. i spent my last stage of “internat” in the department of psychiatry, in versailles hospital, headed by professor serge brion. professor brion was qualified in neurology, neuropsychiatry and neuropathology. he had a critical and insightful mind, a great deal of curiosity and a sense of humor: it was the beginning of a long-lasting collaboration. he was really my mentor when i developed my research at inserm, starting from 1968 and beyond. dementia was the main subject. serge brion with gérard guiot and michel fardeau had developed a syringe-trocar to perform cortical biopsies. at the time, to confirm the diagnosis of dementia, biopsies of cerebral cortex were sometimes prescribed. so, in addition to neuropathological cases, i began to learn transmission electron microscopy. fortunately, i was helped by technicians, to prepare grids and to operate the hitachi microscope. in addition, i also had the pleasure and opportunity to work with jacqueline-godet-guillain. i examined the cerebral biopsies of the diseases for which serge brion was recognized as an expert for creutzfeldt-jakob disease, pick’s and alzheimer diseases. the research center where i worked had been created by neurologist professor garcin, psychiatrist professor delay, and biologist professor couteaux. the main aim of this research lab was to gather researchers from different disciplines to together collaborate. pathophysiology of neuro-muscular diseases were one of the main topics of the group. the center was located at pavillon risler, a very old building in hopital de la salpêtrière. the biologists were working on the neuro-muscular junctions of the torpedo fish. the aquarium was always an object of fascination for one of my children when he or she came with me to the laboratory. when the first “open laboratory days” took place (1968), organized by bernard barataud, the president of the “myopathic association” and michel fardeau, the biologists decided that, as a medical doctor, i had to welcome visitors to whom unfortunately we had very little to show that could have a concrete impact on quality of life of patients. this was the first “telethon”. my work was complemented by an experimental and neuropathological study of the connections of the latero-dorsal (ld) nucleus of the thalamus and its relationships with the limbic system. i began to study twelve anatomo-clinical cases of vascular origin in man. serge brion had noted that in some cases of memory disturbances, not only the hippocampo-mammillo-thalamo-cingular network was involved but the ld was also modified. the experimental study was performed in non-human primates in the laboratory of robert naquet in gif sur yvette. it was shown that the ld is part of a large parieto-cingulo-parahippocampic network with multiple direct and reciprocal connections. advice was provided by professor hgjm kuypers, who asked me to come to amsterdam, to discuss the report i had sent to brain pathology. i was very grateful to him. the studies of the limbic studies had shown the prevalence of the lesions of the ld, over those of the dorso-median nucleus of the thalamus. these results were the object of a controversy upon the hypothesis of victor and adams. they were at the origin of anatomical reports on memory disturbances and the relationships between memory and hemispheric functional specialization. it should be noted that these studies were performed before the practice of functional mri which since have shown more complex networks. i also studied tissue cultures of nervous system, made in maximow slides, to study aging and consequences of extracts of pathological specimens of cortex but survival was too short. professor brion sent me to the meeting of the american association of alzheimer’s disease, created by families and chaired by princess yasmina, daughter of rita hayworth. the committee suggested to create the same type of association in france. as i was not any more a clinician, i proposed to professor françoise forette to create and to chair such an association with a scientific comity. i will remain in the committee of france alzheimer for many years. i was in charge of the diploma of normal and pathological aging (1997-2003). before, i had been in charge of the certificate of “master” of pathology (paris university). figure 1. meetings of the french society of neuropathology in tunis. upper part (from left to right): first row: j. gautron is 1st, p. dreyfus is 3rd, a. rouche is 4th, the author is 5th, m. paturneau-jouas is 6th, m. tommasi is 7th, m. m. berard-badier (blue umbrella) is 8th. second row: m. pluot is 1st, m. chevalley is 2nd, f. tomé is 3rd, r. escourolle is 4th, j. f. pellissier is 5th, m. baudrimont is 6th, p. gaspard is 7th, c. duyckaerts is 8th. background: j. f. foncin (pink umbrella) is 1st, j. c. turpin is 5th, n. baumann is 6th, a. privat is 7th, j. j. hauw is 8th. lower part: first row: m. ben hamida is 1st, his wife c. forestier is 2nd, j. flamand is 3rd, c. duyckaerts is 4th, m. baudrimont is 5th, o. robain is 6th, f. dubas is 7th, m. m. rouchoux is 10th, m. coquet is 11th. d. henin is sitting in front in the middle. second row: m. b. delisle is 3rd, f. chapon is 4th, j. lapresle is 6th, c. vedrenne is 7th, m. pluot is 9th, the author is 10th. i was elected member of the french society of neuropathology. founded as a club in 1964 (madeleine bérard-badié, sege brion, raymond escourolle, edith farkas, jean françois foncin, jean gruner, jean lapresle, gilles lyon, maurice toga, michel tommasi), it became a scientific society in 1989. i chaired the society from 1990 to 1992. the meetings of the french society, before the pandemic, took place either in paris in winter or in another city in spring, with international participation or as joint meetings with our european colleagues or extra-european colleagues, especially from canada. these meetings have created productive relationships between members of the society and allow to enjoy social life together. i have participated to concerted actions on aids chaired by professor françoise gray and prion human diseases, chaired by professor herbert budka. i would like to emphasize the importance of these concerted actions. they were the source of scientific progress and collaborative research. each of us strived to show new results. many reports and books were later published, many friendships took place and strong links were established between neuropathologists. each meeting was in a nice location and enabled to socialize in a very pleasant environment. i shall say the same for congresses and conferences. nowadays, for different reasons accelerated by covid-19 pandemic, most meetings are organized with a computer and internet systems. it is difficult to be satisfied by an orwellian evolution of neuropathology. i had the pleasure to see and to listen to the main famous colleagues, to participate in poster sessions extremely rewarding and stimulating. furthermore, to become a member of neuropathological societies, it was necessary to present at least twice. to illustrate the spirit of this community combining scientific excellence and ability to enjoy life, i shall tell a short story. as the international congress was taking place in perth, chaired by professor byron kakulas, i naively asked to professor colin masters, one of the organizers, if bali was far from australia. the answer was “about 2 hours of flight!” inspired by the question he followed up with “great idea! let us organize a small pre-meeting in bali focused on brain tumors and alzheimer’s disease, from neuropathology to molecular biology”. this enabled us to go deep into some specific scientific subjects and provided a unique opportunity to visit indonesia and to discover borobodur temple at sunrise and bali island. our small group of french lady neuropathologists (m. baudrimont, c. dumas, m. n. delisle, c. lacroix and myself) had herbert budka as improvised volunteer bodyguard. figure 2. hiv concerted action, cork, ireland. first row (from left to right): k. majtényi is 2nd, the author is 3nd, f. gray is 4th, k. keohane is 5th, i. elovaara is 6th. second row: j. artigas is 1st, f. scaravilli is 3rd, p. liberski is 4th, f. razavi is 6th. third row: p. trotot is 1st, f. morinet is 2nd, d. boche is 4th, j. bell is 6th. last row: r. gherardi is 1st, h. goebel is 2nd, a. aguzzi is 5th, h. budka is 6th. in april 1991, delegates from the national neuropathological societies of the european community met in paris as the future existence of neuropathology in europe appeared compromised. i had proposed that the meeting took place in my department as paris was easily accessible from different countries. the delegates were the following: professor dr. h. h. goebel, professor dr. w. schlote, professor dr. w. wechsler (germany), professor r. cotrufo, professor n. rizzuto, professor d. schiffer (italy), dr. d. troost (the netherlands), professor d. i. graham, dr. janice r. anderson, professor r. o. weller (united kingdom), professor f. gray, professor j. j. hauw, professor j. f. pelissier (france); dr. s. j. balogiannis (greece) had apologized. professor david graham chaired this first session and asked a representative from each national society to give an account of the practice and status of neuropathology in their country, including training, examinations and accreditation. that was pivotal for the future creation of euro-cns. figure 3. euro-cns meeting. upper part: the author and d. graham lower part (from left to right): h. goebel is 1st, the author is 2nd, n. rizzuto is 3rd. in 1992 david graham was appointed as the representative and the chairman of our group. he went to brussels and obtained to have a representation on the board of pathology. our small group worked a lot and met with other members of the european community who had become eu (european union) and efta countries (european free trade association) including austria, finland and switzerland. the confederation of the neuropathological societies or euro-cns, term proposed by roy weller, was established in june 1993, including delegates and deputies. a constitution of euro-cns was written by david graham and janice anderson in 1993 and multiple sessions involved the research inventory, the rules of training and examination, the clinical practice. a logo was created. in 1994, david graham was elected president of euro-cns to whom i succeeded, when i was organizing the vth congress of neuropathology in paris, under the auspices, for the first time, of euro-cns. to summarize a long process, in march 1997, there was an official recognition of the subspeciality of neuropathology by the management council of uems (union européenne des médecins spécialistes). it needed to be reactivated in 2003 but was rejected in 2004. in 2005, the european parliament voted that recognition must comply with the 2/5 rule, that is in 10 countries out of 25 (25 at the time). we finally never succeeded to obtain the extension of the recognition of neuropathology at european level and it only continued at a national level when previously established. we probably should have had more support from our clinical colleagues. details were reported in an overview on neuropathology in europe (2006). i would like to pay tribute to dirk troost for his instrumental role in the organization of exams and courses as well as hans goebel and to ilja huang, the essential euro-cns secretary. nowadays, neuropathology is recognized only in a few countries but euro-cns is still active, organizes courses and examinations, and patronizes every four years the european congress whose abstracts are published in clinical neuropathology. figure 4. european meeting of neuropathology. upper part (from left to right): p. kleihues is 1st, the author is 2nd, j. artigas is 3rd, g. gosztonyi is 4th. lower part: the author is 1st, roy weller is 2nd, f. gray is 3rd. after my retirement, daily life was not easy. suddenly my timetable had changed. i am not exactly a housewife having been so active professionally. i went to romania with professor h. goebel to teach neuropathology, invited by professor m. alexianu, under the auspices of euro-cns. i started the inventory of the slides of the dejerine foundation, which led me to publish historical reports about this foundation, the life of augusta dejerine-klumpke, the original slides of facioscapulohumeral myopathy and dejerine-roussy syndrome. i have updated the history of pick’s disease. fortunately, dr. j. p. deslys, director of the neuroprion sepia unit, françois jacob institute, at cea proposed to join his group to study experimental prion diseases. i was back to the lab! he wished to have someone with a clinical and experimental experience. thus, i have a very constructive exchange with the members of the team, biochemists, veterinarians, research engineers, technicians, of very different backgrounds and ages from mine. these fruitful intergenerational confrontation of concepts with a wide range of extremely diverse experiences gave me the opportunity to better understand the strains, the transmissions and to publish, recently a paper which, i believe, provides some compelling data to outstanding questions. figure 5. department of pathology hôpital lariboisière paris. the author is in the middle of the front row and m. polivka is in the last row, the 5th from left to right. i feel you cannot do this job if you are not fully engaged in the research. i had the privilege to practice neuropathology at a golden age when there were too many silos. i believe it would be difficult for me to focus exclusively on a specific disease like oncology, although i have studied a lot of glial tumors, lymphomas, pituitary adenomas now almost exclusively dependent on molecular biology. i had chosen serge brion as a mentor because he was not always looking for what was exactly in mainstream thinking. having a deep understanding of the existing knowledge is key but combining different disciplines with new way of thinking often enables to address challenging unresolved biomedical issues and go to the next level. moreover, even if the incents of administration promote overconcentration between hospitals, it is also necessary to avoid any situation of monopoly which leads to a single thought whereas diversity is always a factor of progress. next september, i shall be 85 years old. from my internship until now, i have been practicing neuropathology in a life science laboratory, which i feel a real gift and has brought me a lot of happiness. references mikol j, weller r: neuropathology in europe: an overview. clinical neuropathology: 2006-25;7-13. copyright: © 2021 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. neuronal intermediate filament inclusion disease may be incorrectly classified as a subtype of ftld-fus feel free to add comments by clicking these icons on the sidebar free neuropathology 1:9 (2020) original paper neuronal intermediate filament inclusion disease may be incorrectly classified as a subtype of ftld-fus kevin f. bieniek1, keith a. josephs2, wen-lang lin3, dennis w. dickson3 1 department of pathology & laboratory medicine, university of texas health science center, san antonio, tx, usa 2 department of neurology (behavioral neurology & movement disorders) mayo clinic, rochester, mn, usa 3 department of neuroscience (neuropathology), mayo clinic, jacksonville, fl, usa corresponding author: keith a josephs, md, mst, msc · mayo clinic · 200 1st street s.w. · rochester, mn, 55902 · usa · tel: +1-507-538-1038 · fax: +1-507-538-6012 josephs.keith@mayo.edu submitted: 02 february 2020 accepted: 05 march 2020 copyedited by: aivi t. nguyen published: 11 march 2020 https://doi.org/10.17879/freeneuropathology-2020-2639 keywords: fus, tdp-43, atypical frontotemporal lobar degeneration, nifid, electron microscope abstract background: the majority of cases of frontotemporal lobar degeneration (ftld) are characterized by focal cortical atrophy with an underlying tau or tdp-43 proteinopathy. a subset of ftld cases, however, lack tau and tdp-43 immunoreactivity, but have neuronal inclusions positive for ubiquitin, referred to as atypical ftld (aftld-u). studies have demonstrated that ubiquitin-positive inclusions in aftld-u are immunoreactive for fused in sarcoma (fus). as such, the current nosology for this entity is ftld-fus, which is thought to include not only aftld-u but also neuronal intermediate filament inclusion disease (nifid) and basophilic inclusion body disease. objective: to compare pathological features of cases of aftld-u and nifid. methods: we reviewed the neuropathology of 15 patients (10 males and 5 females; average age at death 54 years (range 41-69 years)) with an antemortem clinical diagnosis of a frontotemporal dementia and pathological diagnosis of aftld-u (n=8) or nifid (n=7). sections were processed for immunohistochemistry and immunoelectron microscopy with fus, tdp-43, and α-internexin (αinx) antibodies. results: eight cases had pathologic features consistent with ftld-fus, with severe striatal atrophy (7/8 cases), as well as fus-positive neuronal cytoplasmic and vermiform intranuclear inclusions, but no αinx immunoreactivity. five cases had features consistent with nifid, with neuronal inclusions positive for both fus and αinx. striatal atrophy was present in only two of the nifid cases. two cases had αinx-positive neuronal inclusions consistent with nifid, but both lacked striatal atrophy and fus immunoreactivity. surprisingly, one of these two nifid cases had lesions immunoreactive for tdp-43. discussion: while fus pathology remains a prominent feature of aftld-u, there is pathologic heterogeneity, including rare cases of nifid with tdp-43rather than fus-positive inclusions. introduction the pathological term frontotemporal lobar degeneration (ftld) assumes the presence of focal frontal and anterior temporal lobar atrophy(1). histologically, the majority of ftlds are pathologically classified into two broad categories of tau-positive ftld (ftld-tau) and tdp-43-positive ftld (ftld-tdp) based on the presence of tau and tdp-43 immunoreactive inclusions, respectively(2, 3). a subset of ftld cases however, lack tau and tdp-43 immunoreactivity, and instead have neuronal inclusions that are immunoreactive to ubiquitin(4). such cases have been referred to in the literature as atypical ftld with ubiquitin inclusion (aftld-u)(5). in the past decade, immunohistochemical studies have revealed that the ubiquitinated protein in cases of aftld-u is the fused in sarcoma (fus) protein(6). interestingly, there are two other relatively rare ftld pathological variants that also have neuronal inclusions that are immunoreactive to fus(7, 8). these include the entity neuronal intermediate filament inclusion disease (nifid)(9), previously known as neurofilament inclusion body disease(10), and basophilic inclusion body disease(11) that have also been referred to as the generalized variant of pick’s disease(12). as a result, aftld-u, nifid and basophilic inclusion body disease are all currently classified as subtypes of ftld-fus(3, 13). in keeping with pathologically lumping these three entities as ftld-fus, is the fact that all three subtypes of ftld-fus, particularly aftld-u and nifid, are strongly associated with a clinical presentation of the behavioral variant of frontotemporal dementia (bvftd)(13-15). basophilic inclusion body disease is more strongly associated with the juvenile form of amyotrophic lateral sclerosis(16). in addition, aftld-u and nifid have both been found to be associated with striatal atrophy on mri(17, 18). hence, aftld-u and nifid have a lot of features in common. one study has directly compared aftld-u and nifid(19). hence, little is known about which clinical and pathologically differences between the two variants may further help to distinguish them, and whether all cases of aftld-u and nifid do indeed show fus immunoreactivity. in this study, we set out to address these two unknowns in a cohort of 15 ftld cases that including aftld (n=8) and nifid (n=7). materials and methods subject selection the neuropathological databases at the mayo clinic, jacksonville, florida were queried to identify all cases of ftld that had been given a pathological diagnosis of nifid or aftld-u. a total of 15 cases were identified. all 15 cases were evaluated by a single expert neuropathologist (dwd). clinical data the medical records of all 15 cases were reviewed by one clinician with expertise in neurodegenerative diseases (kaj) to abstract demographic and clinical information. data abstracted included sex, age at onset, prominent symptoms during the disease course, family history of any neurodegenerative diseases and final clinical diagnosis prior to death. pathological methods all 15 cases underwent histologic and ultrastructural evaluation. tissue sections were stained with hematoxylin and eosin, luxol fast blue-periodic acid schiff (lfb-pas) and bielschowsky silver stains. immunohistochemical staining was performed using standard methods. the deparaffinized and rehydrated sections were steamed in distilled water for 30 min and immunostained in batches to assure consistency with a dako autostainer (dako, carpinteria, ca) using 3, 3’diaminobenzidine as the chromogen. after immunostaining, the sections were lightly counterstained with hematoxylin. the following antibodies were used: phosphorylated neurofilament (smi-31, 1:20,000; covance, berkeley, ca); ubiquitin (mouse monoclonal ubi-1, 1:40,000; encor biotechnology, alachua, fl; rabbit polyclonal ubq(20), 1:500 and rabbit polyclonal uh-19(21), 1:2,500); phospho-tau (cp13, 1:100; peter davies, albert einstein college of medicine, bronx, ny); alpha-synuclein (nacp(22), 1:3,000), alpha-internexin (1:100; encor biotechnology, alachua, fl); tdp-43 (mc2085, dr. petrucelli, 1:1500), ptdp-43 (s409/410, cosmo bio co., 1:5000) and rabbit polyclonal anti-fus (1:500; hpa008784; sigma, st. louis. mo and bethyl lab; a300-302a; montgomery, tx). the sigma antibody gave consistent and better staining and was used throughout the study. the presence or absence of motor neuron disease was assessed and defined as previously described, including stains for activated microglia(23). electron microscopy small pieces of formalin-fixed brains were immersed in 2.5% glutaraldehyde-0.1 m cacodylate buffer overnight at 4°c. after washing in buffer, they were post-fixed in aqueous 2% osmium tetroxide for 1 hr, washed and fixed in 1% uranyl acetate-50% ethanol for 30 min, followed by dehydration in 70%, 80%, 95%, 100% ethanols and propylene oxide. they were infiltrated and embedded in epon 812. thin sections were stained with uranyl acetate and lead citrate and examined in a philips 208s electron microscope fitted with a bottom-mount ccd camera (orius 831, gatan, pleasanton, ca). immunoelectron microscopy small pieces of formalin-fixed brains were dehydrated in serial washes of 30%, 50%, 70%, 90% ethanol for 10 min each, infiltrated and embedded in lr white resin. they were polymerized in a vacuum oven at 50°c for 2 days. thin sections were collected on formvar-coated nickel grids. grids were floated with section-sides down on citrate buffer, ph 6.0, in a 100°c oven for 10 min, cooled to room temperature for 15 min followed by immunogold labeling. the sigma anti-fus was used at 1:20 in pbs. results demographics and clinical data for all 15 cases are shown in table 1. there were 10 males and 5 females with median age at death of 54 years (range 41-69 years). the median disease duration was 5 years (range 3-13 years). the most common final clinical diagnosis in this series was behavioral variant of frontotemporal dementia (bvftd)(4, 24), rendered in 10 (67%) cases. for the other 5 cases, the final clinical diagnoses were corticobasal syndrome in two cases(25), and one each diagnosed with progressive supranuclear palsy(26), primary lateral sclerosis(27) and multiple system atrophy-parkinsonian type (msa-p)(28). table 1: demographic and clinical features of all 15 cases als = amyotrophic lateral sclerosis; bvftd = behavioral variant frontotemporal dementia; cbs = corticobasal syndrome ocb = obsessive compulsive behavior; psp = progressive supranuclear palsy, snd = striatonigral degeneration † previously published (josephs et al. acta neuropathol 2008; 116: 159-167) ‡ previously published (josephs et al. brain 2003; 126: 2291-2303 * previously published (josephs et al. acta neuropathol 2005; 109: 427-32) the median age of onset of the nifid cases was 52 years old (range: 41-61 years) while for aftld-u it was 54 years old (range: 42-69 years). disease duration in nifid was only 3.5 years (range: 2.0-5.0 years) and was much shorter than the median disease duration of the aftld-u group which was 10 years (range: 4-13 years) (p<0.05). the clinical diagnoses were heterogeneous in the nifid cases, with three cases (43%) diagnosed as bvftd. on the other hand, of the aftld-u cases all but one (88%) had been diagnosed with bvftd. the nifid cases were more likely to have had pyramidal tract signs and motor dysfunction compared to the aftld-u cases; myoclonic jerks and excessive startle were also observed in nifid but not aftld-u. pathological findings gross examination all 15 cases had evidence of frontal and temporal lobe atrophy, and hence all met criteria for ftld. striatal atrophy was observed in nine of the 15 cases (table 2, figure 1). table 2: nci immunohistochemical profile in 15 aftld-u and nifid cases β diffuse cytoplasmic fus staining with mini-pick body-like nci na = not able to find any neuronal intranuclear inclusions (nii) figure 1: striatal atrophy on gross examination striatal atrophy was not seen in many nifid cases (a; case 5) and was only observed in two cases (b; case 6). conversely, only one aftld-u case lacked marked striatal atrophy (c; case 14) while the rest of the aftld-u cases demonstrated severe atrophy (d; case 13) [bar: 1 cm]. light microscopy results of the light microscopic examination are shown in table 2 and figure 2. none of the 15 cases had evidence of tau deposition, and none met criteria for alzheimer’s or lewy body disease. all 15 cases were immune-reactive to ubiquitin. thirteen of the 15 cases had neuronal inclusions that were immune-reactive to fus. seven of the 15 cases had eosinophilic inclusions that were seen on hematoxylin and eosin and were immunostained for α-internexin in keeping with their pathological diagnosis of nifid. none of the remaining eight cases that had been diagnosed as aftld-u showed α-internexin immunoreactivity. seven of the eight aftld-u cases (88%) showed severe striatal atrophy with fus-positive neuronal cytoplasmic and vermiform intranuclear inclusions. of the seven nifid cases, five cases had neuronal inclusions that were immunoreactive to fus. unlike in aftld-u, striatal atrophy was only present in only 2/7 (29%) of the nifid cases, both of which were fus positive. hence, neither of the two fus negative nifid cases had striatal atrophy. therefore, 9/13 cases with fus immunoreactive inclusions showed striatal atrophy while 0/2 without fus showed striatal atrophy. surprisingly, one of the two nifid cases without fus immunoreactivity had lesions that were immunoreactive for both α-internexin and tdp-43 (figure 3). figure 2: alpha-internexin and fus pathology hematoxylin and eosin (a/e/i/m), α-internexin (b/f/j/n) and fus (c/g/k/o) in the frontal cortex and fus (d/h/l/p) in the hippocampus of cases 4 (a-d), 5 (e-h), 7 (i-l), and 15 (m-p) [bar:100 μm] figure 3: fus negative tdp-43 positive nifid case neuronal eosinophilic (a/b) inclusions in the hippocampus (a/c/e/g) and frontal cortex (b/d/f/h) of case 3 are negative for fus (c/d) but positive for tdp-43 (e/f/h), as well as αinx (g/h). α-internexin (brown) and tdp-43 (blue) colocalizes on double-labeling immunohistochemistry (h)[bar:100 μm] electron and immunoelectron microscopy results of the electron microscopic examination are shown in figure 4. in the fus-positive nifid cases, we found that fus was localized to loose granulofilaments that were in close proximity to intermediate filament inclusions that contained tightly packed uncoated filaments unlabeled by fus antibody. the two types of filaments did not mix. the compact intermediate filament inclusions were similar to those we previously reported(29). in the tdp-positive nifid case (#3), the tdp-positive ncis were composed of granulofilaments in tightly packed bundles or loose orientations. cytoplasmic organelles, e.g. mitochondria, were occasionally encompassed by these inclusions. figure 4: electron microscopy variable α-internexin (a/d/g), fus (b/e/h), and tdp-43 (c/f/i) immunoreactivity on electron microscopy in the neuronal inclusions of cases 3 (a-c), 2 (d-f), and 9 (g-i)[bar: 2 μm main; 0.3 μm inset] these granulofilaments were not labeled with fus. the characteristic compact intermediate filament inclusions were not as widespread as the tdp-positive inclusions in this case. importantly, they were located in separate neurons. in addition to their ultrastructural difference from the tdp-positive inclusions, these intermediate filament inclusions were not labeled by tdp-43. immunohistochemistry showed that the intermediate filament inclusions were immuno-negative for fus. the tdp-negative, fus-negative nifid case (#4) had compact intermediate filament inclusions similar to those described above. these compact intermediate filament inclusions were immuno-negative to fus and tdp-43. neuronal cytoplasmic inclusions in all the aftld-u cases consisted of granulofilaments inclusions in loose arrangement and all were immuno-positive for fus. discussion in this study we found pathological evidence for aftld-u to be a homogeneous entity that is strongly associated with a clinical diagnosis of bvftd, and pathologically by fus immunoreactivity and striatal atrophy. on the other hand, we found nifid to be more heterogeneous with more variable clinical presentations. furthermore, nifid does not always appear to be associated with fus immunoreactivity and is typically not associated with striatal atrophy. interestingly, we found evidence of overlap between a case that would meet criteria for ftld-tdp as well as nifid. in this study, aftld-u was a very homogeneous entity and the evidence supports aftld-u being classified as ftld-fus. from a clinical standpoint aftld-u is strongly associated with clinical features of bvftd as previously reported(14, 30) and hence should be considered in patients presenting with bvftd especially in the presence of striatal atrophy. indeed striatal atrophy has been reported in aftld-u on antemortem mri imaging(17). nifid, on the other hand, as currently defined does not appear to be as distinct an entity clinically and pathologically as aftld-u. supportive of this statement is the fact that of the seven nifid cases in this study, patients were given five different clinical diagnoses at the last evaluation prior to death. secondly, striatal atrophy, although present in two nifid case was absent in the rest. interestingly, both nifid cases with striatal atrophy showed fus immunoreactivity, one with a clinical diagnosis of bvftd. it therefore appears that striatal atrophy is predictive of fus, but fus is not necessarily predictive of striatal atrophy. regardless, nifid should also be considered in the differential diagnosis of bvftd with striatal atrophy and should suggest the presence of ftld-fus. the one feature that may be helpful in predicting ftld-fus nifid from ftld-fus aftld-u in patients with bvftd and striatal atrophy may be the rapidity of progression, with faster progression being more suggestive of ftld-fus nifid. to understand what may be happening with nifid it is worth further discussion. neuronal intermediate filament inclusion body disease is a type of ftld with previously reported clinical presenting features of bvftd, corticobasal syndrome, and motor neuron disease, especially the primary lateral sclerosis variant(10, 31, 32). typically, patients with nifid have a relatively rapidly progressive course, becoming mute and unable to ambulate, dying around 3 ½ years after onset(10). these features were observed in this cohort of seven nifid cases. from a pathological standpoint, in nifid, neuronal cytoplasmic inclusions (ncis) are easily visible on hematoxylin and eosin, show variable staining to ubiquitin and silver stains but are strikingly immunoreactive to type iv intermediate filaments, including neurofilament and α-internexin(9, 10, 33, 34). these nci, however, are not morphologically homogeneous and it has been known from the original description of nifid(10) that nci appeared to separate into two types: rounded inclusions that are similar to pick bodies, hence called ‘pick-body like (pbl) inclusions,’ and smaller more compact inclusions with a glass like appearance called ‘compact hyaline inclusions’(7, 10, 19, 29). could this nci inclusion type difference be playing any role in the heterogeneity we observed in this study? the ultrastructual analysis in our seven nifid cases shows that these two different types of nci also have different ultrastructual appearances. the pbl inclusions were ultrastructually granulofilamentous while the compact hyaline inclusions had a more tightly compact appearance. with immunoelectron microscopy we found that the granulofilamentous inclusions were immunoreactive to fus while the compact hyaline inclusions were immunoreactive to intermediate filament. interestingly all seven nifid cases had compact hyaline inclusions, and all seven showed α-internexin immunoreactivity. however, unlike in previous reports(35, 36), two of our seven nifid cases did not show fus immunoreactivity and one of these two nifid cases showed predominantly, almost exclusively, compact hyaline inclusions (case #4). this nifid case was immuno-negative to fus, suggesting that the absence of the granulofilamentous inclusions may be the explanation for the absence of fus immunoreactivity. the other fus negative case (case #3) is also unique. in this case, both granulofilamentous inclusions and compact hyaline inclusions were present. as expected, the presence of the compact hyaline inclusions was associated with α-internexin immunoreactivity. surprisingly though, the granulofilamentous inclusions in this case (case #3) showed immunoreactivity to tdp-43 but not fus. it is therefore possible that this is a case of ftld-tdp in which compact hyaline inclusions happen to also be present and hence accounts for the α-internexin immunoreactivity observed. this would not be counterintuitive since granulofilamentous morphology is the typical appearance of the ncis in ftld-tdp(37). on the other hand, we cannot exclude the possibility that this is a case of nifid with tdp-43 immunoreactivity given that tdp-43 immunoreactivity has been described in many different diseases(38). compared to our nifid cases, all eight aftld-u cases showed a homogeneous pattern of fus immunoreactivity similar to what has been previously reported(6, 8, 19, 36). in our eight aftld-u cases, ultrastructural analysis demonstrated granulofilamentous inclusions as we previously reported(4), and immunoelectron microscopy revealed fus immunoreactivity. of note is the fact that compact hyaline inclusions were absent in all eight aftld-u cases. this finding would also support our hypothesis that fus immunoreactivity in nifid and aftld-u is associated with the presence of the granulofilamentous inclusions while α-internexin immunoreactivity is associated with the presence of the compact hyaline inclusions. it remains unclear however; why some granulofilamentous inclusions show exclusive fus immunoreactivity (e.g. aftld-u) and others show exclusive tdp-43 immunoreactivity (e.g. ftld-tdp). only one study to date has reported both fus and tdp-43 immunoreactivity in the same ncis(39). overall, the data from this study support the importance of fus in the pathogenesis of aftld-u and the classification of aftld-u as an ftld-fus. however, the role of fus in nifid is less clear, with evidence supporting nifid being somewhat different from aftld-u with a concern that nifid, at least not all cases, may be incorrectly classified as a subtype of ftld-fus. supporting this statement is the fact that another fus negative case with α-internexin positive inclusions has been described(40). in this other case, however, a sod1 mutation was identified and some inclusions were immunoreactive to sod1. on a different note, the presence of intermediate filament inclusions and tdp-43 immunoreactive inclusions in the same case makes one contemplate the current sub-classification of ftld into strict categories. the relationship between fus and intermediate filament is reminiscent of the relationship between alzheimer’s disease pathology and lewy body disease. alzheimer’s disease pathology can occur in isolation, as can lewy body disease, but there are instances in which both alzheimer’s disease and lewy body disease co-occur. it would be naïve to argue that when two 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update feel free to add comments by clicking these icons on the sidebar free neuropathology 3:26 (2022) review neurotrauma: 2022 update david s. priemer1-3, daniel p. perl1,2 1 the department of defense/uniformed services university brain tissue repository, bethesda, md, usa 2 department of pathology, f. edward hébert school of medicine, uniformed services university, bethesda, md, usa 3 henry m. jackson foundation for the advancement of military medicine, inc., bethesda, md, usa corresponding author: daniel p. perl · department of pathology · f. edward hébert school of medicine · uniformed services university (usu) · 4301 jones bridge rd · bethesda, md 20814 · usa daniel.perl@usuhs.edu submitted: 24 october 2022 accepted: 01 november 2022 copyedited by: shino magaki published: 15 november 2022 https://doi.org/10.17879/freeneuropathology-2022-4495 keywords: traumatic brain injury, chronic traumatic encephalopathy, tau, interface astroglial scarring, diffuse axonal injury, multidimensional mri abstract the year 2021 was highlighted by many notable advancements in the field of neurotrauma and associated neuropathology. after a thorough review of the new literature, we call attention to what we feel are among the most impactful studies and publications. in brief, 2021 was marked by published consensus papers related to the diagnosis of chronic traumatic encephalopathy (cte) and its clinical counterpart, traumatic encephalopathy syndrome. there was also progress toward our understanding of the impact of traumatic brain injury (tbi) on the general population, and how strongly cte pathology may, or may not, commonly underlie long term clinical sequelae following tbi. next, a critical new study has identified that acetylated tau protein, which has been found to be increased in the brains of alzheimer’s disease and cte patients, can be induced by tbi, is neurotoxic, and that its reduction via already-existent therapeutics is neuroprotective. there are also several important updates that pertain to military and blast tbi, particularly as they pertain to establishing causality of interface astroglial scarring. in addition, and for the first time, a specific signature for diffuse axonal injury has been identified in ex vivo tissues using multidimensional magnetic resonance imaging, providing promise for the clinical diagnosis of this lesion. finally, several important radiologic studies from 2021 have highlighted long-standing structural reductions in a number of brain regions following both mild and severe tbi, emphasizing the need for neuropathologic correlation. we end by highlighting an editorial piece discussing how tbi is portrayed in entertainment media and how this impacts public perception of tbi and its consequences. introduction through the emergence of new viral variants, and the waxing and waning of health measures taken by both governments and private industry, the covid-19 pandemic remained an obstacle to ongoing research work related to neurotrauma in the year 2021. however, a review of the published literature in 2021 reveals that interest and advancement has not been slowed, but rather appears to have increased. for example, a simple search for the term ‘traumatic brain injury’ (tbi) on pubmed reveals 5,387 results for the year 2021, which is the most of any year on record. correspondingly, a number of important papers appeared that are of considerable impact to our neuropathology-oriented readership. here, we present our admittedly biased list of highlight papers that were published in the year 2021, with commentary on the information they provided and why we chose to include them in this year’s listing. we are quick to note that a few of these papers have involved our own participation; we do not apologize for these choices, as we think others tasked with the judgement of important contributions to the field of neurotrauma would have similarly considered them. updates to neuropathological criteria for chronic traumatic encephalopathy (cte) and its proposed clinical syndrome the light that is the study of cte burned as brightly as ever in 2021, and perhaps is best represented by the publication of the highly awaited conclusions of the second national institute of neurological disorders and stroke (ninds)/ national institute of biomedical imaging and bioengineering (nibib) consensus meeting to define neuropathological criteria for cte.1 five years in the making, this paper details the results of the reconvening of the ninds/nibib consensus panel in the year 2016 to refine the diagnostic criteria for cte that were set after the first panel meeting in 2015. through the evaluation of 27 cases of different tauopathies, the panel of neuropathologists was able to identify and discern cte according to the original diagnostic criteria, with high rates of agreement while both blinded and unblinded to gross neuropathological findings and clinical histories. therefore, the panel agreed to largely uphold the original definition of the pathognomonic lesion of cte, but with some refinement that can be appreciated in the comparison below: definitions of the pathognomonic lesion for a minimal diagnosis of cte: first ninds/nibib consensus meeting (2015)2: “p-tau aggregates in neurons, astrocytes, and cell processes around small vessels in an irregular pattern at the depths of the cortical sulci.” second ninds/nibib consensus meeting (2016, published 2021): “p-tau aggregates in neurons, with or without glial tau in thorn-shaped astrocytes, at the depth of a cortical sulcus around a small blood vessel, in deeper cortical layers not restricted to subpial and superficial region of the sulcus” as can be seen, the updated definition for the pathognomonic lesion maintains that a diagnostic cte lesion must be at a cortical sulcal depth and that it must display perivascular neurofibrillary changes, but now more strongly emphasizes neuronal involvement as necessary, and details that p-tau pathology cannot be limited to the superficial aspects of the cortex. these refinements were made in the hopes that they will lead to more reliable delineation between cte and other entities, such as age-related tau astrogliopathy (artag) (see figure 1). figure 1. comparison of chronic traumatic encephalopathy (cte) and sulcal tau astrogliopathy. a and b (black bars are 900µm and 200µm, respectively): pathognomonic cte lesion with tau aggregates in neurons (and glia) around small blood vessels at the depth of a sulcus, with tau pathology not restricted to superficial cortical layers. c and d (black bars are 600µm and 200µm, respectively): in contrast, glial tau aggregates in a sulcal depth without perivascular predilection and limited to superficial cortical layers is not diagnostic for cte, but is rather most fitting of tau astrogliopathy. in addition to a refinement of cte minimal diagnostic criteria, the second ninds/nibib consensus panel on cte introduced a working protocol/workflow for the neuropathological evaluation of a brain for cte, which it hopes to be of service to neuropathologists in the community. further, the panel ambitiously proposed the first consensus-based scheme for staging the severity of cte pathology as either “low cte” or “high cte” according to a checklist of criteria. on the clinical side, the year 2021 also saw the publication of the details of the first ninds consensus workshop to define the diagnostic criteria for traumatic encephalopathy syndrome (tes),3 the clinical syndrome which is intended to correspond to cte neuropathology. the workshop was intended to improve upon the first proposed diagnostic criteria for tes that were published in 2014.4 as part of the workshop, a multispecialty panel of experts in tbi and its sequelae reviewed all published cases of neuropathologically confirmed cte, and where possible, carefully assessed predictive validity of clinical features in relation to cte pathology in 298 cases, and used a modified delphi method to agree upon a restructured, stepwise process for the clinical diagnosis of tes. in short, a diagnosis of tes foremost requires a substantial history of repetitive head impacts and cognitive and/or neurobehavioral dysregulation (core clinical features) with a progressive course unexplained by another disorder. supportive clinical features for tes which the panel concluded had insufficient predictive value to be included as core clinical features include delayed onset of symptoms, motor symptoms (e.g. parkinsonism), and relatively non-specific psychiatric features (e.g. anxiety, depression, paranoia). beyond this, the panel also agreed upon provisional criteria (for research purposes) for determining the level of certainty of cte pathology in a clinical case based on meeting tes criteria, varying levels of impact tbi history (particularly long-standing contact sports history), presence or absence of supportive clinical features as described above, and severity of clinical dementia. there is no evidence, as yet, supporting the predictive value of these clinical criteria based on subsequent neuropathology evaluation. time will judge the success of the neuropathological diagnostic criteria and staging scheme for cte, and also of the new proposed clinical criteria for tes. almost certainly, new issues will arise. however, the continued pursuits of consensus agreement in the face of rapidly evolving, and not uncommonly controversial, data are a promising sign for the future. additional consensus meetings to evaluate newly emerging data will clearly be needed. cte in the community crucial and yet unanswered questions with respect to cte concern its frequency in the general community, and its impact on members of the general public with a history of tbi (concerns largely raised by frightening media accounts). in the almost two decades since the resurgence of tbi and cte in the eyes of the public and the scientific community because of its discovery in american football players and other contact sport athletes, collection of materials and data by a number of research centers is starting to bear fruit. with the use of the national health and nutrition examination surveys (nhnes), whose participants are selected to be representative of the civilian general population, and who undergo a rather thorough interview process, physical examination, and blood and urine collection, schneider and colleagues reported a large scale analysis of 7,390 participants over the age of 40 years to determine a prevalence estimate of prior head injury with loss of consciousness (loc) and associated disability.5 of the 7,390 participants, 944 had a history of self-reported head injury with loc. of those 944 with this degree of head injury, 47.4% were noted to be living with a disability in at least one domain of functioning (e.g. activities of daily living, work limitation, memory and confusion limitation, etc.), which was significantly higher (p<0.001) than those without a history of head injury. extrapolating the data to a prevalence estimate for the general public, the study estimated that there are 11.4 million individuals above the age of 40 with a history of head injury and loc and who suffer disability in at least one domain of functioning. it should be pointed out that this figure is more than twice the current prevalence estimated for alzheimer’s disease. as neuropathologists we naturally wonder what pathologies could underlie this staggering new public health data, and perhaps many of us would suspect cte. however, evidence that has been published from a community cohort in the last year seems to suggest otherwise. in a study entitled “the delayed neuropathological consequences of traumatic brain injury in a community-based sample” published in frontiers in neurology,6 authors postupna et al. reported the neuropathological findings of 532 brains from deceased elderly individuals (average age at death: 87 years) consecutively donated to the adult changes in thought study, which itself is focused on aging and dementia in community-dwelling individuals. one hundred seven of these cases had a history of at least one remote head injury associated with loc (most participants sustained their first tbi with loc at less than 25 years of age). of the 532 cases, only 3 (0.6%) brains had diagnostic cte lesions. further, and more surprisingly, none of these 3 cases were among the 107 subjects with a history of tbi and associated loc. while it may be inappropriate to draw conclusions about the general population from this particular study, when viewed in juxtaposition with the aforementioned nhnes study, it appears that disability relating to one or even a few past instances of tbi with loc in the general community (as opposed to cohorts selected for repetitive neurotrauma, such as contact sports athletes) is not widely attributable to cte, and that different mechanisms are likely involved. future study is necessary. reducing acetylated tau is neuroprotective following brain injury the literature has long established that tbi is associated with increased risk of developing clinical alzheimer’s disease and dementia in general, albeit with extremely limited neuropathologic correlation studies.7,8 nonetheless, this suggests that tbi may potentiate or exacerbate neurodegenerative disease and therefore that neurodegenerative proteins may be a therapeutic target for the prevention of long-term cognitive sequelae of tbi. however, precise pathophysiologic links between tbi and neurodegeneration have proven rather elusive. using studies that pointed to acetylated tau as increased in cte, alzheimer’s disease, and other tauopathies as a basis for their research, shin et al. conducted a hallmark study to determine whether tbi induces acetylated tau and thus establish a potential link between tbi and neurodegenerative disease, to establish that acetylated tau is neurotoxic, and to investigate whether inhibition of tau acetylation could be neuroprotective after tbi.9 the authors used a multimodal mouse model for tbi, and showed that acetylated tau protein (acetylated at positions k263 and k270, corresponding to k274 and k281 in humans) was rapidly induced by tbi in both the mouse cerebral cortex and hippocampus, selectively in neurons, and that this was dose-dependent. further, they demonstrated that acetylated tau levels not only rose acutely in injured mice, but remained elevated for months following injury. in the same study, the authors additionally analyzed human frontal cortex specimens of elderly individuals and demonstrated that acetylated tau accumulation was significantly higher in alzheimer’s disease cases when compared to controls, and amongst the alzheimer’s disease cases acetylated tau levels were significantly higher in those who had a remote history of tbi(s) versus those who did not. in a separate component of the study, the authors sought to determine if acetylated tau was directly neurotoxic. they conducted both in vitro and in vivo genetic studies on cultured human neuroblasts exposed to acetylated tau and transgenic mice with mutations that mimic tau acetylation at k263 and k270, respectively. in the cultured human cell lines the authors found that tau acetylation specifically increased neuronal cell death, and in the transgenic mice there was a significant degree of axonal degeneration particularly in the cerebral cortex and hippocampus compared to controls after one year of life. in a translation of their work to therapeutics, the authors experimented with 3 agents that are established inhibitors of processes that promote tau acetylation or promotors of tau deacetylation: cgp3466b omigapil (gapdh nitrosylation inhibitor), salsalate (non-steroidal anti-inflammatory drug and p300/cbp acetyltransferase inhibitor), and aminopropyl carbazole p7c3-a20 (nampt activator that increases preservation of nad+). administration of all three agents blocked tau acetylation, protected against axonal degeneration, and provided significant protection from neurocognitive deficits following tbi in mice. as if the above data were not enough, the authors finally also demonstrated that acetylated tau levels in mouse plasma were elevated following tbi, establishing the potential for acetylated tau to serve as a blood biomarker for neurodegenerative risk following tbi, and noted that the aforementioned neuroprotective therapies decreased concentrations of acetylated tau in plasma following tbi. collectively this laudable study, published in cell, establishes a potential relationship between tbi and neurodegeneration in the form of acetylated tau, demonstrates that acetylated tau is neurotoxic, and provides a basis for the assessment for and protection from neurodegenerative risk after tbi. updates from the battlefield published 100 years after frederick mott’s seminal reporting of the gross neuropathologic features of acute blast exposure in the brains of world war i military personnel who had died from high explosives,10 a new entity was posited to represent a long-term neuropathological consequence of blast exposure in 2016: interface astroglial scarring (ias).11 ias describes a pattern of glial scarring, visible by glial fibrillary acidic protein (gfap) immunohistochemistry, at brain interfaces (e.g. subpial glial plate, around penetrating cortical vessels, grey-white matter junctions, and structures lining the ventricles). at the time of publication, it was suggested that injury at brain interfaces was compatible with the biophysics of blast waves passing through tissues, though this was presented without experimental data in the brain. as expected, this newly described entity was met with criticism,12 much of which was valid and could not be addressed without more study. the year 2021 brought two important publications in support of ias as a novel entity that is caused by blast exposure. in the journal of neuropathology and experimental neurology, schwerin and colleagues reported the results of a ferret model of blast exposure and provided the first animal correlate of blast-induced ias.13 ferrets were chosen because, unlike lissencephalic rodent brains with relatively limited translational capability, the brain of a ferret is more similar to humans in that it is gyrencephalic, has a high white-to-grey matter ratio and a well delineated grey-white matter junction, and has a ventrally positioned hippocampus (see figure 2). in the study, ferrets were anesthetized and exposed to compressed air shock waves, mimicking exposure to a primary blast wave, and sacrificed after survival periods of 1, 4, or 12 weeks. immunohistochemistry for gfap, particularly beginning at 4 weeks survival and in multiply exposed ferrets (four blast exposures compared to one), showed striking astrocyte immunoreactivity precisely at brain interfaces, including the subpial plate, grey-white matter junctions, and surrounding penetrating vessels, thereby reproducing the pattern of ias that was published in human blast cases. further, in demonstrating that blast exposure produces astrogliosis in a similar distribution to that seen in human blast cases, the study transversely also provides evidence that ferrets may serve as a translatable animal model for human tbi. figure 2. comparison of ferret brain with human and other animal species. though relatively small, ferret brains show striking structural similarities with that of primate and human brains, particularly relative to the brains of rodents which themselves are lissencephalic, lack clear grey-white matter interfaces, and have substantially less white matter. the biophysics of a blast wave producing stresses at brain interfaces has also now been supported using an artificial head model. in a study entitled “localizing clinical patterns of blast traumatic brain injury through computational modeling and simulation” miller et al. describe a human head model – simplified but nonetheless complete with distinct skull, cerebrospinal fluid, white and grey matter forming gyri and sulci geometrically similar to human brains, vasculature, and ventricles – which was blast-loaded at three different overpressures and from three different directions (front blast, side blast, and a more complex but true-to-life “wall blast” in which a reflecting surface was introduced for the blast wave to bounce back at the head).14 what the authors found was that strain from blast exposure within the model was most significant in perivascular regions, the subpial plate, and the periventricular regions, i.e. an interface pattern of mechanical stress from the blast wave. simply put, the investigators demonstrated, in an idealized human head model, support for the hypothesis that tbi from blast injury primarily concerns structural interfaces in the brain. further study of different forms of tbi, especially impact tbi, using this model will be critical to additional understanding and establishing its utility in the study of human tbi. a radiologic signature for diffuse axonal injury in what may come as a surprise to some neuropathologists who are quite familiar with this entity, diffuse axonal injury (dai) was purely a pathologic diagnosis without a sensitive or specific radiologic correlate prior to the year 2021. dai is essentially invisible to conventional computed tomography (ct) and magnetic resonance imagining (mri) scans. in a landmark study, authors benjamini et al. applied multidimensional mri to ex vivo samples of human brain with and without neuropathologically confirmed dai (10 cases in total).15 multidimensional mri is an emerging imaging technique that encodes multiple contrasts (e.g. t1, t2) together to provide a “multidimensional” distribution of these components combined with artificial intelligence to allow for enhanced separation of different biological elements within a heterogeneous tissue sample. using this new technique, the authors were able to identify a unique mri signature that allowed them to produce radiologic findings in tissue that precisely mirrored the distribution of app immunohistochemistry on sections cut from the same samples, and further allowed them to blindly differentiate all of the dai and non-dai cases. though this study was performed on ex vivo tissues and utilized preclinical mri technology, the authors have nonetheless provided a bedrock for the potential future clinical detection of dai and more subtle axonal injury in living patients. with further improvements of the multidimensional mri modality, studies scanning whole brains and brains in vivo, and advancements in clinical mri system technology, this imaging breakthrough may soon redefine the clinical assessment of tbi. long-term structural changes in the brain following tbi with clinical correlation there were a number of imaging studies published in 2021 that assessed the volume and/or integrity of a variety of brain structures in the subacute and chronic stages in mild tbi patients. churchill et al. describe reductions of cingulate gyrus blood flow, particularly in the posterior cingulate chronically (1 year after return-to-play) in adult contact sport athletes with a history of concussion, along with increased mean diffusivity in the corpus callosum (cc) (toward the splenium), as compared to control subjects, indicating potential long-term effects of mild tbi on these midline brain structures.16 in a related study, wang et al. conducted diffusion tensor imaging and functional mri on 42 mild tbi patients and 42 matched controls.17 in the tbi patients, they found evidence of structural impairment in the cc which expanded from the anterior-to-midbody of the cc in acute/subacute phases following tbi into both more anterior and posterior regions of the cc in chronic phase (6-12 months), with corresponding evidence of impairment in interhemispheric connectivity. further, these findings correlated to reduced executive function parameters on clinical testing of the patients. finally, authors meier et al. assessed the hippocampus via mri in a group of 106 collegiate athletes.18 they report that hippocampal volume was reduced in athletes with a history of concussion(s), in comparison to those without, and further that hippocampal volume was inversely correlated with the number of previous concussions. this was observed along with a greater association of various neuropsychiatric symptoms. as it pertains to severe tbi, authors tomaiulo et al. describe the long-term mri findings in a group of 25 patients with a history of severe non-missile tbi but without large focal lesions taken at one year and nine years following head injury. they report significant volume reduction in both grey matter (frontotemporal region, crests of gyri, amygdala, hippocampus, basal ganglia, and thalamus) and white matter (cc, fornix, parasagittal white matter, cerebral peduncles) at one year following injury.19 at nine years, there were no significant increases in grey matter reduction, but white matter reduction continued particularly in the posterior body of the cc, and in the white matter under-surface of several cerebral lobes. the above studies, all image-based, are alarming and should serve as a call to action for neuropathologists interested in studying tbi. the need to pathologically characterize and corroborate the growing myriad of long-term structural changes that occur in the brain from tbi as suggested by radiologic evaluation is obvious. in closing as you have read, there were several important developments in the field of neurotrauma in the year 2021. given the sheer volume of publications produced in the field over this time, this article could easily have continued on toward a tome’s length, and admittedly it was quite difficult to refrain from discussing more. however, we hope that you as the reader have enjoyed this summation of the literature that we chose to highlight. to conclude this writing, we would like to bring to attention a poignant short article entitled “the banality of head injury in the punisher”, written by daniel m. donaldson et al. and published in lancet neurology in 2021.20 in this article, the authors examined 26 episodes of the actionand violence-packed tv series the punisher, which focuses on an antihero from the marvel comic universe who employs vigilante, and usually lethal, methods to fight crime. in their assessment, the authors identified 125 instances of head trauma inflicted on characters in the show, including 12 inflicted on the punisher himself, and determined the severity of the tbi events (as best it could be achieved) by the glasgow coma scale. by their assessment, 62% of the head injuries depicted in the show were severe, with an initial glasgow coma scale of 3 in most cases. however, in almost all individuals who survived these severe tbi events, neurologic symptoms following the initial neurological impairment were virtually nonexistent (i.e. no abnormality in mental status after return of consciousness, no lasting neurocognitive deficits, etc.). indeed, all 12 tbi events suffered by the punisher, even when severe, resulted in no neurologic deficits following a return of consciousness. further, significant or long-term structural damage to the face/skull was not appreciated in all but 1 case, even in circumstances where there was substantial disfigurement of other parts of the body from the same general traumatic events. of course, this phenomenon is far from unique to the punisher, as consumers of action tv series and movies are well aware. instead, the authors’ analysis of this one tv show highlights how inaccurately violence, particularly tbi, is presented in media productions and thus to the public as a whole. the authors suggest that the portrayal of the severity (or lack thereof) of tbi in media may augment our perception of tbi and expected outcomes from tbi in real life, and we agree with this supposition. what can be done about this is uncertain, but as neuropathologists and tbi researchers we are uniquely suited to have a seat at the table. disclaimer the information/content, conclusions, and/or opinions expressed herein do not necessarily represent the official position or policy of, nor should any official endorsement be inferred on the part of, uniformed services university, the department of defense, the us veterans administration, the u.s. government or the henry m. jackson foundation for the advancement of military medicine, inc. conflicts of interest the authors do not have any conflict of interest to declare. references 1. bieniek kf, cairns nj, crary jf, et al. (tbi/cte research group). the second ninds/nibib consensus meeting to define neuropathological criteria for the diagnosis of chronic traumatic encephalopathy. j neuropathol exp neurol. 2021;80(3):210-219. https://doi.org/10.1093/jnen/nlab001 2. mckee ac, cairns nj, dickson dw, et al. the first ninds/nibib consensus meeting to define neuropathological criteria for the diagnosis of chronic traumatic encephalopathy. acta neuropathol. 2016;131(1):75-86. https://doi.org/10.1007/s00401-015-1515-z 3. katz di, bernick c, dodick dw, et al. national institute of neurological disorders and stroke consensus diagnostic criteria for traumatic encephalopathy syndrome. neurology. 2021;96(18):848-863. https://doi.org/10.1212/wnl.0000000000011850 4. montenigro ph, baugh cm, daneshvar dh, et al. clinical subtypes of chronic traumatic encephalopathy: literature review and proposed research diagnostic criteria for traumatic encephalopathy syndrome. alzheimers res ther. 2014;6(5):68. https://doi.org/10.1186/s13195-014-0068-z 5. schneider alc, wang d, gottesman rf, selvin e. prevalence of disability associated with head injury with loss of consciousness in adults in the united states: a population-based study. neurology. 2021;97(2):e124-e135. https://doi.org/10.1212/wnl.0000000000012148 6. postupna n, rose se, gibbons le, et al. the delayed neuropathological consequences of traumatic brain injury in a community-based sample. front neurol. 2021;12:624696. https://doi.org/10.3389/fneur.2021.624696 7. li y, li y, li x, et al. head injury as a risk factor for dementia and alzheimer's disease: a systematic review and meta-analysis of 32 observational studies. plos one. 2017;12(1):e0169650. https://doi.org/10.1371/journal.pone.0169650 8. shively s, scher ai, perl dp, diaz-arrastia r. dementia resulting from traumatic brain injury: what is the pathology? arch neurol. 2012;69(10):1245-1251. https://doi.org/10.1001/archneurol.2011.3747 9. shin mk, vázquez-rosa e, koh y, et al. reducing acetylated tau is neuroprotective in brain injury. cell. 2021;184(10):2715-2732.e23. https://doi.org/10.1016/j.cell.2021.03.032 10. mott fw. the effects of high explosives upon the central nervous system. lecture i. lancet. 1916;187(4824):331–338. https://doi.org/10.1016/s0140-6736(00)52963-8 11. shively sb, horkayne-szakaly i, jones rv, kelly jp, armstrong rc, perl dp. characterisation of interface astroglial scarring in the human brain after blast exposure: a post-mortem case series. lancet neurol. 2016;15(9):944-953. https://doi.org/10.1016/s1474-4422(16)30057-6 12. hoge cw, wolf j, williamson d. astroglial scarring after blast exposure: unproven causality. lancet neurol. 2017;16(1):26. https://doi.org/10.1016/s1474-4422(16)30342-8 13. schwerin sc, chatterjee m, hutchinson eb, et al. expression of gfap and tau following blast exposure in the cerebral cortex of ferrets. j neuropathol exp neurol. 2021;80(2):112-128. https://doi.org/10.1093/jnen/nlaa157 14. miller st, cooper cf, elsbernd p, kerwin j, mejia-alvarez r, willis am. localizing clinical patterns of blast traumatic brain injury through computational modeling and simulation. front neurol. 2021;12:547655. https://doi.org/10.3389/fneur.2021.547655 15. benjamini d, iacono d, komlosh me, perl dp, brody dl, basser pj. diffuse axonal injury has a characteristic multidimensional mri signature in the human brain. brain. 2021;144(3):800-816. https://doi.org/10.1093/brain/awaa447 16. churchill nw, hutchison mg, graham sj, schweizer ta. acute and chronic effects of multiple concussions on midline brain structures. neurology. 2021;97(12):e1170-e1181. https://doi.org/10.1212/wnl.0000000000012580 17. wang z, zhang m, sun c, et al. single mild traumatic brain injury deteriorates progressive interhemispheric functional and structural connectivity. j neurotrauma. 2021;38(4):464-473. https://doi.org/10.1089/neu.2018.6196 18. meier tb, españa ly, kirk aj, et al. association of previous concussion with hippocampal volume and symptoms in collegiate-aged athletes. j neurotrauma. 2021;38(10):1358-1367. https://doi.org/10.1089/neu.2020.7143 19. tomaiuolo f, cerasa a, lerch jp, et al. brain neurodegeneration in the chronic stage of the survivors from severe non-missile traumatic brain injury: a voxel-based morphometry within-group at one versus nine years from a head injury. j neurotrauma. 2021;38(2):283-290. https://doi.org/10.1089/neu.2020.7203 20. donaldson dm, holtmann h, nitschke j, et al. the banality of head injury in the punisher. lancet neurol. 2021;20(7):509. https://doi.org/10.1016/s1474-4422(21)00160-5 copyright: © 2022 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. neuronal transcriptome from c9orf72 repeat expanded human tissue is associated with loss of c9orf72 function feel free to add comments by clicking these icons on the sidebar free neuropathology 1:23 (2020) original paper neuronal transcriptome from c9orf72 repeat expanded human tissue is associated with loss of c9orf72 function elaine y. liu1, jenny russ1, edward b. lee1 1 translational neuropathology research laboratory, university of pennsylvania, philadelphia, pa, usa corresponding author: edward b. lee, m.d., ph.d. · 613a stellar chance laboratories · 422 curie blvd · philadelphia, pa 9104 · usa · 001 (215) 898-0908 edward.lee@pennmedicine.upenn.edu submitted: 20 july 2020 accepted: 13 august 2020 copyedited by: lauren walker published: 21 august 2020 https://doi.org/10.17879/freeneuropathology-2020-2911 additional resources and electronic supplementary material: supplementary material keywords: dementia, transcriptome, frontotemporal, amyotrophic lateral sclerosis, c9orf72, tdp-43, facs, rna-seq, repeat expansion, denn, frontotemporal lobar degeneration, frontotemporal dementia abstract a hexanucleotide g4c2 repeat expansion in c9orf72 is the most common genetic cause of familial and sporadic cases of amyotrophic lateral sclerosis (als) and frontotemporal degeneration (ftd). the mutation is associated with a reduction of c9orf72 protein and accumulation of toxic rna and dipeptide repeat aggregates. the accumulation of toxic rna has been proposed to sequester rna binding proteins thereby altering rna processing, consistent with previous transcriptome studies that have shown that the c9orf72 repeat expansion is linked to abundant splicing alterations and transcriptome changes. here, we used a subcellular fractionation method and facs to enrich for neuronal nuclei from c9orf72 repeat expanded post-mortem human als/ftd brains, and to remove neuronal nuclei with tdp-43 pathology which are observed in nearly all symptomatic c9orf72 repeat expanded cases. we show that the c9orf72 expansion is associated with relatively mild gene expression changes. dysregulated genes were enriched for vesicle transport pathways, which is consistent with the known functions of c9orf72 protein. further analysis suggests that the c9orf72 transcriptome is not driven by toxic rna but is rather shaped by the depletion of pathologic tdp-43 nuclei and the loss of c9orf72 expression. these findings argue against rna binding protein sequestration in neurons as a major contributor to c9orf72 mediated toxicity. introduction amyotrophic lateral sclerosis (als) and frontotemporal degeneration (ftd) are fatal neurodegenerative diseases with overlapping clinical, pathologic and genetic features. als is a motor neuron disease that primarily affects the upper and lower motor neurons in the motor cortex and spinal cord, respectively, whereas ftd affects the frontal and temporal lobes, thereby affecting cognition. the most common genetic cause of familial and sporadic cases of als and ftd was identified as a hexanucleotide g4c2 repeat expansion found in the gene c9orf72 (1, 2). the repeat expansion is found in the first intron of c9orf72 and can be transcribed into pre-mrna that contains the repeat. c9orf72 protein has been predicted to be a denn rab gtpase, and shown to be involved in vesicular trafficking (3-8). three potential mechanisms of toxicity have been implicated in contributing to disease: haploinsufficiency due to reduction of c9orf72 protein; rna foci and titration of rna binding proteins; and dipeptide repeat protein aggregates (reviewed in (9)). it is currently unclear which mechanism contributes most to toxicity. gain of toxic function via rna foci or dipeptide repeat proteins have been seen in cellular and animal models. indeed, several rna binding proteins have been shown to colocalize with repeat containing rna within rna foci, suggesting that rna binding protein titration may be mediating toxicity (10-12). models overexpressing dipeptide repeat proteins and/or mutant rna in both cell and animal systems have shown that the repeat expansion and dipeptide proteins may confer toxicity via a variety of pathways including nucleolar stress, nucleocytoplasmic transport defects, heterochromatin abnormalities, and dna damage (13-32). however, rna foci and dipeptide repeat proteins do not correlate with neurodegeneration in human tissues and are not toxic in some experimental models (13-16, 33-36), raising questions about the relevance of whether these pathologies contribute to disease. the loss of c9orf72 protein has been substantiated by the fact that c9orf72 expansion carriers have reduced c9orf72 mrna and protein expression (1, 2, 37, 38). thus, it is possible that loss of c9orf72 protein may contribute to disease. however, knockout mouse models of c9orf72 do not show neuronal defects but, rather, exhibit an inflammatory phenotype (39-41), although reduction of c9orf72 protein appears to exacerbate phenotypes in mice overexpressing mutant c9orf72 rna (42). we have shown that a subset of c9orf72 expansion carriers exhibit c9orf72 promoter methylation, and this is associated with reduced c9orf72 associated pathology, later age at death in ftd patients, slower rates of cerebral atrophy, and improved cognitive ability (43-45). moreover, the gnomad human genetics database indicates that missense and loss of function c9orf72 mutations are tolerated and present in control populations (46). consequently, the pathogenic role of the loss of c9orf72 protein in als/ftd is not entirely clear, and overall it remains unclear which mechanism of toxicity contributes most to disease. efforts to better understand these mechanisms include transcriptomic studies to determine whether rna dysregulation can contribute to neurodegeneration. indeed, frontal cortical and motor neuron transcriptome analysis from als patients showed splicing changes, dysregulation of rna processing pathways, and alternative polyadenylation insporadic and c9orf72 expansion carriers (47-49). more recently, a large transcriptome study of c9orf72 cases found changes in genes linked to vesicular transport (8). these studies are sometimes complicated by the complex cellular composition of brain tissue which includes neuronal subtypes, oligodendrocytes, microglia, astrocytes, vascular cells and other cell types. laser capture microdissected tissue studies have been very useful (50) but is labor intensive and often is technically difficult due to relatively low yield and preprocessing steps that can affect rna quality (51). single cell rna sequencing methods have recently addressed the issue of complex cellular mixtures within tissue but suffer from low coverage and data sparsity. we have previously reported the use of fluorescence activated cell sorting (facs) to identify transcriptome signatures in neuronal nuclei without tdp-43 from post-mortem brain in als/ftd patients (52). here, we have used the same method to determine the role of the c9orf72 repeat expansion in post-mortem neurons from als/ftd patients. compared to the profound transcriptome changes associated with the loss of normal nuclear tdp-43 protein (52), we found that the c9orf72 mutation was associated with relatively mild gene expression changes and mild splicing changes. moreover, the molecular signature is largely associated with a loss of c9orf72 function, and not a gain of toxic rna or dipeptide repeat aggregates. materials and methods clinical and pathologic assessment human autopsy tissue was obtained from the university of pennsylvania center for neurodegenerative disease research brain bank as described (53). informed consent from next of kin was obtained for every case. isolation was performed exactly as previously described (52). briefly, mid-frontal neocortex of all cases were dounce homogenized using pestil b (kimble chase) in 0.25m sucrose and adjusted to final molarity of 1.6m sucrose in tkm. the homogenate was spun on a 1.8m sucrose cushion on the beckman coulter xpn-80 ultracentrifuge at 40,000g for 40 minutes at 4°c (beckman coulter inc, indianapolis, in, usa). isolated nuclei were stained with alexa fluor 647 conjugated to 2089 (rabbit polyclonal c-terminal anti-tdp-43 antibody, center for neurodegenerative disease research, university of pennsylvania), alexa fluor 488 conjugated neun (emd millipore, billerica, ma, usa), and dapi (invitrogen, carlsbad, ca, usa). alexa fluor 647 was conjugated to 2089 according to the apex alexa fluor 647 antibody labeling kit protocol (thermo fisher scientific, waltham, ma, usa). stained nuclei were sorted for single cells containing tdp-43 and neun on the bd facsaria ii (bd biosciences, san jose, ca, usa) at 20 psi on 100µm nozzle. rna isolation and rna-seq library generation isolation and library generation was done as previously described (52). briefly, rna was extracted using the standard protocol within the allprep dna/rna micro kit (qiagen, germantown, md, usa). rna quality from sorted nuclear rna was determined based on bioanalyzer picochip analysis (agilent, santa clara, ca, usa). isolated rna was amplified, made into cdna, sheared, and libraries were made. the library was quantified using the qubit dsdna kit (invitrogen) and kapa library quantification kit (kapabiosystems, boston, ma). cdna libraries were pooled, clustered on the cbot and subject to 100 or 125 base pairs paired end reads on the hiseq 2000 or 2500 (illumina, san diego, ca, usa). pre-processing, mapping and filtering of rna-seq data rna-seq analysis was done as previously described (52). briefly, raw sequencing reads were demultiplexed through the upenn functional genomics core and analyzed for quality control using fastqc. reads were mapped to the human genome (grch38, gencode release 22) using star and only uniquely mapping reads were selected for further analysis. ribosomal and mitochondrial reads were removed and sam files were converted to bam files using samtools view and bam files were sorted by coordinate with samtools sort. creation of non-overlapping gene, exon and intron annotations annotations were generated as previously described (52). briefly, annotations were based on the comprehensive gene annotation file of the gencode release 22 (grch38.p2). gtf file was loaded into r (version 3.2.2; r core team (2015): “r: a language and environment for statistical computing”, r foundation for statistical computing, vienna, austria) and converted into a transcriptdb object. from the transcriptdb object, all annotated ensembl genes and their exons were pulled out using exonsby (by=”gene”) and ensembl gene ids were replaced by official gene symbols with biomart (version 2.26.1). genes and introns were defined as previously described in the above chapter. regions shared by overlapping genes were removed to count reads that map to one gene or to genic elements (exon or intron) from one gene. exons and introns annotations were used for subsequent analyses in the r package dexseq (1.16.10). differential gene and genic element expression analysis the genomic annotation used for read counting was done as previously described (52). the mapped, filtered rna sequencing reads were counted using a custom r script including the r packages rsamtools (1.22.0), genomicfeatures (1.22.8) and genomicalignments (1.6.3). briefly, sorted bam files were loaded into r (3.2.2) and the number of reads mapping to genes, exons or introns, was computed. genes were analyzed for differential expression using the r package deseq2 (1.10.1). analysis was used to determine differences between nuclei from both c9orf72 expansion cases and non-diseased controls (option design=~experiment in deseqdatasetfrommatrix tool where experiment is the subject id). sex effects were removed with the r package sva (3.18.0) according to the deseq2 vignette. manual annotation of gene descriptions was performed using gene descriptions on ncbi. differential genic element expression analysis was done using dexseq (1.16.10). briefly, the sorted bam files were used to count the number of reads mapping to exons and introns using the exon and intron annotation generated above and computed using findoverlaps and countsubjecthits. reads mapping to exon-intron junctions were excluded. sex was controlled for by including it in the linear model used in the analysis by adding the term “sex:exon”. the following linear models were used full model = ~sample + exon + sex:exon + experiment:exon and reduced model = ~sample + exon + sex:exon + experiment:exon and reduced model = ~sample + exon + sex:exon, where sample is the sample id and experiment the subject id. in addition, dispersions were estimated using the tool estimatedispersions with option fittype=’local’. changes in expression were significant if bonferroni-hochberg multiple testing adjusted p-values were less than 0.05. alternative splicing analysis splicing analysis was performed as previously described (52). briefly, all fastq files were trimmed to 100bp, aligned to grch38 using star; ribosomal and mitochondrial mapped reads were removed. sam files were converted to sorted bam files and rmats.3.0.9 was run using default parameters with the following options (-t paired –len 100 –c 0.05 – analysis u). significant alternative splicing events were used if bonferroni-hochberg multiple testing adjusted p-values were less than 0.05. rna binding protein clip analysis clip analysis was done as previously described (52) using published tdp-43 iclip data from sh-sy5y cells (54) and hnrnp a, a2b1, f, m, u clip data from hek293 cells (55). briefly, hg18 bowtie files were converted to fastq files, and aligned to hg38. pipeclip was run using the python script. using r, the “genomicranges” package with ‘findoverlaps’ option was used to determine which bins had rna binding protein sites. chi-square analysis was done to determine whether there was a significant enrichment of bins with rna binding protein sites within the significantly differentially used bins linked to the c9orf72 mutation. principal component analysis principal component analysis was done using the log transformed read count dataset in r with the ‘stats’ package and ‘prcomp’ function. the coordinates were retrieved and used to plot principal component 2 (pc2) vs principal component 1. to determine the relationship between pc2 vs c9orf72 expression, the coordinates of pc2 for each sample was plotted against the normalized c9orf72 expression calculated from deseq2 from each sample. pearson’s correlation was performed to determine the correlation between pc2 and c9orf72 expression. gene ontology analysis of the top 1% of all genes that contribute to pc2 was done using webgestaldt (56) with the overrepresentation enrichment analysis using the ‘geneontology’ functional database with a fdr corrected significance level < 0.05. methylation correlation methylation levels for the c9orf72 promoter in patients were determined as previously described (45). briefly, genomic dna from the cerebellum was extracted using the qiagen dneasy blood and tissue kit and subject to overnight digestion with hhai and haeiii (double-digested) or just haeiii (mock) alone. a small aliquot of dna was amplified using primers flanking the hhai cutsite within the c9orf72 promoter region using 2x faststart sybr green master (roche) on the abi steponeplus machine. the difference in cycles to threshold amplification between double and mock digested dna was calculated as methylation values. spearman’s correlation was calculated using gene counts for each gene and methylation values for each c9orf72 mutation case. r package ‘lsr’ with ‘correlate’ function (with options corr.method=”spearman” and p.adjust.method=”fdr”) using the spearman’s correlation and fdr adjusted p-values was used. only correlations from genes with hugo gene symbols were calculated and plotted against gene fold change for each gene that was calculated by deseq2. this was done using both significantly differentially expressed genes linked to the c9orf72 mutation (deseq2 fdr p-value < 0.05) and genome-wide using all expressed genes. results fluorescence activated cell sorting and rna sequencing of sorted neuronal nuclei the c9orf72 mutation leads to tdp-43 pathology which includes the formation of neuronal cytoplasmic inclusions and the loss of normal physiologic nuclear tdp-43 protein. we have previously shown that the loss of normal nuclear tdp-43 protein has large effects on the nuclear transcriptome (52). to determine whether the c9orf72 mutation leads to transcriptomic alterations independent of tdp-43 pathology, we isolated neuronal nuclei with intact tdp-43 expression. mid-frontal neocortex from 7 post-mortem c9orf72 expansion carriers and 6 neurologically normal controls were used to isolate nuclei for transcriptome-wide analysis. the autopsy cohort characteristics can be found in table 1. nuclei were immunostained for neun and tdp-43 and subjected to fac sorting to isolate neun positive, tdp-43 positive nuclei from controls (circled, fig 1a) and c9orf72 expansion cases (circled, fig 1b). rna from equal numbers of tdp-43 positive neuronal nuclei (35,000-100,000) was extracted and amplified to generate barcoded cdna libraries for 100 or 125bp paired end sequencing on the illumina hiseq 2000/2500. sequences were mapped to the human genome (gencode grch38) using star algorithms (version 2.2.4). there were 1.8 billion reads of which 1.065 billion reads mapped uniquely between the 13 libraries, with an average of 90 million uniquely mapped reads per library. these reads were filtered to remove ribosomal and mitochondrial reads and the resulting reads were used for downstream analysis to evaluate differential expression of genes, genic elements and alternative splicing. table 1: autopsy cohort characteristics. als = amyotrophic lateral sclerosis; ftld = frontotemporal lobar degeneration with tdp-43 inclusions; mnd = motor neuron disease; nl = normal; pmi = post-mortem interval; rin = rna integrity number fig 1: c9orf72 repeat expansion contributes to relatively mild transcriptome changes. flow cytometry plots of control (a) and c9orf72 expansion cases (b) with tdp-43 positive neuronal nuclei circled as collected samples. (c) principal component analysis using all expressed genes where shape denotes sex and color denotes condition. (d) principal component 2 strongly correlates with c9orf72 expression (pearson’s r = -0.8205; p=0.0006). (e) ma plot showing gene fold change as a function of mean normalized gene counts with red dots being significantly differentially expressed genes. (f) pie chart distribution of all alternative splicing events between c9orf72 expansion carriers and non-diseased controls. se=skipped exons, mxe=mutually exclusive exons, ri=retained intron, a3ss=alternative 3’ splice site, a5ss=alternative 5’ splice site. functional transcriptomic alterations associated with the c9orf72 hexanucleotide repeat expansion to verify that the variation within the dataset is linked to the mutation status, principal component analysis (pca) was performed using the read counts of the entire dataset (fig 1c). principal component #1 (pc1) explained 21% of the variation whereas pc2 explained 19% of the variation (fig 1c). based on the clustering of the dataset, sex explains the variation in pc1 while the c9orf72 repeat expansion explains the variation in pc2. indeed, pc2 strongly correlated with c9orf72 mrna expression (pearson’s r=-0.8205; p=0.0006), suggesting that c9orf72 expression is the major underlying biological variable that contributes to the variation in pc2 (fig 1d). to identify whether there were enriched pathways within the genes that contribute most to pc2, gene ontology analysis was performed using the top 1% of all expressed genes (318 genes) that contribute to pc2. one pathway that is of interest is the cytoplasmic vesicle part or cytoplasmic vesicle membrane (table 2) and is consistent with the functional role of c9orf72 as a vesicle trafficking protein and rab gtpase (3, 5-7). table 2: gene ontology (go) of genes related to top 1% of all genes that contribute to variation in pc2. previous studies have found that the g4c2 rna foci may colocalize with different rna binding proteins (rbps), suggesting that sequestration of these rbps can result in aberrant rna processing (10-12). differential gene expression analysis showed that there are 323 significantly differentially expressed genes with 202 upregulated and 121 downregulated genes linked to the c9orf72 mutation (fig 1e, supplemental table 1). similar to a previous transcriptome analysis on c9orf72 expansion carriers on the frontal cortex, there were more upregulated genes than downregulated genes within the differentially expressed genes (49). notably, the number of differentially expressed genes linked the c9orf72 repeat expansion was relatively mild compared to the massive transcriptome-wide alterations we previously described associated with tdp-43 pathology in these same cases (5,576 significantly differentially expressed genes, described in (52)). further annotation shows that these genes are generally involved in synaptic vesicle fusion or vesicle formation (table 3). additional genes related to vesicle transport and endosomal trafficking are also dysregulated. interestingly, nine out of these 11 genes are upregulated. additional genes that were upregulated were involved in protein aggregation. dnajb2 is almost exclusively expressed in neurons and has been shown to resolve tdp-43 aggregates by interacting with heat shock protein 70 (57). mgrn1 has been shown to confer cytoprotective effects in an als mouse model and can suppress chaperone associated misfolded protein aggregation and toxicity (58). given that c9orf72 expansion cases exhibit protein aggregates in the form of dipeptide repeat protein and tdp-43 inclusions, it is possible that these genes are upregulated in response to these aggregates. lastly, there were also genes that are involved in dna repair (faap20, fam175a, fancb, hmgn1) (59-62) and response to dna damage (kdm4b and kin) (63-65). annotation of these genes reveal multiple themes relevant to c9orf72 including synaptic vesicle formation, endosomal trafficking, chaperone associated protein aggregation, and dna damage (table 3). c9orf72 mutation is associated with mild splicing alterations previous studies have shown that there are abundant alternative splicing changes that are linked to the c9orf72 mutation (49). the finding of rna binding proteins that colocalize with rna foci also support the idea that rna binding protein sequestration and subsequent splicing alterations may contribute to mutant c9orf72 toxicity. to evaluate splicing changes associated with the c9orf72 mutation, rmats was used to align junction reads to annotated junctions from grch38 assembly. within our dataset, there were a total of 112 events that were significantly alternatively spliced which included 84 skipped exons (se), 9 mutually exclusive exons (mxe), 9 retained introns (ri), 8 alternative 3’ splice sites (a3ss), and 2 alternative 5’ splice site (a5ss) events (fig 1f, supplemental table 2). these events affected 111 genes, of which only 3 were also significantly differentially expressed. gene ontology analysis of 111 genes did not result in any significant enriched pathways. therefore, we observed relatively mild splicing changes associated with the c9orf72 mutation. this may be due in part attributable to technical differences where the use of nuclear rna in our study likely reduces sensitivity in terms of identifying splicing alterations. table 3: annotation of significantly differentially expressed genes with general terms relevant to c9orf72 mutation. fdr = false discovery rate. fig 2: the c9orf72 mutation is associated with differential usage of genic elements with rna binding protein sites. (a) ma plot of dexseq differentially used genic elements with red being significantly differentially used elements. (b) genic elements (5’ utr, exon, intron, 3’ utr) distribution associated with c9orf72 mutation and all expressed bins. (c) differentially used elements that are bound by tdp-43 and hnrnp (χ2=5.617; p=0.0178). c9orf72 mutation is associated with differentially used 3’ utrs altered rna binding protein function has been proposed to contribute to c9orf72 toxicity. rna binding proteins can bind to different genic elements including exons, introns and untranslated regions (utr). if rbp activity is altered via titration of these proteins, it is possible the regions that may be bound by rbps are also differentially expressed. thus, dexseq was used to determine whether the presence of the repeat expansion resulted in differential usage of genic elements. there were a total of 865 significantly differentially used elements linked to the repeat expansion affecting 791 genes. specifically, there were 610 significantly downregulated elements and 255 significantly upregulated elements (fig 2a). when the elements were annotated as either 5’ utr, exon, intron or 3’ utr, there was a significant enrichment of differentially used 3’ utrs due to the c9orf72 mutation (13.35% vs 9.78%, χ2=12.50, p=0.0004) (fig 2b). given the role of rbps in binding to 3’ utrs, we hypothesized that the significantly differentially used elements were enriched for rbp binding sites. thus, hnrnp binding sites, including that of tdp-43 and other hnrnps were identified using pipe-clip algorithms applied to published data sets (54, 55, 66). indeed, there was an enrichment of differentially used genic elements that contain tdp-43 and hnrnp binding sites (χ2=5.617, p=0.0178, fig 2c). thus, the c9orf72 expansion is associated with some differentially used genic elements with rbp binding sites. transcriptomic signature due to depletion of tdp-43 pathologic nuclei given that neurons with tdp-43 inclusions have died during the course of disease and our experimental design includes removal of neurons with tdp-43 pathology via fac sorting before rna sequencing, we next considered the possibility that the transcriptomic alterations associated with the c9orf72 mutation in this dataset would in part reflect the depletion of neurons vulnerable to tdp-43 proteinopathy. indeed, within the neocortex, tdp-43 proteinopathy often preferentially affect neurons in superficial cortical laminae (52, 67). as shown in fig 3a, neurons with tdp-43 pathology including loss of nuclear tdp-43 (white) are depleted in c9orf72 expansion cases, while control brains have the normal complement of neurons (blue, fig 3b). as a result, we hypothesized that the depletion of diseased nuclei would contribute to the transcriptomic alterations associated with the c9orf72 mutation. thus, we expected that common genes that were downregulated in c9orf72 expansion cases would be upregulated in nuclei without tdp-43 and vice versa. indeed, among the 118 common significant differentially expressed genes linked to tdp-43 loss (52) and the c9orf72 mutation, a majority of them were followed this pattern (fig 3c, supplemental table 3). a linear regression analysis showed there was a negative correlation between the gene fold change linked to tdp-43 loss and the gene fold change linked to the c9orf72 mutation (pearson’s r=-0.5708; p<0.0001). furthermore, this relationship was extended transcriptome-wide where we observed a negative correlation in all commonly expressed genes derived from both data sets (fig 3d). for example, igsf11 and pvrl3 expression, preferentially observed in superficial neocortical layers and therefore increased in neuronal nuclei without tdp-43 protein (52), is significantly reduced in the c9orf72 transcriptome. conversely, syt6 and col6a1 expression, preferentially observed in deep neocortical layers and therefore decreased in neuronal nuclei without tdp-43 protein (52), is significantly increased in the c9orf72 transcriptome. this suggested that depletion of the diseased nuclei in c9orf72 expansion cases may explain some of the transcriptomic alterations observed in c9orf72 expansion cases. fig 3: c9orf72 transcriptome is linked to depletion of tdp-43 pathologic nuclei. (a) in c9orf72 expansion cases, there are nuclei with tdp-43 (blue) and those without tdp-43 (white). for our transcriptome analysis, only nuclei with tdp-43 were collected (red box), thereby depleting nuclei without tdp-43. (b) in non-diseased controls, nuclei with tdp-43 were collected (red box). (c) gene fold changes of genes that were common significant differentially expressed linked to tdp-43 loss and the c9orf72 mutation were plotted, showing that genes that were downregulated due to the c9orf72 mutation were upregulated due to tdp-43 loss, suggesting that depletion of diseased nuclei (without tdp-43) can partially explain the transcriptomic changes observed in this dataset (pearson’s r=-0.5708, p<0.0001). (d) genome-wide gene fold changes that were expressed in both datasets linked to tdp-43 loss and c9orf72 mutation showed a similar pattern. global c9orf72 associated transcriptome changes are linked to loss of function of the c9orf72 protein current hypotheses of c9orf72 toxicity include loss of c9orf72 protein or gain of toxic rna foci and dipeptide repeat aggregates (9). while some transcriptome changes which appear to be related to c9orf72 gain of toxic function (genes associated with proteostasis and dna damage, dysregulation of transcripts with hnrnp 3’utr binding sites), other changes appeared to be linked to the loss of c9orf72 protein (genes associated with vesicle membranes and endosomal trafficking). however, at a global transcriptome level, it was unclear whether the overall changes associated with the mutation reflect changes linked to a toxic gain of c9orf72 function or the loss of c9orf72 protein. c9orf72 rna foci and dipeptide repeat protein aggregates are seen in c9orf72 repeat expansion carriers but absent in neurologically normal controls. we have also demonstrated that within c9orf72 mutation carriers, c9orf72 promoter methylation is negatively correlated with the accumulation of rna foci and dipeptide repeat protein aggregates in human brains (43-45). based on this, we developed a theoretical framework (fig 4a-d) wherein statistical analysis was performed within c9orf72 expansion cases versus statistical analysis between c9orf72 expansion cases and controls. as a theoretical example, using c9orf72 itself as a gene that contributes to the loss of c9orf72 protein, we predicted that within c9orf72 expansion carriers, methylation of the c9orf72 promoter would negatively correlate with c9orf72 expression (fig 4a). moreover, we predicted that between-group analysis would show that c9orf72 expression is downregulated compared to controls (fig 4b). this would be an example of a “concordant” gene which provides evidence that the altered gene expression is linked to the loss of c9orf72 protein. conversely, using similar logic, if a gene contributes to the gain of toxic function such as the formation of toxic rna, we would predict that within c9orf72 expansion carriers, methylation of the c9orf72 promoter would negatively correlate with toxic rna (fig 4c). however, between groups, this gene would be upregulated in c9orf72 expansion carriers compared to controls (fig 4d). this would be an example of a “discordant” gene, which provides evidence that altered expression of this gene is associated with a gain of toxic function. fig 4: c9orf72 transcriptome reflects the loss of c9orf72 function. (a) theoretical analysis within c9orf72 mutation carriers, we predict that c9orf72 promoter methylation would negatively correlate with c9orf72 expression. (b) theoretical analysis between c9orf72 mutation carriers and non-diseased controls, we predict that c9orf72 expression is downregulated compared to controls. this would be an example of a “concordant” gene that suggests this gene is linked to loss of c9orf72 protein. (c) theoretical analysis within c9orf72 mutation carriers, we predict that c9orf72 promoter methylation would negative correlate with toxic rna. (d) theoretical analysis between c9orf72 expansion carriers and non-diseased controls, we predict that toxic rna expression would be upregulated. this would be an example of a “discordant” gene that suggests this gene is linked to gain of toxic rna. (e) within the actual data, there was a negative relationship between c9orf72 expression and methylation (pearson r=-0.6958, p=0.0825). (f) c9orf72 expression is downregulated in c9orf72 expansion carriers compared to controls (t-test, p=0.0003) as predicted in genes associated with the loss of c9orf72 protein. (g) within c9orf72 mutation carriers, there was a strong correlation between the expression of syp and c9orf72 methylation (pearson r=0.8835, p=0.0083). (h) between c9orf72 expansion carriers and controls, there was a significant upregulation of syp in expansion carriers (t-test, p=0.03). (i) using significantly differentially expressed genes, the methylation correlation within c9orf72 mutation cases was plotted against gene fold change between normal c9orf72 mutation cases and controls. there were more concordant genes than discordant genes, reflecting that the transcriptome was associated with loss of c9orf72 protein. (h) genome wide analysis of methylation correlation vs gene fold change shows the same trend. using the actual data, a correlation analysis was performed comparing c9orf72 expression and methylation of the c9orf72 promoter within our c9orf72 expansion cohort (pearson’s r=-0.6958; p=0.0825) (fig 4e). moreover, between-group analysis revealed that c9orf72 expression is downregulated in c9orf72 expansion carriers compared to controls as expected (t-test, p=0.0003) (fig 4f). another example of a “concordant” gene is synaptophysin (syp), a synaptic vesicle protein. the same correlation analysis was done which showed a positive correlation within c9orf72 expansion carriers (pearson’s r=0.8835; p=0.0083), and an increase in expression in c9orf72 expansion carriers compared to controls (t-test, p=0.03) (fig 4g-h, supplemental table 4). thus, we are able to show that “concordant” genes tend to reflect genes that may be related to c9orf72 protein function. this analysis was extended to all significantly differentially expressed genes where the “within c9orf72 mutation cases” methylation correlation value was plotted as a function of “between c9orf72 cases and controls” gene fold change values (fig 4i). across all significantly differentially expressed genes, there were 267 genes found within the concordant quadrants (blue circles) compared to only 56 genes found in the discordant quadrants (red circles) (fig 4g). similar results were found when this analysis was further expanded transcriptome-wide, suggesting that the global transcriptome changes linked to the c9orf72 mutation are associated with loss of c9orf72 protein (fig 4h). many “concordant” genes were genes involved in synaptic vesicle formation (i.e. clathrin light chain b, syntaphilin, synaptophysin) and vesicle trafficking (i.e. multivesicular body subunit 12a, rab40b, member ras oncogene family). genes labeled as “discordant” include genes related to inflammatory or apoptotic processes including tec protein tyrosine kinase, tyrosine kinase, and ring finger protein 152. thus two different factors appear to shape this global c9orf72 transcriptome dataset: (1) the depletion of neurons with tdp-43 pathology prior to rna-sequencing and (2) the loss of c9orf72 function. to estimate the relative contributions of these two factors, a multivariate linear regression analysis was performed wherein significant changes in gene expression associated with the c9orf72 mutation were related to (1) the changes in gene expression we previously identified due to tdp-43 pathology and (2) the loss of c9orf72 gene expression due to methylation of the c9orf72 promoter. together, these two factors explained the majority of the variance in this dataset (r2=0.5138; βmethylation=0.838, p<2e-16; βtdp-43=-0.499, p<2e-16). discussion here we report that in using post-mortem neuronal nuclei without tdp-43 pathology from c9orf72 expansion carriers, we were able to identify transcriptome changes that link the c9orf72 mutation with loss of c9orf72 function. we found that the top 1% of genes that contribute most to changes in c9orf72 expression were involved in cytoplasmic vesicle trafficking. significant gene expression changes were linked to the c9orf72 mutation and featured upregulated genes related to synaptic transmission and endosomal/lysosomal trafficking. indeed, c9orf72 has been shown to be involved in endosomal or lysosomal trafficking and may be a rab gtpase, and recent whole tissue transcriptome studies have linked the c9orf72 mutation to changes in vesicular transport genes (3-8). nine of the 11 genes related to trafficking were upregulated, potentially reflecting a compensatory mechanism to counteract the loss of c9orf72 and loss of this trafficking related protein. further analysis correlating methylation and gene expression changes confirmed that the transcriptome changes are linked to loss of c9orf72 expression. to parse the relative contributions of c9orf72 protein loss or gain of toxic rna, a methylation correlation between methylation values and expression data was calculated. this correlation analysis indicated that the transcriptome observed in neuronal nuclei from c9orf72 expansion cases was driven in large part by the loss of c9orf72 protein as opposed to a toxic gain of function. in contrast, we observed minor splicing alterations and relatively subtle changes in terms of alterations in rna binding protein function. thus, the global alterations appear to be most reflective of a loss of c9orf72 function rather than toxic rna mediated effects. however, it should be noted that c9orf72 methylation was used as a proxy for dpr and rna foci levels. a more direct analysis of the effects of dpr or rna foci would require specific isolation of dpr or rna-foci containing neuronal nuclei which has not yet been possible using fac sorting approaches. additional future studies can also include comparisons between als/ftd cases with and without the c9orf72 mutation. genes related to dna repair either as a response to dna damage or important to employ dna repair were also dysregulated. interestingly, recent papers have shown that dna damage is activated by the c9orf72 repeat expansion in both als patients and experimental models (23, 25, 26, 30, 68). dna damage has been a consistent feature among trinucleotide repeat expansion diseases and the dysregulation of dna repair related genes is consistent with dna damage contributing to neurodegeneration (69). furthermore, alterations in proteostasis either by failure to fold proteins properly or failure to degrade proteins likely contribute to protein aggregation and are a common theme among all neurodegenerative diseases (70, 71). indeed, overexpressing proteostasis factors in animal and cell als models prevents protein accumulation and aggregation (57, 72, 73), suggesting that altered proteostasis activity contributes to neurodegeneration. thus, although the global transcriptome appears to be driven by the loss of c9orf72 function, embedded within the c9orf72 transcriptome are changes in gene expression, which may relate to the toxic functions of c9orf72. in comparing the data from previous transcriptome analyses from c9orf72 expansion carriers, several differences can be observed. previous transcriptome analyses have identified pathways involved in inflammation and defense responses among dysregulated genes in the frontal cortices from c9orf72 expansion carriers (49). within our dataset, we did not find enriched inflammatory pathways, which is likely due to the fact that only neurons were being sequenced. it is possible that because c9orf72 is expressed in microglia, c9orf72 protein function may have a larger effect on microglial populations (41). given the evidence supporting that c9orf72 is involved inflammatory processes, further cell-specific analyses may prove beneficial to better understanding the effects of mutant c9orf72 on non-neuronal cells in driving disease pathogenesis. previous studies have found large numbers of splicing changes in c9orf72 expansion carriers and found motifs within these splicing changes that correspond to hnrnp h binding (49). however, we did not detect such large changes and instead found milder splicing changes with motifs that do not correspond to any known rna foci colocalization partners (data not shown). current studies debated over the disease relevance of rna foci as some animal models overexpressing the repeat induce rna foci but do not show motor or cognitive defects (13-16). the lack of splicing changes in our dataset does not support the model wherein neuronal toxicity is linked to the titration of rna binding proteins within rna foci. overall, our findings argue against rna toxicity attributed to sequestration of rna binding proteins. other disease-related pathways, such as protein aggregation and dna repair, were observed in this dataset, supporting a potential role for dipeptide repeat protein aggregates and nuclear dna damage in disease, consistent with experimental studies showing that overexpression of dipeptide repeat proteins are toxic both in cells and in animal models (13-32). while the transcriptome appeared to be shaped in large part by the loss of c9orf72 function, it is unclear whether the loss of c9orf72 function contributes to disease and thus understanding whether these changes are functionally linked to disease pathogenesis requires additional experimental studies and validation. finally, our analysis highlights the complexities associated with molecular studies of post-mortem human tissue wherein issues related to cell identity, experimental design, and admixtures of both gain and loss of function effects can be seen concurrently. contributions and acknowledgements these studies were supported by a grant from the judith & jean pape adams charitable foundation, a clinical scientist development grant from the doris duke charitable foundation, and nih grants (t32 ag000255, r21 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this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. neuropathology through the ages – personal reflections: the golden era of neuropathology feel free to add comments by clicking these icons on the sidebar free neuropathology 1:15 (2020) reflections neuropathology through the ages – personal reflections: the golden era of neuropathology herbert budka medical university vienna corresponding author: herbert budka · medical university vienna · department of neurology · division of neuropathology and neurochemistry · währinger gürtel 18-20 · 1090 vienna, austria herbert.budka@meduniwien.ac.at submitted: 19 may 2020 accepted: 25 may 2020 copyedited by: biswa ramani published: 04 june 2020 https://doi.org/10.17879/freeneuropathology-2020-2817 the author´s cv has been attached as electronic supplementary material: cv supplementary material keywords: neuropathology, institute of neurology vienna, personal reflections contents introduction the first family schooling why medicine? why neuropathology? the early years the second family going international and something other than work the hiv onslaught a midlife crisis national and international neuropathology the prion decades success and recognition the third family zurich retirement and aftermath epilogue references addendum if i have seen farther than others, it is because i was standing on the shoulders of giants. ---isaac newton if i have not seen as far as others, it is because giants were standing on my shoulders. ---hal abelson introduction at my current age of 73, the ability to see – somewhere into the mid-distance between the quotations above – has been shaped by mentors, colleagues, students and trainees, friends and family. i started to write this since the second week of the covid-19-related lockdown. virtually nobody was seen outside, a bizarre experience that reminds me of my first memories as an infant in post-war occupied vienna when people tried to avoid public encounters, particularly with patrolling soviet soldiers. in mid-march 2020, nobody could foresee how the sars-cov2 pandemic would evolve. as my wife, an active hospital nurse, and our 12-year-old daughter at school had and have some risk to get infected, we agreed to temporarily separate. now i stay in self-imposed isolation with our dog gorry in a small rented apartment in the beautiful vienna woods. like others who, during the present lockdown, have a chance to re-consider their way of life, i have ample time to reflect on my life and on neuropathology. i write this just based on my memories, as most of my written documents are either back in my home or have been destroyed after i retired from my directorship of the (clinical) institute of neurology, formerly neurological institute (ni, obersteiner institute) in vienna. without doubt, the reader will detect in these memoirs the characteristic reminiscence bump of psychology, i. e. the strongest memories date back to adolescence and early adulthood, and emotionally positive memories dominate. however, i consider my whole professional life as extraordinary privilege to have done what i enjoyed most, having made many friends and met great personalities including true giants in medicine, science and research. moreover, i believe to have witnessed the golden era of neuropathology, spanning from rather subjective interpretation of classical morphology to unprecedentedly detailed molecular diagnoses and fascinating understanding of aetiologies and pathogenesis of diseases of the nervous system. i will keep this fascination forever. the first family my family had the almost obligatory migrant background of viennese. probably my later commitment for refugees and human rights was the subconscious recognition of that fact. my father’s budka (meaning small hut or kiosk in slavic languages) family originated from the double town of cieszyn (in polish) / těšín (in czech) or teschen (in german), at the polish-czech border in silesia, then part of the austrian empire. my grandfather migrated to vienna where my father was born. i have only faint memories of my grandfather – the most vivid one is an old photograph depicting him, with a huge moustache and in a typical knee-long body jersey next to a huge and heavy iron bike, as winner of an austrian bicycling championship. another memory is my first visit with him to the prater entertainment park that had just re-opened. in a haunted house train taking us around many horrifying corners that i passed by firmly closing my eyes, a ghost took grandpa’s hat away. grandpa became very angry and tried to hit the culprit ghost with his stick. i was greatly impressed by such courage – even against ghosts! – also resulting in getting the hat back at the entrance. my mother’s panoš family had lived in olomouc (in czech) or olmütz (in german) where she was born. her father was a recognised expert of beer brewing, then as now a classical czech talent, who became employed by the then well-known nussdorf brewery in vienna. my fondness for beer seems to be another subconscious recognition of my heritage. i was born in 1946 in a small village in upper austria where my parents had to relocate after wwii bombs had destroyed their home in vienna. however, we returned to vienna when i was 3, and rented an apartment in an area where most buildings had been either razed to the ground or heavily damaged. for a boy, this was paradise: the building debris of ruins just across the street was an ideal adventure playground for all kids of the neighbourhood, including slopes for first skiing attempts and sledging, hiding places to play “doctor” and have your first smoke – not cigarettes, of course, but twigs of elder bush. retrospectively, i think we were extremely lucky not to have found one of the many explosive war relics. my father worked as a salesman, and compared to others we did relatively well in the 1950s. he was a highly talented piano and violin player, and i still retain recordings of his usual training in bach and beethoven sonatas that accompanied my school homework. moreover, we had weekly visits of friends who played string quartets or piano trios with my father. at the end of his life, he suffered from alzheimer’s disease (ad), and one striking experiment confirmed more recent findings in brain research that the musical reminiscence bump survives longest in ad and arises strong emotions: in the chronic care institution where my father finally lived, a piano was existing, and once i took my father, then already in a very advanced disease stage, had him sit down and put his hands on the keys. as in the old times, he started immediately to play flawlessly, starting with bach, then beethoven and finally switching into a more popular viennese waltz. all other patients and visitors in the hall immediately stopped to talk and listened. after some two to three minutes, my father suddenly stopped and appeared to be confused what was going on. there was thundering applause, a final salute to this great musician, and tears were running down his face. he died soon thereafter. unfortunately, i was much less gifted in musical instruments, but my father forced me to exercise every day at least for one hour, hoping to have me become a famous pianist that was his own but failed life vision. always thinking of something else more interesting during my daily practicing lessons at the piano, i can still hear him shouting corrections like “c sharp, not c” at the doorstep when he entered the house after work. it took me several years to convince him to allow me to stop playing, a decision that i regret very much now. i would love to play mozart or beethoven like i did as a child, but the rigid enforcement by my father erased or blocked all former training imprints in my brain. according to the traditional role model, my mother stayed at home and took care of my elder sister monika and me (fig. 1). moreover, she was a housewife in the famous bohemian tradition, with a cookbook that she wrote by hand after recipes told by her mother, an apparently outstanding cook. i am still hunting for that old bohemian cookbook in our family records, as i have become more and more interested in perfecting my own cooking. fig. 1. the family in the mid-1950s. my father was also a good tennis player, and with tennis he was more successful to get me interested than with the piano. indeed, i became a quite good junior player who could play competitively in a tennis club and even in the austrian boy championship (age bracket 12-14) where i was able to get into the quarterfinals. the tennis league tournament between clubs always included one slot for a junior (up to an age of 18). unfortunately, i was the only junior of my club interested to play competitively, and this already at an age between 12 and 14. at that early age, i already had to compete with 18-year-old players. i mostly lost such matches, as my competitors were much taller and shot much stronger serves than i was able to do. this was one, but not the only, reason why i increasingly lost interest in tennis; the other was my awakening interest in girls that kept me more and more occupied in the later teenage years. schooling my primary school, then of course gender-separated, was also in the heavily damaged quarter where we lived first in vienna, and i was fortunate to get a highly motivated and skilled teacher, mr. pokorny. i had no problems at all at school; already in kindergarten i had learnt unintentionally everything by listening and looking how my sister in her first class learnt to read and write. later, i just listened during school lessons and remembered everything, without a need to repeat. at my age of 11, after the first year in a municipal high school, my father was so proud of my school performance to have me switching to an élite gymnasium, the theresianum that was founded in the 18th century by empress maria theresia for the higher education of kids (of course only boys!) of aristocrats and public officials. at the time, it just re-opened again after wwii with a newly composed team of qualified teachers. it was a private boarding school where about one half of the kids – the interns – lived and slept there and had only occasional permission to visit their families elsewhere in the country. the other half including me – the half-interns – stayed there for the day and went back to the family in the evening, then a highly unusual schooling schedule. focus was on languages: starting with english at age 10, latin was added after two years, french after two more years, and finally russian after one more year. apparently subconsciously, i decided that three foreign languages should be enough – my schoolmates obviously felt the same –, and took russian quite lightly. now i would be most happy if i could read tolstoy in the original version, but all what has remained is the cyrillic alphabet and a beautiful poem by lermontov that we had to learn by heart. we had opportunities to practice all types of sports, including soccer and athletics fields in the huge park, and swimming in an indoor pool. so every early afternoon we rushed out for about two hours, mostly for playing soccer; this was a feeder for my lifelong love for sports and physical exercise in general. however, we also had some 2 or 3 hours of obligatory study time in the late afternoon. we were about 25 boys in our class. while most teachers were real experts in their field, some were less competent in paedagogic terms and even helpless in front of a bunch of merciless juveniles. the same went for the social knit among classmates – there were the usual fluctuating cliques that bullied outsiders. now, after more than five decades, i still feel ashamed not to have intervened then and would want to apologise to bullied classmates and teachers. personally, i tried to steer clear of cliques without becoming an outsider, a habit of independence that i have cherished through my whole life. in 1964, at 18, we had our final exam (matura, abitur) where i got excellent marks. our class quickly distributed to all corners of the world, and i met most classmates again only later at reunions after 50 and 55 years. some proved to have learnt little from life, playing the same clique stereotypes as during their teenage years. astonishing but probably not so unusual. why medicine? we had nobody with a medical background in the family. however, since i was some 10 years old, it was absolutely clear to me that i would become a medical doctor. i cannot remember a reason or triggering event, it was a matter of course. my career in (neuro)pathology started with the section of a sparrow who broke its neck by flying against a glass window, and that of a goldfish found dead in an aquarium. i just wanted to know how the inside looked like, and what was the very reason for death. this hunger for knowledge also accompanied my medical studies. i was totally immersed and – although i consider myself a true product of the 1968 generation in attitudes and spirit – in fact did not take much note of, nor participated in, the upheavals and demonstrations that paralysed university life in 1968. i kept this no-commitment tradition in politics for long, with one notable exception: having just graduated as an md, i participated in a demonstration by doctors for better pay and working conditions. i felt it somehow obligatory to participate, now that i was a doctor myself. a few hundred white-coated people ceremoniously marched down the famous ringstrasse with banners and slogans, but increasingly my attention was drawn to what happened between demonstrators and onlookers. it was impossible to overlook the strong emotions elicited in surrounding spectators, shouting curses at doctors who were considered privileged over common people and earning too much anyway. it was frightening indeed: a spirit of outright hatred had seized many. with a feeling how easy control might be lost, i decided to never again go to the streets, valid until today. medical study at the time had a relatively free schedule; you just had to pass some practical exercises and a defined sequence of about 20 rigorous oral examinations, rigorosa. it was up to you how quickly you proceed. an important first step was the practical course in anatomy, with careful dissection of conserved corpses donated for teaching and scientific purposes. a group of 6 to 8 students worked collectively on one corpse during most of the first year, dissecting muscles, nerves and blood vessels in the smelly atmosphere of the phenol-formol fixative. we also had a special brain anatomy course with some dissection work that i found drab and uninspiring. thank god, this impression did not last. sitting for long daily hours around the corpses to study and dissect, it was the place to get familiar and make lifelong friends like i did, or, for many, to find partners to marry. the second year included histology, and i soon realised that i had some talent for that. i even gave a crash course to my best friend who initially was unable to recognise anything in microscopic preparations other than coloured spots. later he became a radiologist – maybe he unconsciously fled into a predominantly black-and-white world. after two and a half year, students switched to the clinical phase, starting with pathologic anatomy, pharmacology and then all major clinical disciplines. i became aware that i had no favourite field at the time. in contrast, many mates were confident where to specialise. at exams, there were only three marks given, excellent, sufficient, and fail, and it was good or bad luck which examiner you got. i can remember almost every single rigorosum. the first one was in medical physics, after the first year. we were a group of no less than 12 examinees, and it was surprising to me that i got a “sufficient” already after a single question that i answered satisfactorily. i did not consider it relevant enough to make an effort to improve the mark. the next exam was in chemistry, with a professor who was probably the most feared examiner of the medical faculty. he was reportedly proud of having a low pass rate, particularly with female students, in my memory the only gender bias that i encountered when studying. my first question was about coenzyme a, a large molecule that i could describe quite well. then it was me who asked whether i should draw the formula – the professor reacted surprised and agreed. i did it perfectly but was unsure if the professor liked it or not. after quite some more demanding questions, he gave me an “excellent”, my first one in a row until the end, as i could manage to pass all subsequent rigorosa with the best mark. this appeared to be relevant; i had been told that i could become a candidate for a “promotio sub auspiciis praesidentis rei publicae” and be rewarded with employment in a public institution of my choice, at a time when many medical graduates were working as “guest doctors” without salary. unfortunately it finally turned out that the single lower mark in the initial physics exam did not allow such a distinction. however, i made my way without it. at summer holidays, i had some 3 months to spend until autumn when university life would start again. i had bought a small rubber boat with outboard motor and took unforgettable weeks with a group of friends on the greek island of thassos. days were filled with sunbathing, retsina and ouzo drinking, tavli playing, swimming, snorkelling and fish hunting with a handy harpoon, then still allowed in the mediterranean. we even vowed to ourselves to live only on what we would be able to hunt. however, fish were mostly lucky, as we said, in fact much too fast for us, and escaped our clumsy attempts. once we mistook a seagull for a tasty duck and were disgusted to finally have an after-feathering tiny bird to feed four hungry pals. thus the vow had to be broken soon after arrival, and we raided the next inn to engulf mountains of souvlaki meat with tsatsiki. in addition to these summer delights, i worked as tour guide during other holiday breaks. usually i had a busload of austrian tourists, mostly high school teachers, a rather demanding clientele. i was required to execute by myself the local travel organisation with hotels and restaurants, as well as to provide the explanations at sightseeing spots. of course i had to make detailed preparations and wrote my own manuscript for the whole travel schedule; thus i learnt a lot about countries like italy, france, spain, portugal, morocco, usa, mexico and guatemala. a great personal interest in everything connected with history has emerged from these activities. my favourite trips were those to the absolutely stunning sites of pre-columbian cultures in central america, in particular places like the then recently discovered palenque, tikal or teotihuacán, and the unbelievable archaeological museum in mexico city. something else that i learnt during the several weeks of staying together with up to 50 people was group dynamics and how to handle critical situations and conflicts that emerged quite regularly. christmas, easter and parts of summer holidays were spent that way over subsequent years, and i could earn enough money to buy my own car, first a fiat cinquecento and later a volkswagen beetle, something quite unusual for a student at the time. why neuropathology? while it was obvious to me since childhood to have medicine as my lifeblood, the choice of neuropathology was just by chance. my exam in neurology and psychiatry, then still combined and scheduled about half a year before graduation, was decisive for my further career. the professor, a highly respected psychiatrist with a remarkable empathic style, seemed to be pleased by my performance and asked about my future plans; if i were interested in his field, i should meet him again after the end of my study. of course i was more than delighted, as officially recognised training positions in a given speciality were then almost impossible to get, and i envisaged myself already as renowned psychiatrist. soberingly, when i came back half a year later with high expectations, he did not appear to remember and emphasized the existence of a long cue of candidates lining up for still unpaid positions. however, i was categorical in that i would never, never work without pay, as i was still living with my parents and wanted desperately to escape from that crowded setting. hearing that, he asked for my further interests and recommended to make a visit to the neurological institute (ni) where my preference should be met best, in his opinion. the ni of the medical faculty occupied a full floor in a building shared with histology at schwarzspanierstrasse, in a block where most pre-clinical institutes were located, in about mid-distance between the main university building at ringstrasse and the general hospital (akh). since ni was then not involved in teaching to medical students, i had passed by its huge glass-and-white wooden doorway and large lettering of its name many times without entering, when i was on my way to attend histology courses upstairs. none of us students had any idea what was going on behind its doors. anyway, i made an appointment with its director, professor franz seitelberger, and he welcomed me in his office, with his desk at the very end of an impressively large room. he asked about my interests, and i told him that at the moment i was reading a booklet about biological psychiatry. i read there the description of a (chromatographic, i learnt later) “pink spot” in urine that was claimed as specific for schizophrenia. wow, this was it! i was absolutely fascinated – an extremely complex psychopathology condensed into a single spot. what could be more exciting to study?! prof. seitelberger listened patiently and asked me about many additional private topics like family, hobbies, sports etc. that i considered completely irrelevant. finally he said, yes, there is a position available. while he mentioned that my pink spot fascination was in neurochemistry, an area also covered by the institute, he emphasised that i would be required to start in neuropathology, a discipline that analyses structural changes in the nervous system during disease, something absolutely basic to all studies of nervous system disorders. of course i had nothing to object, and we agreed upon my entry to the institute later that autumn. surely, i remembered to have already liked histology during my study, but wasn’t aware then which chance i would get from the very start: it would become an absolutely perfect fit for my interests and abilities – or did neuropathology choose me? looking back on my professional life, i cannot imagine something better suited to satisfy a hunger for knowledge and understanding, to experience case histories to evolve like a thriller, to feed any curiosity, to keep your inner fire burning and to help transmitting scientific fascination to others. and the best thing was that it was not only much fun and satisfaction but – very important for me as a young guy who would soon have his own family – you are even paid for it! the early years fig. 2. bust of heinrich obersteiner, having its traditional place immediately behind the glass-wooden doorway of the neurological institute (ni). my small kids occasionally visited the ni and were greatly impressed by this bust. after asking once to be allowed to tweak the stony cold nose, they did it again every time they visited. after ni’s move to the general university hospital (akh) in 1993, the statue was re-located to the honour courtyard of the university of vienna building at ringstrasse. when i started to work in the ni on oct. 1st, 1971, nobody could foresee that i would stay there uninterruptedly until my retirement, on the day exactly 40 years later, a straightforward but highly unusual curriculum vitae for a scientific researcher. i realised only later the eminence of the ni as the first multidisciplinary institution of the neurosciences in the world. founded in 1882 by heinrich obersteiner (fig. 2) and blossoming under otto marburg until the collapse of the medical faculty by the advent of nazism, it had become a model for such institutes elsewhere, from fukuoka in japan to montreal, new york and philadelphia. absolutely unique was ni’s library that contained an unrivalled treasure of neuroscience books, in particular from the 19th century. it was franz seitelberger’s merit to have re-built the ni after wwii, from practically scratch with only a single half-time position, to a nationally and internationally recognised centre of excellence when he became professor emeritus in 1986. at my entry to the ni, it was a composite of several disciplines led by distinguished scientists (fig. 3). tumour and autopsy neuropathology was led by kurt jellinger; neuromuscular neuropathology by elfriede sluga who mainly worked the ni’s own electron microscope; neuropathology of the vegetative nervous system by gustav lassmann; neurochemistry by hans (hanno) bernheimer; neurophysiology by hellmuth petsche; and neurolinguistics by karl gloning. soon another young aspiring researcher arrived, hans lassmann, to establish a lab of experimental neuropathology. fig. 3. people working at the ni in the early 1970s. 1st row, sitting from left: lassmann sen., petsche, seitelberger, jellinger, gloning; 2nd row: 4th from right, sluga; 3rd row at left auff; last row, 2nd from left budka, 4th bernheimer. unnamed persons include the indispensable workforce of technicians and secretaries. note the distinctive traditional hierarchy, as evident from the arrangement of this photograph, that then imbibed ni’s atmosphere everywhere. in the ni, i was supposed to become kurt jellinger’s assistant and, over some 3 years, was given a unique chance to familiarise with neuropathology in my very personal way, by re-examining the huge histopathological collection and compare my fresh impressions with the available written reports. this autodidactic learning style was well in agreement with both my and kurt’s self-absorbed approach to work. for about half a year, i did not really know how to appropriately re-examine an autopsy case with very large-sized microscopic sections. it took me half a day to microscope such a section, as i mainly used high magnifications and even the oil immersion objective! no wonder that i saw an astonishing new universe of structures of amazing shapes, sizes and colours. after about a couple of weeks of such microscoping, i saw something really bizarre, a rounded body with a strange and multicoloured structure. consulting not only oil immersion but also textbooks, i could not find anything that resembled this discovery. i got very excited and immediately informed prof. seitelberger who, after a shared microscoping session in his office, agreed about the novel character of this strange body. he even suggested to publish it together, and advised me to take microphotographs. of course i had no idea about microphotographing and admitted that. no problem, he replied and pointed to a small mountain of something hidden under a huge dusty cover in one corner of his room. he would do it himself, as that photomicroscope was the very best in the world. i should come back in about one week. so i asked after one week and weeks thereafter, but he always answered to have been too busy to do it. then, there was no need anymore to document the strange body: i found out by myself that it was a corpus amylaceum, an extremely common structure produced by astroglia, occasionally looking a bit unusual. as working space was scarce in and between the lab rooms crammed with instruments and equipment, i was given a desk and microscope within the lecture hall, a huge room with large windows and old-fashioned seating rows (fig. 4). already a postdoc from japan had arrived, riki okeda, a new friend who later became professor of neuropathology at tokyo medical and dental university. another close friend, ferenc garzuly who became head neurologist in szombathely, hungary, completed our trio there. it was much fun – we had small parties, riki used to play the violin, and we took everyday a nap after lunchtime snoring in the seat rows. when i recently told ferenc that i am writing up my memories, he obliged me to mention my first day in the lecture hall when i drew onto the chalkboard a cell nucleus to demonstrate a “wonderful” change, but both ferenc and riki could not detect anything unusual. yes, already on my first day i thought to see something special and difficult for others to see. moreover, according to ferenc, i must mention to have had a green metal box with some gadgets that made enough noise to prevent both of them from sleeping in the seat rows. fig. 4. the lecture hall of the ni in 1991 during an informal meeting. hans lassmann at far left, the author at right in front, thomas berger, the present director of the neurological clinic of the medical university vienna, in rows at right, 3rd from front. indeed, it was a great – and by then rather unusual – tradition to always have several postdocs at the ni, in particular from japan. this started already in the early 20th century when the famous poet saitṓ mokichi stayed for 2 years at the ni and wrote some haiku poems there, including so-called tankas that are limited to 31 syllables. one tanka was written about prof. obersteiner (translated from german to english by myself): "unexpectedly stepped the old teacher next to me with great simplicity he encouraged me." "unerwartet trat der alte lehrer neben mich mit großer einfachheit munterte er mich auf." another tanka was written about prof. marburg: "in front of my eyes stands professor marburg, whom with trembling heart i express my thanks.” "vor meinen augen steht professor marburg, dem ich mit bebendem herzen meinen dank ausdrücke" over several decades, numerous postdocs have been trained in neuropathology in the ni (a long list of names that i can remember is attached at the end of this article). in addition to many japanese colleagues (who have organised a “vienna party” whenever i made a visit to japan), we intentionally kept traditional connections to our neighbouring countries, in particular to those that were behind the iron curtain at the time, such as hungary, poland and yugoslavia. my first student was a bearded guy always wearing black spectacles, pawel p. liberski from łodz, poland, who was interested to study a rare disease named after creutzfeldt and jakob since his first visit in 1979; he became a friend and collaborator during his regular subsequent visits. two more persons to mention are takeshi kurata, professor at the nih in tokyo, with whom i published work on virus detection in brain and who also became a friend and regular visitor until today, and ichiro akiguchi, professor of neurology at the university of kyoto, another regular visitor and collaborator on the neuropathological work-up of the community-based vita study on ageing. he became a friend and invited me several times to stay for up to two weeks in kyoto, in addition to several trips that i made to all parts of that captivating country. this was to pour oil onto my fire for traditional japanese lifestyle and culture such as food and onsen baths, as well as buddhist temples, shinto shrines and amazing japanese gardens. after a couple of years, i was allowed to write histopathological reports on neurosurgical biopsies in kurt’s absence, and autopsy reports on less complicated cases, almost exclusively strokes. i believe there are few neuropathologists in the world (of course with the exception of charles miller fisher) who had the opportunity and gusto to study stroke cases in great detail from the start of their career, and my first book chapter publication was on the neuropathology of cerebrovascular diseases. i am still proud of that early article written in german in the pre-mri era, now forgotten since decades, because it comprises interesting and original data on the infarct patterns resulting from verified occlusions in the carotid-media system (fig. 5), demonstrating high interindividual variability and lack of any predictable correlation between site of infarct(s) and site of occlusion [1]. at that time, i was unaware of the importance 1) to publish in english, 2) to publish in a peer-reviewed journal rather than in an obscure multi-author book, and 3) of the relevance to consider bibliometric data such as the impact factor. fig. 5. topographical patterns of brain infarcts in 196 autopsies with verified occlusions in the carotid-media vascular supply. among the 23 patterns that highlight the eminent individual variability by site and extent of brain infarction, the percentage of the four most frequent patterns (total, subtotal, central and peripheral media infarcts, in sum 71%) are indicated in red. vascular areas: a = anterior cerebral artery, m = media, p = posterior, cha = a. choriodea anterior, cp= a. communicans posterior. modified from ref. [1]. in that early time, one of my few responsibilities was taking macroscopic photographs during brain cutting sessions, taking place every wednesday morning (fig. 6). i made several experiments to optimise that type of documentation: the best background for colour contrast was finally settled as a dark blue, and whole brains and brain slices were arranged on a glass plate some 10 cm above the background cloth, in order not to have any interfering shadows when the illumination was from both sides. fig. 6. brain cutting session with prof. seitelberger in the 1980s. note the numerous fixed brains in glass jars in the back – and the formal ties everybody wore then during work. one more responsibility was to visit the department of pathology every early morning, in order to determine which autopsy brains would be interesting and important enough to be fixed for neuropathology. at that time, autopsies still numbered more than 2000 per year, and not every brain could be fixed and examined. i still remember vividly the boss of the autopsy suite, prof. kucsko, in the beautiful old building of the institute of pathology (now the institute of brain research) where already carl von rokitansky had worked. kucsko was a gaunt chain smoker always with a cigarette butt on his lips during a whole section. he was an extremely experienced pathologist who taught me great respect for the autopsy as golden standard and one of the most difficult tasks in medicine, less with regard to skills but more to generalised knowledge, experience and “feeling”, a talent somewhat difficult to explain by words. another lesson i learnt from him is the importance of a very detailed macroscopic examination – what you do not see or at least suspect at gross dissection, you have little chance to find by microscopy. initially he had little consideration for that young guy interested only in the brain. however, once i told him that i found small cancer metastases in the brain when the body section by him had not found anything. can’t be, he snapped, and went back to the inner organs that fortunately had been fixed and were still available. he called me back in the afternoon to confirm that, indeed, no tumour was found in the body. when i stayed firm with my metastases, he went back again to the fixed organs and finally found, after 3 days of cutting almost every millimetre, a tiny scar carcinoma of the lung. after that i had some reputation with him and was free to express my wishes for neuropathology. this came in handy when kurt ordered me to freeze fresh brain slices from then relatively many creutzfeldt-jakob disease cases autopsied in vienna. i had to send them on dry ice to d. carleton gajdusek at the nih in bethesda, for transmission experiments into non-human primates. i did that without any special safety precautions that then were unheard of, and retrospectively i am not sure whether i touched these brains by bare hand or not. i was later very proud to have contributed some part, even when minuscule, to carleton’s nobel prize in 1976. kurt jellinger left the ni in 1975 for a position of head of a department of neurology in the municipal lainz hospital in vienna where he could do both clinical neurology and neuropathology. of course, this was a severe blow for the ni, but in particular for me, as suddenly all responsibilities for diagnostic neuropathology were mine. kurt was the collector type of neuropathologist who always had large case numbers available for his research. during my years with him, the number of neuropathological autopsies was as high as 600 per year, and neurosurgical biopsies numbered around 1000. only he could handle such a volume alone or with one assistant, and when he left, i – a newcomer with only 3.5 years in business – was almost alone to shoulder such tremendous quantities. i decided quickly to give priority to tumour pathology, and for several years i was unable to keep up with the flow of autopsy cases even when, by necessity, they had to be reduced to a significant extent. to lose kurt at that time was still more painful to me, as i just had entered into clinical training. traditionally all mds of the ni had training in a clinical speciality (to become facharzt or consultant), usually neurology rather than pathology. neuropathology became a speciality of its own in austria only later, a development that i could push forward to a significant extent. to become a specialist in neurology and psychiatry, i started a year of internal medicine in 1974, psychiatry in 1975, and neurology in the following 4 years. it had been agreed beforehand with the directors of the respective clinics that i would do clinical training in the mornings until noon or early afternoon, and then continue my work in the ni where i kept my official position. under these circumstances, it is not difficult to imagine what workloads i had to face when returning to the ni in the afternoon and working into the night. i soon became unsure whether i could, and should, carry on this double burden at all, a feeling of uneasiness and uncertainty that dominated the second half of my 1970s. even under these extremely difficult circumstances, i soon realised that research was pre-eminent in importance for the career. as a relative newcomer, i was not sure in which topic i should invest, in preparation for the next step in career building, the habilitation (lectureship, to become dozent). i had spent most of my year in internal medicine in haemato-oncology, then led by the very amicable prof. josef kühböck, and could witness the successes as well as complications of then modern aggressive and even prophylactic chemotherapy of leukaemia and lymphoma. in parallel, i could collect a small but very interesting and then timely series of brains of patients who unfortunately had died with such complications. thus i published “the pathology of encephalopathies induced by treatment or prophylaxis of neoplastic lesions of the nervous system”, again as a book chapter that i was proud of but became quickly forgotten; however, this time i wrote in english [2]. another potential topic that i considered for habilitation was the neuropathological substrate of collagen vascular disease, again a publication that i was very happy with but went practically unnoticed, even in an american journal [3]. finally, publication of the first description of the adult type of adrenoleukodystrophy (ald), what is now called adrenomyeloneuropathy (amn), became a highly cited paper for the first time [4]. in order to be successful, it would be particularly important to introduce to neuropathology, as performed at the ni, new investigations, such as csf cytology, and techniques such as immunohistochemistry (ihc), in addition to an interesting topic and adequate material to study. i developed csf cytology with a special sedimentation device, examining csf samples that i brought to the ni from patients with leukaemia or lymphoma. we counted the number of cells, sedimented them and made a giemsa stain, later also occasional ihc labellings. for ihc, i started with visualisation of immunoglobulins (igs), because good antibodies were then available, to be used in direct or indirect immunofluorescence. astrocytes were usually full of igs, but soon i realised that no really interesting problem was to be solved with these antibodies. fortunately, at that time larry eng had just purified glial fibrillary acidic protein (gfap) as astroglia-specific marker and raised antibodies against it, and lucien rubinstein – then with larry in stanford – had successfully made first immunostainings on brain tumours. thus the opportunity emerged to introduce ihc for gfap to the institute, at the start still with immunofluorescence, soon thereafter using ludwig sternberger’s elegant peroxidase-antiperoxidase (pap) technique. over the next decades, ihc (and to a lesser degree nucleic acid in situ hybridisation, ish) would dominate most of, if not all, my diagnostic and research work in neuropathology, in particular in neural tumours, infections and neurodegeneration. of course ihc was also essential for neuromuscular biopsies, but in the old ni they were elfriede sluga’s domain, and i entered that field only after she moved, like kurt earlier, to a position of head of a department of neurology in the municipal wilhelminen hospital in vienna. much of this work on new techniques must be credited to the indispensable help of our amazing laboratory technicians who patiently and persistently developed perfect (immuno)visualising systems in pioneer experiments: mrs. helga flicker, ni’s all-time good spirit and helping hand who virtually accompanied my whole career in vienna, mrs. irene leisser who arrived only shortly later, and from the millennial years onwards mrs. gerda ricken. while they kindly agreed to co-author the publication of some of their successes, in fact they should have been on the author ticket of all respective papers. having said this, any thanks to them would be incomplete without the recognition of work that was accomplished also by other technicians, secretaries and support staff – i was extremely lucky to have such people to work with. despite all troubles, my early years in neuropathology ended successfully, in 1980 with recognition as clinical specialist (facharzt) in neurology and psychiatry, a step that also promoted me from assistant to consultant (oberarzt), and further on in 1982 with my habilitation (lectureship) in neuropathology. the title of my habilitation thesis was “viral diseases of the nervous system” and combined 3 original papers [5-7] on viral antigens in herpes simplex virus (hsv) encephalitis, rabies and subacute sclerosing panencephalitis (sspe). i was more than happy that the exhausting time of riding two horses at the same time has ended, even when i liked clinical work very much. so it was only later, during my midlife crisis, that i considered to go back to clinical medicine. the second family the turbulent first decade of my professional life coincided with establishment of my own new family. i met christine at the csf lab of the neurological clinic where she worked as technician, and we married one year later. philipp was born in 1974, julia in 1977, daniel in 1979, and rafael in 1980. with these kids born in a short period, my wife had stayed at home and it was not that easy for me, as university assistant with a very mediocre salary, to sustain a family of 6. our financial situation improved somewhat when prof. seitelberger allowed me to make forensic expertises that are remunerated additionally. that activity turned out to be demanding but most rewarding intellectually and scientifically, and i have continued it up to this day – something that always has enabled me to keep my fascination for neuropathology alive. with all my obligations, it was very difficult to spend time with the family. i devoted all weekends and holidays to them, but during the week i used to come home from work when most if not all kids were already asleep. we tried to remedy the situation by establishment of a weekly family day when i would come home earlier, play, do storytelling and reading with the kids. nevertheless, i used to have a guilty conscience about my inability to dedicate enough time that a family with children would deserve. moreover, we always had dogs that became full family members as well, the wire-haired dachshund ilko at start, followed by polski owcarek nizinny (polish lowland sheepdog) betsy and later hovawart (“farm watcher”) debbie. although dogs snip away further bits from your time, they are beautiful and most grateful creatures enriching and rounding up any family; they love you unconditionally, whether you deserve it or not. all my kids grew up to become successful professionals and, more importantly of course, happy, amicable and easygoing personalities to be proud of: philipp became an established lecturer and researcher in social and cultural anthropology at the university of vienna and free university berlin, specialising in the sociocultural consequences of digital media and technologies, living in a harmonious partnership with julia, an online media editor. my daughter julia became an internationally highly respected egyptologist who repeatedly has performed excavating campaigns in egypt and sudan and has won two extremely competitive erc research grants; she is now professor of egyptology at the ludwig-maximilian-university munich and in a partnership with tanja, a kindergarten supervisor. i admire and envy her, as she is rapidly running up to the number of entries on the internet that i have. daniel became an expert, art-prize-winning graphic designer and video producer, happily married to caroline, a hospital physician; they have delightful ellie and are expecting their second child soon. rafael succeeded in establishing his own media and marketing company to organise various types of events, from business celebrations to pop concerts, living in partnership with nathalie, a journalist, and their two gorgeous girls lori and noemi. looking back when getting old, it is most gratifying to see a sequence of generations that carry on the spirit and ideas that you have valued highly during your life, even when my small wish, to have one of my kids enter medicine, has not realised (but i still have such hopes for my fifth child, natalia). the difficult family years had one casualty, as it turned out only later. my marriage broke apart, fortunately only when all kids had already left our home. certainly a divorce, and mine was a bitter and battled one, is a sad event in any life. however, to quote paul watzlawik, there is nothing bad in life that doesn’t have something good as well: my longstanding guilty conscience about the inability to dedicate enough time to the family fell apart quickly, and i suddenly felt completely free, a feeling of happiness and contentment that i had missed since decades. of course it is a very selfish thing to be back to independence, but my advice to everybody is to listen to one’s very gut feelings how you really sense your being. i discussed the situation that i went through with friends, and some of them had similar constellations; a couple of them did the same as i – and these made identical experiences. in the following years i was free to work as i wanted, to travel around as i wanted, and to have no private commitments at all. the most important matter, however, was continuation of my good relation with all children despite the divorce. going international and something other than work totally focused on work and a growing family, i had neither time nor interest for the many distractions young people had at the time. a somewhat more relaxed schedule was possible when i went to scientific meetings and congresses outside of vienna. from early on, i tried to attend international congresses of neuropathology (icns), the biggest event in our field. my first icn was in 1974 in budapest, and the technique that dominated most investigations was electron microscopy, with “virus-like structures” almost everywhere. brain tumour pathology, for the very last time, had retained a strong influence by spanish and latin american researchers using sophisticated gold and silver techniques as basis for classification – we dubbed a symposium chaired by the famous professor moises polak in budapest as “metal mining” meeting. within the few following years, the centre of gravity shifted to the anglo-american tumour tradition, mostly represented by the landmark book editions by dorothy s. russel and lucien j. rubinstein, then the bible and only comparable in impact to the present who classification of tumours of the central nervous system. when the icn in 1982 was held in vienna, i was charged with organising a tumour slide seminar together with lucien. it was an unbelievable luck and privilege to meet, discuss and work with such a giant. i visited him in charlottesville, va, well before the icn in order to select interesting candidate cases to present. what impressed me most was his memory with the incredibly huge collection of ”similar” cases that he had at his fingertips – whatever you showed and asked, he immediately answered convincingly with demonstration at the microscope of a similar case. by the way, he was equally impressive as an impeccable british gentleman (although originally from belgium), almost seeming misplaced in the us (he could have been the topic of sting’s song ”englishman in new york”, in particular the quote “if manners maketh man, as someone said, he's that hero of the day”), as a true polyhistor with amazing knowledge of art (he decorated his home with stunning huge tapestries by jean lurçat), and as husband to a most charming and warm-hearted wife, mary m. herman, also a neuropathologist and his closest collaborator, with particular em expertise. in addition to icns that are only held every 3 or 4 years, i regularly attended annual meetings in austria’s great neighbour, germany, i.e. those of the german society of neuropathology and neuroanatomy. in addition to a much broader scientific scope than what we had in small austria, those meetings were nice social events to meet new and old friends, in a relaxed atmosphere that sometimes culminated in rather hilarious sessions, unexpectedly in business sessions of society members. i remember some colleagues with unbelievable humour, such as an extremely witty guy from frankfurt, prof. thomas. everybody was already holding back laughter when he started to speak and made bizarre suggestions. one of the early german meetings that i attended, in 1979, was held together with the polish society of neuropathology in cracow. i went there by car, then quite a trip across countries behind the iron curtain, and highways were still non-existing on that route. i went there also to have a glimpse of cieszyn / těšín / teschen from where my grandfather had migrated to vienna. i arrived there late in the evening, and the impression was appalling, as the town appeared completely dark, depopulated and dilapidated; in my thoughts i congratulated my grandfather to have escaped from there. for fairness: when i came back 40 years later, it was a very nice and well-maintained central european town with a huge historical main square, deserving a visit. the meeting in cracow was nice, the city very beautiful and full of history, but i looked also elsewhere and soon found out that auschwitz was close. i thought that it would be obvious, even obligatory, to make a visit to such a historical site nearby. therefore i asked at the german society members’ business meeting about the tour that certainly would be organised there and whether a place was free for me to join. totally naïve as a youngster who was spared from the horrors of the nazi period by the blessing of late birth, i did not expect that my question would raise any nerve, but it did. there was icy silence, and nobody wanted, dared, or was interested, to answer. my god, i thought, this was then at least 34 years ago. good lesson for my naïvety. it was only later that i learnt of the involvement of some austrian and german neuropathologists in nazi atrocities, but also of the admirable efforts by some, in particular by prof. jürgen peiffer from tübingen, to elucidate and document the disastrous deviation of our discipline during that period. twenty years later, after i became director of the ni, i had to re-visit and clean up such involvement in our own backyard. to some degree, the visit to auschwitz changed my life. the main camp, auschwitz i, had been restored as a museum that was well maintained and diligently documented, with all the horrors of gallows, execution walls, mountains of hairs, spectacles or children’s shoes, all difficult or impossible to stomach. however, the extermination camp proper, auschwitz ii – birkenau, was different; it was kept as it was, without any restoration, additions or changes, giving it a surreal atmosphere of the still present, villainous evil as well as persistent agony. i spent the whole day there, wandering around completely distraught, aimlessly and disoriented, sometimes hallucinating victims’ whispers. it took me a few days to get over this experience, and i decided for myself to contribute my very own, very personal, however small, part to have that never to happen again. back to vienna, i identified amnesty international (ai) as perfect fit for my abilities and intentions, and founded the first ai medical doctors’ group there. ai’s remit is to fight human rights abuses, torture and death penalty everywhere in the world. then it was still cold war – the fight against lawbreakers and offending countries was three-tiered, politically balanced between the west, countries behind the iron curtain, and those in the developing world. medical doctors had a definite role in that fight. as examples, our group documented objective signs of torture in victims who were lucky to have escaped to austria during and after the iranian revolution or from latin american dictatorships. i also participated in missions that investigated and reported on conditions of prisons in countries accused of abuses in jail, another disturbing personal experience. since ai does not provide direct treatment to affected people, siroos mirzaei, a radiologist and amicable friend with iranian roots, and i decided later to proceed with an organisation called hemayat (meaning help or protection in arab and persian) that was to provide free psychological and physical treatment for victims of torture in vienna. the balkans war of the 1990s created many, often too many, victims in need of help. in this year, hemayat celebrated its 25th anniversary – and is still necessary to exist, maybe now more than ever. the hiv onslaught by the first half of the 1980s, i had investigated the regional and cellular tropism in a range of viral infections involving the nervous system, but nobody had previously assumed that this knowledge would become very helpful when, since 1981 in the usa and then in europe, a new disease appeared apparently out of the blue, acquired immune deficiency syndrome (aids). in austria, we had our first autopsies from early 1983, and we were well aware that appropriate safety measures had to be applied, although only later that year luc montagnier (with françoise barré-sinoussi, also sharing with her the nobel prize in 2008) isolated the culprit as lymphadenopathy-associated virus (lav), or htlv-iii as bob gallo called it, or human immunodeficiency virus (hiv) since 1986. i could meet and listen to these two eminent researchers, both of whom gave the immediate impression to be characters as different and irreconcilable as possible to imagine, maybe one of the reasons for their bitter fight for recognition as hiv discoverers and exploiters. when i had examined a small series of aids brains, i realised there was something going on in addition to the by then well known opportunistic infections, an inflammatory process with multinucleated haematogenous cells as hallmark. i published that in 1986 [8], after having described with paul kleihues from zurich another characteristic type of pathology, a progressive diffuse leukoencephalopathy [9]. in those years, i was busy with presenting my hiv data at lectures and meetings. after such a meeting in germany, a well-known professor took me aside and asked: “well, i also have a series of hiv brains but have never found the multinucleated cells you describe!”. i replied by suggesting to show me slides of whatever case he wanted to discuss, and it was perfect luck for me to find almost immediately these cells in his own slides. “oh, they look like this, i didn’t expect that!” was his reaction. yes indeed, these cells may look somewhat strange occasionally and differ from other types of multinucleated cells. one lesson that i took from that and similar situations was my absolute belief in the old wisdom that there is a lot to see when you look, and in old-fashioned, analogue microscoping for training, when one can play around with focus over several planes, something that is difficult to impossible to experience with simple pictures or even more advanced digital slide processing. so up to today, i refuse to give diagnoses just on cabled images. exactly at that time i met gianriccardo trabattoni, an extremely big-hearted neurologist from parma, italy, having striking similarities in physiognomy and humour with woody allen. he asked whether i would be interested to study autopsy brains from the aids epidemic in italy. an enormous tragedy was then occurring in milan, with many deaths every day. now this sad history repeats itself there, with the difference that sars-cov-2 kills preferentially old people, whereas hiv killed indiscriminately, including many youngsters. yes, no question, i was interested to study those brains, and gianriccardo spent subsequent weeks in smuggling caseloads of formol-fixed brains in jam glass jars by rail across the brenner frontier from italy into austria. at that time, there were still border controls, and i wondered what would happen if he would have had his luggage controlled – i had nightmares of press headlines roaring “band of brain smugglers arrested” or, still worse, “scientists uncovered as graverobbers”. almost immediately we had several dozens of brains, and in 1987 we published our combined neuropathological experience from 100 aids autopsies, including most beautiful em photographs of hiv virions provided by silvia cristina, a very gifted pathologist [10]. the next step was visualisation of hiv products in the brain, as i had done previously with other viral infections, and detailed determination of cell tropism in the brain [11]. as already suggested in my work on the multinucleated giant cells of hiv encephalitis, productive infection was seen exclusively in microglia and macrophages, a new paradigm of infection and inflammation in the brain, with neural elements proper not participating in virus replication and production. however, neurons are secondarily affected: in collaboration with prof. haug’s neuroanatomy group in lübeck, we demonstrated by morphometry neuronal loss in aids brains [12], in addition to hiv encephalitis and leukoencephalopathy another substrate of hiv-associated neurological disorders (hand). a midlife crisis my career in neuropathology was successfully under way when, almost exactly at midterm of my period in the ni and at the age of 44, the confidence in my future was shattered. in 1990, i had two invitations for meetings in india, one to speak at the world neurology congress in new delhi on hiv and the nervous system, and another to contribute to a specialised satellite symposium on sspe at the famous christian medical college in vellore, a more than 100 years old foundation in a city in the south, originally for women only, with an affiliated hospital, by now one of the premier such institutions in india. whenever participating at scientific meetings, i always try to squeeze off a few days for my own sightseeing, on and off the beaten track, in particular when it was the first time for me in that area. then it was my first time in india, and i was shocked to verify myself the amount of poverty and suffering on the streets, with people lying and dying on crammed sidewalks in the north. to some contrast, the south seemed better off, with less pressure by sheer numbers of people and a friendly tropical climate. the meeting in vellore was organised by dr. jacob john, already then an eminent virologist who fortunately makes his expert opinion still heard now on covid-19. i was much impressed, but still more when he took us foreign participants to the hospital ward to meet patients with infections in india. as neuropathologists, usually we do not see individual fates and histories behind our fascinating stains and slides; so first-hand experience by meeting patients with diseases of our study topics is a completely different dimension. they had several boys with sspe in the ward, for most of us now an interesting but extremely rare if not exotic condition; however, i would wish that anti-vaccinationists could see the devastating and deadly disease that is preventable by measles vaccination. other interesting cases included what i discussed with dr. john as tropical spastic paraparesis, then a new retrovirus-mediated disorder, tuberculoma and many unclear conditions. i became speechless seeing all that. apparently this experience well coincided with what was emotionally my midlife crisis – i spent the following nights and days seriously considering to drop out of my safe harbour of academic work, career and family life and start again from scratch with clinical work in a place like southern india, where your personal contribution to alleviate sufferings could really make a difference. i turned pros and cons in my mind, realising that the youngest of my kids was already 10 years old, so it would be not that damaging for their upbringing if i would leave, whereas their economic future might become uncertain. the longer i pondered these considerations, the paler the memory of south india became, and finally these ideas faded away. apparently i had no guts for a complete new start, and it was much more convenient to follow in the prepared footsteps of one’s comfortable life. do i regret it? i am not sure, even now when i am very happy with my life track, and probably any present thoughts on what-would-have-been are more coquetry than deep-hearted wishes. national and international neuropathology the 1990s were an important period for the old ni. after more than 100 years, its facilities moved in 1993 from the old building at schwarspanierstrasse to the newly built university hospital, the new allgemeines krankenhaus (akh) where ni was renamed as clinical institute of neurology (kin) and located in the ground floor at the southeast corner of the enormous main building. i had been involved in planning for the new location since more than a decade and was proud to have achieved a consented plan offering unique facilities for the future. the move itself was one of my worst experiences, and i commuted for weeks from the old to the new building. kin was provided in the new quarters with generous space and top-class laboratories featuring most modern equipments for neuropathology (including an own em and tissue culture), neurochemistry and molecular neurobiology. what was still missing i could manage to have added in later years, such as a dedicated prion bsl-3** lab, laser scanning and confocal microscopes and laser microdissecting device. the downside was that the other multidisciplinary but non-clinical parts of the old ni were not transferred as well, but moved to a brand-new institute of brain research in the completely refurbished building of the former institute of pathology, with hans lassmann appointed as professor of neuroimmunology and first director. for the clinically working parts of the old ni, now the kin, the environment of a modern university hospital turned out to be most beneficial, not only for diagnostic service, but also for research and teaching. after i became full professor and director of kin, i followed the research output by publications year by year, and from very modest scores in the early 1990s, a regular increase of cumulative impact factors per year became evident, surpassing 100 in the last years that i served as director until 2011. the move to the akh was also favourable for teaching. although ni had been involved in some practical courses to undergraduates, kin became increasingly involved with teaching lessons for clinics of neurology, psychiatry and the institute of pathology. usually selected topics of the neuropathological spectrum of diseases of the nervous system were lectured, but i always tried to include some of my very own concerns for future mds. one general was functional neuroanatomy that i found rather underrepresented in students’ knowledge and understanding, and another specific was brain trauma, in particular by voluntary activities such as boxing, with the aim to inflict – or at least tolerate – brain damage to others, an issue that should prevent its performance as sport. such new success did not happen without shadows. when describing above my early experience at a german congress in poland with regard to a visit to auschwitz, i mentioned also the need to clean up the institute’s backyard. it was only very late, around the turn of the millennium, that efforts were made by the university of vienna and its medical faculty (from 2004 the new medical university of vienna) to finally clarify, document and purge any remains of the nazi past. for the institute, it turned out that the neuropathological archive contained brain samples, mostly processed histological slides and blocks, of “handicapped” persons in mental institutions, mostly kids, who had been murdered within the nazi “euthanasia” program. in vienna, the psychiatric institution “am spiegelgrund” was the place where these killings occurred, and brains of victims were collected and neuropathologically examined – later unbelievably supported by public funds – by heinrich gross who had also a role in the killings. for further research, some samples were later brought by gross to the ni, and much later i had to identify and de-archive such specimens that were then ceremoniously laid to rest in a dedicated memorial tomb at the vienna central cemetery. additional data by jürgen peiffer from tübingen revealed that brains with a peculiar neuropathological disease from three brothers, murdered in the landesanstalt görden in brandenburg, germany, were first described by franz seitelberger as connatal type of pelizaeus-merzbacher disease (known today to be due to missense mutations in the plp1 gene) in his thesis for habilitation. the samples had originally been archived in the max-planck-institute for brain research in gießen, germany, headed by julius hallervorden who had been heavily involved in the neuropathological work-up of brains of victims of nazi “euthanasia”. seitelberger had spent a postdoc stint in gießen and brought these samples to vienna and the ni. again, i identified these samples in the archive and sent them to hamburg where they were ceremoniously laid to final rest. as a small discipline, neuropathology relies very much on international contacts, interdisciplinary discussions, connections with colleagues and friends, and communication at meetings. having realised this soon, i have fostered such contacts by personal visits, invitations and correspondence. in addition to diagnostic matters, it was research that was, and still is, most in need of international networking. an overlapping field encompassing both diagnostic work and research like neuropathology has classification of disease as a necessary but also possibly arbitrary component of biomedical science and practice. nowhere this was more evident than in the field of pathology of tumours of the nervous system that has been based on histogenetic concepts since long but clouded by complexity over generations. during medical studies, most of my colleagues just skipped the several pages of brain tumour types in our learning documents, as the classification given there was considered counterintuitive, intricate, confusing in places and simply too cumbersome to absorb. this fact has been recognised by the who that reacted by publication of the blue book series on tumour pathology. the 1st blue book on “histological typing of tumours of the central nervous system” was edited in 1979 by klaus joachim zülch, an eminent neurologist-neuropathologist in cologne-merheim, and appeared, at least to outsiders, as compromise between zülch’s classical german tradition of neuropathology and the anglo-american school represented by lucien rubinstein. as example, it included the monstrocellular sarcoma, one of zülch’s dearest entities as sarcoma because of a distinctive network of reticulin fibres, as well as the monstrocellular glioblastoma championed by rubinstein because of gfap immunopositivity, both the very same tumour type. thus most of the complexity remained. as i had to diagnose large numbers of neurosurgical biopsies, i applied my immunohistochemical expertise from my studies on viruses to cns tumours and always tried to keep abreast of current trends. much credit is due to paul kleihues, then professor of neuropathology in zurich, to make an effort to renew brain tumour classification in a modern system that took account of molecular and genetic characteristics and did not only rely on morphology. a meeting was convened in zurich in 1990, with 26 distinguished experts in neurooncological pathology, almost all male, including me, and only two renowned female researchers, lucy b. rorke and ana lia taratuto. one giant was notably missing – lucien rubinstein had died from brain haemorrhage briefly before. the result of intense deliberations – to include new entities, in particular the concept of primitive neuroectodermal tumour (pnet), and delete obsolete ones like the monstrocellular sarcoma – was finally published in 1993 as 2nd edition of the blue book. the 3rd edition in 1997 and its update of 2000 followed meetings in lyon in 1997 and 1999, were re-titled as “pathology and genetics of tumours of the nervous system”, had expansion on genetics and were published by the international society of neuropathology (isn) together with the international agency for research on cancer (iarc) that was then directed by paul kleihues. the 4th edition in 2007 and its revision in 2016 returned to the title “who classification of tumours of the central nervous system” of the old blue books. all these editions proved to be an indispensable tool for neuropathologists not only in their daily diagnostic work, but also in use of a common language with clinical colleagues and basic researchers. i have contributed to all editions and was mostly happy with the outcome, with one exception: i remained sceptical about the scientific rigour and value of the who grading system that applied four uniform prognostic categories across all types of tumours and was carried on unchanged since the very start in 1979. we had heated discussions at all meetings on that issue, but paul was a passionate and dominant defender of who grading. moreover, he was clever enough to put discussions on this topic to the very end of the agenda – usually there was not enough time left for debates in depth. based on my experience with tumour neuropathology, i realised the importance of working with established national and international bodies with regard to diagnostic matters, research and fostering of the discipline. thus i have served for several periods as president of the austrian society of neuropathology. in europe, the supranational european confederation of neuropathological societies (euro-cns) is the contact for professional matters, and i became president in the early 2000s. the international society of neuropathology (isn) is the global representative of our discipline, and i have served as national councillor, as vice-president and finally as president from 2010 to 2014. many scientists have disgust for such officer honours and “committology” in general, as they feel it much more rewarding to spend time in the lab than in discussions at formal administrative meetings. indeed, such meetings may produce little more than hot air, but my experience tells it depends on you – yes, on you in whatever function you attend – how productive or not your participation will be. moreover, don't forget: if you don’t commit yourself to meetings, there is always a possibility of non-transparent agreements negotiated in back rooms by parties attempting to over-rule others, something nobody wishes (or claims so…). the prion decades in the early 1990s, i realised that the type of hiv neuropathological studies, as i had done for almost a decade, was unlikely to contribute much novelty to our future understanding. so where to look next? in the field of infections, a new paradigm was emerging that overlapped with neurodegeneration – something called prion, an infectious proteinaceous particle, a name coined by the later nobel laureate stan prusiner for agents causing transmissible spongiform encephalopathies, then still hotly debated and regarded by many as viruses or virus-like agents. in austria, we had a disease dubbed as “austrian kuru” by franz seitelberger who studied the neuropathology of several cases of a family condition first described by josef gerstmann, ernst sträussler and i. scheinker in 1936, now usually abbreviated as gss. scheinker's first name initial would mean isaac, as i found out, a name not appropriate in germany under nazi rule. featuring prominent amyloid plaques, the pathology indeed resembled that of kuru, the new papuan disease that d. carleton gajdusek transmitted to chimpanzees, like he did later also with creutzfeldt-jakob disease (cjd). again, i stepped into that research field more or less by chance – or by another good luck? fortunately, i had some reputation with the then professor of neurology in the university hospital vienna, herbert reisner. he sometimes called for my opinion when he suspected strange and rare clinical cases. as example, i could diagnose the first clinical case of progressive supranuclear palsy, a disease then practically unheard of by clinicians in vienna. once he even took me as company when he got a call from the veterinary university to consult on a horse with a strange gait disorder. we came to the stable holding the horse, to have a look how the animal moved. the horse was there, but prof. reisner not – he had immediately hidden behind the door and told me to examine the horse in his absence. i learnt later that, possibly as sequel of war trauma, he hated horses and was terrified by them. the horse suffered from what veterinarians called a “wobbler”, a yet rather unspecified ataxic condition, and i had to decide myself what to recommend. guess what my, a neuropathologist’s, recommendation was? euthanasia and neuropathological examination to get a final diagnosis, something that turned out not to be feasible because of severe squeezing artefactual damage of the thick equine spinal cord when the mortuary assistants pulled it out like a rope. in 1990 prof. reisner asked me for an opinion on a rare case that some considered as cjd. the middle-aged lady had indeed symptoms and signs indicative of cjd, but additional spinal long tract signs were unusual, and i had a yet unspecified feeling of something very special. normally, i never think of it, but here i looked for the maiden name of the patient. bingo! it was “h”, the name of a family that i knew very well from earlier studies of important cases in the archive of the ni – i had discovered a new case of the original austrian gss kindred! it had been completely lost to follow up since some 30 years, as family members were dissatisfied by their experience with medical doctors who were unable to help them; some even told them to belong to a “syphilitic family”, since spinal long tract signs are characteristic of tabes dorsalis. in the small villages south of vienna where most of the family lived, this was not something to like to hear or speak about. anyway, the re-discovery enabled us to complete the full pedigree to 221 members in nine generations from the late 18th century onward, with at least 20 patients suffering from definite gss, our last proposita presenting a switch from classical gss to a cjd-like phenotype [13]. we tried over the following years to find out why and how a disease with the very same genetic aberration in the same family was able to change the phenotype, but this has remained elusive. anyway, it was reassuring to find the salient p102l mutation in the prion protein gene prnp also in the original gss family, in collaboration with hans kretzschmar from munich [14]. from 1994 onwards, initially well before the advent of variant cjd (vcjd), i succeeded in coordinating a series of large european networking projects funded by the european commission (ec). the aim was to establish and apply diagnostic criteria in the neuropathological assessment of human prion disease, to determine their clinicopathological phenotypic spectrum and work on specific research issue related to tissue pathology. these projects perfectly complemented other ec projects coordinated by bob will from edinburgh on clinical surveillance of human prion diseases. it was a pleasure and privilege to collaborate with bob for many years on these challenging issues, and to make many good friends in the prion surveillance system, including heino diringer, paul brown, james ironside, maurizio pocchiari, fabrizio tagliavini, inga zerr, pawel liberski…. the scientific collaboration within such networks was strengthened by meetings at regular intervals; my own ec projects had their meetings usually in the small town of baden near vienna, a historical spa type of place popular already in the austro-hungarian empire. the huge bonus of these meetings were the limited number of participants (usually between 50 and 100), lectures on timely and emerging prion issues by top researchers followed by enough time for detailed discussions, and half a day reserved for socialising, walking and hiking. following these simple rules, these meetings have become almost legendary in retrospect – whenever i meet people who had attended, it becomes a nostalgic exchange of memories. baden is amidst a wonderful, wine-growing landscape at the very eastern end of the alps, where pleasant hills of the viennese woods come down to the pannonian planes, so hiking there was always a most enjoyable activity – and sometimes a true escapade to remember. as the baden area was not too familiar to me and we had to keep our time schedule, i wanted somebody to guide us on our hiking tours. i have a friend who lives in the area, erich, a retired high school teacher who described himself as knowing every rock in the vicinity and was enthusiastic to take over the organisation and guidance of our hike. it was an unusually hot day in early june, and the first part of the track was steeply uphill for about 45 minutes. erich calmed down early wishes for a break by promising a mountain lodge on top. when we arrived, thirst was significant, as was disappointment: the lodge was closed for restauration. no problem for erich: as he knew all hidden details of that part of the forest, he promised to get us to a spring with wonderful fresh water. of course, in order to get there, we had to leave the marked trail and step into the dark forest. i had advised everybody in the 40-plus-numbering group of hikers to wear strong walking shoes, but some ladies apparently misunderstood that as invitation to test the most modern and glitzy slippers then available. as to expect, it was not that easy for such a poorly equipped party to walk across thickly covered forest floor. finally arriving exhausted at the spring, another disappointment and still more reason for thirst: the spring had dried up, and erich had some trouble to explain why he had not been aware of an unusually prolonged dry period in the immediate past. feelings of despair were palpable, and i decided in a revolutionary mood to take command. i phoned our bus driver, ordered him to buy a supply of boxes with hectolitres of water bottles and come to the nearest parking spot where a bus could go. it was still some more 45 minutes to walk there, but fortunately downhill below shady trees, so a full rebellion by the party could be avoided by repeated announcements how near the bus parking already was. when we arrived at the bus, exsiccated and with ragged clothes, everybody offered a fortune for a drop of water. i threw away my chances to get such a fortune, like always, and gave the water for free. finally, whatever worries people had, they disappeared suddenly and were replaced by everlasting memories of an exciting adventure. the work on human prion diseases coincided with emergence since the late 1980s of a new animal disease, bovine spongiform encephalopathy (bse), aka mad cow disease. hundreds of thousands of bovines were affected, had to be slaughtered and disposed of in the uk. this economic disaster accompanied loss of public confidence in politics, a phenomenon that spilled over also to continental europe where bse became a problem only delayed, from around 2000. in 1996, the high water mark broke when uk government officials had to concede that bse had transmitted to humans in form of another new disease, variant cjd (vcjd). proof for that, of course, came by neuropathology: james ironside in bob will’s group demonstrated characteristics of a new prion neuropathology. immediately, some hysterical press releases forecasted an armageddon of millions of victims, and feverishly reactions and solutions were deliberated, in the uk as well as in the european union (eu). this was the hour of experts – however, at the time few were there, as prion diseases had been the domain of a small group of basic researchers in biochemistry, of veterinarians working on scrapie, the prototype prion disease in small ruminants, and of medical specialists in epidemiology, neurology and – you are right again in guessing – neuropathology. surveillance for human prion diseases became quickly established throughout europe and needed neurologists, laboratory specialists and neuropathologists. in austria, the austrian reference centre for human prion diseases (örpe) was formally established at the ni and funded by the ministry of health. örpe served as national focal point as well as representative for international contacts in the quickly enlarging prion arena. i was head of örpe from 1996 until 2012 when i left to work in zurich. at the time of the bse/vcjd meltdown, the top scientific advisory body in the eu was the scientific steering committee (ssc) that quickly established its own prion advisory panel, the tse/bse ad hoc group with about 20 members with experience in all aspects of these diseases. i had the privilege to be chosen as member, and from 1997 we had meetings in brussels every month or even more often. for preparation of these meetings we were sent exhaustive documents, sometimes numbering hundreds of pages. the pressure was considerable, but the panel was highly successful to recommend a series of comprehensive measures to mitigate the risk, most notably with regard to the safety of animal-derived products, identification for destruction of specified risk materials in the food and feed chains, quantification of the residual bse risk in food, and country-wise evaluation of the geographical bse risk according to a standardised set of parameters [15]. it was a most interesting experience, as everything had not only public health and economic, but also political implications. in contrast to some members of the ssc, the tse/bse ad hoc group appeared to me more reluctant and cautious, and in the group i worked most closely with dominique dormont and hans kretzschmar. they were not only wonderful scientists and friends, but had the very same cautious approach to a situation with many unknowns. we even insisted on a minority report when we disagreed with the ssc on a pivotal issue. now, years after they have died, i still miss them. when it became clear in 2000 that bse was present in continental europe, we also had in austria our first bse cases that put the government into a panic-stricken work mode. restaurants in vienna were particularly affected by the bse crisis, as boiled beef from tafelspitz, a peculiar meat cut, is a traditional delicacy that, as the saying goes, was never missing at francis joseph’s imperial table (* note to gourmets: my personal preference is not the famous tafelspitz that i find too dry, but some cuts with slightly fatter composition like schulterscherzel or really fat beinfleisch, or kruspelspitz with a small cartilage. this must be served within a beef broth garnished with slices of bone marrow tasting like paradise on toasted dark bread). for a few weeks, i was interviewed many times by tv and radio, and in the midst of this turmoil i got a phone call from a governmental secretary to invite me to a “beef dinner” with the austrian federal chancellor, the head of government, the federal minster of agriculture – and me. although having never been a member of a political party or movement, i smelled that the aim was to stage a televised public event at which the safety of austrian beef should be verified beyond doubts by shared politico-scientific consumption of tafelspitz. i respectfully declined the invitation and explained that i loved boiled beef but that i was in science and medicine but not in political activism. retrospectively, some might argue that i missed a chance to climb up the career ladder, but i was happy with what i got – and then indeed did not rise higher. after 2003, scientific advice and risk assessments were continued by the newly established european food safety authority (efsa); prion-related issues were handled by its biohazard panel. again, i became a member, later its vice-chairman, until 2012. monthly two-day meetings were held, at first in brussels and, after completion of the new efsa headquarters, in parma, italy. numerous opinions on various aspects of food safety were the outcome of our work there. with regard to bse, vcjd and other human prion diseases, it became clear that scientific advice on risk mitigation has resulted in almost complete disappearance of classical bse, a real success story, whereas other prion problems persist [16]. during the long years with efsa the beautiful and historical city of parma became dear to me, as did the colleagues in the panel – it was a constructive and open atmosphere that i enjoyed very much. as parma is the very food capital of italy, we had of course splendid dinners there. my favourite was il bollito misto de parma, a famous dish with meats from cheek, tongue, tail, belly and head, mostly beef, some stuffed but all just boiled, sounding like components of scottish haggis, but reminding me more of the typical viennese tafelspitz. as the habsburgs used to export princesses as monarchic wives all over europe, maria luisa, the daughter of austrian emperor francis ii, who had been napoleon’s second wife and thus empress of france, was later made duchess of parma. maria luisa had great impact on parma, and there are still austrian souvenirs in parma, with the bollito probably as one of the most prominent. upon request, i can advise on the best ristorante for bollito. success and recognition how do you measure success in science? depends. as always, money counts also in research, no question. thus it is essential, already early in a career, to acquire research funds from whatever source possible, something that needs to be trained, as amounts of funds collected for research, patents etc. have become one hard currency of measuring success in science. the other is publications; there are now bibliometric parameters that determine work, career and even the very existence of scientists. this can be considered good or bad, but it definitely is not all what counts. in the present world of multi-authorships, sometimes diluted up to 100 co-authors or more, i am proud to have been one of the very few who wrote many articles as single author, without the safety net of helping hands. of course, this has become different nowadays – present science by necessity has become teamwork, even a world-spanning network. another aspect is the range of research, whether it is narrow and limited to a single problem or wider. for an academic career – and i have witnessed that from sitting on a plethora of search committees for academic positions – it is important to prove both depth and breadth in research. as a now almost extinct species in this regard, i am also proud of having published in virtually every field of the discipline, from developmental neuropathology over metabolic and toxic, inflammatory, infectious and vascular diseases to neurodegeneration, tumours and neuromuscular disorders. many papers have been highly cited, and i have achieved a high hirsch index – the beauty with that, in my view, is its steady growing, even when you publish little at present, as i do. however, what is really important is to have made new contributions to the general body of knowledge, such as i did with first observations or conceptual outlines, as our report in collaboration with kenji kosaka about a case series with neocortical lewy bodies characterising a new disease, now called lewy body dementia [17]; or neuropathological diagnostic criteria for cjd [18], neuropathological features [19] and other aspects of prion diseases [13, 14 and many others] including the subcellular localisation of disease-associated prion protein [20] and characterisation of a new disease in wild-type animals by synthetic prions [21]; the neuropathology of hiv infection [10, 22 and many others], viral products in tick-borne encephalitis [23]; gfap in oligodendrogliomas [24], the clinical relevance of meningioma subtypes [25]; a new glial globular tauopathy [26], nigral burden of α–synuclein as a correlate of striatal dopamine deficit in parkinson’s disease [27], morphological evidence of α–synuclein propagation in the human brain [28], the basis of biomarker diagnostics in neurodegeneration [29], the frequent mixture of neurodegenerative pathologies in the aging community [30], and transmission of aβ by dural grafting [31]. after transfer of the old ni into the akh, we established several clinical routine processes to support our daily work in an expanded environment, such as weekly conferences with the whole staff, research reporting meetings and time-fixed daily microscoping sessions around a multi-ocular device that was able to accommodate up to 10 viewers (fig. 7). it was where we had most of our diagnostic and scientific discussions, in particular when we welcomed distinguished visitors such as john j. kepes (fig. 7) or bob d. terry (fig. 8). and it was always fun and a pleasure, as well as a place where to preserve the good working atmosphere. fig. 7. daily microscoping sessions were always held around the multi-head viewing microscope, here in the second half of the 1990s when an old and cherished friend and neuropathological giant, john kepes with wife (in foreground) was visiting from kansas city. sitting behind them, haberler at left and budka at right, standing from left, hainfellner, hussun (a pathologist from yemen), jarius, wanschitz. john had briefly stayed at the ni after the failed hungarian revolution of 1956. fig. 8. bob terry, another giant of neuropathology, with the author at an alzheimer conference in southern styria, a wine-growing region, nov. 2007. kurt jellinger in the centre at back. in the early 2000s, a young clinical neurologist arrived from budapest, gabor g. kovacs, to learn some neuropathology. his boss at semmelweis university, prof. szirmai who had stayed as postdoc at the ni two decades before, was reluctant to let him go. soon i found out why: gabor became the very best trainee i ever had, with an unbelievable eye for morphology, a sound biological understanding and intelligent translation of neuropathological features into clinical significance, extreme diligence and last but not least, in agreement with albert einstein’s dictum that it is character that makes the scientist excellent, an open, warm-hearted nature that made him soon everybody’s darling at the institute. it was gabor who soon covered most of the research work, most prominently in the prion field but also elsewhere in his broad interests. it is sad for me to realise that he left – in my belief was forced to leave because of the circumstances – two years ago to become full professor in toronto, canada, a brain-drain consequence of the turmoils suffered by the institute in recent years, but definitely a deserved recognition for this fine man and scientist. in the years of my directorship, i was indeed lucky to have an excellent staff that was about equally balanced between neuropathology and neurochemistry (fig. 9). around the same time when gabor arrived, ellen gelpi, another young researcher, joined from barcelona. with her warm-hearted mediterranean-style approach to all matters, irrespective of how important or not they were, she soon became an indispensable hand in diagnosis and research, from prion diseases to tick-borne encephalitis. we lost her for some years to an attractive position as head of a brain bank in her home city, but fortunately she is now back and remains the very soul of the institute. another indispensable mind and soul is romana höftberger who trained with hans lassmann in neuroimmunology in vienna, and with josep dalmau and francesc graus in barcelona. she has high reputation in research on antibody-mediated disorders of the cns, actually relevant for both neuropathology and neurochemistry, and thus is now the no-longer-missing-link between the two major areas of work in the institute. last but not least, christine haberler has made her name known in the field of paediatric tumour neuropathology, something that needs very specialised expertise. for me, aging as an “old white man”, it was a pleasure to see a highly successful female touch in achieving the most recent accomplishments of the institute. fig. 9. the kin staff in 2006. 1st row, from left: hainfellner, regelsberger, budka, ströbel, gelpi, pipp, haberler, höftberger, bernheimer, trabattoni, preusser; behind, the dedicated laboratory and secretariat staff committed to the success of the institute. with increasing age, and success of kin in general, i was awarded some prizes that recognised our work in some specific areas. already as far back as 1982, i had received the moore award for the best paper on clinico-pathological correlation at the american association of neuropathologists (aanp) meeting; it was for my work on ihc in pml, and i was proud of it because i received the award despite the session chairman’s attempts to disturb my presentation. he insisted to limit the number of slides at my talk; so this was a very distinguished virologist who arbitrarily decided that a youngster had two slides too many than what he personally considered appropriate. no, sir, i explained politely but firmly to him that i would definitely show all my slides, that numbers of slides don’t matter as long the allotted time frame is kept, but that he could, of course, stop me if i would go overtime. so good arguments do help youngsters against forceful professors. for explanation of the then highly publicised bse and prion situation, i frequently gave scientific statements to the public by press, tv and radio. these were obviously well received, as i was awarded with the title of “austrian scientist of the year 1998”; the prize included also the naming of a star after the awardee in one of the far corners of the universe. in 2004 i received an honorary doctorate from the medical academy łodz in poland, and in 2008 i had the honour to give the dorothy russell memorial lecture at the british neuropathological society meeting in london (fig. 10). fig. 10. lecture at the award ceremony to receive the dorothy s. russel prize by the british society of neuropathology in london 2008, in front of a memorizing image of my all-time hero, lucien j. rubinstein (photo courtesy of bns). in 2007, we celebrated the 125th anniversary of the institute by a dedicated symposium [32]. many friends, including some from overseas, came to follow a series of lectures and posters on ni/kin’s history and our more recent achievements. it appeared then that the future of kin was safe and expected to prosper, and i even speculated whether i would live to see the institute’s 150th anniversary in 2032. one of my concerns was to maintain and foster a good relation to the non-medical and non-scientific world that is funding everything we do. one possibility was the children’s university when kids who visited kin were told about our work and, most importantly, even could touch a human brain if they dared to (fig. 11). fig. 11. since about 2010, kin participated in the “children’s university” with demonstrations of brain anatomy to primary schoolchildren. all are exited to see a real brain – some even do not dare to look. the third family i met my second wife ivana in 2003 by chance, during a summer most europeans probably remember, as it was extremely hot and beautiful. after my divorce i used weekends and holidays to make long trips by bike (or by car when it was too hot) and stop in between for a refreshing swim, once even in the big danube river when a most beautiful young lady caught my eyes. i was immediately hooked. she is czech, worked as a nurse in mobile care in an area northwest of vienna, and then took a dip in between like i did. she soon decided to take a job offer in a big municipal hospital in the post-surgery recovery room according to her training in anaesthesia. our daughter natalia was born in 2007, and this time circumstances were much more family-friendly than with my earlier family. i had more time to spend with the family, of course never enough in a job such as mine, and it was – and is – an absolute delight to see natalia growing up. when i went to work in zurich, ivana wanted to accompany me with natalia, but i was unsure how it would work out in a completely different setting, with a foreseeable deadline and much higher costs for schooling and living than in vienna. so we arranged that the family would stay in vienna, and in the “guest-worker” tradition of europe i would live over the week in switzerland, with commuting flights every friday afternoon to vienna, and early monday morning from vienna. after i came back from zurich at the end of 2016, i had of course plenty of time to spend with the family. ivana was ambitious to do a master’s study of three years and is now a recognised specialist for pain nursing. two years ago we gave in to natalia’s dearest wish, and gorry joined our family, a female portuguese water dog, which is admired not only by us, but also by practically everybody we meet when walking her (fig. 12). fig. 12. the family in dec. 2019 at a dog exhibition when gorry became austrian junior champion. zurich at an age of 65, i felt too active to do little to nothing after retirement in vienna, so i looked elsewhere and found an announcement for a position of consultant at the institute of neuropathology of the university hospital zurich, headed by prof. adriano aguzzi. i knew adriano from several meetings, as he is a renowned prion researcher. adriano was delighted when i phoned him, and we quickly agreed on my start in zurich from may 2012. i commuted between vienna and zurich by flights on mondays and fridays, almost always with the same bunch of expats in the plane. i tried a few times to drive by car, but it is some 750 km and more than 7 hours driving time, a rather exhausting adventure. i lived for my first two years in a hostel tower for university visitors, students and postdocs, high enough to have a spectacular panoramic view of the city, before renting a small apartment near the limmat river. it was wonderful to walk every morning from there alongside the river to the hospital and back in the evening, watching a lot of waterbirds like cranes, encountering youngsters jumping into the water or balancing on slacklines, and passing by still existing public river baths made entirely of wood, cherished by the locals as “badis”. in comparison with vienna, zurich has a well preserved medieval centre, but is much smaller, much is within walking or biking distance (adriano turned out to be a passionate mountain biker daily climbing up the ütliberg overlooking the city), the river is in the very centre, and the lakeside part of town has an almost mediterranean feeling. so it is a gorgeous place to live indeed. i must admit that i came to switzerland with some trepidation, as i was unsure how the relation with adriano, who was well known as strong personality, would work out. in fact, it turned out to be an extremely pleasant and convenient situation: since an eternity, i had suddenly no or few administrative duties but could concentrate on what i like most (and probably do best), studying and diagnosing interesting neuropathological cases. as adriano was busy with his big experimental lab that comprised some 30-plus postdocs and graduate students in addition to a basic staff of researchers and technicians, he was happy to leave the clinical diagnostic service to me and elisabeth rushing, a very friendly and easygoing colleague and most experienced neuropathologist with whom it was a delight to harmoniously share supervision of a young team of motivated and gifted trainees. elisabeth had specialised in tumour and muscle neuropathology and had been the last head of neuropathology at the legendary armed forces institute of pathology (afip) in washington, dc, before its closure in 2011. together with adriano, i was also responsible for the swiss national reference centre for human prion diseases (nrpe). overall, my time in zurich was the very best indeed that could happen to an aging guy at the end of his career. i am most grateful to adriano – who might have had also some trepidation at start, as i have been considered by some as strong personality as well – to have given me such an opportunity. i learnt a lot there, most notably in experimental neuropathology where i had little experience, but also how to organise and run a successful institute in a way differing from what i was used to do. in fact, after passing the age of 70, it was with a heavy heart that i had to stop my stint in zurich. retirement and aftermath for a long time, i thought that formal retirement in vienna, due for oct 1st, 2011 (fig. 13), was still a long shot in the future. big error, it came quicker than i had imagined. i had made some plans that would be options for that remote time, but in fact, believe it or not, it took me almost by surprise. somehow lost, i suddenly had to look for attractive possibilities to continue at least some of my activities. so i made a visit as honorary professor to the naval general hospital in beijing, on invitation by prof. xiaokun qi, a neurologist with keen interest in neuropathology. in addition, i visited the brain bank, karolinska institutet, huddinge hospital, stockholm, sweden, as guest consultant for several times, on invitation by prof. bengt winblad, caroline graff and inger nennesmo, the local neuropathologist. fig. 13. commemorating plaque given to me by the kin personnel at my farewell party in 2011. it is in the style of a shop sign in the habsburg tradition. its text means “highly laudable neuropathologist in non-retirement”. however, i hoped most to be able to contribute in some function for kin in the future. for a few months i was given a room that was soon converted into a secretariat, and i could keep only a desk elsewhere that, again, was taken away after a short time. after these disappointments, i looked for something else and found the position in zurich described above. after coming back to vienna from 2017, i continued to work on a few forensic cases every year, giving detailed reports to state attorneys and courts, a good possibility to keep active and in touch with neuropathology. of course the number of my publications has dwindled down, and at the end of my scientific career i came back to my neuropathological love affair, viruses: most recently, i wrote a chapter on flaviviruses and tick-borne encephalitis (tbe) in an isn-sponsored book on infections of the nervous system [33]. in view of the present unique global challenge by sars-cov2, i hope to be able still to contribute my experience with neuroviral pathology to research on covid-19. with regard to the future of kin, i expected that a search committee would be appointed soon after my retirement by the rector and senate of the medical university vienna, to fill the vacant position of full professor of neuropathology and institute director. such a committee is common practice in most if not all universities when a professor retires, and its aim is to find the best candidate; my personal favourite was gabor, of course. for unclear reasons – at least for me – this did not happen, and year by year went by while i was in zurich. my former deputy, hans hainfellner, was keen to head the institute but remained formally only deputy director until 2019 when, till the end of that year, the director of the institute of pathology was intermittently appointed to head kin. reasons to appoint an external director were, as i was told, financial frictions between the medical university vienna (muv) and akh about charging for kin diagnostic services; in a hybrid fashion, kin has been funded by both muv and akh. finally and sadly, it was decided to formally degrade the traditionally independent institute on jan. 1st, 2020, after a highly successful history of 137 years, to a division of neuropathology and neurochemistry of the neurological clinic, as a muv-only organisational unit. this was only the second best option to avoid the worst, i.e. complete disbanding when neuropathology would become part of pathology, and neurochemistry would be incorporated into laboratory medicine. why the by far very best option was not pursued, i.e. the further existence of such a renowned and successful institution, has remained a mystery, not only for me, but also for other muv professors. fortunately romana höftberger, an associate professor of kin with excellent knowledge of both neuropathology and neurochemistry, was made head of the new division; i am sure she will do her best to steer it into calm waters. similarly, prof. thomas berger, the director of the neurological clinic and now responsible for the old institute becoming new division, promises to keep it alive as much as possible. epilogue at the end, it is time to balance. was all indeed as positive as these reflections appear, or is it just due to an extended version of the reminiscence bump? for my professional and family lives, there is definitely nothing to regret. with regard to the role of my beloved neuropathology, i cannot find a better summary definition than what lucien rubinstein wrote: “it is that neuropathology is an exquisitely enjoyable pursuit”. the only bitter pill, however, is my personal finale – i never dreamed of becoming the very last director of the original institution that has shaped my whole life. what about the future? for a long time, i used to give a special farewell gift to postdocs and graduate students who trained with me, santiago ramón y cajal’s “advice to a young investigator”. it is an interesting and amusing reading, not only because he was one of the most eminent neuroscientists in history, but also because his advice penetrates virtually every relevant issue of life. a hilarious example is cajal’s criteria for selecting the best wife for a scientist: she should be unattractive, because otherwise she would be too distractive, and she should be rich, to make the husband financially independent in his research. i don’t have such down to earth advice (and i did not follow that advice). however, i believe in an attitude that seems to imbibe also ramón y cajal’s writing and can be put in a nutshell of three words: competence, solidarity and trust. then, now and forever, they are as essential for a scientist or medical practitioner as for any individual’s life. still more, they are pivotal in the fight against crises, including what we are now enduring. if we respect and follow them, there is no reason to await the future with trepidation. references 1. budka h: morphologische aspekte zerebrovaskulärer erkrankungen. in: die zerebrale apoplexie, hrsg. g. s. barolin, pp. 62-79. stuttgart : enke 1980 (1. auflage, 2. auflage 1983, 3. auflage 1985). 2. budka h: pathology of encephalopathies induced by treatment or prophylaxis of neoplastic lesions of the nervous system. in: treatment of neoplastic lesions of the nervous system. j. hildebrand, d. gangji, eds. (europ. j. cancer clin. oncol. suppl. 3) pp. 45-50. pergamon press, oxford-new york 1982. 3. budka h: brain pathology in the collagen vascular diseases. angiology 32: 365-372 (1981). 4. budka h, sluga e, heiss w-d: spastic paraplegia associated with addison's disease: adult variant of adreno-leukodystrophy. j. neurol. 213: 237-250 (1976) 5. budka h, popow-kraupp th: immunohistological studies in viral encephalitis. acta neuropathol. suppl. vii: 142-144 (1981) 6. budka h, popow-kraupp th: rabies and herpes simplex virus encephalitis. an immunohistological study on site and distribution of viral antigens. virchows arch a 390: 353-364 (1981) 7. budka h, popow-kraupp th: immunmorphologische untersuchungen viraler antigene bei verschiedenen enzephalitiden. verh. dtsch. ges. pathol. 65: 181-185 (1981) 8. budka h: multinucleated giant cells in brain: a hallmark of the acquired immune deficiency syndrome (aids). acta neuropathol. 69: 253-258 (1986) 9. kleihues p, lang w, burger pc, budka h, vogt m, maurer r, lüthy r, siegenthaler w: progressive diffuse leukoencephalopathy in patients with acquired immune deficiency syndrome (aids). acta neuropathol. 68: 333-339 (1985) 10. budka h, costanzi g, cristina s, lechi a, parravicini c, trabattoni r, vago l: brain pathology induced by infection with the human immunodeficiency virus (hiv). a histological, immunocytochemical, and electron microscopical study of 100 autopsy cases. acta neuropathol. 75: 185-198 (1987) 11. budka h: human immunodeficiency virus (hiv) envelope and core proteins in cns tissues of patients with the acquired immune deficiency syndrome (aids). acta neuropathol 79: 611-619 (1990) 12. ketzler s, weis s, haug h, budka h: loss of neurons in the frontal cortex in aids brains. acta neuropathol 80: 92-94 (1990) 13. hainfellner ja, brantner-inthaler s, cervenáková l, brown p, kitamoto t, tateishi j, diringer h, liberski pp, regele h, feucht m, mayr n, wessely p, summer k, seitelberger f, budka h: the original gerstmann-sträussler-scheinker family of austria: divergent clinicopathological phenotypes but constant prp genotype. brain pathol 5: 201-211 (1995) 14. kretzschmar ha, honold g, seitelberger f, feucht m, wessely p, mehraein p, budka h: prion protein mutation in family first reported by gerstmann, sträussler, and scheinker. lancet 337: 1160 (1991) 15. vossen p, kreysa j, goll m (eds): overview of the bse risk assessments of the european commission’s scientific steering committee (ssc) and its tse/bse ad hoc group adopted between september 1997 and april 2003. pp. 18-34. european commission, published online june 5, 2003; https://ec.europa.eu/food/sites/food/files/safety/docs/sci-com_ssc_out364_en.pdf 16. budka h, will rg: the end of the bse saga: do we still need surveillance for human prion diseases? swiss med wkly 145:w14212 (2015) 17. kosaka k., m. yoshimura, k. ikeda, h. budka: diffuse type of lewy body disease: a progressive dementia with numerous cortical lewy bodies and senile changes of varying degree a new disease? clin. neuropathol. 3: 185-192 (1984) 18. budka h, aguzzi a, brown p, brucher j-m, bugiani o, gullotta f, haltia m, hauw j-j, ironside jw, jellinger k, kretzschmar ha, lantos pl, masullo c, schlote w, tateishi j, weller ro: neuropathological diagnostic criteria for creutzfeldt-jakob disease (cjd) and other human spongiform encephalopathies (prion diseases). brain pathol 5: 459-466 (1995) 19. budka h: neuropathology of prion diseases. brit med bull 66: 121-130 & plate x (2003) 20. kovács gg, preusser m, strohschneider m, budka h: subcellular localization of disease associated prion protein in the human brain. am j pathol 166: 287-294 (2005) 21. makarava n, kovacs gg, bocharova o, savtchenko r, alexeeva i, budka h, rohwer rg, baskakov iv: recombinant prion protein induces a new transmissible prion disease in wild type animals. acta neuropathol 119/2: 177-187 (2010) 22. budka h: neuropathology of human immunodeficiency virus infection. brain pathol 1: 163-175 (1991) 23. gelpi e, preusser m, garzuly f, holzmann h, heinz fx, budka h: visualization of central european tick borne encephalitis infection in fatal human cases. j neuropathol exp neurol 64: 506-512 (2005) 24. herpers mjhm, budka h: glial fibrillary acidic protein (gfap) in oligodendroglial tumors: gliofibrillary oligodendroglioma and transitional oligoastrocytoma as subtypes of oligodendroglioma. acta neuropathol. 64: 265-272 (1984) 25. maier h, öfner d, hittmair a, kitz k, budka h: classical, "atypical" and anaplastic meningioma: three histopathological subtypes of clinical relevance. j neurosurg 77: 616-623 (1992) 26. kovacs gg, majtenyi k, spina s, murrell jr, gelpi e, hoftberger r, fraser g, crowther ra, goedert m, budka h, ghetti b: white matter tauopathy with globular glial inclusions: a distinct entity of sporadic frontotemporal lobar degeneration. j neuropathol exp neurol 67: 963-975 (2008) 27. kovacs gg, milenkovic ij, preusser m, budka h: nigral burden of a-synuclein correlates with striatal dopamine deficit. mov disord 23 (11): 1608-1612 (2008) 28. kovacs gg, breydo l, green r, kis v, puska g, lőrincz p, perju-dumbrava l, giera r, pirker w, lutz m, lachmann i, budka h, uversky vn, molnár k, lászló l: intracellular processing of disease-associated α-synuclein in the human brain suggests prion-like cell-to-cell spread. neurobiol dis 69: 76-92 (2014) 29. kovacs gg, botond g, budka h: protein coding of neurodegenerative dementias: the neuropathological basis of biomarker diagnostics. acta neuropathol 119: 389-408 (2010) 30. kovacs gg, milenkovic i, wöhrer a, höftberger r, gelpi e, haberler c, hönigschnabl s, reiner-concin a, heinzl h, jungwirth s, krampla w, fischer p, budka h: non-alzheimer neurodegenerative pathologies and their combinations are more frequent than commonly believed in the elderly brain: a community-based autopsy series. acta neuropathol 126: 365-384 (2013) 31. frontzek k, lutz mi, aguzzi a, kovacs gg, budka h: amyloid-β pathology and cerebral amyloid angiopathy are frequent in iatrogenic creutzfeldt-jakob disease after dural grafting. swiss med wkly 146:w14287 (2016) 32. kreft g, kovacs gg, voigtländer t, haberler c, hainfellner ja, bernheimer h, budka h: 125th anniversary of the institute of neurology (obersteiner institute) in vienna. “germ cell” of interdisciplinary neuroscience. clin neuropathol 27: 439-443 (2008) 33. budka h: flaviviruses 1. general introduction and tick borne encephalitis. in: chrétien f, wong kt, sharer lr, keohane c, gray f (eds) infections of the central nervous system: neuropathology and genetics. international society of neuropathology (isn) book series pathology and genetics, chapter 14, pp 131-146. wiley, hoboken, nj 2020 addendum list of neuropathological research visitors and postdocs who trained in neuropathology in the ni/kin: masanori tomonaga, professor in tokyo, riki okeda, professor in tokyo, takeshi kurata, professor at the nih tokyo, ichiro akiguchi, professor in kyoto, toshihiko suenaga, yoshitomo shirakashi, yasuhiro kawamoto, hiroshi sugiyama, toru kimura, kyoko ozawa, all kyoto, masahiro yoshimura-yasuhara, hitoshi yamanouchi, both tokyo, noburo kochi, tatsuo morimura, both hyogo, kazuko sato-matsumura, sapporo, sami khoshyomn, burlington vt, silvia cristina, milano, gianriccardo trabattoni, parma, carlos lima, lisboa, mara popovic, professor in ljubljana, raf sciot, louvain, marcel jhm herpers, professor in maastricht, ferenc garzuly, szombathely, tibor hortobágyi, professor in szeged, felicia slowik, budapest, andras guseo, székesfehérvár, pawel p. liberski, professor in łodz, beata sikorska, professor in łodz, radek kordek, łodz, maria barcikowska, łodz, radek matej, professor in prague, marin guentchev, professor in sofia, ognan kalev, sofia, later linz, yervand karapetyan, armenia, hussun, yemen, sashine tolunay, bursa, karl rössler, professor in vienna, christian bancher, horn, eduard auff, professor in vienna, stefan klöppel, professor in freiburg i. br, peter pilz, salzburg, hans maier, innsbruck, christine wüstinger, johannes preiser, elisabeth wondrusch, claudia radbauer, peter mazal, oskar koperek, christa jarius, elisabeth lindeck-pozza, anna c rudnay, roland sedivy, ute laggner, irene pipp, ivan j milenkovic, harald stefanits, all vienna, michael huemer, schwarzach-st. veit, reza yassari, chicago il. i apologise to those whom i missed, as i could not cross-check the list with documents in the institute. past and present neuropathological researchers who have worked for prolonged periods in the ni/kin include ellen gelpi, johannes hainfellner, christine haberler, romana höftberger, gabor g. kovacs, matthias preusser, manfred schmidbauer, sabine urbanits, julia wanschitz, adelheid wöhrer. copyright: © 2020 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. pigmented ependymoma, a tumor with predilection for the middle-aged adult: case report with methylation classification and review of 16 literature cases feel free to add comments by clicking these icons on the sidebar free neuropathology 3:16 (2022) review pigmented ependymoma, a tumor with predilection for the middle-aged adult: case report with methylation classification and review of 16 literature cases alexander s. himstead, bs1,2, mari perez-rosendahl, md2, gianna m. fote, phd1, angie zhang, md1, michael g. kim, md1, david floriolli, md3, martha quezado, md4, kenneth aldape, md4, drew pratt, md4, zied abdullaev, phd4, edwin s. monuki, md, phd2, frank p. k. hsu, md, phd1, william h. yong, md, fcap2 1 department of neurosurgery, university of california, irvine school of medicine, orange, ca, usa 2 department of pathology and laboratory medicine, university of california, irvine school of medicine, orange, ca, usa 3 department of radiology, university of california, irvine school of medicine, orange, ca, usa 4 laboratory of pathology, center for cancer research, national cancer institute, national institutes of health, bethesda, md, usa corresponding author: william h. yong, md, fcap · department of pathology and laboratory medicine · uc irvine health, school of medicine · 101 the city drive south · orange, ca 92868 · usa yongwh@hs.uci.edu submitted: 09 june 2022 accepted: 21 june 2022 copyedited by: mónica miranda published: 08 july 2022 https://doi.org/10.17879/freeneuropathology-2022-4076 additional resources and electronic supplementary material: supplementary material keywords: pigmented; ependymoma; lipofuscin; fourth ventricle; case report; posterior fossa; methylation abstract ependymomas have rarely been described to contain pigment other than melanin, neuromelanin, lipofuscin or a combination. in this case report, we present a pigmented ependymoma in the fourth ventricle of an adult patient and review 16 additional cases of pigmented ependymoma from the literature. a 46-year-old female showed up with hearing loss, headaches, and nausea. magnetic resonance imaging revealed a 2.5 cm contrast-enhancing cystic mass in the fourth ventricle, which was resected. intraoperatively, the tumor appeared grey-brown, cystic, and was adherent to the brainstem. routine histology revealed a tumor with true rosettes, perivascular pseudorosettes and ependymal canals consistent with ependymoma, but also showed chronic inflammation and abundant distended pigmented tumor cells that mimicked macrophages in frozen and permanent sections. the pigmented cells were positive for gfap and negative for cd163 consonant with glial tumor cells. the pigment was negative for fontana-masson, positive for periodic-acid schiff and autofluorescent, which coincide with characteristics of lipofuscin. proliferation indices were low and h3k27me3 showed partial loss. h3k27me 3 is an epigenetic modification to the dna packaging protein histone h3 that indicates the tri-methylation of lysine 27 on histone h3 protein. this methylation classification was compatible with a posterior fossa group b ependymoma (epn_pfb). the patient was clinically well without recurrence at three-month post-operative follow-up appointment. our analysis of all 17 cases, including the one presented, shows that pigmented ependymomas are most common in the middle-aged with a median age of 42 years and most have a favorable outcome. however, one patient that also developed secondary leptomeningeal melanin accumulations died. most (58.8%) arise in the 4th ventricle, while spinal cord (17.6%) and supratentorial locations (17.6%) were less common. the age of presentation and generally good prognosis raise the question of whether most other posterior fossa pigmented ependymomas may also fall into the epn_pfb group, but additional study is required to address that question. introduction primary pigmented tumors of the central nervous system (cns) are rare entities and often contain melanin.1 primary pigmented intracranial tumors include melanoma, melanocytoma, schwannomas,2 meningiomas,1 and, less commonly, gliomas,3 neurocytomas,4 ependymomas and choroid plexus tumors.2 ependymomas are glial neoplasms found, most commonly, intracranially in the posterior fossa of children and within the spinal cord of adults.5 prognosis primarily depends on location, with intracranial ependymomas harboring worse outcomes than spinal variants.6,7 pigmented ependymomas are most commonly melanotic,8 although these neoplasms are often incompletely characterized in the literature. furthermore, cases of pigmentation with neuromelanin and lipofuscin have been previously described.2,9 nowadays, subtyping of ependymomas includes genetic and methylation profiling, therefore several methylation profiles have emerged which may correlate with clinical prognosis.10 here, we describe a pigmented ependymoma of the fourth ventricle that contains lipofuscin and has a methylation profile consistent with a posterior fossa group b ependymoma (epn_pfb). we also review the available published cases pertaining to pigmented ependymomas by characterizing their age distribution, location, clinical features and outcomes. case history a 46-year-old hispanic female presented with hearing loss for one year and worsening headaches and nausea for two months. her physical examination revealed no focal neurological deficits. magnetic resonance imaging (mri) revealed a 2.2 x 1.5 x 2.6 cm mass involving the floor of the fourth ventricle, which showed marked intrinsic t1 hyperintensity, variable t2 signal (with areas of both hyperintensity and hypointensity), predominantly hyperintense fluid-attenuated inversion recovery (flair) signal and areas of contrast-enhancement (fig. 1). after informed consent to surgery and involvement in research (irb ethics approval deferred as university of california, irvine policy permits reports of up to 3 individuals), she underwent a suboccipital craniotomy for resection of the mass. intraoperatively, the tumor appeared as a grey-brown cystic mass within the fourth ventricle that was tightly adherent to the posterior inferior cerebellar arteries and lower brainstem (fig. 2). radiographic gross-total resection (gtr) was achieved. the patient’s postoperative course was uncomplicated, and she was without radiographic recurrence and satisfied with her treatment at oneand three-month follow-up appointments (fig. 3). figure 1. a. sagittal pre-contrast t1 demonstrates an intraventricular mass involving the floor of the fourth ventricle and heterogenous t1 signal with areas of prominent intrinsic t1 hyperintensity (blue arrow), areas isointense to brain (red arrows), and scattered areas of low signal. b. fat saturated sagittal post-contrast t1 demonstrates enhancement of the previously isointense portions (red arrows) with persistent areas of low signal. c. axial fat saturated t2 at the level of the lesion demonstrates cystic areas of high t2 signal (red arrow) which correspond to low intensity of pre-contrast t1. d. a prominent low signal area on t2 is intrinsically bright on t1 (blue arrow), suggesting high protein and/or lipid contents. e. axial flair demonstrates predominantly isoto hyperintense signal. figure 2. intraoperative photographs of the tumor demonstrating: a. dissection of arachnoid bands tethering the tumor to the floor of the fourth ventricle and b. the macroscopic appearance of the lesion with notable grey-brown color (blue arrows). figure 3. patient care timeline. pathology intraoperative smears showed pigmented cells often with swollen granular cytoplasm mimicking macrophages (fig. 4a), as well as a population of cells with ovoid nuclei and a moderate amount of cytoplasm with variably epithelioid features (fig. 4b). intraoperative frozen sections showed perivascular chronic inflammation, abundant pigmented cells and sheets of tumor cells with epithelial surfaces mimicking glands, but reflecting ependymal rosettes or canals (arrowhead) (fig. 4c). the pigment was fine and grey-brown in micrographs of the frozen section (fig. 4d). figure 4. staining is with hematoxylin and eosin (h&e) unless otherwise specified. a. (400x) smearswollen pigmented tumor cells. b. (400x) smearunpigmented and pigmented tumor cells. c. (100x) frozen section“epithelial” surfaces resembling glands (red arrowhead). d. (400x) frozen sectionfine, grey-brown pigment. figures e-i are from permanent sections. e. (40x) cellular tumor (right fragment) and areas reminiscent of subependymoma gliopil (left fragment). f. (100x) ependymal canal (blue arrowhead) and pseudopapillary configuration (red arrowhead). g. (200x) perivascular pseudorosette (red arrowhead). h. (100x), i. (400x) swollen pigmented cells. j. (200x) fontana-masson staintumor cells negative; few perivascular macrophages positive (blue arrowhead). k. (600x) pas stain. l. (600x) fluorescence microscopy shows autofluorescence. m. (200x), n. (400x) gfappigmented cells immunopositive (red arrowheads). o. (200x) cd163tumor cells negative; scattered microglia immunopositive. p. (200x) h3k27me3loss in subset of tumor cells. clicking into the figure will lead you to the full virtual slide. permanent sections showed tumor tissue that was mostly cellular (right fragment) with minor areas where the background consisted of an eosinophilic gliopil (left fragment) (fig. 4e). ependymal canals (blue arrowhead) were focally prominent, sometimes suggesting a papillary configuration (red arrowhead) (fig. 4f). perivascular pseudorosettes could also be seen (fig. 4g). swollen cells with grey-brown pigment were abundant in some areas (fig. 4h, i). the pigmented cells were negative for melanin and neuromelanin by fontana-masson (fig. 4j); only scant focal often coarser and/or larger black-staining pigment was seen extracellularly and in a few perivascular macrophages (arrowhead). these cells stained strongly with pas (fig. 4k) and were autofluorescent with fluorescence microscopy (fig. 4l). concerning the gfap staining, the tumor was diffusely and strongly positive (fig. 4m), as were the swollen pigmented cells, which showed gfap staining at the cytoplasmic periphery (arrowheads) consistent with a glial origin (fig. 4n). both unpigmented and pigmented tumor cells were negative for the macrophage marker cd163; with only scattered, interspersed, small darkly staining microglia (fig. 4o). collectively, these findings indicated a lipofuscin-pigmented ependymoma. an h3k27me3 immunostain showed immunonegativity in approximately 20-30% of tumor cells, but did not show global loss (fig. 4p). whole slide digital images of frozen h&e (sfig. 1), permanent h&e (sfig. 2), pas (sfig. 3), gfap (sfig. 4) and h3k27me3 (sfig. 5) slides are available in the supplement section. dna methylation arrays and analysis methylation profiling was performed at the national institutes of health (nih), bethesda, md, usa. genomic dna was extracted from formalin-fixed paraffin-embedded (ffpe) tissue sections on slides using an allprep dna/rna ffpe kit (qiagen, venlo, netherlands). the dna was bisulfite-converted using the ez dna methylation kit (zymo research, irvine, ca, usa), and processed to create a beadchip (850k methylation sites) using the infinium methylation epic kit (illumina, san diego, ca, usa). the beadchip was then scanned on the iscan reader (illumina). methylation profiling was carried out through an nih pipeline using umap, the nci-epic methylation classifier, and the heidelberg classifier (brain, versions 11b6 and 12.5), obtained from the german cancer research center in heidelberg, germany. the sample was classified as ependymoma, posterior fossa group b with a score of 0.47 on version 11b6, and the same methylation class with a higher score of 0.98 on version 12.5 of the heidelberg classifier. on the umap, this sample embedded with the epn_pfb cluster (fig. 5). the consensus methylation profile, in the context of the clinical and histopathologic features, supported epn_pfb, cns world health organization (who) grade ii. figure 5. methylation analysis results. a. genome-wide copy number profile generated from dna methylation array signal intensities. the copy number profile shows numeric whole chromosome changes, which are typical for epn_pfb group. b. the umap embedding of dna methylation array data for select tumor groups. sample tumor embeds with epn_pfb tumor cluster. discussion ependymomas are glial neoplasms with a slight male predominance that represent, approximately, 2-6% of all intracranial tumors and 50% of intramedullary spinal tumors.11,12 much less common are pigmented ependymomas.2 these rare tumors may contain three types of pigment: melanin, neuromelanin, or lipofuscin.13 the most common pigment in pigmented ependymomas is melanin, a tyrosine derivative present in the choroid of the eye and, sometimes, in the meninges.13 rarely, pigmented ependymomas contain neuromelanin, which is likely produced by nonenzymatic oxidation of dopamine or lipofuscin. lipofuscin arises from iron-catalyzed peroxidation of membrane lipids and lipoproteins within lysosomes, and tends to accumulate with cellular aging.2,13 a recent case report and literature review on pigmented ependymomas describes only one previous case, in which lipofuscin was discovered to be the sole pigment contained within a pigmented ependymoma in a 16-year-old male.2 here, we report on an additional case of lipofuscin-pigmented ependymoma of the fourth ventricle in a 46-year-old female with the epn_pfb subtype. given the rarity of this entity, a literature review was performed using the pubmed and scopus search engines to identify previously described cases of pigmented ependymoma. keywords used included “pigment”, “melanin”, “lipofuscin”, “neuromelanin” and “ependymoma”. this yielded 8 total studies2,8,9,14-19 reporting on 16 total patients with pathologically confirmed pigmented ependymoma (table 1). melanin was the most common pigment found in these ependymomas (n=12, 70.6%), followed by lipofuscin and neuromelanin (n=2, 11.8%) and lipofuscin alone (n=2, 11.8%). in one case, the pigment composition was not reported.16 only two studies reported the tumor grade using the who classification.8,15 six cases were who grade ii, and two were who grade iii anaplastic ependymoma according to the who schema at that time. pigmented ependymomas tend to occur in young adults, most commonly in the 4th ventricle, and present with symptoms caused by local mass effect. the primary treatment described was resection variably with adjuvant radiation therapy, and gross-total resection was reported in all but 5 cases (29.4%). the average age at time of diagnosis was 38.6 years and the median age was 42 years (range 13-52) (fig. 6). there was a slight male predominance with 9 out of 17 male patients (52.9%). the most common location was the fourth ventricle (n=10, 58.8%), followed by the cervical spine (n=2, 11.8%), the thoracic spine, temporal lobe, frontoparietal lobe, and sella turcica (n=1, 5.9% each) (table 2). figure 6. histogram demonstrating age distribution of patients with pigmented ependymomas. the primary clinical presentation was based on mass effect from the tumor location. several patients with fourth ventricular tumors presented with symptoms of obstructive hydrocephalus (n=4). one patient was asymptomatic. another developed right upper extremity numbness and right lower extremity weakness. only one noteworthy case described a posterior fossa melanotic ependymoma complicated by secondary pial accumulation of melanin, resulting in dysarthria, hearing loss and death of the patient twelve days after subtotal resection.14 this was the only death reported in the literature and may be related to the secondary leptomeningeal pigment accumulation. the typical treatment for these tumors was attempted gtr, with adjuvant radiation therapy in six cases. there was one case where the patient was intact after resection, but developed hydrocephalus in the setting of adjuvant radiotherapy and devolved into a coma.8 in all other cases, the patients were alive without recurrence at last follow-up, with a mean follow up of 43.7 months. there are three distinct molecular variants of posterior fossa ependymoma, including posterior fossa group a ependymoma (epn_pfa), epn_pfb, and subependymoma, distinguished by their dna methylation profile. patients with epn_pfb are typically adults, whereas epn_pfa is more likely to be diagnosed in infants. given the age of onset (adult) and typical lack of recurrence, we hypothesize that the methylation profiles of the posterior fossa pigmented ependymomas described in the literature are likely to be epn_pfb in most cases. the molecular subgroup has been demonstrated to be a powerful independent predictor of outcomes.10 a recent study found that epn_pfb was associated with an excellent 10-year overall survival (os) rate of 96.1% after gtr, whereas epn_pfa, the other posterior fossa variant, had worse outcomes; 5-year os after gtr was 65.2% in females and 45.5% in males. furthermore, epn_pfa was a highly significant predictor of poor progression-free survival (hazard ratio [hr] 2.14; 95% confidence interval [ci] 1.31 to 3.49; p=0.002) and overall survival (hr 4.30; 95% ci 1.88 to 9.87; p<0.001) in this cohort.10 in addition to differences in methylation profile, epn_pfa and epn_pfb differ by protein expression of trimethylated histone h3k27 (h3k27me3).20,21 a study of 112 childhood ependymomas showed that 100% of epn_pfa ependymomas had h3k27me3 loss defined as staining in less than 80% of cells.20 in contrast, only 1 out of 40 epn_pfb ependymomas (2.5%) had less than 80% h3k27me3 immunopositivity.20 in a later study, two out of 15 epn_pfb tumors (13.3%) showed h3k27me3 loss with immunopositivity in 10% and 60% of cells respectively.22 the specificity for epn_pfb is 100%, but sensitivity is only 86.7% using the 80% immunopositivity cutoff.22 while all 29 epn_pfa tumors showed h3k27me3 loss, the amount of “loss” was quite variable. a majority (62%) of epn_pfa tumors showed h3k27me3 immunopositivity that ranged from 5-50%; another 38% of epn_pfa tumors expressed h3k27me3 in less than 5% of cells.22 our case, which demonstrated an estimated 70-80% h3k27me3 immunopositivity, is in the vicinity of the original 80% cutoff for epn_pfb and is at a level of expression above that of most epn_pfa tumors. the only previous case of ependymoma containing only lipofuscin pigment was reported by malhotra et al. in 2021.2 they describe a fourth ventricular mass in a 16-year-old male that demonstrated perivascular pseudorosettes, ependymal canals, and tumor cells with plump cytoplasm filled with a greyish granular pigment and a high mitotic count (5 in 10 high-power fields in hotspots). similar to the present case, the cells stained positively for pas, but were negative for fontana-masson. in addition, the cells autofluoresced. two 4th ventricle tumors demonstrated the presence of both lipofuscin and neuromelanin. the basis of the excessive intracellular lipofuscin accumulation is unclear. ultimately, the prognostic significance of lipofuscin-pigmented ependymomas has yet to be determined given the small number of cases and limited long-term follow-up. nonetheless, this literature review suggests a generally good prognosis in pigmented ependymoma, especially if gtr is achieved. conclusion here, we present an additional case of lipofuscin-pigmented ependymoma of the fourth ventricle and review the literature pertaining to these unique, uncommon neoplasms. pigmented ependymomas occur most commonly in the 4th ventricle and in middle-aged patients, typically contain melanin, and seem to be associated with good long-term outcomes, especially when gross-total resec-tion is achieved. secondary leptomeningeal melanin accumulations may portend a worse prognosis. further study, molecular profiling, and long-term follow-up are required to better understand the natural history and recommended treatment for this rare central nervous system neoplasm. acknowledgments we thank dr. jack reid for assistance in creating whole slide digital images. references 1. smith ab, rushing ej, smirniotopoulos jg. pigmented lesions of the central nervous system: radiologic-pathologic correlation. radiographics. 2009;29(5): 1503-1524. https://doi.org/10.1148/rg.295095109. 2. malhotra a, rao s, santhoshkumar r, muralidharan n, mitra s, shetty s. pigmented ependymoma of the fourth ventricle-a curious entity: report of a rare case with review of literature. international journal of surgical pathology. 2021;29(1): 80-84. https://doi.org/10.1177/1066896920926700. 3. vajtai i, yonekawa y, schäuble b, paulus w. melanotic astrocytoma. acta neuropathol. 1996;91(5): 549-553. https://doi.org/10.1007/s004010050465. 4. ng th, wong ay, boadle r, compton js. pigmented central neurocytoma: case report and literature review. the american journal of surgical pathology. 1999;23(9): 1136-1140. https://doi.org/10.1097/00000478-199909000-00019. 5. brown nj, wilson b, lien bv, et al. citation analysis of the most influential ependymoma research articles illustrates improved knowledge of the molecular biology of ependymoma. neurosurgical review. 2021;45: 1041-1088. https://doi.org/10.1007/s10143-021-01579-1. 6. boström a, von lehe m, hartmann w, et al. surgery for spinal cord ependymomas: outcome and prognostic factors. neurosurgery. 2011;68(2): 302-308; discussion 309. https://doi.org/10.1227/neu.0b013e3182004c1e. 7. cage ta, clark aj, aranda d, et al. a systematic review of treatment outcomes in pediatric patients with intracranial ependymomas. journal of neurosurgery: pediatrics. 2013;11(6): 673-681. https://doi.org/10.3171/2013.2.peds12345. 8. yang c, li g, fang j, wu l, deng x, xu y. clinical analysis of primary melanotic ependymoma in the central nervous system: case series and literature review. acta neurochirurgica. 2013;155(10): 1839-1847. https://doi.org/10.1007/s00701-013-1810-1. 9. chan ac, ho lc, yip ww, cheung fc. 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cancer. 1976;37(5): 2373-2379. https://doi.org/10.1002/1097-0142(197605)37:5<2373::aid-cncr2820370529>>3.0.co;2-2 18. ogawa y, watanabe m, jokura h, tominaga t. primary intrasellar melanotic ependymoma successfully treated by combined transsphenoidal and gamma knife surgeries. international journal of clinical and experimental medicine. 2016;9 (1): 371-375. 19. rosenblum mk, erlandson ra, aleksic sn, budzilovich gn. melanotic ependymoma and subependymoma. the american journal of surgical pathology. 1990;14(8): 729-736. https://doi.org/10.1097/00000478-199008000-00005. 20. panwalkar p, clark j, ramaswamy v, et al. immunohistochemical analysis of h3k27me3 demonstrates global reduction in group-a childhood posterior fossa ependymoma and is a powerful predictor of outcome. acta neuropathologica. 2017;134(5): 705-714. https://doi.org/10.1007/s00401-017-1752-4. 21. bayliss j, mukherjee p, lu c, et al. lowered h3k27me3 and dna hypomethylation define poorly prognostic pediatric posterior fossa ependymomas. science translational medicine. 2016;8(366): 366ra161. https://doi.org/10.1126/scitranslmed.aah6904. 22. fukuoka k, kanemura y, shofuda t, et al. significance of molecular classification of ependymomas: c11orf95-rela fusion-negative supratentorial ependymomas are a heterogeneous group of tumors. acta neuropathologica communications. 2018;6(1): 134. https://doi.org/10.1186/s40478-018-0630-1. copyright: © 2022 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. between two worlds life in neuropathology and beyond feel free to add comments by clicking these icons on the sidebar free neuropathology 1:22 (2020) reflections between two worlds life in neuropathology and beyond peter lantos professor emeritus, king’s college london address for correspondence: peter lantos · www.peter-lantos.com · peter.lantos@btinternet.com submitted: 10 august 2020 accepted: 12 august 2020 published: 17 august 2020 https://doi.org/10.17879/freeneuropathology-2020-2933 keywords: hungary; middlesex hospital, london; neuropathology; institute of psychiatry/maudsley, london; papp-lantos inclusions; bergen-belsen; memoir, fiction and plays introduction on a rainy october afternoon in 1968, as my plane landed at heathrow airport from budapest, i did not know that i would never return to the country of my birth and early youth. i arrived with a small suitcase and five pounds in my pocket. i was a fortunate and happy recipient of a wellcome research fellowship at £1,200 per annum, which had been awarded two years earlier. the hungarian authorities, however, for reasons known only to themselves, did not give me an exit permit. my visit was on a knife edge until the last minute, for on the day when i was going to the british consulate to apply for a visa, the hungarian state radio announced that the armies of the warsaw pact (including hungary’s) were giving “brotherly help” to the people of czechoslovakia. translating this orwellian newspeak, it meant invading a friendly, neighbouring country. arrival in london my first impression was compensation for all the preceding stress: my future boss, professor stanley holt, met me at the airport and brought one of his assistants, who happened to also be a hungarian and who left after the revolution of 1956. holt (fig 1), or “holtie” to his friends, was a cell biologist of international reputation. his department of histochemistry of the courtauld institute of biochemistry within the middlesex hospital medical school, london, w1, which despite its small size, was a beehive of activity with young scientists from all over the world. he originally excelled in histochemistry and later applied electron microscopy to the demonstration of enzymes – a methodology i learned from him. this expertise stood me in good stead one year later when i started to work on my phd. he had lexicographic knowledge of a wide range of subjects (including an in-depth personal expertise of scottish single malt whiskies), and was an excellent teacher and a most entertaining raconteur. fig 1 professor stanley holt in the late summer of 1969, as my fellowship was coming to an end, i realised that, whatever happens, i was not returning to hungary. the reason for this decision was complex, but at its core was the discovery of personal freedom with the liberating feeling that i was in charge of my own destiny. having experienced a new way of life, i felt that i could not conform to the rules and regulations of life under communism. “defection”, my trial and prison sentence in hungary when i told holtie of my decision, his response was rapid and efficient as he went to see the director of the institute and the secretary of the medical school. the next couple of months remain memorable for the kindness and help of people who allayed my way of getting permission to stay in the united kingdom, to find accommodation and, most importantly, a job. in the meantime i had to write an official letter of resignation to my workplace, the institute of morbid anatomy and histopathology, medical university in szeged, hungary. it was not an easy task. the response was immediate and brutal. the rector of the university ordered a disciplinary action on the basis that i refused to return to hungary. since i failed to appear personally to present my case, i was summarily dismissed from employment. but this was not the end. i was also indicted with a criminal charge of refusing to return to hungary. the trial took place, in my absence, on 9 february 1970 in the district court of szeged. i was sentenced to 16 months imprisonment as principal punishment and complete confiscation of all my belongings as secondary punishment. the latter i viewed with a sense of bitter irony, for it was for the third time in less than three decades that we lost all our material possessions: first, the fascists took away everything in 1944, then the communists closed down the family’s enterprise in 1949 and finally i lost everything in 1970, for my flat in szeged had been sealed and sold with its contents. for many weeks after my “defection”, i had a recurring nightmare. i was getting on the plane at budapest airport and, as i was fastening my seatbelt, a stewardess called my name and frogmarched me off the plane. i was terrified; i would never fly to london. the nightmare always ended the same way i awoke in a pool of sweat, but happily realising that i was in london after all. many years later in 2015, i learned that the hungarian state security (avo) had a file on me (fig 2). at my request its archives sent a copy which is now in my possession. it consists of 39 pages, with all the details of my trial and some other information that had been kept for decades. “big brother” had a long memory. fig 2 copy of my photograph in the file of the hungarian state security, taken circa 1966 the middlesex hospital medical school, london my first appointment was a research assistant in the bland-sutton institute of pathology of the middlesex hospital at an annual salary of £2,160. not knowing at the time, i was the first to be employed from the so-called “biscuit money.” in the 1960s, garfield weston, multi-millioner owner of fortnum and mason (the queen’s grocery shop) and many other outlets, donated a large sum of money to promote research into neurological diseases at the middlesex hospital. this donation was, by the way, the seed from which the reta lila weston institute of neurological studies at university college london later blossomed. professor george dick, director of the bland-sutton, and dr. helen (“wendy”) grant, a consultant neuropathologist (fig 3), were looking for a research worker who would examine ultrastructural abnormalities in progressive multifocal leukoencephalopathy (pml). this was an appointment clearly tailored for me, nevertheless i was formally interviewed. fig 3 dr. helen (wendy) grant in 1987 i started to investigate post-mortem brains with pml, but soon realised that the information we could get would not add a great deal to the existing knowledge. after an extensive literature search and discussions with professor dick and dr grant, i suggested that my project be in the field of experimental brain tumours. two british biochemists, peter magee and john barnes, had discovered the carcinogenic actions of nitroso compounds in 1956. subsequent extensive investigations revealed that two nitrosoureas, methyland ethyl-nitrosourea, have preferential carcinogenic actions in the nervous system. ethyl-nitrosourea (enu) when injected in a single dose to pregnant rats after the 15th day of gestation induces tumours at a rate of nearly 100% in the nervous systems of offspring. this is of paramount importance in carcinogenesis; because the precise time of interaction between the carcinogen and the cells is known, it became possible to study carcinogenesis and the evolution of tumours from their inception. phd on brain tumours it was at this time that i met a young german neuropathologist who expressed his ideas with enthusiasm and some force. we started to collaborate and became friends: his name is paul kleihues and he came to work with magee, a professor at the courtauld institute of biochemistry. i started my project on the development of enu-induced tumours of the nervous system in bd-ix rats in 1970. the tumour yield of the offspring approached 100% and i sequentially followed their growth from the earliest abnormal cell proliferations to fully developed malignant tumours. pleomorphic gliomas, resembling human glioblastoma multiforme, have originated from undifferentiated cells of the subependymal plate. my first collaborator who joined me was also funded from the “biscuit money”: geoffrey pilkington, 21 years old at the time at the time of our collaborations, recently retired at the end of 2019 as professor of cellular and molecular neuro-oncology. i submitted my phd thesis to the university of london with the title, the fine structure and enzyme cytochemistry of tumours induced by n-ethyl-n-nitrosourea and was awarded in 1973. it might have been the last word in the field, but leafing through it now, its two volumes are fossils of a bygone era. while working on the phd, i was also doing the occasional neurological post-mortems and looking at brain biopsies. at the advice of senior colleagues, i decided to prepare and sit for the mrcpath examination. to learn the “trade,” i visited many neuropathological departments in england and abroad. on the basis of my phd and previous experience in hungary, i was exempted from the primary and got successfully through the final examination in the winter of 1975. this i celebrated by treating myself to a round-the-world air ticket. in the summer of 1976, i was appointed senior lecturer in neuropathology at the middlesex hospital medical school and honorary consultant to the hospital. i already had my small research team working on experimental brain tumours and my first phd student, who later became a professor of neurology in athens, greece. the changing ethos of medicine the middlesex hospital exists no more. first it fused with university college hospital london (uchl) to become “unisex” for a couple of years while the new uchl was built. it was later demolished to give space to modern blocks of flats. only its beautiful chapel – an oasis of peace and a place for reflection – has survived as a protected building. while i worked there from 1968 to 1980, it was a most pleasant and stimulating environment; the competence of its consultant staff was matched by the excellence of its academic life. medicine by that time might not have been a vocation, but it was still a profession and not the business to which it has been degraded today. it was run by doctors for patients and not by managers for profit and quotas. when the uk went through turbulent economic and financial difficulties, we were encouraged to raise funds for research; yet the basic principle that it was the university which should provide funds for salaries and infrastructure still prevailed. this changing world was reflected even in the everyday attitude of employers. young doctors of today would not believe that, for many years, it was a daily tradition at the middlesex to lay out tea with cakes and sandwiches in the senior common room – free. this is a far cry from now, when staff are expected to pay for parking at their work place. the institute of psychiatry/maudsley in 1979, the chair of neuropathology at the institute of psychiatry (iop) was advertised and i applied. to my surprise (and perhaps to everybody’s), i was appointed in october of that year; exactly ten years after i had decided to remain in england. in the immediate euphoria and surprise, i did not realise that i arrived at the wrong place, at the wrong time. the chair at the iop was, at the time, one of the two established chairs of neuropathology in the uk, created to acknowledge the work of professor alfred meyer who escaped from nazi germany and built up the neuropathology laboratory at the maudsley hospital, london (fig 4). i was the fourth incumbent of the chair and did not realise that i was going to face an uphill struggle. in the late 1970s the uk was literally bankrupt and, after the “winter of discontent” when the country was paralysed by strikes, margaret thatcher had won the election for the conservative party in may 1979. the new government embarked on an austerity policy that did not spare universities. the vacant jobs of two senior lecturers and one lecturer, advertised with the chair, were “frozen” – the dreaded term i heard for the first, but unfortunately not the last time. the department was decimated; there was one full-time and one part-time consultant, one senior registrar and a group of three neurophysiologists. however, there was no shortage of advice, with the best coming from lucien rubinstein in his letter of congratulation. i will quote the penultimate sentences, for it is wise advice even for future generations: “the main thing is to plan carefully and deliberately, and recognize where action is possible and where it is futile. if you insist, however, long enough, often enough, and nicely enough, you will, we are sure, get your own way.” and this i tried to follow during the next 22 years, with more or less success. fig 4 professor alfred meyer i made a plan for the first five years and decided that i would resign if i did not succeed. first, with a major grant from the mrc and with geoff pilkington’s help, who came with me from the middlesex hospital, we redesigned and upgraded the electron microscope unit. this was followed by modernising the histopathology laboratory and creating laboratories for immunohistochemistry, molecular biology, genetics and tissue culture. with the late professor david marsden, we successfully applied to the mrc for a grant to establish an alzheimer’s and parkinson’s diseases brain bank. after he was appointed to the chair of neurology at the institute of neurology and neurosurgery in london, we had a friendly “divorce.” the parkinsonian component moved with him, whilst the larger share stayed at the iop to become a major resource of research. this brain bank is still flourishing under the directorship of professor safa al-sarraj, head of clinical neuropathology. in 1985, with the spreading of hiv, the mrc funded an aids brain bank which became one of the national centres of aids research. by this time, the department of neuropathology expanded to occupy two floors in the iop and had a foothold in the new clinical neuroscience building of king’s college hospital. research during the 1980s and 1990s thrived, chiefly for two reasons. first, the department was fortunate to attract talented young researchers, including trainees in neuropathology, other clinicians (including psychiatrists), and a large group of non-medical neuroscientists. there were several independent, but collaborating research groups in neurodegeneration, hiv/aids, prion diseases, brain tumours, neuro-immunology, schizophrenia and autism. second, although there was never enough money to cover everything we wanted to do, the department was successful in getting longand short-term grants. by this time the department had some 50 members, including four consultants and three secretaries. when the iop merged with king’s college london as part of the major re-structuring of medical schools and postgraduate institutes in london, i became the clinical director (lead clinician) of neuropathology services covering a catchment area of 3.5 million people. i also held honorary consultant contracts with the maudsley, king’s college, guy’s and st thomas’ hospitals. in 1992, i received the degree of dsc of the university of london, and in 2001 i was elected fellow of the academy of medical sciences. my main research interest was in neurodegeneration, particularly in familial and rarer dementias and prion diseases. this included successful collaboration with psychiatrists and neurologists of the iop/maudsley and elsewhere, both in england and abroad. i was also invited to participate in three editions of the who classification of brain tumours (1993,1997 and 2000), the clinico-pathological definition of pick’s disease, the european network of brain tissue bank (european union), hiv infection of the nervous system diseases and creutzfeldt-jakob disease (international society of neuropathology), and the definition and diagnostic criteria of corticobasal degeneration, supranuclear palsy and multiple system atrophy (national institutes of health of the usa). it is in multiple system atrophy in which we have made a significant contribution to the diagnosis and definition of disease. the discovery of glial cytoplasmic inclusions, also (unfortunately not) known as papp-lantos inclusions in 1987, mátyás papp, professor of neurology and neuropathology at the semmelweis medical university of budapest (who in the early 1980s spent one year in my department), sent me several cases of striatonigral degeneration, olivopontocerebellar atrophy and shy-drager’s syndrome. he observed, by using the gallyas’ technique, tangle-like inclusions which occurred predominantly in oligodendrocytes. in london, we further characterised these inclusions by electron microscopy and immunocytochemistry (a state-of-the-art technique available at the time) and established that they were present in all three clinical syndromes. later, it was found that all three processes also contain ɑ-synuclein. the fundamental importance of glial cytoplasmic inclusions are twofold. first, they are the hallmark cellular lesions – not in any way less important than lewy bodies – by which these elusive disorders can be confidently diagnosed. second, their presence in all three clinical syndromes demonstrated that they are a single nosological entity: multiple system atrophy. editing greenfield’s neuropathology when professor david graham asked me in 1992 whether i would co-edit the sixth edition of greenfield’s neuropathology, i had no hesitation to accept. whilst editing textbooks never appealed to me, two titles were tempting: “greenfield’s” and “russell and rubinstein”. earlier, i was approached by the publisher of the latter, but the transatlantic negotiations came to a halt and i felt better not to be involved. the first edition of greenfield’s was published in 1958 and the fifth edition of 1992 was not an unqualified success. we decided to have a different concept, content and design from the previous editions. with the support of georgina bentliff, this was the beginning of our harmonious collaboration of some ten years with the publishers: first edward arnold, then hodder and stoughton and finally hodder headline. the sixth edition, published in 1997, was well received, collected a couple of prizes and had to be reprinted. the seventh edition appeared on the market in 2002 and must have been regarded as a success, for the publisher gave a special, leather-bound copy to the two editors. the british neuropathological society british neuropathologists have two excellent forums dedicated to neuropathology: the british neuropathological society (bns) and the bimonthly scientific journal, neuropathology and applied neurobiology. in the past, the bns had two annual meetings: in the winter in london with rotating venues and in the summer in the country at clinical neuroscience centres. the meetings were open to everybody who paid the registration fee. they were attended by neuropathologists and neuroscientists from other countries – mainly from continental europe. at their best, the meetings lasted two days and were frequently preceded by a symposium with invited speakers on selected, topical subjects. these were occasions as informative and stimulating scientifically as they were enjoyable socially. i joined the bns in 1972, becoming its vice-president in 1993-1995 and president in 1995-1997. the bns first published neuropathology and applied neuropathology in 1975 under the editorship of professor john cavanagh, an outstanding neuroscientist with razor-sharp critical sense. he edited the journal until 1988 when, under professor roy weller’s editorship, the journal continued to flourish as it does to the present day. the problems of british neuropathology as head of a large department of neuropathology, i was aware of three chronic problems of our specialty. first, the complex and sometimes difficult relationship with histopathology, as reflected by the chronic “independence” fight with the royal college of pathologists for recognition as a separate specialty. this was less of a problem for departments which were part of a large clinical neuroscience centre, but more difficult for those which were de facto part of a histopathology department. the royal college of pathologists eventually recognised neuropathology as a subspecialty, organised a sub-committee of neuropathology of which i was chairman (1987-1990), and allowed the final examination to be taken in neuropathology, overseen by a panel of examiners which i also chaired (1985-1990). second, irrespective of location, the cost of neuropathology services were always a concern – increasingly so with the advent of managers and during the endless reorganisations. the argument that the post-mortem examination of brains and the diagnosis of biopsies from the nervous system may exceed the average cost was not always readily accepted. the third problem was recruitment. unfortunately, neuropathology is a small specialty and during my time the number of consultants did not exceed 50 in the uk. choosing neuropathology as a career in medicine had the potential difficulty of finding a consultant appointment after the completion of training – let alone getting one at a desired location. this clearly was a limiting factor, although both the mrc and the wellcome trust were most helpful by funding training positions in neuropathology. despite the rumours: there is life after retirement the publication of the seventh edition of greenfield’s in 2002, coupled with other reasons, seemed to be the ideal time for my retirement. i also remembered my fore-teacher at school who advised to leave the party when one enjoys it most. whilst i admire my colleagues who do not seem ever want to retire, i knew that i was not going to select this avenue, despite a couple of tempting offers for part-time work. i did love neuropathology and do not think, if i were starting again, i would choose another specialty within medicine. overall i enjoyed my work, both at the middlesex hospital and at the iop, but, apart from working pro bono for medical charities, retirement was a closure of my professional life. however, even after retirement, i followed the careers of those who worked with me. it is with some satisfaction to record that all my phd students (with the exception of one who has a leading position in the british pharmaceutical industry) became professors in their fields in this country, the usa, ireland, hungary and greece. one is now executive dean of the iop, which is still one of the leading centres of its type and now appropriately renamed the institute of psychiatry, psychology and neuroscience. some of my young colleagues and trainees also had stellar careers: two at the institute of ophthalmology and one as the director of the wellcome sanger institute, having been knighted a few years ago. immediately after retirement, i enrolled in a two-year theology course on judaism, french and hebrew classes and short cookery courses and demonstrations. however, these were only diversions, for i had a project to pursue. bergen-belsen in the summer of 1944, as a child of five, i was deported with my parents first to austria and then to bergen-belsen concentration camp in germany. i was prisoner 8431, my mother 8517 and my father 8432. my father died there of starvation; my mother and i survived (fig 5). before the british army reached the camp, we were put on a train with destination theresienstadt, another concentration camp outside prague. on the way our train was bombed by the allies for they thought that it was carrying german troops, but finally we were liberated by the 30th division of the 9th us army on 13 april 1945 outside a small village in eastern germany. after travelling across war-torn europe, we arrived home to an empty family house at the end of august 1945. my mother never talked about this experience – not until i was a teenager when i started to ask questions. fig 5 with my mother in 1946 life under the communists after the war, hungary was a democratic country with a multi-party political system, free elections and an increasing living standard. however, at the end of 1949 our normal life had abruptly changed. insidiously increasing its power with the assistance of the red army, which by then had changed from a liberating to an occupying force, the communist party came to power to inaugurate years’ of terror. the revolution of 1956 brought a few days’ of hope and freedom, only to be squashed by the red army and with the introduction of reinvigorated oppression. in 1958, after some difficulties and at the protestation of my mother, my social classification was upgraded from “class alien” (which meant social death) to “other” (a dubious category with which one could just get by). i was admitted to the medical university of szeged (now named after albert szent-györgyi, the nobel prize-winning scientist who worked there). despite oppression until 1960 – for example we knew there was at least one “student” who regularly reported on us to the police – i greatly enjoyed my university years. the main reason was the quality of teaching. there was an extensive and demanding curriculum, including a good balance between theory and practice in the form of lectures and practical classes. despite the “iron curtain,” medical and scientific journals were available from all over the world, and from the late 1950s senior staff could apply for fellowships in western countries. and this is how some years later i received a wellcome research fellowship in 1966, as a lecturer in the institute of morbid anatomy and histopathology. revisiting belsen i realised that as an adult, i knew very little of what had happened to us. after all, 21 members of my family perished in the holocaust. the aim of the project i embarked on was to fill this gap. however, i encountered a formidable obstacle at the very beginning in that most of the family documents had been lost during the war. those which survived had been left, and presumably destroyed, in my flat in szeged. to rekindle memories, during 2003 and 2004 i traced the footsteps my family made 60 years earlier in 1944 and 1945. it was an exciting and rewarding time – a sort of research i had not done before: diving into the archives of four different countries and meeting other survivors and witnesses. two witnesses, amongst the many, were unforgettable: the late george gross, the american tank commander who liberated our train and whom i visited in san diego, california (fig 6) and the late professor john hankinson, professor of neurosurgery in newcastle, who as a last-year medical student volunteered to help in belsen after liberation. fig 6 with george gross in san diego in 2003 for his work, the british army presented him with a beautiful silver pocket watch which he passed on to me when i visited him in newcastle. this is one of my most cherished possessions (fig7). fig 7 the silver pocket watch from bergen-belsen on the top of my bureau reinventing myself as a self-employed author i had not planned to write a book about my childhood. however, with a large amount of material from my travels, i started to make notes as a sort of aide memoire. the experience at the beginning was quite unsettling, for there was an element of emotional involvement, completely different from the impersonal, objective and sparse style of medical/scientific writing – something i had practised for four decades. however, in the end, i had a manuscript and one of my friends, an editor, found a small publisher. parallel lines – a journey from childhood to belsen was first published in 2006 by arcadia books london (fig 8a). it was well received both in this country and abroad; it was translated into hungarian (sínek és sorsok, scolar 2009), italian (tracce di memoria, giunti 2015) and german (von ungarn nach bergen-belsen und zurück, wallstein 2017). after attending medical/scientific meetings, symposia and workshops, it was a great change and exciting to be invited to literary festivals and book launches in this country, the usa, germany, hungary, australia and new zealand. after parallel lines i wrote a novel, closed horizon, also published by arcadia books in 2012 (fig 8b). it is a dystopic novel which takes place in the surveillance state of the near future and it is a clash between the individual and the darker power of the state. “the play’s the thing” (shakespeare) i never had any intention of writing plays and it was only the love of theatre which lead me to a short course, organised by the writing academy of the publisher faber and faber, on writing plays in 2012. the course was good fun with two excellent tutors: one being a well-known playwright and the other the artistic director of a central london theatre. thus, my playwriting “career” is entirely accidental, but i enjoyed the idea of trying to bring characters to life by creating inner tensions and external conflicts. the research, which was essential for the historical background and characters of the plays, was very similar to the excitement of discoveries of medical research. yet the world of theatres, actors, directors and agents is far removed from the medical/scientific environment. it was not any less revelatory and exhilarating, yet far more uncertain and changeable – as if someone attempted to enter a mirage. nothing exemplifies this more than the author relationships with publishers. whilst medical publishers came to ask, or even begged, authors to write for them, one has to find a publisher in the in the literary world where there are over 160,000 titles a year. the first three play were collected into a volume, stolen lives to form a trilogy (regent books london 2018; fig 8c). all three take place in hungary against historical turning points: the visitor during the second world war; distorting mirrors in the 1956 revolution and stolen years at the fall of communism in 1989. in all three, the source of drama is the same: human tragedy, in the form of an irredeemable conflict between vulnerable individuals and brutal authoritarian dictatorships relentlessly motivated by an obnoxious ideology, be it fascism or communism. i have completed two more plays. light and shadow is the extraordinary love affair which shook puritanical victorian london between the french painter james (jacques) tissot and kathleen newton, a catholic irish divorcée with an illegitimate child. my last play, bread from air: the strange case of dr haber has perhaps the most complex of characters: dr fritz haber the nobel laureate in chemistry (1918), who by producing ammonia from hydrogen and nitrogen, the basic substance of fertilisers, is credited with saving the lives of two out of the seven billion people alive today. yet, it was also he who produced (on a scientific basis) poisonous gases, one of which was first introduced in 1915 against french troops at ypres. his private life was as contradictory and colourful as his professional career. he lived long enough to be refused entry by the nazis into the institute he founded and that bore his name. fortunately, he did not survive to witness the death of some of his relatives in auschwitz, who were murdered by the same poisonous gas his team had experimented with in the 1920s. these two plays were published in a volume, love and obsession (regent books, 2019; fig 8d). fig 8 covers of my books all these plays had professional rehearsed readings with actors – mostly in the theatre and one in the tate gallery – but none has had a full production yet (fig 9). now, with the pandemic creating grave consequences for theatres, it is even less likely that this will occur in the near future. nevertheless, i am working on my sixth play which, through its extraordinary heroine, brings together my country of birth and my adopted country. fig 9 at the reading of one of my plays in 2015 epilogue or final curtain after 21 years of absence, i arrived at budapest airport on a sunny september day in 1989. the city was more beautiful than i ever remembered and was floating on a wave of expectation and hope. the still “socialist” government had just opened the border to austria and this was the “beginning of the end” of communism, for the berlin wall came down two months later in november. for me this journey was of great importance, for it built a bridge between the two worlds in which i have lived: hungary of my childhood and youth and england of my adult life. in the sunshine of euphoria and wellbeing, i felt that a new golden age was dawning over hungary, europe and the world. looking back now, i could not have been more wrong. yet we should hope that common sense and decency will still prevail over madness and outrage; liberal democracy over dictatorships and populism (fig 10). fig 10 peter lantos today copyright: © 2020 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. neuropathology and epilepsy surgery: 2022 update feel free to add comments by clicking these icons on the sidebar free neuropathology 3:12 (2022) review neuropathology and epilepsy surgery: 2022 update ingmar blümcke1 1 department of neuropathology, university hospital erlangen, germany corresponding author: ingmar blümcke · department of neuropathology · university hospital erlangen · schwabachanlage 6 · erlangen · germany bluemcke@uk-erlangen.de submitted: 17 march 2022 accepted: 25 april 2022 copyedited by: vanessa goodwill published: 03 may 2022 https://doi.org/10.17879/freeneuropathology-2022-3813 keywords: brain, seizure, malformation, brain tumor, molecular diagnostics abstract the impact of a precise histopathology diagnosis and molecular workup for surgical patient management remains a controversial issue in epileptology with a lack of diagnostic agreement as root cause. very recent advances in genotype-phenotype characterization of epilepsy-associated developmental brain lesions, including the first diagnostically useful dna methylation studies, opened new avenues and will help to finally resolve these issues. a series of most recent articles were decisively selected by the author to exemplify the areas of improvement in neuropathology and epilepsy surgery. these topics include the progress in genotype-phenotype association studies of focal cortical dysplasia (fcd) leading to the discovery of new molecularly defined entities, i.e. mild malformation of cortical development with oligodendroglial hyperplasia in epilepsy (moghe), slc35a2 altered. these studies also triggered the first update of the international fcd consensus classification scheme from 2011, which will hopefully support diagnostic agreement in clinical practice and research. the dilemma of new tumor entities proposed by the 5th edition of the who classification primarily associated with early seizure onset yet not well introduced to the epileptology community will also be discussed in the light of emerging experimental evidence when transfecting the developing murine brain with the single most important genetic alteration for both carcinoand epileptogenesis, i.e. braf v600e. introduction the impact of a precise histopathology diagnosis and molecular workup for patient management remains a controversial issue in epileptology 1, i.e., the decision making for surgical treatment when a patient with focal epilepsy is considered drug-resistant and prediction of postsurgical seizure freedom should be based on the underlying cause. this discussion continues despite large enough patient series suggesting a positive prediction of scientifically defined brain lesions for long-term postsurgical seizure control for more than 5 years2. a root cause of these inconsistencies is likely due to lack of diagnostic agreement across histopathology laboratories around the world, a fact widely recognized in the scientific literature3-7. the introduction of molecular diagnostics has helped to overcome this issue in the field of neurooncology with the best renowned examples of abandoning mixed oligo-astrocytomas and introducing molecularly defined and clinically/therapeutically relevant tumor subtypes. literature published in 2021 about the topic of neuropathology and epilepsy surgery elicited new hope for epilepsy centers around the world to better connect with molecular diagnostics and integrated genotype-phenotype pathology diagnosis, which will finally move the field toward personalized medicine and targeted treatment options. the following chapters will address ten topics of interest to the author based on papers published in 2020-2022 and with reference to other older topic-related key data. topic 1: the molecular phenotype of human epilepsies at the single cell level topic 2: brain somatic mutations in cortical malformations (mcd) topic 3: dna-methylation classifier for malformations of cortical development topic 4: new clinico-pathologically and genetically defined entities: moghe and fcd1a topic 5: toward a first update of the international consensus classification of fcd topic 6: news from low-grade epilepsy-associated brain tumors (leat): human studies topic 7: news from leat: animal models topic 8. what is new about hippocampal sclerosis: human herpes virus infection and the impact of inflammatory infiltrates on neuronal cell loss topic 9. the fine structure of the epileptogenic neocortex and white matter topic 10: what is still missing to further the advancement of neuropathology and epilepsy surgery? topic 1: the molecular phenotype of human epilepsies at the single cell level the inception of single cell biology has completely revolutionized the study of neurological diseases and has the potential to answer many of the yet unresolved questions in the neurosciences8. the advancement in molecular-genetic barcoding technology (figure 1) at the single cell level including single-cell genomics and transcriptomics finally reached the realm of epileptology. human surgical tissue samples of clinically well-characterized patients have ever been a valuable resource for research, although vast differences in the patients’ genetic background, clinical histories and medical treatment pose major challenges to comprehensive data analysis. focusing on single cell genomics, transcriptomics, and finally also epigenomics in focal epilepsy research, will circumvent the averaging artifact when studying bulk brain tissue and will reveal the kind of granularity that is needed for investigating the consequences of network alterations at the single cell level8. figure 1: single-cell genomic applications using single cell combinatorial indexing or droplet based cell isolation with barcoding 8 single cell combinatorial indexing (sci) and droplet-based cell isolation are the most popular barcoding strategies used by most high-throughput single cell genomic applications. sci uniquely tags the nucleic acid molecules in each cell through serial mixing, splitting, and barcoding. the higher the number of barcoding steps, the higher the number of cells that can be uniquely tagged in each experiment. droplet-based techniques rely on physical isolation of individual cells and engineered barcoded beads in nanoliter droplets, which limits their scalability but they produce less noisy results. with permission from the author 8. whereas single cell genomics have already been applied using human epilepsy surgery brain samples9, pfisterer and colleagues described in 2020 for the first time single nucleus transcriptomics in human temporal lobe epilepsy and non-epileptic subjects10. they deciphered dysfunctional neuronal subtypes in the seizure active cortical region and found that the largest transcriptomic changes occurred in distinct subtypes of principal neurons, as classified by the layer-specific marker genes cux2, rorb, themis, and fezf2, and gabaergic interneurons, as classified by the marker genes pvalb, sst, vip, and id2. as a result, fezf2 positive neurons of layers 5 and 6 and cux2 neurons of layers 2 and 3 were more affected than other subtypes of the same families. similarly, parvalbumin and somatostatin interneurons showed more of an epileptogenic signature than other gabaergic interneurons. furthermore, the subtypes with the largest epilepsy-related transcriptomic changes belong to same circuits. in addition, glutamate signaling exhibited a strong dysregulation in epilepsy, as indexed by layer-specific transcriptional changes in multiple glutamate receptor genes and upregulation of genes coding for ampa receptor auxiliary subunits10. overall, these data confirmed neuron-specific molecular phenotypes in the epileptic neocortex, which may become novel targets for anti-epileptic precision medicine in the near future. over the past 5 years dna-methylation has gained significant interest and momentum in neuropathology. the development of dna-methylation based classifier for epilepsy-associated brain malformations will be discussed further below (see topic 3 for further reading). dna methylation has not yet been addressed at the single cell level in human neurosurgical specimens though. in the mouse brain, however, a first comprehensive assessment of the epigenomes of mouse brain cell types was published in 202111. the researchers used single-nucleus dna methylation sequencing from 45 regions of the mouse cortex, hippocampus, striatum, pallidum and olfactory areas and identified 161 cell clusters with distinct spatial locations and projection targets11. furthermore, they constructed taxonomies of these epigenetic types, annotated with signature genes, regulatory elements and transcription factors. these features revealed the repetitive usage of regulators in excitatory and inhibitory cells to determine cellular subtypes. furthermore, an artificial neural network model was developed to predict neuron cell-type identity and their spatial brain localization. the creation of a comprehensive dna methylation-based atlas will establish the epigenetic basis for neuronal diversity and spatial organization throughout the mammalian brain, being also instrumental when developing a human methylome brain atlas in the near future, including epilepsy surgery brain samples from various disorders, e.g. cortical malformations and hippocampal sclerosis. topic 2: brain somatic mutations in cortical malformations fifty years ago, in 1971, taylor and colleagues coined the term focal cortical dysplasia (fcd) for peculiar lesions causing drug resistant epilepsy12, which is now the most common cause for epilepsy surgery in children13. the study of resected human fcd tissue using advanced genomic technologies (figure 1) has led to remarkable advances in understanding the genetic basis of fcd (figure 2)14. mechanistic parallels have emerged between these malformative lesions and low-grade and epilepsy-associated developmental brain tumors (leat). two comprehensive review articles published in 2021 and 2022 recapitulated the avenue from recognition of somatic variants in genes related to cell growth, i.e. the mtor pathway in fcd type 2, which were acquired in neuronal progenitors during neurodevelopment14,15. the timing of the genetic event and the specific gene involved during neurodevelopment is likely to drive the nature and size of the lesion, and is maybe also related to the lobar localization. the proposed two-hit mechanism in patients presenting primarily with germline mutations, however, e.g., depdc5, has been scientifically approved but remains often difficult to detect with standard technologies. along these lines, lee and coworkers newly reported pathogenic brain-specific somatic ras homolog enriched in brain (rheb) variants in three patients with cortical malformations16. interestingly, the somatic variant load directly correlated with the size of the malformation and upregulated mtor activity was confirmed in dysplastic tissues. laser capture microdissection showed enrichment of rheb variants in dysmorphic neurons and balloon cells, i.e. fcd type 2b. these findings added rheb as an additional mtor pathway gene to the diagnostically relevant gene panel for mcd. that the extent of dysplastic brain directly correlated with the somatic variant load suggested that cortical malformations with cytopathological features, i.e. fcd type 2, represent a disease continuum from a regionally localized dysplasia to lobar or full blown hemimegalencephaly. figure 2: “one brain – many genomes” 17 somatic variants are spontaneously acquired during neurodevelopment. all the somatic variants in a progenitor cell are passed down to its daughter cells. the number of cells carrying a specific variant is an indirect marker for the developmental time point at which it was generated. with permission from the author 8. brain mosaicism, however, can only be detected in tissue obtained from autopsy or brain biopsy. liquid biopsy using cell-free dna derived from cerebrospinal fluid (csf) could yet be another source for genetic testing, as successfully proven in malignant brain tumors. in 2021, ye and coworker published a proof of principle study demonstrating that csf liquid biopsy is valuable in investigating mosaic neurological disorders where brain tissue is unavailable18. first, they sequenced csf cell-free dna (cfdna) in 28 patients with focal epilepsy and 28 controls (using droplet digital polymerase chain reaction (pcr)). they detected somatic mutations in three patients, i.e., the lis1 p.lys64* variant at 9.4% frequency in one patient with subcortical band heterotopia; the tsc1 p.phe581his*6 variant at 7.8% frequency in another patient with fcd and thirdly the braf p.val600glu variant at 3.2% frequency in a patient with ganglioglioma. they also determined that cfdna was brain-derived by using whole-genome bisulfite sequencing and demonstrating an enrichment of brain-specific dna methylation patterns. in a second parallel study and publication, same results were obtained from csf collected during epilepsy surgery19. somatic variants were detected in cfdna from 3 epileptic patients with known somatic mutations previously identified in brain tissue (out of 12 patients included in the study). both proof-of-principle studies provide evidence that brain mosaicism can be detected in the csf-derived cfdna and open future avenues for detecting the mutant allele driving epilepsy in csf. topic 3: dna-methylation classifier for malformations of cortical development the histopathological work-up of epilepsy surgery specimens is often based only on routine hematoxylin and eosin (h&e) staining with its many difficulties and pitfalls in reliable identification of anatomical landmarks and cortical layering. this applies particularly to samples not resected anatomically en bloc, or when compromised by peri-operative artefacts or diagnostic procedures such as intracranial electroencephalography (eeg) recordings, laser ablation or thermo-coagulation. it has been a long-standing effort in histopathology, therefore, to support the microscopic diagnoses with objectifiable diagnostic measures. indeed, the same issue applied to the scenario in neuro-oncology before the discovery and introduction of predictive and prognostic genetic markers for the differential diagnosis of high-grade glioma and embryonal brain tumors, which helped to develop a reliable and widely used open-access dna methylation-based brain tumor classifier20. jabari and coworkers presented such a first dna methylation classifier for cortical malformations in 2021, which opens the avenue for an objective genotype-phenotype approach in epilepsy surgery using routinely processed (archival) formalin-fixed and paraffin embedded (ffpe) tissue samples21. this work included a series of 308 histopathologically defined samples to cover the broad spectrum of mcd, including most common fcd subtypes, as well as polymicrogyria, hemimegalencephaly, tuberous sclerosis complex, mild malformations of cortical development (mmcd), and moghe specimens as well as non-mcd epilepsy samples and non-epilepsy post-mortem controls. the possibility to apply such a dna methylation classification for non-neoplastic lesions has been questioned and is an ongoing challenge due to the often low content of abnormal cells and admixture with architecturally normal-appearing cortical areas. the possibility to microscopically dissect an area of the lesion with the most cell dense abnormality from ffpe tissue blocks was an important foundation, therefore, to the success of this project. the study did also follow a classifier approach different from the well-established heidelberg classifier for brain tumors. in fact, their paper disclosed a graph in the supplemental material showing how all of their mcd samples would assemble with the heidelberg classifier (figure 3). this highlights the challenge but also the benefit of advanced bioinformatics for developing a dna methylation classifier. it also highlights the need for a disease-specific pipeline addressing structural brain lesions in epilepsy and potential confounders. as an example, seizures themselves alter the dna methylation map as demonstrated in a recent in vitro culture model “epilepsy in a dish”22. the variable seizure burden in a given patient, the affected brain region (i.e. grey and/or white matter), and the surgical tissue sample (e.g., representing the lesion or its periphery), present an ongoing issue when using the dna methylation methodology for human brain specimens. assessing as many potential confounders in the new pipeline published by jabari et al. helped to separate the histopathologically labelled groups into well distinguishable entities e.g., taking into account the different referral centres, seizure onset, disease duration before surgery, cellular heterogeneity, batch effects, and sex. finally, a test cohort of 43 independent surgical samples from different epilepsy centres was used to test the precision of the dna methylation-based mcd classifier. amongst this cohort were 19 samples previously reviewed by an international consensus expert panel (will be discussed further below). all samples from the test cohort were accurately assigned to their histopathologically agreed disease classes by the algorithm. this report provides a first dna methylation-based mcd classification scheme suitable across major histopathological disease entities observed in epilepsy surgery. there is still a lot of work to do, however. the impact of increasingly recognized brain somatic mutations in fcd and other mcd entities (see chapters above), were not yet addressed in this study and need further attention. figure 3: dna methylation-based classifier using a new deep learning algorithm adapted for epilepsy surgery brain samples (on the left) compared to the heidelberg brain tumor classifier (on the right) on the left: umap uniform manifold approximation and projection of the decision boundaries of the trained deep learning algorithm showing the distribution of 308 datasets (taken from 21; this is an open access article distributed under the terms of the creative commons cc by license). colors correspond to the following coding ctrl – post-mortem control, ncx – neocortex, wm – white matter; fcd – focal cortical dysplasia type 1, 2 and 3 according to ilae classification scheme; moghe mild malformation with oligodendroglial hyperplasia in epilepsy, pmg polymicrogyria, hme hemimegalencephaly, tle – temporal lobe epilepsy without cortical dysplasia, tsc tuberous sclerosis complex, mmcd mild malformations of cortical development. on the right: t-sne – “t-distributed stochastic neighbour embedding” of the brain tumor classifier from heidelberg of the same samples shown on the left. topic 4: new clinico-pathologically and genetically defined entities: moghe and fcd1a in 2021, we witnessed the exciting rise of a new, genetically defined cortical malformation associated with early onset focal epilepsy. the term “mild malformation with oligodendroglial hyperplasia in epilepsy” (moghe) was already coined in 201723 but received only little attention by the histo-/neuropathology community. when epileptologists described its mri fingerprint in 201924, many more reports became available cumulating in 2021 as a distinct clinico-pathological moghe pattern in children with early seizure onset and mostly affecting the frontal lobe25-28. two other papers in 2021 almost simultaneously assigned brain somatic mutations in the udp-galactose transporter slc35a2 gene to moghe6,29. the slc35a2 mutation was first discovered in a cohort of focal epileptic brain lesions in 2018, replicated in 2019 and originally assigned to either mmcd or fcd ilae type 1 entities30,31. notwithstanding, both of these differential histopathology diagnoses are difficult and poorly defined. bonduelle and coworker specifically searched for slc35a2 mutations in histopathologically confirmed moghe and detected pathogenic mutations in 9 out of 20 new cases included in their series (45%). the frequency of somatic variants were usually above 10%, ranging from 1.4% to 52%. droplet digital pcr of microdissected cells from one moghe case confirmed variant enrichment in abnormally clustering oligodendroglial cells and heterotopic neurons, suggesting that the mutation targeted neuroglial progenitors during brain development. these data provide compelling evidence for a consistent genotype-phenotype correlation in moghe with slc35a2 mosaicism as a diagnostic and probably also prognostic marker, as more than 60% of moghe patients benefit from gross total resection of the lesion25. the slc35a2 gene opens another avenue for targeted treatment in mcd and is outside of the well-recognized mtor pathway associated with fcd ilae type 2 lesions15. a recent report showed a clinical improvement in response to oral d‐galactose supplementation in several slc35a2-congenital disorder of glycosylation (cdg) patients, caused by monoallelic pathogenic variants in slc35a2 on chromosome xp11.23, resulting in reduction of seizure frequency 32. evaluating this therapeutic approach in patients with brain mosaic mutations of slc35a2 and moghe, and for whom surgery is not an option or failed to reduce seizures due to the large extent of the lesion, could offer a personalized treatment strategy. yet, there is no targeted animal model of slc35a2 brain mosaicism to clarify the precise pathogenicity. it also remains to be shown how a primary defect in glycosylation in neuroglial progenitors leads to the histopathological features described in moghe, i.e. patchy oligodendroglial hyperplasia and heterotopic neurons in the white matter (figure 4). figure 4: simplified representation of pathophysiology and neuropathology findings in slc35a2-related moghe cases on the left: slc35a2 loss-of-function variants cause a defect in protein/sphingolipid glycosylation in the cell. on the right: loss of slc35a2 function leads to a moghe phenotype, with clusters of increased density of olig2-positive cells in the white matter and deep cortical layers. l1-l6: layers 1-6; wm: white matter; figure taken from 29, an open access article distributed under the terms of the creative commons cc by license. in 2021, another comprehensive study described the clinico-pathological and molecular presentation of fcd ilae type 1a33. holthausen identified a cohort of 19 young children with drug-resistance from seizure onset and severe eeg abnormalities almost always emphasized in the posterior (temporo-parieto-occipital) quadrant. the lack of focal neurological deficits but severe cognitive impairment was another hallmark of holthausen’s disease. important for the diagnostic work-up is the mri signature presenting as subtle hypoplasia of the epileptogenic area with focus on the white matter. abundance of cortical microcolumns, best visible by immunohistochemical stainings using neun antibodies and detected in all 19 cases assigned holthausen’s disease to fcd ilae type 1a (figure 5), molecularly confirmed by dna methylation analysis. these defining features coined the provisional term “multilobar unilateral hypoplasia with severe epilepsy in children (muhsec) and is referred to holthausen’s disease herein. to the best of my knowledge, this case series is a first comprehensive electro-clinical and anatomo-pathological description of a pure fcd ilae type 1 cohort in the contemporary peer reviewed literature. the three fcd1 subtypes have been almost hypothetically defined by the ilae classification scheme as architectural abnormality of the 6-layered neocortex without frank signs of cytopathology, i.e., dysmorphic neurons or balloon cells. up to date, there is no clear description available for either fcd 1b (horizontal dyslamination) or fcd 1c (horizontal and vertical dyslamination), although the latter is often associated with developmental vascular lesions, e.g., meningeal angiomatosis of sturge-weber34, or perinatal stroke, and assigned to fcd 3c or 3d, respectively (figure 5)6,35. there was always concern that different patterns of cortical dyslamination are caused by different aetiologies and pathophysiological mechanisms, which will have implications for the diagnostic work-up as well as for the therapeutic management36. yet, we have to await further studies until the issue of fcd type 1 subtypes and their clinical relevance for patient management and treatment options will be finally resolved. figure 5: fcd subtypes of the ilae classification scheme of 2011 as visualized with neun immunohistochemistry upper panel with the pial surface always presented on top. neun immunohistochemistry with hematoxylin counterstain. scale bar on upper left = 250 μm, applies to all images of upper panel. scale bar on lower panel = 2500 μm. fcd1a: 2-year old girl with right temporo-parieto-occipital seizures. the histograph reveals abundance of microcolumns. this is even more visible on the low-power magnification of the same patient in the lower panel with arrows indicating several large regions with microcolumns and heterotopic neurons in the white matter. fcd 2b: 5-year old boy with left frontal lobe epilepsy. note the large (dysmorphic) neurons in an otherwise unlayered neocortex. fcd 3a: 28-year old male patient with left-sided temporal lobe epilepsy and hippocampal sclerosis (not shown). note the pathognomonic narrowing of cortical layer ii and depletion of pyramidal neurons in layer iii. fcd3c: 20-year old male patient with sturge-weber syndrome. note the meningeal angiomatosis on top and the abundant microcolumnar arrangement of neurons in the neocortex. adjacent cortical areas also revealed disorganization of cortical layers in a horizontal direction (although not visible by this magnification). fcd3d: 14 year old girl with seizures and intrauterine stroke. note the architectural abnormalities in cortical layers ii-v. topic 5: toward a first update of the international consensus classification of fcd fcd are the most common malformations of cortical development recognized in epilepsy surgery case series2,13. clinical diagnosis is usually based on the presurgical evaluation including highor ultra-high-resolution mri37, but should be always confirmed histopathologically from surgically resected brain samples. hence, the agreement of microscopic diagnoses remains ever challenging, which has been documented many times in the scientific literature3-7. in 2021, a new approach was launched by a consortium nominated by the ilae task force of fcd. they iteratively reviewed a consecutive cases series of 22 patients from a single center in the united states and with each patient being clinically (presurgically) suspicious for any type of fcd6. in addition, five independent laboratories performed genetic testing with fcd-specific gene panel sequencing from tissue and blood samples of the very same 22 patients. four rounds of histopathology agreement were performed amongst 20 internationally renowned colleagues from 16 countries. the group started with a series of 196 h&e stains made available through an open access digital slide review platform. notably, agreement was very low (kappa value = 0.16), and all reviewers were also asked to propose useful immunohistochemical stains from sections with the most prominent histopathologic changes. antibodies directed against the neun, map2 and non-phosphorylated neurofilament epitopes were requested most often and can thus be considered as most valuable for the histopathology work-up of epilepsy surgery specimens6. notwithstanding, the panel of recommended immunostainings cover many more epitopes, which may become necessary in the differential diagnosis of difficult-to-classify epileptogenic brain lesions38. this panel has been recently extended including mutation-specific antibodies directed against brafv600e and idh1r132h, as well as the ps6 epitope to visualize an activated mtor pathway (najm et al. the ilae fcd classification update 2022, in revision). with the addition of requested immunohistochemical stains the agreement increased to a kappa-value of 0.35 in the second round. during the third round, all previous answers were anonymously disclosed using the delphi consensus method. mri and histographs showing specific features of each lesion were also disclosed for the consensus discussion. this increased the diagnostic agreement to a kappa value of 0.5. in the final round, the results from genetic testing were available, i.e., pathogenic mtor, depdc5, akt3, nprl3 and slc35a2 mutations in 7 cases. the final agreement based on all available information was substantial with a kappa value of 0.686. this study will define the path towards an integrated genotype-phenotype diagnosis in fcd and the authors proposed a number of amendments to be recognized in an update of the 2011 ilae classification scheme (table 1). most importantly, the authors suggested to include two new entities, which were predominantly related to the white matter and already described in the scientific literature, but not yet well recognized in the neuropathology work-up of epilepsy surgery specimens. the moghe entity has been already discussed above and the association with pathogenic slc35a2 brain somatic mutations was confirmed in the presented case series. yet there is no hotspot mutation nor any immunohistochemical surrogate marker available for slc35a2. the other entity is that of mmcd with abundance of heterotopic neurons in the white matter as a defining hallmark. this lesion type will surely remain a matter of concern and discussion, as few heterotopic neurons are also present in the normal white matter of the non-epileptic cortex, as well as in several other epileptogenic disease conditions, first and foremost in fcd ilae type 1a (holthausen’s disease) and moghe. in addition, the term mmcd should be used only when any other pathology finding was ruled out, including any other principal lesion such as hippocampal sclerosis, developmental brain tumors or glial scars acquired early during life. the authors further suggested to include a panel of immunohistochemical markers to confirm the diagnosis of fcd or when no lesion can be microscopically defined6. table 1: proposed amendments to the ilae classification scheme of fcd fcd, focal cortical dysplasia, hs, hippocampal sclerosis, mmcd, mild malformation of cortical development, moghe, mmcd with oligodendroglial hyperplasia in epilepsy; * mmcd: not associated with any other principal lesion, such as hippocampal sclerosis, brain tumor, or vascular malformation; for reference values see39-42. ** no definite fcd on histopathology: a descriptive report is recommended to highlight anatomical ambiguities (if applicable). table modified from 6, an open access article distributed under the terms of the creative commons cc by license. the proposed amendments to the classification scheme presented in table 1 were taken from the discussed publication6, whereas the actual ilae classification update is still under peer review and expected to be published in due course of 2022 (najm et al. the ilae fcd classification update 2022, in revision). this fcd classification update will propose for the first time a multi-layered integration of all available diagnostic information, including histopathology (layer 1), genetics (layer 2) and imaging (layer 3), in order to achieve a final diagnosis. this approach may also compensate for the lack of accuracy or agreement shown for each diagnostic layer on its own, due to the aforementioned issues and challenges in the histopathology assessment6. it will then be the task of the treating physician to assemble all these data to a final genotype-phenotype diagnosis. a likely scenario to foster and promote this final diagnosis would be a post-surgical patient management conference, including the epileptologists, neurosurgeon, neuropathologist, neuroradiologist and neurogeneticist. the proposition to add the level of genetic testing to a comprehensive, reliable and integrative genotype-phenotype diagnosis represents an important step towards precision medicine in the realm of epileptology, as has been successfully implemented in neuro-oncology. genetic testing should ideally be performed from dna extracted from a microscopically assessed surgical brain tissue and paired blood sample, to facilitate the identification of brain mosaicism that represent post-zygotic mutational events. whether or not such genetic testing should become mandatory in clinical practise or should be applied foremost in scientific research will be a matter of further discussion and will always depend on available resources in each epilepsy (surgery) centre. the possibility to detect brain somatic mutations in cell-free dna obtained from cerebrospinal fluid using targeted droplet digital pcr was discovered in 2021 and described by two papers18,19. these findings offer the opportunity to establish a genetic diagnosis even before surgery, or in patients not eligible for surgery, and to guide the implementation of targeted therapies into the arena of epileptology. topic 6: news from low-grade epilepsy-associated brain tumors (leat): human studies the fifth edition of the who classification of brain tumors is now available online and in print43. this 5th edition introduced six new entities pertinent to the arena of epileptology (out of 22 overall new tumor types, 27%), namely the many variants and subtypes of glio-neuronal tumors and low-grade gliomas, i.e., paediatric type diffuse low-grade gliomas, mapk pathway-altered; diffuse astrocytoma, myb or mybl-altered; polymorphous low-grade neuroepithelial tumor of the young; diffuse glioneuronal tumor with oligodendroglioma-features and nuclear clusters; myxoid glioneuronal tumor; and multinodular and vacuolating neuronal tumor. their molecular-pathologic features have been scientifically defined but the epileptological disease condition with early seizure onset and drug-resistant epilepsy as clinical hallmarks and their long-term seizure outcome following gross surgical resection were rarely addressed at a comprehensive level. as a result, most of the new tumor entities are not yet introduced to the clinical arena of epileptologists and a careful review of published histopathology images in the new who classification suggests that these new entities mimic well-established tumors of the leat family (leat – low-grade and epilepsy-associated tumors)43. one example is that of diffuse astrocytoma, mybor mybl1-altered, which was first described in 2004 as isomorphic astrocytoma in a series of 19 patients with long-term and drug-resistant epilepsy44,45 or the polymorphous low-grade neuroepithelial tumor of the young (plnty), which shares many structural (i.e., glio-neuronal) and molecular (i.e., cd34, braf v600e) similarities with gangliogliomas (gg)43,46. a recently published genotype-phenotype study from beijing, china addressing 30 patients with gg confirmed the presence of braf v600e in the majority of their gg cases (77%) when submitted to targeted next-generation sequencing using a panel of 131 genes47. there was no low-grade paediatric type diffuse glioma, plnty, nor any other of the new subtypes specified above present in their series. along these lines, a group of french neuropathologists have asked the question whether the staggering number of new leat entities make a difference in the clinical management of epilepsy48. they focused on a subgroup of 72 leat cases, predominantly presenting with an oligodendroglial phenotype, and tried to classify them according to the 2021 who classification scheme. based on rna-sequencing, multiplexed digital pcr, dna methylation analysis and histopathology review they identified only two major clusters of biological significance, with cluster 1 being enriched with dysembryoplastic neuroepithelial tumors (dnt) at histology, belonging to the lgg-dnt methylation class with cd34 negativity and fgrf1 alterations; while cluster 2 was classified histopathologically as gg, belonging to the lgg-gg methylation class, with braf v600e mutation and cd34 positivity. the same conclusion was already drawn in 2019 by an international leat review panel of 25 experts from 18 countries when studying their series of 30 leat7. the current situation questions, therefore, the clinico-pathological relevance of the various types recognized by the who within the spectrum of leat48. it will remain a continuous debate and negotiation to bring together all experts, including histopathologists, epileptologists and neuro-oncologists, to define a successful strategy for best patient management. these tumors rarely progress into malignancy but offer the opportunity for long-term seizure freedom when neurosurgically resected following a comprehensive epileptology work-up2. this will require new measures and parameters for the definition of leat when addressing the specific needs of patients with early onset drug-resistant epilepsy. topic 7: news from leat: animal models braf v600e is the most common genetic driver in leat and is now variably assigned to several tumor entities listed in the 2021 who classification update (see above). notwithstanding, none of these entities share an easy to classify histopathology phenotype and the failure to establish and agree upon the diagnosis across countries and laboratories is well known from published literature49. intuitively, one would expect that a gene driver mutation such as braf v600e would establish a single diagnostic entity rather than being implicated in separate neuroepithelial tumors, i.e., paediatric type diffuse low-grade gliomas, mapk pathway-altered; plnty; pleomorphic xanthoastrocytoma; gg, dnt, or multinodular and vacuolating neuronal tumor. when braf v600e is experimentally introduced in utero into the developing murine brain by intraventricular electroporation, the resulting phenotype was consistently described as biphasic gg by koh et al. in 201850. the resulting lesion also showed intrinsic epileptogenic properties in the neuronal cell lineage, whereas tumorigenic properties were attributed to the proliferation active glial cell lineage. rna sequencing analysis of patients’ brain tissues with the mutation revealed that braf v600e-induced epileptogenesis is mediated by re1-silencing transcription factor (rest), which is a regulator of ion channels and neurotransmitter receptors associated with epilepsy50. moreover, they found that seizures were treatable in mice with the fda-approved braf v600e inhibitor vemurafenib, as well as various genetic inhibitors of rest. accordingly, this study provides direct evidence of a braf somatic mutation contributing to the intrinsic epileptogenicity in paediatric brain tumors and suggests that braf and rest could be treatment targets for intractable epilepsy. cases-cunillera and co-worker have used the same technique of in utero electroporation to extend these studies towards braf v600e mutations with doubleand triple-hits51. in their most recent animal model, aberrant braf expression in murine neural progenitors generated benign glio-neuronal neoplasms of the gg phenotype only in concert with active mtor-signaling, i.e. by phosphorylated akt (pakt). additional somatic trp53-loss generated anaplastic gg, a grading scale and terminology not supported anymore by the current who classification scheme. functionally, only braf/pakt altered tumors showed substantial neuronal activity which spread to the adjacent neocortical tissue, which was interpreted as correlate of increased epileptogenicity51. in both animal studies, however, cd34 immunoreactivity was an immunohistochemical surrogate of the experimentally induced gg50,51. such experimental leat models, which reproduce the variable genetic and histopathologic tumor phenotypes, will be important tools to further our knowledge and finally assess targeted therapies. topic 8. what is new about hippocampal sclerosis?: human herpes virus infection and the impact of inflammatory infiltrates on neuronal cell loss hippocampal sclerosis (hs) remains the single most common cause of focal epilepsy amenable to epilepsy surgery13. its etiology remains, however, to be further clarified with most researchers probably agreeing with the statement that hs is an end stage resulting from multiple etiologies. this may also be reflected by the different histopathologic patterns of segmental neuronal cell loss in hs being recognized in the ilae consensus classification scheme of 201352. however, inflammation remains a major topic of interest in the pathogenesis of mesial temporal lobe epilepsy (mtle) with or without hs. i will briefly discuss three publications highlighting this topic. tröscher and colleagues analyzed the presence of t cells in various mtle subgroups to answer the question of how much inflammation is present and whether the presence of t cells is associated with seizures and/or neuronal cell loss in the hippocampus53. their detailed histopathological investigation of the involvement of t cells in various mtle subgroups, i.e., gangliogliomas, febrile seizures, postinfectious encephalitis and rasmussen encephalitis w/o hs, suggested that t cell numbers correlated with the degree of neuronal cell loss rather than seizure frequency or disease duration. their quantification also showed that t cell numbers were significantly elevated in all mtle groups compared to healthy post-mortem controls. however, cd3+ as well as cd8+ t cell numbers varied among their mtle subgroups and nearly all mtle groups revealed elevated numbers of t cells years after the precipitating injury. hippocampal infection with human herpes virus 6 (hhv-6) has been demonstrated in patients with mtle already in 200354 and confirmed thereafter in several scientific publications. it can be envisioned, therefore, as one key etiological cause of mtle with hs. this knowledge gains further momentum from the publication of theodore and coworkers published in 2021, detecting hhv-6a and hhv-6b strains in fresh human tissue samples obtained from 87 patients with drug-resistant epilepsy submitted to epilepsy surgery55. twenty-nine of their 54 patients with hs (54%), six of 23 with fcd (26%), and one of three with a history of encephalitis (33%) were positive for hhv-6. this contrasts the overall low percentage of hhv-6 dna detection in only 6% of human brain samples with non-neurologic illness56. a febrile seizure history was not significantly associated with hhv-6 detection. however, patients with hhv-6 positive hs had significantly lower age at seizure onset than those with other pathologies. also, there was a trend for hhv-6 positive patients to have higher binding of [11c]pbr28 in positron emission tomography (pet), the latter being a marker for reactive astrocytes and activated microglia, suggesting an inflammatory pathomechanism. indeed, these findings reinforce the potential role and impact for hhv-6 in the etiology of mtle with hs. however, the same group of researchers published a neuroimaging approach in 2020 to address the effects of hhv-6 on hippocampal volume in patients with hippocampal sclerosis57. they used mri post-processing to segment cortical structures and to obtain an asymmetry index between hippocampal volumes ipsilateral and contralateral to the seizure focus when comparing between hhv-6 positive and negative patients. in this study, however, hhv-6 negative patients had significantly greater asymmetry and lower total hippocampal volume ipsilateral to the seizure focus compared to hhv-6 positive patients. disease duration and age of onset did not affect these results. their data suggest that hhv-6 still play a role in mtle but with hhv-6 having a less severe effect on hippocampal damage. this controversy will be ongoing, however, and in need of more studies to clarify the issue. topic 9. the fine structure of the epileptogenic neocortex and white matter gray-white matter blurring of the anterior temporal lobe (gwmb) is a common neuroimaging finding in patients with hs58. it is often assigned to fcd, i.e., fcd ilae type 1, although systematic correlation studies of in vivo and ex vivo mri with histopathology showed disturbed axonal myelination of the affected white matter as an underlying structural correlate rather than any signature of cortical (neuronal) malformation58. whether or not such white matter alterations were due to secondary axonal degeneration, e.g., neuronal cell loss in the ipsilateral hippocampus, has yet to be clarified but was already suggested as severity aggravates with disease duration58. this topic was once again addressed in a study by demrath and co-worker in 2021, when studying mri-histopathology correlations in twenty patients with unilateral temporal lobe epilepsy, gwmb and hs59. anterior temporal lobe white matter t1 relaxation times and diffusion measures were analyzed on the side of hs, on the side contralateral to hs, and in ten normal controls. resected brain tissue was further evaluated at the ultrastructural level from three patients without gwmb and four patients with gwmb addressing axon density and diameter, the relation of the axon diameter to the total fiber diameter, and the thickness of the myelin sheath59. as a result, hs with gwmb of the anterior temporal lobe was related to prolonged t1 relaxation and axonal loss. a less pronounced reduction in axonal fraction was also found on imaging in gwmb-negative temporal poles compared to normal controls. contralateral values did not differ significantly between patients and normal controls. reduced axonal density and axonal diameter were histopathologically confirmed in samples with gwmb compared to temporal poles without gwmb. the authors concluded that gwmb should be considered as an imaging correlate for disturbed axonal maturation that can be quantified with advanced diffusion imaging. in my view, however, the data does not provide true evidence for “disturbed axonal maturation” in the sense of a maldevelopmental feature. similar findings may occur as secondary retrograde damage and/or repair of the axonal compartment as suggested previously58. in 2021, another study addressed the fine microstructure of the epileptic neocortex using capricious golgi-impregnations to further analyze synaptic networks of glutamatergic and gabaergic axon terminals60. alterations in dendritic morphology and spine loss have been reported both in epilepsy animal models and in human brain tissues from patients with epilepsy61. however, it is still unclear whether these dendritic abnormalities relate to the cause of epilepsy or are generated by seizure recurrence. rossini and coworker investigated these fine neuronal structures in cortical specimens from 28 patients with different neuropathologically defined etiologies, i.e., fcd type 1a and type 2, and non-lesional neocortex obtained from tle with hs. autoptic brain tissues were used for comparison. three-dimensional reconstructions of golgi-impregnated neurons revealed severe dendritic reshaping and spine alteration in the core region of fcd type 2. dysmorphic neurons showed increased dendritic complexity, reduction of dendritic spines and occasional filopodia-like protrusions emerging from the soma (figure 6). surprisingly, the intermingled normal-looking pyramidal neurons also showed severe spine loss and simplified dendritic arborization. no changes were observed outside the dysplasia in perilesional tissue or in neocortical tissue obtained from the other patient groups. these data confirmed a rather normal appearing fine morphological aspect of neurons and dendritic spines in the epileptogenic neocortex, with the exception of type 2 dysplastic lesions and argue against the concept that long-lasting epilepsy will produce per se any dendritic pathology. figure 6: filopodia-like protrusions in dysmorphic neurons of fcd 2b low power microphotographs of golgi-impregnated dysmorphic neuron from a fcd 2b on the left, showing the presence of numerous short filopodia-like protrusions emerging from the soma. higher magnification on right. modified from 60 with permission from the author. the missing topic 10: what is still missing to further the advancement of neuropathology and epilepsy surgery? we are already facing another major advancement in neuropathology, which will soon be recognized as the era of digital neuropathology. artificial intelligence (ai)-based disease classifier and ever growing digital slide suites will substitute the routine work-up and may be able to also predict essential molecular features of the underlying disease condition to inform precision medicine treatment. these topics have not yet been publicized in peer reviewed journals with the exception of a deep learning-based algorithm to differentiate fcd type 2 from cortical tuber on routine h&e-stained glass slides in 202062. ai-based algorithms may even be able to replicate histochemical and immunohistochemical stainings from unstained slides without actually performing the staining procedures63,64. this potential is yet to be fully explored, including within the field of neuropathology and epilepsy surgery, and we will await upcoming publications addressing this fascinating matter as it may well reshape our next future in neuropathology. conclusion during the past decade, there have been considerable advances in understanding the genetic and morphogenic processes underlying cortical malformations and developmental brain tumors15. focal brain malformations result from somatic (post-zygotic) variants in several genes related to the mtor pathway in focal cortical dysplasia type 2, akt3, depdc5, mtor, nprl2/3, pik3ca, rheb, tsc1/2 and others, or in the galactose transporter gene slc35a2 in moghe, which were acquired early during cortical development. the timing of the genetic event, the specific gene involved and the targeted precursor cell population will determine the nature and size of the lesion, whether developmental malformation or a brain tumor. there is also emerging evidence of epigenetic processes underlying a ‘molecular memory’ in epileptogenesis22, which also helped to establish a diagnostic dna methylation classification scheme21. this knowledge will finally accumulate into a better understanding of why and how patients with these lesions have epilepsy and to move toward precision medicine in patients with drug-resistant focal epilepsy. these topics will be continuously addressed and discussed also at the annual ilae summer school of neuropathology and epilepsy surgery (https://www.ilae.org/congresses). please address your application to bluemcke[at]uk-erlangen.de references jehi l, braun k. does etiology really matter for epilepsy surgery outcome? brain pathol. 2021 jul;31:e12965. lamberink hj, otte wm, blümcke i, braun kpj. seizure outcome and use of antiepileptic drugs after epilepsy surgery according to histopathological diagnosis: a retrospective multicentre cohort study. lancet neurol. 2020 sep;19:748-757. chamberlain wa, cohen ml, gyure ka, kleinschmidt-demasters bk, 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learning. nat biomed eng. 2019 jun;3:466-477. de haan k, zhang y, zuckerman je, liu t, sisk ae, diaz mfp, et al. deep learning-based transformation of h&e stained tissues into special stains. nat commun. 2021 aug;12:4884. copyright: © 2022 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. neuroinflammation: 2022 update feel free to add comments by clicking these icons on the sidebar free neuropathology 3:3 (2022) review neuroinflammation: 2022 update hans lassmann1 1 center for brain research, medical university of vienna, austria address for correspondence: hans lassmann · center for brain research · medical university of vienna · spitalgasse 4 · 1090 wien · austria hans.lassmann@meduniwien.ac.at submitted: 20 january 2022 accepted: 08 february 2022 copyedited by: shino magaki published: 10 february 2022 https://doi.org/10.17879/freeneuropathology-2022-3790 keywords: covid-19, multiple sclerosis, alzheimer’s disease, microglia, neurodegeneration abstract besides important progress in the understanding of the pathological substrate of covid-19-associated brain disease, major insights into mechanisms of neurodegeneration in human disease have been provided in neuropathological studies published in 2021. recently developed techniques, which allow the simultaneous detection of a large battery of different molecules within single cells, have proven useful in the analysis of disease mechanisms in experimental and human neuroinflammatory conditions. they have elucidated protective and detrimental effects of activated microglia, which act in a stage and context-dependent manner in the induction and propagation of neurodegeneration. in addition, they emphasize the importance of synaptic damage and of selective neuronal vulnerability in the respective diseases. the results provide important new insights with high clinical relevance. introduction many manuscripts published in 2021 focused on neuropathological aspects of brain diseases, and it is thus difficult to select studies for a review article providing an update on their immunopathology. the manuscripts were identified by the frequent screening of the journals of neuropathology, neuroimmunology and clinical neurology, supplemented by the search for articles in pubmed, dealing with immunopathology of human diseases. obviously, all aspects related to the covid-19 pandemic raised major attention, and key elements of this work deserve to be presented here. in addition, i have focused this year mainly on aspects of neurodegeneration, which not only describe a specific feature in a single disease, but have relevance for a broad spectrum of different disorders. covid-19 and the brain not quite unexpected, a major focus in neuropathological research in 2021 focused on the changes in the central nervous system (cns) present in patients with covid-19. insight into the nature of the respective damage in the nervous system is of critical importance to develop strategies to prevent neurological disease complications. in addition, it may provide a new understanding of the pathogenetic substrate of the long covid syndrome. consensus was reached during 2021 that two different pathologies dominate in patients who died in the course of covid-19: vascular hypoxic/ischemic brain lesions and global microglia activation (matschke et al. 2020, maiese et al. 2021, pajo et al. 2021, al-sarraj et al. 2021). vascular pathology included endotheliitis, associated with a variable degree of focal ischemic or hemorrhagic lesions and diffuse or focal hypoxic alterations of neurons and glia (thakur et al. 2021, lee et al. 2021, fabbri et al. 2021). microglia activation was most prominent in the brain stem and the hippocampus. it consisted of a diffuse increase of microglia density with cellular activation and the appearance of microglia nodules (thakur et al. 2021, maiese et al. 2021). overall, neither the vascular changes nor the patterns of microglia activation are considered specific for sars-cov-2 infection, but can also be found in other virus infections of the cns, in conditions of systemic immune activation or sepsis and in patients dying in intensive care units (thakur et al. 2021, al-sarraj et al. 2021). thus, systemic cytokine storm in the course of the disease together with episodes of general hypoxia or metabolic derangement are likely to be at least in part responsible for the respective damage in the nervous system. since the mean age of the patients in the larger autopsy series was beyond seventy years, it was not a surprise that about half of them exhibited pathologies related to age-related neurodegenerative diseases. all these pathological changes are also in line with the overall clinical appearance of neurological complications of covid-19 (thakur et al. 2021). the prevalence and potential role of direct sars-cov-2 infection in the cns is still controversial (al-sarraj et al. 2021). in the majority of cases no virus rna or antigen in the cns has been detected, and when it was present, the virus titers were low even in cases with persistent active infection in the respiratory organs (maiese et al. 2021, thakur et al. 2021, fabbri et al. 2021). this, however, does not exclude a transient endothelial infection, which triggers endotheliitis and vascular occlusion at early lesion stages (kirschenbaum et al. 2021, nalugo et al. 2021). another potential route of virus invasion of the brain may occur through the olfactory system. however, a recent very systematic analysis of experimental and clinical data on this topic came to the conclusion that brain infection via olfactory nerves and a trans-neuronal spreading of infection through the olfactory system is unlikely (butowt et al. 2021). yet, neuronal spread of the virus by retrograde axonal transport in sensory nerves from the infected mucosa is still an option in some cases. importantly, this study also pinpoints to the limits of experimental research in this topic, since those models, which mimic human disease best, are induced in animal species, which are only poorly suitable for currently available immunological and molecular research tools (butowt et al. 2021). an important question is whether structural brain damage in the disease course of covid-19 could in part explain long-term sequelae of the disease (long covid; baig 2021). ischemic changes and microglial activation were seen in 40% and 80% of brains, respectively. however, only 17% of these patients had neurological manifestations that were severe enough to involve a neurological consultant during life (thakur et al. 2021). this suggests that brain damage in the course of covid-19 is more frequent and widespread than reflected in clinically overt neurological disease. this is in line with the results of a very large clinical study on a cohort of more than 10,000 covid-19 patients from the philippines, which describes a high prevalence of mild neurological deficits (espiritu et al. 2021). clinically, mild cognitive impairment, encephalopathy and cerebrovascular disease were the most common manifestations. the high incidence of microglia activation in the hippocampus and brain stem may be related to neuropsychiatric symptoms in covid-19 survivors, including memory disturbances, somnolence and fatigue (thakur et al. 2021). the immunopathology of the covid-19 brain the studies discussed above describe the basic neuropathology of the central nervous system of patients with covid-19, but they have not addressed the phenotype of the immune reaction within the brain, and its relation to a possible infection with sars-cov-2. this gap has recently been filled by a broad and systematic study, which was performed on a large sample of brain autopsies from covid-19 patients, in comparison to patients who died in intensive care units under conditions of respiratory distress or to patients with multiple sclerosis (schwabenland et al. 2021). the study used imaging mass spectrometry, which allows simultaneous detection of multiple antigens, their spatial distribution in relation to lesion architecture and their vicinity to other immune or resident cells within the tissue. searching for sars-cov-2 antigen within the brain tissue, the study reached similar conclusions, as described above, by showing only sparse virus antigen in the central nervous system. when present, it was mainly seen in endothelial cells of cerebral vessels. displaying leukocyte-related molecules revealed a remarkable inflammatory reaction in the brain. it was characterized by cell clusters, representing different stages of microglia activation and perivascular myeloid cells recruited from the circulation. most profound pro-inflammatory microglia activation was seen in the microglia nodules. microglia and myeloid cell activation was associated with infiltration of the tissue by t-lymphocytes with a dominance of cd8+ cells, but a very low number of b-cells and plasma cells. in comparison to other diseases, neuroinflammation was more pronounced than in patients who died in intensive care units under respiratory distress only, but the inflammatory reaction in patients with multiple sclerosis was much more severe. considering the immensely rich spectrum of immunological details described in this study, a number of conclusions have been reached. there is apparent evidence for a sars-cov-2 infection in the vessel walls, in particular affecting endothelial cells, and this is associated with a perivascular inflammatory reaction with blood brain barrier damage, mainly composed of cd8+ t-lymphocytes and recruited myeloid cells. however, in contrast to other previous studies thrombotic changes in inflamed vessels were absent. the inflammatory process concurred with global microglia activation and the formation of microglia nodules. the latter were the sites for most intense microglia activation and within the nodules cd8+ t-cells closely interacted with activated microglia, possibly providing pro-inflammatory as well as immunoregulatory signals. whether such microglia nodules are a secondary phenomenon of the profound inflammatory reaction in the tissue or drive the inflammation through (virus) antigen recognition remains unresolved. analysis of virus antigen expression did not provide evidence for virus entry into the brain through retrograde axonal transport from mucosal sites or through olfactory nerves. despite the ground-breaking nature of this study, there are still a number of limitations. the major open question relates to the mechanisms of microglia activation. although the authors did not find positive evidence, a potential role of direct sars-cov-2 infection in the brain tissue is not completely excluded. to finally prove or disprove a neuronal transport of virus into the brain and its direct role in microglia activation and neurodegeneration, the analysis of much earlier stages in the infectious process, disease stages which are rarely encountered in human autopsies, may be required. similarly, a final view on similarities and differences of the inflammatory process between covid-19 and multiple sclerosis requires the analysis of much larger numbers of samples from different disease and lesion stages. finally, there are still some technical issues which have to be resolved in the future. the technology used here depicts phenotypic cell clusters, but whether these separations are functionally meaningful has yet to be shown. furthermore, when the expression profiles of the different markers in imaging mass spectrometry, as shown for instance in the supplementary figure 3b in this study, are compared with the experience obtained during the last decades with conventional immunocytochemistry, there are some obvious discrepancies, which raise some questions regarding specificity controls for each individual marker used in the imaging mass spectrometry panel. selective vulnerability of neuron subpopulations in inflammatory or demyelinating conditions acute and partially reversible neurological deficit is a common feature of inflammatory brain diseases. this can be mediated through disturbance of electrical conduction or through demyelination or synaptic loss, and can be compensated by remyelination or synaptic plasticity. in contrast, neuronal damage and loss is the prime substrate of permanent and irreversible neurological deficit. an interesting but controversial issue is whether there is a selective vulnerability of certain neuronal subpopulations, and when this is the case, what its mechanisms and consequences are. as an example, in cortical lesions in multiple sclerosis (ms) several groups have provided evidence that the gabaergic interneurons are prime targets for neurodegeneration (dutta et al. 2006, clements et al. 2008), while in another study a predominant degeneration of cux2 positive excitatory neurons was observed and gabaergic interneurons were unaffected (schirmer et al. 2019). these contradicting data need to be resolved. two large and very well-performed studies have re-addressed the question of selective vulnerability of cortical neurons in inflammatory demyelinating disease in 2021, and both came to the conclusion that the specific subpopulation of gabaergic parvalbumin-containing inhibitory interneurons is lost preferentially in the ms cortex (magliozzi et al. 2021, zoupi et al. 2021). this is in agreement with previous human and experimental studies, which also showed a preferential loss of parvalbumin positive interneurons in autoimmune encephalomyelitis and in toxic demyelination (falco et al. 2014, lapato et al. 2017). importantly, the loss of these neurons was associated with a significant and selective reduction of gabaergic presynaptic terminals in the cortex (zoupi et al. 2021). in an attempt to clarify the underlying mechanisms, the authors also analyzed an experimental rodent model with focal primary cortical demyelination, induced by a chronic application of a lysolecithin-containing hydrogel. the authors conclude that demyelination triggers the loss of these particular neurons, and this may be related to their particular features of axonal myelination (zoupi et al. 2021). in the other study (magliozzi et al. 2021) it was shown that the extent of loss of parvalbumin-containing neurons correlated with the extent of meningeal inflammation. since meningeal inflammation also determines the extent of cortical demyelination in ms lesions, demyelination may be the driving force of neurodegeneration also in this study. the observation that parvalbumin (pv) containing interneurons are preferentially destroyed in inflammatory and/or demyelinating diseases has clinical implications. parvalbumin is liberated during cell death and reaches the cerebrospinal fluid, where it may serve as a paraclinical marker for active neurodegeneration (magliozzi et al. 2021). furthermore, loss of gabaergic interneurons in the course of inflammatory demyelination results in an altered excitatory/inhibitory balance, clinically manifested by epileptic seizures (potter et al. 2016, lapato et al. 2017). thus, the selective vulnerability of inhibitory interneurons may be one of the explanations for the occurrence of epileptic seizures in multiple sclerosis and other inflammatory brain diseases (koch et al. 2008, nicholas et al. 2016, langenbruch et al. 2019). loss of gabaergic interneurons involves complement and results in specific brain region dependent functional consequences a similar selective loss of gabaergic interneurons was observed in the hippocampus of multiple sclerosis patients, mainly affecting the ca2 subfield. in line with the results discussed above this was also associated with an even more prominent loss of gabaergic synaptic terminals. since excitatory glutamatergic terminals were unaffected, a dysbalance between excitatory and inhibitory synaptic input was noted (ramaglia et al. 2021). essentially similar findings were obtained in the demyelinated hippocampus in the rodent cuprizone model (ramaglia et al. 2021). this allowed a detailed analysis of neurobiological mechanisms of neurodegeneration and its functional consequences. synaptic degeneration was associated with profound precipitation of the complement component c1q, which significantly correlated with synaptic pathology and the extent of demyelination, but was not associated with the presence of pathogenic autoantibodies. a key role of complement in synaptic plasticity and degeneration has been observed in a variety of studies before (schartz and tenner 2020). early complement components, such as c1q or c3, are produced in part in neurons and their production is enhanced in conditions of neurodegeneration. these proteins are transported in the axons to the terminals and focally accumulate at sites of axonal or synaptic damage (michailidou et al. 2016). their removal by microglia is a prerequisite for synaptic plasticity and axonal regeneration. in pathological conditions this mechanism is enhanced (michailidou et al. 2016) and the subsequent complement/microglia interaction may result in substantial axonal and synaptic loss, followed by retrograde neuronal degeneration (schartz and tenner 2020). the neurophysiological consequences of the loss of gabaergic interneurons and synapses in the ca2 region was a disturbance of the excitatory/inhibitor balance and a loss of feed-forward inhibition of pyramidal neurons. in concomitant behavioral investigations it turned out that these functional changes were mainly reflected in a disturbance of social memory tasks, which were not accompanied by a general learning impairment or by changes of motor activities (ramaglia et al. 2021). in summary, this study, too, shows a selective vulnerability of gabaergic interneurons in ms and cuprizone lesions and suggests that these changes are a potential consequence of demyelination. however, no loss of pv-containing gabaergic interneurons has been seen in another study in hippocampal ms lesions despite extensive demyelination (kiljan et al. 2019), suggesting that demyelination alone is unlikely to be responsible. a similar preferential loss of parvalbumin reactive neurons has also been seen in a mouse model of autoimmune encephalomyelitis, which is driven by inflammation, but largely lacks primary demyelination in the cortex (falco et al. 2014). furthermore, hypoxic/ischemic damage is associated with a preferential loss of pv-containing gabaergic interneurons (fowke et al. 2018, stolp et al. 2019, vaes et al. 2020, povysheva et al. 2019) and a similar selective vulnerability has also been observed in a mouse model of alzheimer’s disease (petrache et al. 2019) thus, the key question arises whether there are common mechanisms of brain injury shared between these different conditions. all of these conditions are associated with microglia activation, reflected by the expression of nadph oxidase in activated microglia (zrzavy et al. 2017, 2018). the beneficial effect of its pharmacological blockade indicates a role of oxidative injury in the induction of neuronal damage (yuan et al. 2015) by mitochondrial injury and subsequent energy deficiency (mahad et al. 2015). in such a state of “virtual hypoxia” (dutta et al. 2013) additional demyelination amplifies damage due to the increased energy demand of denuded axons. microglia phenotype in relation to alzheimer’s disease (ad) pathology research performed during the last decade brought microglia into a central position in the cascade of events triggering neurodegeneration. in alzheimer’s disease the concept was originally proposed following the observation that activated microglia cumulate in amyloid plaques (mcgeer et al. 1988) and that a variety of different pro-inflammatory cytokines and mediators are expressed in the ad cortex (eikelenboom and stam 1982, mcgeer et al. 1988, dickson and rogers 1992). more recently, systematic genome wide association studies (gwas) identified numerous risk genes for ad susceptibility, and many of these genes code for molecules involved in microglia function (lambert et al. 2013, lewcock et al. 2020). these results encouraged new studies on the phenotype of microglia in alzheimer’s disease using the new opportunities offered by single cell transcriptomics and proteomics (mathys et al. 2019, lewcock et al. 2020). this allowed the identification of a disease-associated microglia phenotype (dam), which was characterized by the loss of homeostatic phenotypic markers and the expression of a large array of different activation markers (figure 1). figure 1: microglia activation in alzheimer’s disease: microglia in the cortex of ad patients are activated within the amyloid plaques and also in surrounding cortical parenchyma. within the plaques (labeled with *) the activation is more advanced showing an ameboid microglia phenotype and a higher expression of markers associated with phagocytosis (cd68), antigen presentation (mhc), oxidative injury (nadph oxidase and iron storage (ferritin, iron)), and proteolysis (tpp2). a key step in this activation is the induction of the trem2/apoe pathway, which occurs in response to programed neuronal cell death (krasemann et al. 2017). a variety of different molecules, identified by gwas in ad patients, are related to this pathway, and loss of function mutations of trem2 are associated with earlier onset and more severe disease. functional studies revealed that loss of trem2 function arrests microglia in a homeostatic state, reduces phagocytosis and inhibits the focal stabilization of aβ deposits (lewcock et al. 2020). all these data support the view that activated microglia within plaques play a neuroprotective role by inhibiting the spread of soluble aβ oligomers. however, microglia in the ad cortex are also activated outside of aβ plaques, where they are also associated with degeneration and loss of neurons and synapses. whether these microglia cells have other activation profiles compared to the plaque-associated microglia has been addressed in a recent systematic study using single nuclei transcriptomics (gerrits et al. 2021). the authors confirmed the phenotype of the plaque-associated microglia. in addition, however, they identified several new phenotypic microglia clusters, which were associated with the severity of cortical tau-pathology. this shows for the first time that the activation patterns of microglia are heterogeneous in the ad cortex in a context-dependent manner. these data indicate that tau-related neurodegeneration is triggered or associated with microglia activation steps, which differ from those responsible for their protective role in plaques. only a minority of the molecules identified in these neurodegeneration-associated clusters are so far functionally characterized, some of them, however, seem to be related to mechanisms of tissue plasticity and repair. thus, the final role of this new neurodegeneration-associated microglia phenotype is currently unresolved. despite the highly innovative approach of recent studies using single cell transcriptomics and proteomics, such studies have major limitations. they identify a large battery of cluster-associated molecules, but only a small fraction of them is functionally defined at the present time. their functional characterization has to be performed in experimental models, but this approach has limitations. although basic activation steps of microglia are similar between different species, there are major differences in the molecular phenotypes of activated microglia between rodents and humans (wimmer et al. 2018), and further differences in their patterns of activation are seen in human disease in comparison to those in ad models (sobue et al. 2021). another problem is related to the annotation of specific molecules to specific cell types. as an example, molecules of the nadph oxidase complex, which are prominently expressed in human microglia and are clearly associated with oxidative injury in a variety of different diseases (fischer et al. 2012), are surprisingly absent from the microglia activation list in recent single cells transcriptomic studies. why this is the case is not clear. it may be that these molecules are not included in the list of microglia genes, since they are prominently expressed in granulocytes and thus annotated as granulocyte markers. the other possibility is that they are not included, since the gene lists have originally been defined in mouse models, and nadph oxidase, in contrast to humans, is not prominently expressed in mouse microglia. protective or detrimental action of microglia in alzheimer’s disease: context or lesion stage dependent? as discussed above, heterogeneous clusters of activated microglia are prominent in the ad cortex and they are associated with deposits of aβ in the plaques (lewcock et al. 2020). whether activated microglia promote or ameliorate disease is unclear and can only partly be addressed in humans. the identification of trem2 loss of function mutations as risk genes in ad suggests that microglia activation is protective by inhibiting aβ spread into the cortical parenchyma. however, experimental models suggest a more complex role of microglia. thus, microglia depletion before the onset of amyloid deposition in ad mouse models reduces plaque formation, possibly by inhibiting synapse degeneration, the liberation of amyloid precursor protein (app) and its cleavage into aβ fragments in the course of synapse degeneration (delizannis et al. 2021). in addition, microglia seem to play a fundamentally different role in neurodegeneration in the ad brain. this was recently observed in transgenic mice expressing familial alzheimer’s disease and human tau mutations (5xfad/ps19 mice), in which tau tangles were precipitated by stereotactic seed injections of synthetic preformed tau fibrils into the hippocampus (lodder et al. 2021). these animals developed severe aβ plaque pathology in addition to profound tau-tangle pathology in the hippocampus and cerebral cortex, and both pathologies were associated with a disease-associated microglia phenotype (dam). microglia in the central nervous system can be depleted by chronic treatment of the animals with plx 3397, a drug which selectively inhibits the colony stimulating factor 1 receptor (csf1r). interestingly, this treatment had a differential effect on plaque or tangle-associated microglia. while the diffuse microglia infiltration, which was associated with tau-pathology, was completely eliminated, the plaque-associated activated microglia was unaffected. this selective elimination of diffuse microglia in the cortical parenchyma resulted in a significant attenuation of the propagation of tau-pathology and neurodegeneration. from all these data one can conclude that microglia in the ad brain exert different functions, depending upon the stage of the cortical lesions and the nature of the damage. the initial stage of aβ plaque formation is propagated, but the spread of mature plaques into the surrounding tissue is ameliorated by activated microglia. when there is a predisposition for tau phosphorylation, aβ deposition propagates tau-tangles, and this process is further enhanced by activated microglia. this heterogenous scenario of microglia function in ad pathogenesis is a challenge for the development of anti-inflammatory treatment strategies. iron and microglia in alzheimer’s disease microglia activation is seen in human ad as well as in mouse ad models and their basic activation pattern is similar, but not identical between these two conditions (krasemann et al. 2017, keren-shaul et al. 2017, mathys et al. 2019). most importantly, most rodent models of ad differ from human disease in the absence or the extent of neurodegeneration. one difference between human and rodent neuropathology is the age-related accumulation of iron in the brain (connor et al. 1992, hametner et al. 2013), which is largely absent in rodents, at least at ages of the animals generally investigated in ad models. iron accumulation in human microglia is increased in neurodegenerative diseases, and it is associated with a pro-inflammatory activation of the cells (mehta et al. 2013, hametner et al. 2013, van duijn et al. 2017), possibly related to its pro-oxidative function. furthermore, the phenotype of dystrophic microglia, present in human ad but largely absent in rodent models, is restricted to iron and ferritin-loaded cells (lopes et al. 2008, hametner et al. 2013; figure 2). figure 2: iron accumulation and microglia senescence: microglia, identified with the panmicroglia marker iba1 in the cortex of ad patients may display a homeostatic phenotype, expressing p2ry12 and showing a normal ramification of their cell processes. another fraction of microglia contains high levels of iron stored together with ferritin. these cells frequently show a dystrophic phenotype with loss and clumping of cell processes. the insert in the iron figure shows double staining for iron (brown) and the microglia/macrophage marker cd68 (blue). an important missing piece of knowledge was a detailed phenotypic and functional characterization of different microglia populations in the ad brain in relation to their iron content. this information is now provided by a recent study, describing the microglia activation profile in the cortex of ad patients using multispectral immunofluorescence (kenkhuis et al. 2021). as described before, the study confirmed the increased microglia infiltration and activation in the ad cortex, the association of activated microglia with aβ-plaques and the ferritin and iron accumulation in the microglia cytoplasm. fine mapping of microglia was performed by cluster analysis. this revealed microglia clusters, which were mainly associated with ad lesions, and these clusters had in common the high ferritin content and the downregulation of various markers of the homeostatic microglia phenotype. the high ferritin content was associated with a phenotypic change of the cells towards dystrophic microglia. in contrast, microglia in the surrounding normal-appearing brain tissue were grouped in clusters with high or variable expression of homeostatic markers and low expression of ferritin. thus, this study identifies disease-associated microglia through their iron and ferritin content. this was also reflected by the association of this microglia phenotype with disease severity, apo-e genotype and the accumulation of aβ plaques and neurofibrillary tangles in the brain. the major limitation of this study is the selection of ad cases, which is dominated by patients at later disease stage (braak stage v to vi). thus, it remains unclear whether iron accumulation and its binding to ferritin is a secondary phenomenon in the formation of aβ plaques. thus, iron uptake in microglia at the expense of microglia dystrophy may reflect removal of free iron from the extracellular space in an attempt to ameliorate oxidative injury. however, iron in microglia may activate microglia in a pro-inflammatory manner, which contributes to oxidative damage in the lesions. the iron content in the brain can be visualized and quantified in magnetic resonance imaging (mri), using specific sequences such as r2* or quantitative susceptibility mapping (qsm) (bulk et al. 2018). using this technology, the authors also showed that the increased iron content in the sections is also correlated with an increased mri signal (kenkhuis et al. 2021). dynamics of chronic microglia-associated neurodegeneration the possibility to detect accumulation of iron-loaded microglia in vivo by magnetic resonance imaging offers a very attractive tool to monitor the dynamics of active lesions and neurodegeneration in human disease. this is exemplified in multiple sclerosis, where the presence of an iron rim around the lesion identifies it as chronic active (bagnato et al. 2011, mehta et al. 2013, hametner et al. 2013; figure 3). furthermore, such lesions show more pronounced tissue damage and neurodegeneration in comparison to those lacking an iron ring, and the presence of such lesions is associated with an aggressive clinical disease course (absinta et al. 2016, weber et al. 2021). pathological data on the distribution of tissue degradation products in such iron lesions suggested that they expand very slowly, but the time course of their dynamic evolution was unknown. this has changed when prospective longitudinal mri studies using iron sensitive sequences were performed, which provide data on dynamic changes in ms lesions over a time period of up to 7 years (dal bianco et al. 2017, 2021). brain lesions without an iron rim shrank during the first 3 years and then their volume stabilized. in contrast, iron ring lesions slowly expanded over 3 to 5 years, before their size stabilized. these data indicate that chronic neurodegeneration in progressive multiple sclerosis is a very slow process, gradually developing over years, while demyelination and neurodegeneration in acute relapses develop within weeks. figure 3: slowly expanding iron ring lesions in the ms brain can be visualized on mri with iron sensitive imaging sequences. the typical presentation of such lesions reveals high iron signal in one or several central veins and in a rim at the lesion edge, reflecting iron-containing activated microglia. the iron rim (r) demarcates the demyelinated plaque (pl); the abnormal mri signal around the iron rim is due to wallerian degeneration (wd). direct neuropathological analysis of mri scanned tissue blocks showed that the iron ring seen in mri always reflects the iron-loaded microglia at the edge of active plaques, while the t2 signal of the lesions gradually expands outside the ring (dal bianco et al. 2017, 2021). these lesion changes reflect an expanding halo of secondary wallerian degeneration around a destructive lesion (figure 3). thus, the quantitative lesion load, determined on mri, not necessarily indicated lesion load, but also wallerian degeneration in the normal appearing white matter. all these data highlight the importance of studies, directly correlating mri changes with neuropathology and integrating this information into long term longitudinal prospective studies. it is expected that a similar approach will also provide significant new information for age-related neurodegenerative diseases. is there evidence for involvement of t-cells in neurodegenerative disease? neuropathological studies have provided evidence that cells of the adaptive immune system infiltrate the brain, in particular at sites of ongoing neurodegeneration in diseases such as alzheimer’s disease or parkinson’s disease (togo et al. 2002, brochard et al. 2009, evans et al. 2019). these cells are mainly t-cells, including mhc class ii restricted cd4+ and mhc class i restricted cd8+ cells. the key question, which is currently unresolved, is whether these cells actively contribute to tissue injury, are neuroprotective or are just passively recruited bystanders. this question has recently been addressed in an experimental model of parkinson’s disease, induced by local injection of an adeno-associated virus vector, inducing the over-expression of α-synuclein in the substantia nigra of mice (williams et al. 2021). in this model cd4+ and cd8+ t-cells infiltrate the midbrain, and this is associated with activation of myeloid cells, including microglia, and the degeneration and loss of tyrosine hydroxylase positive nigral neurons. the t-cell infiltrates are associated with the local expression of pro-inflammatory and in part also regulatory cytokines. most importantly, t-cell receptor or cd4 deficient mice revealed a reduced activation of immune effector cells and also an ameliorated neurodegeneration in the substantia nigra. this study thus provides evidence that adaptive immune reactions, mainly mediated by mhc class ii restricted cd4+ t-cells, contribute to the propagation of neurodegeneration in this model. the results support the view that attempts to therapeutically modulate the t-cell immune response (weiner et al. 2011, mayo et al. 2016) could be effective. direct support is provided by the observation that fingolimod, a drug which reduces the infiltration of immune cells into the central nervous system and is effective in the treatment of multiple sclerosis patients, ameliorates disease in the respective parkinson’s disease model (williams et al. 2021). despite these findings, which suggest a pathogenic role of t-cells in parkinson’s disease and other neurodegenerative diseases, it remains unresolved whether this is mediated by autoimmunity. no proof is provided that the t-cell immune reaction is directed against a specific autoantigen, such as for instance α-synuclein (williams et al. 2021). thus, local overexpression of α-synuclein may attract non-specifically t-cells into the brain. since the normal immune repertoire contains t-cells against a variety of different brain auto-antigens (wekerle et al. 1996), their activation in the lesions may just represent a non-specific consequence of the inflammatory response. also, the mechanism of damage of neurons in the substantia nigra remains unresolved. involvement of microglia and macrophages, which are activated by the t-cell mediated immune response, is likely to amplify neurodegeneration, but innate immunity alone will not specifically and selectively target dopaminergic neurons (lassmann and van horssen 2011). finally, the amelioration of neuronal damage in animals treated with fingolimod does not necessarily prove immune mediated damage, since the modulation of sphingosine 1 phosphate receptors may also have direct neuroprotective effects (colombo and farina 2022). thus, the study by williams et al. (2021) supports a possible involvement of t-cell mediated mechanisms in neurodegeneration in parkinson’s disease, but further research is necessary to clarify the underlying mechanisms. the local inflammatory response in gliomas: good or bad or irrelevant? it is generally accepted that local inflammation is present in the malignant gliomas, but it is largely unresolved whether this is beneficial or detrimental for the patients (jack and lu 2015). overall, there is an immunosuppressive environment within gliomas due to the production of anti-inflammatory cytokines, such as transforming growth factor β (tgf-β) in the tumor cells (binnewies et al. 2018, brown et al. 2018). the extent of tgf-β expression is, however, variable between patients and one expects that lower levels of immunosuppression is associated with inflammation, immune attack of tumor cells and good clinical prognosis in patients. alternatively, high density of inflammatory cells within the tumor may be secondary to more aggressive tissue damage and associated with a less favorable prognosis. in addition, the inflammatory response itself may be dominated by pro-inflammatory activated cytotoxic effector cells or by regulatory t-cells and myeloid-derived suppressor cells, and this, too, may influence tumor progression and functional outcome (kmiecik et al. 2013, 2014, gabriely et al. 2017). several studies have so far analyzed the phenotype of tumor-infiltrating inflammatory cells and its relation to clinical outcome, but the results are variable and in part contradictory (charles et al. 2012, szulzewsky et al. 2015, orrego et al. 2018, martinez et al. 2009, zhang et al. 2019). this is mainly due to the fact that the studies were performed by immunocytochemistry, which does not allow the simultaneous analysis of a large spectrum of different immune cells. this problem was addressed in a recent study by applying flow cytometry of dissociated cells from a large number of biopsy samples from carefully characterized glioblastoma patients (gonzales-tablas pimenta et al. 2021). by using novel state-of-the-art techniques of multi-antibody and multi-color labeling, a simultaneous quantitative evaluation of all different leukocyte populations was possible. as shown before, the study revealed that on average only a quarter of the total cell population were cd45+ immune cells, two thirds of them being tumor-associated macrophages of hematogenous or microglia origin and granulocytes. the lymphocyte population mainly consisted of mhc class i restricted cd8+ and mhc class ii restricted cd4+ t-cells. the infiltration by regulatory t-cells, b-lymphocytes and natural killer cells (nk cells) was very low. immunosuppressive cytokines, such as tgf-β and interleukin 10 (il10) were mainly seen within tumor cells and in low incidence in myeloid derived suppressor cells. the broad spectrum of markers used simultaneously in this study allowed to define three patient clusters with distinct immune infiltration. in the first, immune cell infiltration was sparse and dominated by tumor-associated macrophages. in the second, the tissue infiltration was profound, but consisted mainly of myeloid cells with little contribution of lymphocytes. in the third, the immune infiltration, too, was high containing high amounts of tumor-associated macrophages, granulocytes and t-lymphocytes. the latter patient cohort showed a significantly worse clinical prognosis compared to the other groups. this study represents an important first step for a comprehensive characterization of the local immune response in gliomas in correlation to clinical prognosis and tumor genotype. this approach will also be essential to interpret the effects of new immunotherapies in gliomas. conclusions mechanisms of adaptive and innate immunity appear to be involved in the propagation of neurodegeneration in many different diseases, including inflammatory conditions such as multiple sclerosis or classical 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departments of pathology and neurological surgery, university of california, san francisco, california, usa corresponding author: char loo tan · department of pathology · national university hospital · 5 lower kent ridge road · singapore 119074 char_loo_tan@nuhs.edu.sg submitted: 12 june 2021 accepted: 11 september 2021 copyedited by: jerry j. lou published: 20 september 2021 https://doi.org/10.17879/freeneuropathology-2021-3397 keywords: ependymoma, zfta, c11orf95, maml2, myogenic abstract ependymomas are glial neoplasms with a wide morphological spectrum. the majority of supratentorial ependymomas are known to harbor zfta fusions, most commonly to rela. we present an unusual case of a 9-year-old boy with a supratentorial ependymoma harboring a noncanonical zfta-maml2 fusion. this case had unusual histomorphological features lacking typical findings of ependymoma and bearing resemblance to a primitive neoplasm with focal, previously undescribed myogenic differentiation. we discuss the diagnostic pitfalls in this case and briefly review the histological features of ependymoma with noncanonical gene fusions. our report underscores the importance of molecular testing in such cases to arrive at the correct diagnosis. supratentorial ependymomas with noncanonical fusions are rare, and more studies are necessary for better risk stratification and identification of potential treatment targets. introduction ependymomas (epn) form a diverse group of morphologically and molecularly heterogeneous glial neoplasms [5, 14]. while the classical histological appearance of epn is well-described, the morphological spectrum is broad and may confound diagnosis. unusual features encountered in ependymomas include chondro-osseous elements [13, 17], neuropil-like islands [6], melanin-containing cells [4], tumor giant cells [8], neuronal differentiation [16], granular cell features [18], et cetera. recent advances in genomic and methylome profiling have identified distinct molecular subgroups characterized by recurrent genetic or epigenetic alterations that may better inform diagnosis, clinical outcomes and risk stratification of epn as compared to conventional light microscopic diagnosis and histological grading [5]. the majority of supratentorial (st) epns have gene fusions involving either zfta (zinc finger translocation associated; previously known as c11orf95) or yap1. these gene fusions characterize the two molecular subgroups of st-epn: st-epn-zfta and st-epn-yap1, respectively [1, 14]. here, we present an unusual case of a st-epn with zfta-maml2 fusion demonstrating diagnostic challenges due to primitive morphology, suggesting an embryonal neoplasm and divergent differentiation, including hitherto undescribed myogenic differentiation. our case underscores the importance of molecular testing in such cases to arrive at the correct diagnosis. clinical summary a 9-year-old boy presented with a visible, enlarging right forehead lump for 2 months. the child complained of an occasional headache but was otherwise well without any focal neurological deficits. brain magnetic resonance imaging (mri) revealed a well-demarcated, right frontal cystic tumor with a mural nodule, surrounding vasogenic edema and midline shift (fig. 1a-c). the tumor showed a high choline peak and high choline:n-acetylaspartate (naa) ratio. the child underwent craniotomy, revealing a well-demarcated bosselated tumor with a clear interface between tumor and brain (fig. 1d-e). gross total resection of the tumor was achieved (fig. 1f), and the postoperative course was uneventful. he received adjuvant radiation therapy followed by temozolomide. the child remained well at follow up 9 months post-resection, with no radiological evidence of recurrence. figure 1. mri findings of the tumor. a) axial, b) coronal and c) sagittal views showing a cystic tumor with an enhancing multilobulated mural nodule in the right frontal lobe with mass effect. d) intraoperative view after lifting of dural flap, prior to resection e) dissection of a well-demarcated multinodular cystic tumor. f) final intraoperative view after gross total resection of the solid tumor showing part of cyst lining with ventricular wall. material and methods the specimen was routinely processed for paraffin embedding and staining with hematoxylin and eosin (h&e). immunohistochemistry (ihc) with commercially available primary antibodies, including gfap, olig2, synaptophysin, desmin, myogenin, myod1, cam 5.2, ema, ini1, brg1, lin28a, bcor, hmb45 and ki67, was performed following locally validated technical protocols. gene fusion detection was performed using a next-generation sequencing-based anchored multiplex polymerase chain reaction (pcr) assay (archer® fusionplex, boulder, co, usa) that can detect and identify gene fusions involving any of 101 covered genes, including rela, yap1, maml2, ncoa1 and ncoa2. briefly, total ribonucleic acid (rna) was extracted from formalin-fixed paraffin-embedded tissue sections of tumor. 150 ng of rna was used for library preparation utilizing the archer® fusionplex kit, according to the manufacturer’s protocol (archerdx, boulder, co, usa). the prepared library was sequenced using an illumina miniseq sequencer. the data obtained was analyzed by the archer data analysis (version 6.2.3) portal. a confirmatory reverse transcription-pcr was designed flanking the breakpoint of the two genes and the pcr product was sanger-sequenced. dna methylation analysis was performed using the illumina infinium human methylation epic beadchip kit according to published protocols [2, 7]. the results of the methylation profiling and copy number variation analysis were obtained using an automated web-based deoxyribonucleic acid (dna) methylation profiling program (molecularneuropathology.org) [12]. the case was also integrated onto the t-distributed stochastic neighbor embedding (t-sne) analysis in the german cancer research center (dkfz) database. results light microscopy sections showed a discrete tumor composed of hypercellular sheets of small, round to ovoid cells with hyperchromatic nuclei and indistinct cell boundaries, imparting a primitive appearance to the tumor. in other areas, the tumor cells formed nodular aggregates, short fascicles and palisades/ trabeculae, separated by fibrous septa imparting a desmoplastic appearance (fig. 2a). a minor subset of tumor cells showed prominent intracytoplasmic fine eosinophilic granules, some with a more globular appearance (fig. 2b). a minor component of the tumor was composed of spindled cells with eosinophilic cytoplasm reminiscent of skeletal muscle fibers (fig. 2c). focally, aggregates of tumor cells with eccentric nuclei and mucinous cytoplasm were also identified (fig. 2d). no other heterologous differentiation was seen. there was a distinct lack of a fibrillary background in the tumor. while some tumor cells appeared to congregate around large thick-walled vessels, no distinctive perivascular pseudorosettes were present. geographic areas of necrosis accompanied by dystrophic calcification were present. the mitotic count reached up to 8 per 10 high power fields (3 per mm2). figure 2. light microscopy and immunohistochemical findings of the tumor. a) low-power appearance of the tumor consisting of nests of tumor cells separated by fibrous septa, imparting a desmoplastic appearance. the bulk of the tumor cells show primitive cytomorphology with minimal cytoplasm, imparting a nondescript “small blue cell” appearance. no perivascular pseudorosettes are seen, although tumor cells aggregate along blood vessels. b) a minor subset of tumor cells showed prominent intracytoplasmic fine eosinophilic granules (arrowheads). c) rare tumor cells showed spindled morphology, resembling skeletal muscle fibers. d) aggregates of tumor cells with eccentric nuclei and mucinous cytoplasm (arrowheads). the tumor cells were positive for e) gfap. very focal areas showed myogenic differentiation, being positive for f) desmin, g) myogenin and h) myod1 (f-h were images taken from same area of the tumor.) clicking the figure will lead you to the full virtual slide (h&e). the tumor cells showed patchy positivity for gfap (fig. 2e), and focal reactivity for synaptophysin, cam 5.2 and ema (cytoplasmic; no dot/ring-like pattern was found). focally, some cells expressed desmin (fig. 2f) and nuclear reactivity for myogenin (fig. 2g) and myod1 (fig. 2h). no neurofilament-positive axons were identified, in keeping with the non-infiltrative nature of this tumor. there was retained expression for ini1 and brg1. olig2, lin28a, bcor and hmb45 stains were negative. the ki67 labeling index reached up to 40%. gene fusion detection and confirmation the archer fusionplex assay detected a zfta (exon 5)maml2 (exon 2) fusion (fig. 3a). this gene fusion was confirmed by rt-pcr with sanger sequencing of the amplicon product (fig. 3b). figure 3. a) archer fusionplex assay detected a zfta (exon 5; previously known as c11orf95)-maml2 (exon 2) fusion, which was confirmed by b) sanger sequencing. methylation analysis dna-based methylation analysis of the tumor revealed a methylation class corresponding most closely to “ependymoma, rela fusion”, with a calibrated score of 0.65. concurrent copy number analysis revealed 1q gain and cdkn2a/b loss (fig. 4a). no chromothripsis of chromosome 11 was present. on the t-sne plot, our case localized to the methylation cluster of st-epn-rela, consisting of 444 cases including our case, out of a total of 3272 cases of ependymomas of different anatomical locations and molecular groups, lending further diagnostic confidence (fig. 4b). a final diagnosis of st-epn with zfta-maml2 fusion was rendered after synthesizing the pathological and molecular findings of this case. figure 4. a) copy number variation profile of the tumor, with 1q gain and cdkn2a/b losses. b) t-distributed stochastic neighbor embedding (t-sne) plot containing a total of 3272 cases of epns for cluster analysis. our case localized to methylation cluster of supratentorial epn with rela fusion (n= 444, including our case) (inset, case indicated in black). clicking the figure will lead you to a high-resolution version. discussion zfta, now recognized as a key player in the oncogenesis of st-epn, confers transforming capabilities upon oncogenic fusion proteins by increasing their translocation to the nucleus and permitting access to the transcriptional machinery [9]. most commonly, zfta is fused to rela [9, 14, 19]. being promiscuous, zfta may also rarely fuse with alternative partners such as maml2, maml3, ncoa1, ncoa2 and ctnna2 [14, 18, 19]. of note, the zfta-maml2 fusion has only been identified in 18 cases of st-epn in the literature thus far, including the current case [14, 18, 19]. tumors with such alternative fusion partners form satellite clusters around the previously known st-epn-rela cluster in the t-sne plot of methylation profiles and show distinct transcriptional profiles [19]. while most st-epn-zfta demonstrate classical features of epn such as perivascular pseudorosettes and fibrillary matrix, a recent paper by zheng et al found that epns with zfta fusions with alternative partners tend to demonstrate unusual histological features. these include sarcomatous features, areas mimicking high grade central nervous system (cns) neoplasms like diffuse high-grade glioma, cns embryonal tumors and other primitive tumors [19]. such atypical histological findings are in keeping with the case we present herein. similar to the case by tamai et al, our case also demonstrated granular cell features [18]. in their case, the granular cells showed eccentrically placed nuclei and cytoplasm filled with eosinophilic granules which were positive for periodic acid-schiff (pas) and alpha-1-antitrypsin stains. in addition, our case showed a small population of tumor cells with unequivocal expression for desmin, myogenin and myod1, consistent with myogenic differentiation, a finding which has not been previously described. initial diagnosis on the basis of light microscopy was difficult because of the atypical morphologic features of the tumor including the conspicuous absence of a fibrillary stroma, the absence of classical epn morphology, and the presence of primitive-appearing cells in a desmoplastic background. coupled with the polyphenotypic immunoprofile, which included desmin reactivity, the differential of desmoplastic small round cell tumor was initially entertained [10]. however, the subsequent molecular finding of the zfta-maml2 gene fusion and the absence of an ewsr1-wt1 fusion clinched the diagnosis of st-epn with zfta-maml2 fusion and led to revision of the histologic diagnosis. recurrent fusions involving zfta are characteristic of epn and fusions involving this gene have only been described previously in chondroid lipoma [15]. no primitive tumor has been described in the literature with this characteristic fusion to date. while lacking in histological elements of classical epn, the circumscribed, non-infiltrative nature of this tumor and the presence of the signature fusion and methylation profile were all in keeping with a diagnosis of st-epn. the novel finding of myogenic differentiation, likely attributable to metaplasia of the neoplastic glial component or the mesenchymal component of the tumor cells, broadens the spectrum of heterologous differentiation in epn [3]. the protean morphological manifestations of epn has the potential to obfuscate morphological diagnosis, particularly when classical epn features are not present. this underscores the limitations of morphological evaluation alone and the necessity of molecular testing. there has been longstanding controversy surrounding the utility of histological grading of epn. while st-epn-rela was found to be associated with adverse patient outcomes in one retrospective study [14], this was not reproduced in another trial-based study [11]. interestingly, mice with epn induced by zfta-maml2 fusion demonstrated a reduced survival compared to those with zfta-rela fusion [19]. out of the 18 cases of st-epn with zfta-maml2 fusion, follow up data was only available in two cases. both cases showed anaplastic features, corresponded to who grade 3. the case reported by tamai et al. showed no recurrence for 30 months without chemoradiotherapy, which may indicate a favorable prognosis [18]. our patient remained disease free at 9 months follow up. owing to the paucity of survival data, it remains to be seen if differences in survival exist in st-epn with alternative fusions. similarly, the utility of assignment of a who grade to molecularly defined epn remains insufficiently characterized [5]. additional studies are necessary to better delineate the behavior of these tumors, which may inform treatment decisions and risk stratification. in conclusion, we present an unusual case of st-epn with zfta-maml2 fusion 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article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. intraoperative confocal laser endomicroscopy for brain tumors potential and challenges from a neuropathological perspective feel free to add comments by clicking these icons on the sidebar free neuropathology 3:24 (2022) opinion pieces intraoperative confocal laser endomicroscopy for brain tumors potential and challenges from a neuropathological perspective theoni maragkou1, karl quint2, bianca pollo3, ekkehard hewer4 1 institute of pathology, university of bern, bern, switzerland 2 quint healthcare, fürth, germany 3 unit of neuropathology, fondazione irccs istituto neurologico carlo besta, milano, italy 4 institute of pathology, lausanne university hospital and university of lausanne, lausanne, switzerland corresponding author: ekkehard hewer · institut universitaire de pathologie · bugnon 25 · ch-1011 lausanne · switzerland ekkehard.hewer@chuv.ch submitted: 16 august 2022 accepted: 21 october 2022 copyedited by: georg haase published: 23 november 2022 https://doi.org/10.17879/freeneuropathology-2022-4369 keywords: confocal laser endomicroscopy, intraoperative consultation, digital biopsies, brain tumors, resection margin, in vivo microscopy abstract confocal laser endomicroscopy (cle) represents a new non-invasive in vivo imaging technique that holds considerable promise in neurosurgery and neuropathology. cle is based on the principle of optical sectioning which uses pinholes placed in the light path to selectively image photons of a specific focal plane by filtering out photons above and below the focal plane. potential indications of cle in neurosurgery and neuropathology include intraoperative tumor diagnosis and staging as well as assessment of tumor resection margins notably in the case of diffusely infiltrating gliomas. cle-based tumor analysis in near-real time may also have a significant impact on future tumor resection strategies. we here discuss the technical features of cle, its potential for wide-field imaging, its role in comparison to established histological techniques for intraoperative tumor assessment and its position in digital pathology and telepathology. based on our group’s experience with a commercially available confocal laser endomicroscope (zeiss convivo), we critically address the current state of intraoperative cle in brain tumor surgery, the applicability of classical histological criteria and the strategies required to further improve the diagnostic accuracy of cle. we finally discuss how a widespread use of cle in neurosurgery may modify the role of neuropathologists in intraoperative consultation, generating both new opportunities and new challenges. introduction intraoperative consultation in the form of frozen section analysis and/or intraoperative cytology is an integral part of neuropathology. its relative importance compared to other diagnostic activities is arguably higher in neuropathology than in most other domains of pathology. this is due to a variety of reasons, including (a) the high percentage of brain tumor surgeries among all surgical procedures, (b) the absence of preoperative biopsy in most cases meaning that intraoperative consultation is usually the first opportunity to obtain a tissue-based diagnosis, (c) the large variety of tumor types with often overlapping imaging features and (d) the difficulty to distinguish tumoral tissue macroscopically from non-tumoral tissue. nevertheless, intraoperative consultation has not benefited from any major improvements for several decades and remains currently afflicted by the drawbacks of established histological and cytological methods. these comprise tissue freezing leading to morphological artifacts, tissue consumption, delays in the transfer of tissue samples from the operating room to the laboratory and difficulties in cell and tissue processing. in vivo microscopy based on various techniques such as confocal laser endomicroscopy (cle) or optical coherence tomography has been used for a long time in ophthalmology and has become available for clinical routine for different indications such as gastrointestinal diseases (1), pulmonary mucosal lesions or urological disorders. our opinion piece is based on our own experience with a commercially available zeiss convivo confocal laser endomicroscope in both preclinical and clinical settings. we here first review technical aspects of cle and then describe the characteristics of the first clinical-grade cle device available for neurosurgical applications. finally we discuss potential advantages and limitations of in vivo cle as well as its impact on diagnostic practice in surgical neuropathology. with this contribution to the scientific literature, we aim to inform potential future users, to stimulate discussion and research on cle, and to discuss the potential benefit of cle in neurosurgery and neuropathology. limitations of histological and cytological methods in neuropathology and neurosurgery traditionally, intraoperative diagnosis in neurosurgery has relied on standard pathological techniques such as frozen sections and intraoperative cytology, which are followed by analysis of formalin-fixed, paraffin-embedded (ffpe) tissue for definite diagnosis. while these latter techniques are still being considered the gold standard, some of their drawbacks are prolonged time for sampling, transferring, processing and interpreting the samples, artifacts and sampling errors, all which limit their applicability for rapid interpretation of tissue samples. in addition, standard pathological techniques involve a multitude of different processes and specialized personnel. in the case of ffpe tissue, they involve tissue fixation, dehydration, rinsing, embedding in hot paraffin, cooling, freezing in preparation for cutting, microtome cutting, transferring to glass slides, and drying. in routine hematoxylin and eosin (h&e) staining, several more steps are being performed: dewaxing, dehydration, hematoxylin staining, differentiation in mild acid, bluing, eosin staining, dehydration, clearing and cover-slipping, with various washing and alcohol incubation steps in between. all these procedures can take up to one full day for basic staining, and several more days for advanced subsequent investigations such as immunohistochemistry and molecular analyses (2–4). for digital pathology, the final slides need to be scanned, processed, analyzed and archived. confocal laser endomicroscopy in neurosurgery the disadvantages of traditional histologic and cytologic techniques are increasingly being overcome by newer technologies. confocal laser endomicroscopy (cle) allows in vivo noninvasive histological imaging using optical sectioning. by placing pinholes in the light path, optical sectioning enables the microscope to selectively image a specific focal depth by mechanically filtering most out-of-focus photons above and below the focal plane. this allows in situ visualization of tissue with higher contrast compared to conventional wide-field microscopy, eliminating out-of-focus background and scattered light. in addition, endomicroscopy allows for three-dimensional image reconstruction and greater variety of imaging parameters. most importantly, this technology enables real-time histopathological analysis with increased diagnostic yield without the traditional histopathological process of excision, fixation and staining. only recently this new technological advancement has been applied to neurosurgery, by using a wide range of fluorescent dyes as contrast enhancers. a single clinical-grade device for cle, the zeiss convivo, is hitherto commercially available with clearance by the food and drug administration (fda) of the united states of america and ce marking in the european union. routine clinical experience with the cle device is currently being investigated in several clinical trials. preclinical research indicates that the cle device could improve tumor visualization at the tumor margin and speed up intraoperative diagnosis. furthermore, the cle device is designed to be used directly in the operating room and to allow telepathology, thus bringing the neuropathologist even closer to the neurosurgeon. confocal laser endomicroscopy beyond neurosurgery standard histology and confocal laser endomicroscopy are not interchangeable nor can one method fully replace the other. tissue samples can be analyzed by standard histology and subsequent elaborated investigations. both techniques are time-consuming which can become limiting in the case of frozen sections. by contrast, confocal laser microscopy of tissues yields almost instantaneous information but is not amenable to traditional subsequent analyses that require a resected physical sample. it might, however benefit from new analytical methods such as artificial intelligence. such methods are being increasingly employed in radiology and termed radiomics or radiogenomics. unlike the situation in neurosurgery and neuropathology, cle has already been adopted by multiple medical specialties including gastroenterology, pulmonology otorhinolaryngology and urology since early the 2000s. it is therefore worthwhile to analyze how cle has been integrated in the workflows of these medical specialties (table 1). in gastroenterology for instance, cle has proven its clinical utility for the diagnosis of esophageal premalignant lesions and neoplasms such as barrett’s esophagus with low-grade and high-grade dysplasia (5,6) and esophageal cancer (7). the usefulness of cle in the detection of esophageal, stomach and colon cancer has moved beyond early proof of concept to large clinical trials (8–14). table 1: selected clinical in vivo studies reporting interobserver agreement for cle imaging and consistency of cle-based diagnosis with histopathology in various precancerous lesions and cancer entities n.r.: not reported, cle: confocal laser endomicroscopy, wlc: white light cystoscopy, fna: sodium fluorescein as for all new methods, interpretation criteria for cle have to be developed, validated and adopted by the medical community. while interpretation guidelines for cle images are in their infancy in neuropathology, various interpretation criteria and classification systems have been developed and validated in urology and gastroenterology owing to the earlier adoption of cle in these areas. these diagnostic systems range from simple criteria such as the presence of dark aggregates of cells as the cle criterion for malignant mucinous lesions of the pancreas (15) to elaborate morphological criteria and diagnostic scores such as the ones published for dysplasia for barrett’s esophagus (16,17), oral squamous cell carcinomas (18) and bladder cancer (19) and others. these diagnostic systems use features of tissue and cellular architecture found in cle images to distinguish normal from malignant tissue, or to distinguish between different types of lesions. scoring methods assign positive and negative scores to specific tissue or cell features. if the score is beyond a predetermined threshold, there is a very high likelihood of malignancy (18). similar to all diagnostic procedures requiring human interpretation, cle is also affected by the pathologist’s subjectivity. in medical disciplines that require interpretation of diagnostic images, such as pathology and radiology, interobserver studies are commonly done to ensure that results are reproducible. diagnostic methods showing higher levels of agreement between observers are deemed more reliable. while this aspect has not yet been fully investigated for brain tumors, reports on interobserver agreement exist in various tumor entities (table 1). confocal laser endomicroscopy in the context of digital pathology and telepathology implementation of cle will not happen in a void, but in the context of existing workflows and current developments. first, cle integrates well into the current movement towards digital pathology. a fundamental challenge of digital pathology is digital microscopy, i.e. the adoption of digital images. this made workflows more complex in almost all applications as glass slides cannot be eliminated but remain the source of the ultimate digital image. this contrasts with the situation in digital radiology where workflows were simplified by the elimination of films. from this perspective, cle as well as other techniques for in vivo microscopy offers the advantage of being intrinsically digital which has a number of positive consequences, including the absence of tissue consumption, the lack of a delay for scanning glass slides and the possibility of teleconsultation. in the conceptual framework of telepathology (table 2), cle is most similar to dynamic telemicroscopy since it is potentially very interactive with the possibility of real-time discussion of findings with the neurosurgeon who holds the cle probe and needs to identify areas of interest, and with a second person who operates the cle device. table 2: paradigms for telepathology and their respective advantages and limitations confocal laser endomicroscopy in the context of intraoperative consultation: strengths and limitations traditionally, frozen section and/or cytological preparations are used in variable combination for intraoperative diagnosis in most centers. both techniques have specific strengths and limitations. while frozen sections provide more information on the tissue architectural level and are more similar to histological slides, cytological preparations provide better nuclear morphological details and are not susceptible to freezing artifacts. given that intraoperative cytology requires only minimal technical equipment, it is performed on-site in some centers which eliminates transfer to the pathology laboratory and accelerates processing. it requires however the presence of neuropathologists on-site or the presence of equipment for telepathology. cle will likely result in faster turnaround times even than on-site cytology while being intrinsically prone to teleconsultation. this may turn out particularly advantageous given the limited availability of neuropathologists in many places. the implementation of cle in clinical routine will however require both neurosurgeons and neuropathologists to adopt a novel conceptual approach. table 3: time requirement for intraoperative microscopic diagnosis using cle and competing techniques the ultimate clinical utility of cle may very well differ in terms of specific indications. when neuropathologists are required to assess sample adequacy for diagnosis and ancillary diagnostic techniques, the highest possible diagnostic accuracy probably obtained with frozen section and cytology will arguably outweigh any advantage in turnaround time or non-invasiveness of cle. conversely, cle may turn out as the method of choice for the identification of residual tumor cell infiltration in the periphery of resection cavities, because its speed and non-invasiveness allow for much more extensive sampling than tissue biopsies. cle thus has the potential to change current paradigms for maximal safe resection of brain tumors. a hybrid approach combining cle and tissue biopsies may be envisioned for intraoperative diagnosis. here, cle would provide a faster response, while frozen section or cytology would serve as confirmatory techniques. table 4: expected potential of cle to supplement or replace frozen sections any novel technique can potentially add something to the existing armamentarium, either by adding precision as historically seen with gene sequencing or methylome analysis or by providing information even if limited more rapidly than existing techniques. current diagnostic practice in neuropathology is characterized by a whole set of different diagnostic modalities. in terms of turnaround times, any technique that is slower than existing ones may be of interest if it adds, either alone or as a complement to other techniques, diagnostic precision. hematoxylin-eosin-stained slides of ffpe tissue will typically be available within one day, immunohistochemical stainings may require an additional working day. based on this, patients may be informed about their diagnosis before they leave hospital. molecular analyses often require around one week which is sufficient for discussion in a multidisciplinary tumor conference before starting adjuvant therapy. frozen sections and cytology are sufficiently fast for intraoperative consultation, which makes a somewhat lower precision than that of histological slides acceptable. their turnaround times are however typically too long for extensive repetitive sampling. therefore, cle may prove clinically highly useful by allowing repeated analysis or analysis of multiple sites, even if its accuracy were ultimately found to be somewhat lower than that of frozen sections (figure 1). figure 1: schematic illustration of turnaround time versus diagnostic precision. an ideal technique would fit in the upper left corner, i.e. offer the greatest possible accuracy with the shortest possible turnaround. nevertheless, any new technique that is either more precise or faster than available techniques is expected to improve the diagnostic armamentarium. in the case of cle, the turnaround time in order of one to few minutes allows for multiple repetitive analyses, which are impractical with frozen sections due to the time constraints for their preparation. for a given question, cle may provide significant benefit by providing fast results even if its precision were lower than that of frozen sections. general aspects of interpretation correlation between confocal laser endomicroscopy and standard histology in vivo confocal endomicroscopy produces a novel representation of tissue structures that neuropathologists are well accustomed to interpret (figure 2). as for any new technique, it will be crucial to learn to what extent previously defined morphological criteria can be applied and to identify the differences as compared to traditional techniques. our experience with cle images matches h&e slides for different types of lesions. the cellular structures most consistently identifiable in confocal laser endomicroscopy are cell nuclei due to their fainter fluorescence as compared to adjacent tissue. nuclear size, nuclear size variation as well nuclear spatial distribution are generally well visualized by cle. on static images, red blood cells are occasionally difficult to distinguish from neural nuclei as they produce a similar type of negative image due to their low fluorescence. we have found their uniform size and round shape useful for their identification. figure 2: example of morphologic patterns in glioblastoma in h&e slides (a, b, c) and in cle (d), illustrating how similar morphological findings of the same lesions can be visualized with both imaging techniques. red circles indicate giant cells, green circles indicate necrotic areas. correlation of cle-based morphology and standard histology in clinical routine will likely be an important basis of continuous training for all neuropathologists, allowing them to refine diagnostic criteria. it should be kept in mind, however, that any sole morphological analysis of cns tumors has significant limitations in the age of a molecular tumor classification. in particular, tumor types defined by molecular alterations cannot be diagnosed with certitude by histology or by any other morphological technique including cle. cle may face similar limitations regarding lesions that are considered high-grade solely due to molecular features. neither of these limitations, however, fundamentally thwart the clinical utility of intraoperative cle. three clinical trials are currently investigating cle in neurosurgery and neuropathology: a 200 patient multicenter trial in germany (invivo, nct04597801), a 200 patient trial in berne, switzerland (clebt, nct04280952) and a trial in milano, italy (besta institute review board, verbal n. 72/2020) focusing on the concordance between cle and definitive histopathological analysis. the results of these trials will clarify the potential clinical benefit of cle in neurosurgery and neuropathology and define the role of cle in the neurosurgical armamentarium. development and refinement of diagnostic criteria for cle cle is a promising method which achieves in vivo imaging during neurosurgery at high magnification and in real-time without tissue manipulation. cle is expected to help to distinguish more easily between healthy and neoplastic tissue, thus addressing a major challenge in brain tumor surgery. by optimizing tumor resection and preventing tumor relapse, cle is expected to offer a significant advantage to patients. in order to achieve these goals, the neuropathologist and/or the neurosurgeon will require basic knowledge of the cytoarchitecture of different tumor types and subtypes imaged by cle. in addition, entity-specific interpretation criteria and tumor classification guidelines are needed, similar to the previous situation in urology and gastroenterology. here, we would first like to discuss and present the general aspects of interpretation of cle images, based on the thorough examination of thousands of cle images and the corresponding histology from different brain tumor samples, and before establishing definite diagnostic criteria for each tumor type and subtype. in general, our cle images revealed features similar to already known tissue architecturea and morphology. the cle images correlated well with the corresponding histology (figure 2). unique aspects of individual cells and other surrounding tumor tissue elements were observed while the cle probe acquired images throughout its focal-depth range. cle images of brain tumors demonstrated high-contrast background with features of hypercellularity, atypic nuclei, microvascular proliferation, necrosis, and/or abnormal cell clustering and grouping (figures 3, 6, 7). by contrast, cle images of normal brain tissue showed a rather quiet, dark and normocellular background (figure 3e). erythrocytes were visualized as abundant, round to oval shaped cells that appeared to be much smaller than tumor cells. these features were already sufficient to allow a quick intraoperative diagnosis and to classify the tissue as normal or pathologic, independent of the exact tumor type and subtype, supporting the intraoperative decision making. figure 3: examples of morphologic patterns in various tumors on in vivo cle images recorded in comparison with microscopically matched h&e slides from the same region of interest. pleomorphic cells and hypercellularity in a case of glioblastoma multiforme (gbm) (a-b), a case of anaplastic oligodendroglioma (c-d), a case of high-grade glioma (hgg) showing a region of normal brain tissue and a highlighted vessel (arrow in h&e slide, bright streak in cle image) adjacent to the neoplastic tissue (e-f), and a case of anaplastic ependymoma (g-h). possible use cases for cle as mentioned previously, cle images showed the presence or absence of typical histomorphological characteristics of high-grade gliomas (figures 2 and 3) as observed on routine histological examination such as microvascular proliferation and/or necrosis in addition to hypercellularity and pleomorphism. distinguishing between a low-grade glioma (cns who grade 1/2) and a high-grade glioma (cns who grade 3/4) is important, because the therapeutic options differ: simple resection in low-grade tumors versus additional radiation and chemotherapy in high-grade tumors. an intraoperative statement regarding the malignant potential of the tumor is often asked, and can help the neurosurgeon decide if further resection should be undertaken or not. however, making a distinction between a low-grade glioma cns who grade 2 and a high-grade glioma cns who grade 3 can be very demanding, since the differentiation between these tumors mostly relies on the presence of mitoses, which are hitherto difficult to identify on cle images. these limitations should however not affect the integration of molecular data into the tumor grading. furthermore, cle is able to access the center of a brain tumor which may be useful for rapid tumor recognition and confirmation. last but not least, cle bears the big advantage to evaluate the borders of the tumor, recognizes abnormal histological features, and optimizes surgical resection in marginal regions if necessary. due to the diffuse growth pattern of most glial tumors, it is often challenging during surgery to distinguish neoplastic tissue from normal or reactive brain tissue without special or enhanced imaging techniques, often resulting in incomplete tumor resection. here, virtual biopsies of gliomas at the tumor margins using cle are of particular interest, because they allow an immediate histological assessment. in such cases, a specific pattern of cellular structures showing delineation of nuclei appearing darker than cytoplasm can often be noticed. moreover, small clusters of tumor cells or individual tumor cells within brain tissue can sometimes be identified, and a tumor border can often be delineated. figure 4: representation of the field of view of the zeiss convivo device superimposed over a low-power histological image of a glioblastoma. it should be pointed out that the field of view of cle images is small, approximately 475 μm x 267 μm in the case of the zeiss convivo (figure 4). using cle to decide whether or not to extend tumor removal can thus be challenging. thus, when cle provides an intraoperative preliminary diagnosis based on high magnification images, and a low-power histological image is not available yet, attention must be paid to avoid a potential pitfall for misleading diagnosis, as the entire lesion cannot be overviewed in a single cle image yet. consequently, the focus should be on detecting abnormal histoarchitectural features in such cases. due to the intrinsically digital nature of cle images, image stitching techniques (figure 5) can compensate the small field of view of current generation cle devices. by scanning the tissue surface of the region of interest, cle can generate a digital map in near real-time. here, the cle probe tip constantly acquires images which are digitally stitched together as they are acquired. the resulting digital map then covers a much greater area and allows a better overview of the region of interest than the individual cle images. figure 5: experimental image processing (a,b) using stitching techniques. serial images with overlapping content can be stitched together and result in a greater field of view. illustrative example of two (a) and three (b) serial images of vasculature with overlapping image content. the images were extracted from an image series recorded over several seconds during which the cle probe was moved along the tissue surface. tilting of the probe tip or tissue compression during image series acquisition can result in perspective distortion, which has been corrected for in these examples. composite of 45 serial images of tumor tissue (c, d): the cle probe was maneuvered along the tissue surface while serial images were acquired. the images were aligned based on overlapping content (c), which results in a much greater field of view compared to the individual images. because the images are stacked onto each other, the borders of the individual images are clearly recognizable, as the images on top of the stack overlay the images further below. further image processing methods, such as blending, can create a seamless tissue map and allow assessment of the tissue in its continuity (d). for clinical purposes it has to be made sure that such methods do not introduce diagnostically relevant artifacts. confocal laser endomicroscopy of specific tumor types in our experience, cle of metastatic tumor lesions presented mostly high-contrast, clear and good quality images with histomorphological features that were highly diagnostic of the tumor type (figure 6). a sharp delineation between the neoplastic and healthy brain tissue as well as a grouped growth pattern of tumor cells displaying a high nuclear-to-cytoplasmic ratio, sometimes with a detectable prominent nucleolus, were easily recognizable in cle images. figure 6: comparative examples of metastases in in vivo and ex vivo cle images and h&e slides. shown are a metastasis of a poorly differentiated carcinoma (a-b) and reactive changes (gliosis and discrete lymphocytic inflammatory infiltrate) at the border of the same lesion (c-d), metastatic adenocarcinoma (e-f) and metastasis of ductal breast carcinoma (g-h, h&e of frozen section). for illustrative purposes, both in vivo and ex vivo cle images are included showing that morphology is represented consistently independent of the acquisition modality (a, c, and e: ex vivo; g: in vivo). imaging of meningiomas often revealed moderate hypercellular tissue with relatively monomorphic tumor cells and patterns characteristic of a subtype, such as a fibrous background, whorls or microcystic changes (figure 7). psammoma bodies were always easily identified when present in the tumor sample. meningiomas of cns who grade 2 and 3 seemed to have increased cellular atypia than cns who grade 1 tumors, although this distinction was mostly hard to draw. figure 7: comparative examples of tumor morphology in ex vivo cle images and matched h&e slides in two distinct cases of meningioma (a-b and c-d) and microcystic meningioma (e-f). while in vivo imaging is the preferred usage scenario of cle, ex vivo imaging is useful for training purposes. it enables recording images over a longer period than what is practicable during surgery. it can also help to point out specific tissue characteristics that might otherwise be more time-consuming to achieve intraoperatively. in the same manner, schwannomas exhibited a fascicular architecture composed of spindled cells with elongated cytoplasmic processes, which were easy to recognize on cle images. features such as antoni a and antoni b patterns or verocay bodies were not always detected, even when present in the matched histological samples. another important aspect to consider when evaluating cle images as a neuropathologist, is the quality of the images obtained by the neurosurgeon. in the beginning, lack of experience in handling the cle probe can cause difficulties in obtaining adequate images for diagnosis. indeed, the area of interest can be obscured by hand instability or by acquisition of images with blood or movement artifacts. at the beginning of the learning curve, we found that ex vivo cle imaging was very useful for training purposes. such ex vivo images can be easily obtained, allow careful selection of the imaging window and artifact-free depiction of the features of interest. when both the neurosurgeon and the neuropathologist gain experience with the cle probe, high quality in vivo images are then guaranteed. potential impact of cle on the role of neuropathologists the future integration of cle in clinical routine may have a significant impact on the role of neuropathologists in the interdisciplinary team and on neuropathologists’ workload. on one hand, the intrinsic suitability of cle for telepathology may facilitate distribution of the workload onto a larger team of neuropathologists possibly working at different hospital sites. this may also increase efficiency since the neuropathologists would no longer need to move to the frozen section laboratory or to stay there between subsequent intraoperative consultations. furthermore, closer interaction due to increased and real-time intraoperative consultation may enhance the visibility of neuropathologists to clinical colleagues and thereby contribute favorably to the role of neuropathologists in the interdisciplinary team. on the other hand, a possible increase in intraoperative consultations may increase the workload of neuropathologists and their limited workforce. some neuropathologists may be sceptic about cle since cle gives neurosurgeons immediate access to a tool of microscopic analysis. there could be fears that the neurosurgeons may be tempted to interpret cle images on their own, which ultimately might be seen to render neuropathologists superfluous. we are convinced, however, that such fears would not be warranted and that neuropathologists have any reason to be confident in the importance of their morphological competences. when neurosurgeons became more and more comfortable in interpreting cle images, this will undoubtedly enhance the quality of the interdisciplinary collaboration and of the entire diagnostic process. challenges in the clinical implementation of cle further challenges to be considered for cle are economic aspects. clinical implementation of cle technology may be initially restricted to prosperous countries able to afford the cost of the novel technology. on a global scale, many people do not have access to adequate neurosurgical care for brain tumors. we deem it easier to implement in vivo cle in an existing setting of micro-neurosurgery, than to build up infrastructure for frozen section and train staff from scratch in neuropathology. in this scenario, an added benefit would be the easier cle technology dissemination of knowledge with, e.g. via joint expert teleconsultations from the operating room, as compared to standard slide-based histology. in many healthcare systems, billing in neuropathology is closely linked to technical analyses of tissues, which means that adequate reimbursement for reading of cle images may be difficult to obtain. furthermore, specific training will have to be developed and implemented in order to permit widespread implementation of cle. additionally, meticulous quality control procedures assessing diagnostic accuracy and turnaround times will be necessary. finally, the respective roles, strengths and limitations of frozen section, intraoperative cytology and cle depending on the clinical context will have to be clarified in order to allow this entire armamentarium of diagnostic modalities to unfold its full potential. conclusion and outlook cle has a significant potential as an additional tool in intraoperative consultation for cns lesions. the main strengths of cle relate to its speed, the possibility to analyze tissue without prior resection, and its intrinsic suitability for telepathology. the commercial availability of a first clinical-grade device will facilitate future widespread application. data obtained through previous experimental use of cle will provide a basis for interpretation in clinical use cases. further refinement of diagnostic criteria and of diagnostic accuracy in routine settings will be paramount. furthermore, cle offers unique perspectives for intraoperative diagnosis at the tissue level, thereby enhancing the role of neuropathologists in the interdisciplinary care team. ultimately, cle promises to complement the intraoperative diagnostic armamentarium in neuropathology and neurosurgery and thereby to contribute favorably to the clinical management of cns tumors. conflicts of interest the departments of neurosurgery in milan and bern receive fees from carl zeiss meditec ag, oberkochen, germany, for lectures at international congresses and support for research using the zeiss convivo device. dr. karl quint receives fees for coordinating the community of researchers and clinicians working with the zeiss convivo device. prof. ekkehard hewer and dr. maragkou have received honoraria from carl zeiss meditec ag. carl zeiss meditec ag has not played any role in analysis or interpretation of the data and has not been involved in the preparation of the manuscript. references 1. goetz m. confocal laser endomicroscopy, current indications and future perspectives in gastrointestinal diseases. endoscopia (2012) 24:67–74. 2. sadeghipour a, babaheidarian p. making formalin-fixed, paraffin embedded blocks. methods mol biol clifton nj (2019) 1897:253–268. https://doi.org/10.1007/978-1-4939-8935-5_22 3. sy j, ang l-c. microtomy: cutting formalin-fixed, paraffin-embedded sections. methods mol biol clifton nj (2019) 1897:269–278. https://doi.org/10.1007/978-1-4939-8935-5_23 4. canene-adams k. preparation of formalin-fixed paraffin-embedded tissue for immunohistochemistry. methods enzymol (2013) 533:225–233. 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farhadi ds, byvaltsev va, eschbacher jm, nakaji p, preul mc. intraoperative confocal laser endomicroscopy ex vivo examination of tissue microstructure during fluorescence-guided brain tumor surgery. front oncol (2020) 10:599250. https://doi.org/10.3389/fonc.2020.599250 30. abramov i, park mt, belykh e, dru ab, xu y, gooldy tc, scherschinski l, farber sh, little as, porter rw, et al. intraoperative confocal laser endomicroscopy: prospective in vivo feasibility study of a clinical-grade system for brain tumors. j neurosurg (2022) 1:1–11. https://doi.org/10.3171/2022.5.jns2282 copyright: © 2022 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. differentiation of primary cns lymphoma and glioblastoma using raman spectroscopy and machine learning algorithms feel free to add comments by clicking these icons on the sidebar free neuropathology 2:26 (2021) original paper differentiation of primary cns lymphoma and glioblastoma using raman spectroscopy and machine learning algorithms gilbert georg klamminger*1,2,3, karoline klein*1,4, laurent mombaerts*6, finn jelke1,4, giulia mirizzi1,4, rédouane slimani5,7, andreas husch6, michel mittelbronn2,3,6,7,8,9, frank hertel1,4,6, felix b. kleine borgmann3,6,7 1 saarland university medical center and faculty of medicine, homburg, germany 2 national center of pathology (ncp), laboratoire national de santé (lns), dudelange, luxembourg 3 luxembourg center of neuropathology (lcnp), dudelange, luxembourg 4 national center of neurosurgery, centre hospitalier de luxembourg (chl), luxembourg, luxembourg 5 doctoral school in science and engineering (dsse), university of luxembourg (ul), esch-sur-alzette, luxembourg 6 luxembourg centre of systems biomedicine (lcsb), university of luxembourg (ul), esch-sur-alzette, luxembourg 7 department of oncology (donc), luxembourg institute of health (lih), luxembourg, luxembourg 8 department of life sciences and medicine (dlsm), university of luxembourg, esch-sur-alzette, luxembourg 9 faculty of science, technology and medicine, university of luxembourg, esch-sur-alzette, luxembourg * these authors contributed equally to this work. corresponding author: felix b. kleine-borgmann · luxembourg center of neuropathology (lcnp) · department of oncology (donc), luxembourg institute of health (lih) · 84, val fleuri· l-1526 luxembourg · luxembourg felix.kleineborgmann@lih.lu submitted: 04 august 2021 accepted: 27 september 2021 copyedited by: vanessa s. goodwill published: 04 october 2021 https://doi.org/10.17879/freeneuropathology-2021-3458 additional resources and electronic supplementary material: supplementary material keywords: raman spectroscopy, pcnsl, glioblastoma, machine learning abstract objective and methods: timely discrimination between primary cns lymphoma (pcnsl) and glioblastoma is crucial for diagnosis and therapy, but also determines the intraoperative surgical course. advanced radiological methods allow for their distinction to a certain extent but ultimately, biopsies are still necessary for final diagnosis. as an upcoming method that enables tissue analysis by tracking changes in the vibrational state of molecules via inelastic scattered photons, we used raman spectroscopy (rs) as a label free method to examine specimens of both tumor entities intraoperatively, as well as postoperatively in formalin fixed paraffin embedded (ffpe) samples. results: we applied and compared statistical performance of linear and nonlinear machine learning algorithms (logistic regression, random forest and xgboost), and found that random forest classification distinguished the two tumor entities with a balanced accuracy of 82.4% in intraoperative tissue condition and with 94% using measurements of distinct tumor areas on ffpe tissue. taking a deeper insight into the spectral properties of the tumor entities, we describe different tumor-specific raman shifts of interest for classification. conclusions: due to our findings, we propose rs as an additional tool for fast and non-destructive tumor tissue discrimination, which may help to choose the proper treatment option. rs may further serve as a useful additional tool for neuropathological diagnostics with little requirements for tissue integrity. introduction the highly malignant and rare non-hodgkin primary central nervous system lymphoma (pcnsl) accounts for only 3% of all brain tumors1. the common practice to confirm the suspicion of a pcnsl is a stereotactic biopsy, according to which it can be histopathologically diagnosed and the following treatment adjusted2. due to similar initial clinical presentations and imaging properties, an important differential diagnosis of primary cns lymphoma is diffuse high-grade glioma, especially glioblastoma, who grade iv. the clinical management and treatment, however, considerably vary between these entities. while gross surgical resection of the tumor followed by combined radio-chemotherapy is standard of care in the treatment of glioblastoma, surgery plays a subordinate role in pcnsl therapy3-4. it is essential to differentiate between these tumors as early as possible5. recent magnetic resonance imaging (mri) studies have shown potential, using, amongst other parameters, the signal of the tumor blood flow (tbf) and diffusion tensor imaging (dti) characteristics preoperatively5. furthermore, discrimination was described using the levels of myo-inositol concentration measured with magnetic resonance spectroscopy6. nevertheless, stereotactic biopsies remain necessary to confirm the diagnosis in the majority of cases. as a promising method in tumor diagnostics, raman spectroscopy (rs) allows fast and non-destructive, label-free tissue classification even perioperatively. so far, it has been successfully used to distinguish different entities and grades of brain tumors, such as meningiomas and gliomas7,8,9,10. due to their inherent ability of tissue recognition, inelastic scattered photons caused by changes in the vibrational state of molecules are used as a “molecular fingerprint” of the examined tissue. rs has already been shown to be capable of detecting intraocular lymphoma cells11. rs is also able to distinguish between diffuse large b-cells and chronic lymphocytic leukemia in blood samples12, as well as to predict the malignancy status of lymph nodes13. assessment of tumor tissue via rs during diagnostic stereotactic interventions may lead to early changes of the intraoperative surgical course by favoring immediate surgical intervention in the case of glioblastoma or rather to cessation of the procedure in lymphoma patients without waiting for more detailed histopathological analysis. rs may further be applied on processed tissue14, such as on formalin fixed paraffin-embedded (ffpe) tissue in the pathology department to distinguish between pcnsl and malignant glioma. here, we applied rs on freshly resected tissue within the operating room (or) and after histopathological diagnosis of ffpe tissue. we then tested linear and nonlinear machine learning algorithms with the goal to create a classifier that in the future may be useful both in the or and in the pathological diagnostics for the differentiation between pcnsl and glioblastoma. materials and methods patient data 75 measurements from 3 pcnsl patients (due to a low prevalence the number of samples remains limited) and 10 glioblastoma patients were carried out intraoperatively on freshly resected tissue without further processing (2 patients) and after formalin fixation (1 patient). 45 measurements from 3 pcnsl patients and 6 glioblastoma samples were carried out on ffpe tissue. table 1 provides a more detailed overview about conducted measurements, supplemental table 1 and 2 contain additional information about the glioblastoma samples. all tumor samples underwent neuropathological diagnostics (histology, immunohistochemistry, epigenetic and genetic analysis), performed by a board certified neuropathologist at the national center of pathology (ncp) at the laboratoire nationale de santé (lns, luxembourg). the patient data were collected between 2018-2020; all patients were part of the insitu® study (nr. 201804/08), which has been authorized by the ‘comité national d’ethique de recherche’ (cner) and was performed according to the 'eu general data protection regulation' gdpr15, as well as the world medical association (wma) declaration of helsinki. 16 data acquisition and tissue preparation tumor samples of pcnsl biopsies and glioblastoma resections were collected during surgery / biopsy and put into vials with physiological saline solution. this intraoperative standard procedure prevents drying of the tissue and further degradation. for intraoperative data acquisition, we then put the tissue samples directly into an aluminum cup. the insignificant spectral attribution of aluminum allows unimpaired examination of biological tissue and minimizes influencing spectra from the surroundings. for the measurement itself, we used a robotized visualization and spectroscopic acquisition system (solais™, synaptive®, toronto, canada) enabling the determination of exact measuring points. all points were distributed uniformly on the sample; we included data that showed representative coverage of characteristic histopathological features of the subsequent pathologic diagnosis. visible light images of the tissue and the selected measurement spots were acquired for comparison and traceability (figure 1). figure 1. overview about our workflow and the different raman devices used for tissue examination. a) tumor tissue is measured in the or using the solais™ device. b) primary cns lymphoma biopsy with intraoperatively set raman measurement points (colored). c) ffpe tissue blocks are cut using a standard microtome and d) diagnosed by a neuropathologist (corresponding histological sample of the tissue in b). e) consecutive cuts are mounted on a caf2 slide and used for raman measurements on processed tissue with the tsi proraman device. for neuropathological diagnosis, the tumor samples were then fixed in a formalin solution and subsequently embedded in paraffin; resulting ffpe blocks were cut with a standard microtome. the first cuts were used for routine diagnosis (hematoxylin and eosin (he) staining and immunohistochemistry). the consecutive cuts (7 μm) were left unstained and placed on a caf2 (calcium fluoride; crystran, poole, uk) slide, allowing for microscopic examination of the section, which is not possible on aluminum with transmitted light. due to a low amount of spectral background (precisely one single peak at 321 cm-1) the caf2 substrate allows appropriate spectroscopical examination of even thin tissue fragments17. to reduce paraffin signals from residual wax within the tissue18, the slides were dewaxed using our in-house dewaxing protocol. this procedure includes a 60 min incubation period (at 60°c) to melt the paraffin and xylene/ethanol baths afterwards (2 x 15 min xylene; 3 x 2 min ethanol) to dewax the tissue chemically. first, areas with vital tumor cells were identified by means of light microscopy on the diagnostic he slides (surrounding blood and fibrin residuals were excluded), then the corresponding area on the unstained caf2 slides was marked (encircled) with a slide marking pen. the raman measurement of the ffpe tissue was carried out repeatedly at several sites within the marked area using the proraman-l high-performance raman spectrometer (tsi, shoreview, usa). additionally, see figure 1 for an overview of our workflow. raman spectroscopy for data acquisition on intraoperative and ffpe tissue, two different raman spectrometers were used. as our permanent tool in the operating room, the solais™ raman system consists of an automatic data acquisition and visualization system. via a robotized stage the tumor tissue can be exposed to the excitation laser (785 nm). the measuring points can be set individually on the tissue using a camera and an integrated coordination system. acquisition parameters were set to 2 seconds with 6-30 averages for each measuring point. for the raman measurement on ffpe tissue in the neuropathology unit, the portable tsi raman spectrometer was used. it also provides an excitation laser at 785 nm and a ccd sensor; the used lens has a 7 mm working distance and a 100 μm laser spot size. with the proraman reader software version 8.3.6 (tsi, shoreview, usa) acquired spectra can be displayed, baseline correction can be applied, and acquisition parameters can be defined (10 seconds, 30 averages, 90 mw) according to the request of the user. data analysis and machine learning to reduce bias, data acquired at different time points and states of fixation were analyzed separately and divided into two data sets. to reduce the impact of patient dependent clustering, the same number of measurements for each patient was carried out in the majority of cases and all of them were recorded with the same acquisition time in the respective systems. using a custom-made python script, data cleaning was carried out on intraoperatively acquired raman measurements (n = 75), including outliers and trend removal, as well as removal of artifacts and standardization of spectra. the mean spectra and the variance were visualized (figure 2). afterwards, three different machine learning algorithms were applied to distinguish between the two tumor entities and performance statistics were calculated (accuracy/sensitivity/specificity/roc/pr curve). starting with the linear algorithm of logistic regression we continued with random forest classification, which may be more favorable in this study due to non-independent data points within a raman measurement. to further reduce the bias of patient-dependent clustering, random forest classifiers were trained with randomly split data distribution and a distribution split by patients; results were internally checked for comparability and suggested no relevant bias. as the third machine learning algorithm, we used xgboost classification, in which parameters could be set individually. figure 2. mean spectra and variance of the perioperatively acquired rs after standardization. the x-axis displays the wavenumbers (in cm-1) of the inelastic scattered photons (= the raman shift) and the y-axis the corresponding intensity value. figure 3 gives an overview of our data processing and statistical approach. we identified characteristic wavenumbers usable for spectral tumor discrimination and compared our findings with the underlying biochemical composition of the tissue. the raman measurements acquired in the neuropathology department on ffpe tissue (second data set, n total = 45) were classified using a random forest algorithm. due to a low number of patients, the data was not split into a training and an external validation set. in order to create a representative sample of the performance of the algorithms, particular attention was instead made on the internal validation (for example intrinsic re-validation based on bootstrap subsamples). classification was validated using specific parameters of algorithm performance (auroc, aupr), as well as internal comparison of the performance based on randomly shuffling of measurement points with a patient stratified distribution. for a deeper technical insight in our way of machine learning (e.g., way of hyperparameter optimization) and a visualization of sample individual raman spectra, see supplementary material. figure 3. workflow of data selection and machine learning for the intraoperative measurements. results efficient differentiation of distinct tumor entities on freshly resected tissue using linear and nonlinear machine learning algorithms for an accurate overview of the performance of each of our classifiers, see table 2. our trained logistic regression algorithm distinguishes with a balanced accuracy of 79.3% between the intraoperative measured pcnsl versus glioblastoma tumor tissue. our random forest classifier discriminates between these two tumor entities with an overall accuracy of 82.4% (balanced), with a sensitivity of 0.89 ± 0.13 and a specificity of 75 ± 0.12. as an indicator of the classifier performance, we used the roc (receiver operating characteristics) curve, in which each threshold is visualized according to the resulting specificity and sensitivity, and the auc (area under the curve) value. the auc value of the random forest classification for the pcnsl class was 0.86 ± 0.07 (figure 4). since the roc curve may be biased due to an imbalance in the data set, we additionally used the pr (precision – recall) curve, where instead the ratio between ppv (positive predictive value=precision) and recall (=sensitivity) of every possible cut-off is displayed. figure 4 shows our resulting pr curve with the resulting auc value (0.66 ± 0.2) for the pcnsl class. figure 4. left: roc curve of random forest classification with auroc value of 0.86 ± 0.07. right: corresponding pr curve with an aupr value of 0.66 ± 0.2. in order to evaluate our trained models in more detail, sensitivity and specificity were calculated for all algorithms and displayed in table 2 according to the optimal threshold (maximizing sensitivity and specificity). for all resulting plots of statistic performance, see supplementary material. biochemical insights from spectral analysis in order to determine certain wavenumbers of raman shift for more detailed analysis of the underlying biochemical substrates, we arranged wavenumbers of interest in descending order, according to the distinct tumor class and importance as a feature coefficient for the logistic regression. table 3 displays the class-defining wavenumbers (i.e., positive and negative weights). as wavenumbers with highest attribution for glioblastoma classification distinct peaks in the region 2450 to 3000 cm-1 can be detected (2791, 2486, 2650, 2562, 2673, 2971, 2823 cm-1); most of them likely representing ch stretching / oh nh ch stretching motions. in particular, peaks occurring in the region between 2700 to 3000 cm-1 are known to be related to underlying methyl groups from phospholipids19,20,21. on the contrary, the wavenumbers of interest for pcnsl classification cannot be assigned to the high wavenumber region. for pcnsl classification, the most important peak at 1831 cm-1 seems to be related to changes in the vibrational state of c=c compounds22; followed by bins at 374 cm-1 and 977 cm-1, respectively. those latter bins are, according to the literature19-20, related to chain expansions and cc-stretching of n-alkanes. other important peaks for pcnsl classification, notably 1085 cm-1 and 316 cm-1, may also be attributed to n-alkanes, whereas 784 cm-1 is caused by ring vibrations – as the spectral region from 600 to 800 cm-1 is mainly contributed to dna molecules21. figure 5 provides an overview of the wavenumbers with highest impact and their distribution in relation to the mean spectra of the examined tumor entities. figure 5. feature importance for classification between primary cns lymphoma and glioblastoma. the ascending bins are important for pcnsl detection; the descending bins for glioblastoma detection. see table 3 for further explanation of the wavenumbers behind the frequency bins. tumor classification on chemically aggressively treated formalin fixed paraffin-embedded tissue to analyze our measurements of the ffpe tumor tissue (second dataset with n = 45), we chose a random forest algorithm, as it showed the best performance on fresh tumor tissue in our analysis. applying rs on histopathological proven tumor areas, we were able to establish a classifier with a balanced accuracy of 94%, a sensitivity of 0.93 ± 0.09 and a specificity of 0.95 ± 0.05. the auroc value was 0.98 ± 0.03; the aupr was 0.97 ± 0.05. for a deeper understanding of our results, we investigated the wavenumber regions used to classify between glioblastoma and pcnsl. we detected two regions (around 690 700 cm-1 and 2820 2890 cm-1) contributing to the classification performance. pure paraffin wax was described to have spectra with peaks at 1133, 1296, and 1441 cm-1 23,18, but we also saw significant paraffin contribution in higher wavenumber regions, especially around 2820 to 2910 cm-1. therefore, we suppose that, in addition to tissue-originating spectra, different and potentially characteristic amounts of residual paraffin or its reaction products with the tumor tissue may play a role, which, together with the possible influence of other materials related to the dewaxing process and experimental setup, needs to be considered when applying rs on ffpe tissue. discussion in this study, we show that raman spectroscopy can be used as a non-destructive, label-free, fast technique for perioperative tissue classification to address the differential diagnosis between glioblastoma and primary cns lymphoma at an early stage in the diagnostic workflow. we suggest rs as a future additional method in the or. in the current application, the method would complement the biopsy procedure by giving a fast-feedback diagnostic tool that would allow for a direct treatment decision without waiting for lengthy histopathological examination. this could be advantageous, for example by giving the surgeon the opportunity to immediately proceed with surgical resection in the case of a diagnosis of glioblastoma. additionally, the tool could be modified in such a way that an optical probe could replace the biopsy needle and yield an invasive but non-destructive stereotactic assessment of the tissue24-25. with our machine learning algorithms, the spectral data obtained from intraoperative tumor samples could be classified with linear (logistic regression) and nonlinear classifiers (random forest), with the latter displaying the highest potential with an overall balanced accuracy of 82.4%. to our knowledge, we describe for the first-time important wavenumbers to distinguish pcnsl from glioblastoma. the most important numbers for the detection of the who grade iv glioblastoma are mainly arising from the spectral region 2450 to 3000 cm-1, which is not in contradiction with previous findings. zhou et al. 10 described three major peaks (2850, 2885, 2932 cm-1) in that specific region associated with healthy brain tissue, and the mainly phospholipid-derived peaks in the high-wavenumber region also showed the potential to distinguish between different who grades of gliomas. our suggested peaks for primary cns lymphoma most likely arise from vibrational changes in n-alkane molecules (316, 374, 977, 1085 cm-1) and partially also from backbone vibration of nuclei acids (784 cm-1). to our knowledge, these peaks have not yet been used for the differentiation between pcnsl and other tumor entities. so far, shiramizu et al. 26 showed the possibility to discriminate between b-cell lymphoma cells and non-neoplastic lymphocytes by using rs and set the fingerprint wavenumber region to 600 – 1800 cm-1. manago et al. 27 described certain raman peaks of non-neoplastic lymphocytes and lymphoma cells (amongst other peaks in the region 700-800 cm-1). they differentiated between non-neoplastic b-cells and three b-cell lymphoma cell lines and tracked changes in the resulting raman spectra during treatment application27. furthermore, our results demonstrate the capability of rs to distinguish tumor entities on highly processed ffpe tissue in the neuropathology department. here, precise regions of the tissue can be measured at the price of aggressive chemical treatment and spectral contamination by residual paraffin wax, fixation and washing agents. since spectral regions initially attributed to paraffin may even play an influencing role in ffpe tissue classification, standardized dewaxing protocols and tried and tested processes are essential. a potential role for rs in the neuropathological toolbox is the examination of tissue fragments with little requirements for tissue integrity and neuropathological expertise. recent studies investigate this possibility14. future studies will show whether the use of this “spectral molecular fingerprint” holds true, and can also be applied on multiclass differentiation of several tissue entities. due to a low prevalence of primary cns lymphoma, the quantity of measurements in our data set remained limited. to deal with resulting imbalance and to minimize the impact of patient-dependent clustering, several efforts have been made. not only were the number of measurements per patient correlated for both tumor groups to reduce insensitivity on imbalanced data, but we additionally evaluated our statistical algorithms with the balanced accuracy, the pr curve, and the corresponding aupr value. while cross validation and bootstrapping techniques have been used to provide an indicative assessment of the model performance in future patients, external validation would be required to implement such a prediction model in clinical practice. although our classification shows reasonably good results and a distinct separation of classes, for practical application, the number of reads of one single patient (and therefore the number of reads related to tumor tissue and not surrounding brain) is key. therefore, multiple reads from one sample, which is also preferable to further reduce the bias of patient depending clustering, will improve the diagnostic accuracy in a real-world perioperative setting. further studies are required to see if our proposed model of tissue classification holds true when using rs for the differentiation of other tumors, such as metastasis, and non-neoplastic lesions e.g., brain abscess. more extensive knowledge of the amount of tumor cells needed to get a tumor-specific raman signal would aid the surgeon to find the tumor borders28. in a next step, rs may also be able to provide early insights into genetic / epigenetic alterations of tissue29; as well as insights into therapeutic effects of chemoor radiotherapy27. rs might also be used in combination with additional spectral analysis such as ir (infrared) or mass spectroscopy as complementary techniques30-31, and in addition as an image-forming technique32. conclusion in conclusion, our study shows that machine learning algorithms can be successfully applied on spectroscopic data of brain tumor tissue and fulfill the need of early differential diagnosis between primary cns lymphoma and glioblastoma to determine an individual clinical treatment at an early stage. disclosures we thank the fondation cancer luxemburg (grant to frank hertel, michel mittelbronn, andreas husch and felix kleine borgmann) for the generous support. michel mittelbronn would like to thank the luxembourg national research fund (fnr) for the generous support (pearl p16/bm/11192868 grant). the authors declare no conflicts of interest. references kerbauy mn, moraes fy, lok bh, et al. challenges and opportunities in primary cns lymphoma: a systematic review. radiother oncol. 2017;122(3):352-361. https://doi.org/10.1016/j.radonc.2016.12.033 louis dn, ohgaki h, wiestler od, cavenee wk, eds. who classification of tumours of the central nervous system. revised 4t. lyon: international agency for research on cancer; 2016. stupp r, mason wp, van den bent mj, et al. radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma. n engl j med. 2005;352(10):987-996. 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discriminant analysis. anal methods. 2013;5(1):89-102. https://doi.org/10.1039/c2ay25544h orringer da, pandian b, niknafs ys, et al. rapid intraoperative histology of unprocessed surgical specimens via fibre-laser-based stimulated raman scattering microscopy. nat biomed eng. 2017;1:0027. https://doi.org/10.1038/s41551-016-0027 copyright: © 2021 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. malignant melanotic nerve sheath tumor with prkar1a, kmt2c and gnaq mutations feel free to add comments by clicking these icons on the sidebar free neuropathology 3:21 (2022) case report malignant melanotic nerve sheath tumor with prkar1a, kmt2c and gnaq mutations merryl terry1, kristina wakeman1, brian j. williams2,4, donald m. miller3,4, müge sak5, zied abdullaev6, marwil c. pacheco1, kenneth aldape6, norman l. lehman1,4,5 1 department of pathology and laboratory medicine, university of louisville, louisville, ky, usa 2 department of neurological surgery, university of louisville, louisville, ky, usa 3 department of internal medicine, university of louisville, louisville, ky, usa 4 the brown cancer center, university of louisville, louisville, ky, usa 5 department of biochemistry and molecular genetics, university of louisville, louisville, ky, usa 6 laboratory of pathology, center for cancer research, national cancer institute, bethesda, md, usa corresponding author: norman l. lehman · department of pathology and laboratory medicine · brown cancer center · department of biochemistry and molecular genetics · university of louisville · 505 south hancock street · louisville, ky 40202 · usa nllehman1@gmail.com submitted: 03 april 2022 accepted: 12 august 2022 copyedited by: jeffrey nirschl published: 26 august 2022 https://doi.org/10.17879/freeneuropathology-2022-3864 keywords: malignant melanotic nerve sheath tumor, mmnst, psammomatous melanotic schwannoma, nerve sheath tumor, pigmented epithelioid melanocytoma, prkar1a, kmt2c, gnaq abstract malignant melanotic nerve sheath tumor (mmnst) is a rare and potentially aggressive lesion defined in the 2021 who classification of tumors of the central nervous system. mmnst demonstrate overlapping histologic and clinical features of schwannoma and melanoma. mmnst often harbor prkar1a mutations, especially within the carney complex. we present a case of aggressive mmnst of the sacral region in a 48-year-old woman. the tumor contained prkar1a frame shift pr352hfs*89, kmt2c splice site c.7443-1g>t and gnaq p.r183l missense mutations, as well as braf and myc gains. genomic dna methylation analysis using the illumina 850k epicbead chip revealed that the lesion did not match an established methylation class; however, uniform manifold approximation and projection (umap) placed the tumor very near schwannomas. the tumor expressed pd-l1, and the patient was treated with radiation and immune checkpoint inhibitors following en bloc resection. although she had symptomatic improvement, she suffered early disease progression with local recurrence, and distant metastases, and died 18 months after resection. it has been suggested that the presence of gnaq mutations can differentiate leptomeningeal melanocytic neoplasms and uveal melanoma from mmnst. this case and others demonstrate that gnaq mutations may exist in malignant nerve sheath tumors; that gnaq and prkar1a mutations are not always mutually exclusive and that neither can be used to differentiate mmnst or mpnst from all melanocytic lesions. introduction malignant melanotic nerve sheath tumors (mmnsts) are rare neoplasms formerly known as melanotic schwannoma [1] and sometimes melanotic malignant peripheral nerve sheath tumor (mpnst). they are renamed in the 2021 who cns tumor classification [2] to better reflect their potentially aggressive nature and align with the 2020 who soft tissue nomenclature [3]. mmnst often carry an inactivating mutation in the protein kinase a inhibitory subunit gene protein kinase camp-dependent type i regulatory subunit alpha (prkar1a) on chromosome 17p22-24, which acts as a tumor suppressor [3, 4]. mmnst occur in some patients with carney complex, a rare syndrome associated with prkar1a mutation, cardiac and skin myxomas, endocrine and gonadal neoplasms, variable endocrine manifestations such as cushing syndrome or acromegaly, increased lentigines, and a type of blue nevus known as pigmented epithelioid melanocytoma [5, 6]. we present a case of an aggressive sacral mmnst in a woman which demonstrated prkar1a, kmt2c, and gnaq mutations, braf and myc copy number gains, and pd-l1 overexpression. we also compare histologic, genetic, and clinical features of conventional schwannomas, mmnst, mpnst, and melanoma. case report a 48-year-old female developed severe lower back and right buttock pain with right thigh numbness over a period of one year. she also reported difficulty urinating and defecating. her past medical history included endometriosis, hypothyroidism, and multinodular goiter. on examination, she had saddle anesthesia and urinary retention. computed tomography (ct) demonstrated a right sacral mass with posterior bony destruction. no local regional lymphadenopathy was appreciated. magnetic resonance imaging (mri) revealed a 7.8 x 8.6 cm heterogeneously enhancing mass with a large soft tissue component (figure 1). figure 1: tumor imaging. (a). axial and (b). sagittal t2 mri of the pelvis demonstrating an intermediate to brightly heterogeneously enhancing mass in the right sacrum and soft tissue with boney destruction. ct-guided core needle biopsy showed a pigmented malignant spindle cell neoplasm. the main histological differential diagnosis was metastatic melanoma, primary cns melanoma and mmnst. positron-emission computed tomography (pet/ct) demonstrated that the sacral mass was centered on the right s2 nerve root, with anterior and posterior soft tissue extensions, and a large lytic component in the right posterior ilium. there was no evidence of metastatic disease. a trans-abdominal, stage i procedure for high sacrectomy, with colectomy and colostomy was performed. this was followed one week later by en bloc resection and sacrectomy with l3-pelvis posterior spinal instrumentation and fusion, and flap reconstruction. intraoperative pathology revealed a pigmented spindle cell, malignant tumor involving the surgical margin. additional tissue was excised with grossly negative margins. figure 2: gross sacrectomy specimen. (a). lateral view, displaying a well-circumscribed tumor involving soft tissue and bone. (b). after inking the specimen blue, sectioning the tumor revealed the typical black “dried tar” appearance of cut surfaces. pathology the resected tumor was 9.0 x 8.5 x 7.0 cm in dimension, centered in soft tissue, and eroded into adjacent bone. it was relatively circumscribed, heavily pigmented with a “black tar” appearance, and partly covered by a thin fibrous membrane. (figure 2a-b). microscopically, the cells were arranged in sheets, lobules, and fascicles (figure 3a-d). click here to view the full virtual slide. large areas of necrosis were present (figure 3a). most tumor cells were spindleto epithelioid-shaped with oval nuclei, vesicular chromatin, and prominent nucleoli (figure 3e). up to 18 mitotic figures were counted in ten 400x fields. scattered melanin was identified within tumor cells and melanophages (figure 3b and 3f). focal areas showed verocay-like nuclear palisading. some areas contained highly pleomorphic cells demonstrating eosinophilic intranuclear pseudoinclusions (figure 3f). the tumor was surrounded by a pseudocapsule containing neurofilament-immunoreactive normal ganglion cells and nerve tissue (figures 3b-c and 4a). multiple areas of pseudocapsular and lymphovascular invasion were noted. occasional psammoma bodies were seen within the pseudocapsule adjacent to the tumor. figure 3: tumor histology. (a). geographic necrosis comprised approximately 30-40% of sampled tumor areas. (b). the tumor was lobular, pigmented, and surrounded by a pseudocapsule containing benign nerve elements. (c). the pseudocapsule incorporated benign ganglion cells (left). some tumor areas were deceptively bland (right). (d). most tumor areas contained spindled cells in a fascicular and vaguely nodular arrangement. (e). nucleoli were prominent and mitoses were abundant. (f). some areas were highly pleomorphic and/or demonstrated nuclear pseudoinclusions. magnification: a-b, 20x; c-d, 100x; e-f 400x. click here to view the full virtual slide. tumor cells were diffusely immunopositive for sox10, hmb-45, and melan-a (figure 4b). s100 was positive in scattered cells. cytokeratins ae1/ae3, braf-v600e, and pax-8 immunostains were negative. ini1 (smarcb1) was positive in tumor cell nuclei. reticulin stain diffusely highlighted spindle tumor cell-associated basement membranes and showed focal lobular staining of small groups of epithelioid cells (figure 4c). ki-67 labeling was 5-10% in most areas and up to 20% focally (figure 4d). pd-l1 was detected by immunohistochemistry (clone zr3; pd-l1 expression score 2, detection cut-off score ≥ 1). figure 4: tumor immunohistochemistry and reticulin stain. (a). neurofilamentimmunoreactive entrapped nerve elements within the tumor (upper left) and ganglion cells and axons within the pseudocapsule (lower right). (b). diffuse positivity for melan-a. (c). pericellular and lobular reticulin staining. (d). ki-67 immunohistochemical staining. magnification: a, 40x; b-d, 200x. next generation sequencing (neogenomics neotype analysis discovery profile) and fluorescence in situ hybridization (neotype discovery solid tumor fish panel) were performed on formalin-fixed paraffin-embedded (ffpe) tumor. two predicted inactivating mutations were detected: a lysine methyltransferase 2c (kmt2c) splice site mutation (c.7443-1g>t) and a prkar1a frameshift mutation (c.1055del, p.r352hfs*89). additionally, a pathogenic missense mutation (c.548g>t, p.r183l) was identified in the g protein subunit alpha q (gnaq) [7]. missense variants of unknown significance were detected in akt2 (c.1013t>g, p.v338g), kmt2c (c.7015a>g, p.n2339d) and setd2 (c.65c>g, p.t22s). nf1 gene mutation was not detected. fish demonstrated additional copies of braf (>2f, 52.0%; negative <25.1%) and myc (3f, 30.0%; negative <16.2%), consistent with gains of braf (7q34) or chromosome 7 and myc (8q24) or chromosome 8. genomic dna methylation analysis was performed on ffpe tissue using the illumina infinium methylation epic beadchip 850k microarray [8]. methylation data was uploaded to the german cancer research center (dkfz) methylation-based classification platform for central nervous system tumors (www.molecularneuropathology.org). results did not match any existing tumor methylation class (calibration scores < 0.9). chromosome copy number analysis confirmed chromosome 7 and 8 gains, as well as gains of 6p, 6q, 11, 12, 18, and 20p, and loss of 1p, 16q, 17, 20q, and 22q, among other changes (figure 5a). uniform manifold approximation and projection (umap) dimensional reduction analysis comparing the tumor methylation data to the dkfz brain tumor reference cohort [8] placed the tumor very near, or within, the schwannoma/melanotic schwannoma group (figure 5b). figure 5: genomic dna analysis. (a). chromosomal copy number data. (b). unsupervised umap dimension reduction analysis of tumor genomic dna methylation data. although the tumor did not match a specific tumor methylation class using the dkfz classifier, it grouped near, or with, schwannoma/melanotic schwannoma by umap (inset). adjuvant therapy and follow-up: postoperatively, the patient received 5940 cgy radiation in 33 fractions to her pelvis. she then received three cycles of pembrolizumab. the fourth cycle was withheld due to autoimmune hepatitis. the patient had a dramatic improvement in pain from a visual analogue scale (vas) pain score of 8-10/10 to 2-3/10. her karnofsky performance score was 90. ct scans five months after surgery demonstrated a new enhancing lesion of the pericardium, new gluteal mass, and new lytic lesions in the t2 and l2 vertebra and the left acetabulum. positron-emission tomography (pet) showed these and multiple rib lesions. core needle biopsy of the right gluteal muscle confirmed tumor recurrence. six months after surgery, immunotherapy with ipilimumab and nivolumab was initiated. she completed her fourth cycle three months later. ct showed new and worsening osseous metastases, as well as innumerable sub-centimeter pulmonary metastases, and enlargement of the pericardial mass. spinal mri revealed a pathologic fracture of t2. the patient was hospitalized with right-sided radiculopathy and underwent surgical stabilization of t2 followed by radiation to residual t2 tumor. she also developed a 3.6 cm right frontal calvarial lesion, which was irradiated. the patient was then treated with doxorubicin. she had further progression at c2 three months after spinal surgery. the patient elected for hospice care and died one month later (18 mo after primary resection). discussion mmnst are slightly more common in females (1.4:1 female to male) [3]. they are most often located paraspinally or in the gastrointestinal tract, but also occur at other sites [9, 10]. their average age of presentation is 33.2 years for sporadic tumors and 22.5 years within the carney complex. grossly, mmnst are typically solitary, partially circumscribed or encapsulated, and heavily pigmented [2]. they range from 0.5 cm to 25 cm in diameter, but most exceed 5 cm [10]. mmnst are associated with nerves or soft tissue and may erode bone. true intraosseous examples are rare [11]. microscopically, they are comprised of short fascicles or sheets of polygonal or spindled cells with a syncytial appearance. vague tumor cell palisading or whorling may be evident [2]. pigment is variable. nuclei are typically round to ovoid with nuclear grooves, pseudoinclusions, and/or prominent nucleoli. marked pleomorphism and nuclear hyperchromasia may be present [12]. associated vessels are usually capillary-like. psammoma bodies are present in ~50% of cases. psammomatous variants may contain lipoma-like fat accumulation and occur more often in carney complex [6, 11]. residual ganglion cells may be identified in paraspinal examples. mmnst usually strongly express s100 and melanocyte markers (sox10, hmb45, melan-a, and tyrosinase) [13]. although, examples of s100 patchy-positivity or s100-negative tumors have been reported [2, 13, 14]. ultrastructurally, tumor cells exhibit both schwann cell and melanocyte features, i.e., elaborate cytoplasmic processes, premelanosomes, and melanosomes [2, 10, 12]. mitotic rate is the only histologic feature found to be predictive of clinical outcome. in one study, a mitotic rate of >2/10 hpf correlated with metastases (p=0.008) [34]. however, >50% of tumors that eventually metastasized did not show increased mitoses. mmnst are biologically distinct from conventional schwannoma. mmnst tend to occur in posterior spinal nerves and ganglia, while schwannomas additionally may involve other nerves, including cranial nerves. the peak age of mmnst onset is a decade younger than for schwannoma [12]. unlike the latter, mmnst generally lack antoni a and b regions and hyalinized blood vessels histologically. gene expression profiling has shown significant differences between mmnst, melanoma, and schwannoma [13]. mmnst exhibited downregulation of genes involved in schwann cell function, e.g., pmp22, pmp2, and mpz, while genes related to melanin synthesis were upregulated in mmnst compared to schwannoma. in contrast to mmnst, conventional mpnst usually arise from peripheral nerves or extraneural soft tissue and are commonly seen in neurofibromatosis type 1 (nf1) [2], while mmnst only rarely occurs in nf1 [10, 15]. mpnst histologic features include monomorphic spindle cells with broad, intersecting, herringbone fascicles, alternating hyperand hypocellular areas, a high mitotic count, and geographic necrosis [2]. mpnst also generally lack pigment and are negative for melanotic markers. reported gene alterations in mpnst include nf1, cdkn2a, cdkn2b, tp53, tyk2, eed, and suz12 mutations and deletions, and egfr, pdgfra, and met amplification [2, 16]. the uncommon epithelioid variant of mpnst arises from a pre-existing schwannoma and harbors smarcb1 mutations [9, 11], whereas smarcb1 has been reported to be intact in mmnst [13]. distinguishing mmnst from melanoma can be difficult. both may demonstrate spindled or epithelioid cells with pigment, violaceous macronucleoli, and positivity for s100 and melanocytic markers [9]. a lower ki-67 labeling index may be suggestive of mmnst [2]. extensive collagen iv or reticulin basement membrane staining supports schwannian differentiation. a lobular/clustered tumor cell arrangement with syncytial-like cytology, psammoma bodies, and fat accumulation may also help distinguish mmnst from melanoma [9]. the genetics of mmnst are not completely defined. the majority of tumors are sporadic. prkar1a mutations and loss of prkar1a protein expression are seen in most cases and should prompt a search for other findings of the carney complex [3, 4]. a complex karyotype including 22q monosomy, recurrent losses involving chromosomes 1, 2, 21, and 17p, trisomy 6p, and ring chromosome 11 have been described in mmnst [3, 4, 10, 17]. melanoma typically shows 6p and 8q gains and 11q loss [17, 18]. mpnst exhibits gains of chromosomes 2, 7p, 8q, 14 and 17q, and loss of 9p, 11q, 13q, 17p, 18 and 22q [11, 19]. schwannoma also shows 17p and 22q loss. this case thus exhibits reported karyotypic changes overlapping mmnst, mpnst, and schwannoma (8q gain, and 1p, 17p, and 22q loss), and mmnst and melanoma (6p and 8q gains) (figure 5a). the case also demonstrates gene aberrations not previously ascribed to mmnst, namely, braf copy number gain and kmt2c splice site, and gnaq r183l missense mutations. braf v600e mutations, not gains, are found in cutaneous melanoma and pigmented epithelioid melanocytoma [20]. notably, the latter also may harbor prkar1a aberrations [21] or loss of heterozygosity [22]. gnaq codon q209 mutations are reported in leptomeningeal melanocytic neoplasms, uveal melanoma, and pigmented epithelioid melanocytoma [20, 21, 23, 24]. gnaq variants have also been described in a malignantly transformed schwannoma (gnaq t96s) [25] and an nf1-associated mpnst (gnaq y101x stop gain) [16] (angela hirbe, personal communication, july 2022). therefore, unlike previously suggested for mmnst [2, 23], neither gnaq nor prkar1a mutations can be relied on to differentiate mmnst from melanocytic lesions. although, prkar1a and gnaq mutations coexist in the mmnst described herein, we found no other documentation of their coexistence in the entities discussed above [16, 20, 21, 23, 24, 25]. however, in an important study comparing mmnsts to melanocytic lesions, only exon 5 of gnaq containing the q209 codon was sequenced [23]. thus, in that study gnaq codon t96, y101, and r183 hotspot mutations within exons 2 and 4, respectively, could not have been detected. gnaq mrna tissue distribution is greatest in the brain [26]. a propensity for gnaq mutations in neoplasms of other neuroectoderm/neural crest-derived tissues, i.e., melanocytes and nerves, is thus not surprising. however, gnaq is also important in vascular development. gnaq r183l occurs in brain endothelial cells of sturge-weber syndrome [27]. gnaq r183l and r183q mutations have been identified in capillary malformations [7, 28, 29] and gnaq q209 mutations in other benign vascular lesions [30]. additional tissues also highly express gnaq and gnaq mutations may occur in endocrine tumors and other neoplasms [31]. achieving long-term survival in mmnst is hampered by its tendency to metastasize and recur, and its propensity to involve the spine or other structures, which preclude complete surgical resection [32]. one study reports a 20-year remission rate of 67% with total resection [33]. however, torres-mora et al [13] reported mmnst local recurrence and metastatic rates of 35% and 44%, respectively (mean follow-up 55 mo), with 73% of metastases occurring in <4 years. metastases are typically to the lung and pleura. radical resection with or without radiation and/or chemotherapy is the mainstay of treatment. although, radiation has not shown a clear benefit [12]. a few reports using traditional chemotherapeutics, e.g., carboplatin and etoposide, showed low response rates and no survival benefit [14]. while immune checkpoint inhibitors are commonly used in cutaneous melanoma, their use in mmnst is not well-established. this patient with documented pd-l1 expression was treated with the checkpoint inhibitors pembrolizumab, ipilimumab, and nivolumab with pain improvement, but no objective response in disease progression. bajpai et al., however, described a patient with recurrent and metastatic mmnst who underwent multiple surgeries, external beam radiation, and nivolumab treatment [34]. ipilimumab was added after disease recurrence. the authors reported a survival of 51 months from initial diagnosis and 35 months after the start of checkpoint inhibitor therapy. vining et al. described a patient with retrocaval mmnst who experienced decreased pain and disease stabilization with pembrolizumab prior to resection [35]. thus, the use of immune checkpoint inhibitors in mmnst is limited to three reported cases, nevertheless, they suggest some evidence that they may provide symptomatic improvement and clinical response in some pd-l1-positive mmnst patients. references louis dn, ohgaki h, wiestler od, cavenee wk, world health organization, international agency for research on cancer (2016) tumours of the cranial and paraspinal nerves. who classification of tumours of the central nervous system. revised 4th edn. international agency for research on cancer, lyon, 213-230 who classification of tumours editorial board (2021) cranial and paraspinal nerve tumours. who classification of tumours: central nervous system tumours. 5th edn. international agency for research on cancer, lyon, france, 259-279 who classification of tumours editorial board (2020) peripheral nerve sheath tumours. who 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https://doi.org/10.1136/bcr-2020-240296 vining cc, hsu pj, miller a, olson dj, gajewski tf, pytel p, bauer bs, millis mj, roggin kk (2021) novel response to neoadjuvant anti-pd1 therapy for a patient with retrocaval melanotic schwannoma. melanoma res 31:92-97. https://doi.org/10.1097/cmr.0000000000000711 copyright: © 2022 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. neuropathology, my friend – on the paths of being and non-being feel free to add comments by clicking these icons on the sidebar free neuropathology 1:21 (2020) reflections neuropathology, my friend – on the paths of being and non-being ferenc garzuly markusovszky university teaching hospital, university of pecs, 5 markusovszky street, szombathely, hungary corresponding author: dr. ferenc garzuly · markusovszky university teaching hospital · university of pecs · markusovszky street 5 · szombathely · hungary garzuly@t-online.hu submitted: 02 august 2020 accepted: 03 august 2020 published: 11 august 2020 https://doi.org/10.17879/freeneuropathology-2020-2934 the author´s cv has been attached as electronic supplementary material: cv supplementary material keywords: neuropathology, szombathely, personal reflections contents introduction flight dictatorship and university the mirror of sufferings black water, swirling time neuropathology laboratory – without a boss as chief physician of the department of neurology deus ex machina under the wings of pécs and budapest a young man slips through the iron curtain – the neurologisches institut steps towards rare diseases efforts to diagnose an undiagnosable disease establishment of the hungarian society of clinical neurogenetics, szombathely, 1997 autopsy at night changing times – changing diseases among patients and colleagues – establishment of the west pannon neurological forum, szombathely, 1998 spotlight on rare diseases the music of the 21st century “the dead teach us” (hic mortui vivos docent)   prof. dr. györgy romhányi (1905-1991)   markusovszky hospital szombathely, department of laboratory and pathology (1946-51) and institute of pathology, medical university of pécs (1951-1976) introduction let me start off my recollections by saying that it had never occurred to me that i would ever become a doctor. i was interested in the arts and humanities, history, literature. when i was in high school, back in the early fifties, i participated in a literature competition and wrote a piece called conversation with the moon. it was about our escape from bratislava (preßburg, pozsony). we fled at night by boat on one of the river branches of the danube in august 1945, in the last year of world war ii. i was eight years old. our possessions in bundles on a cart, our destination was a small hungarian town called mosonmagyaróvár. fig. 1. bratislava, 1945 source: tasr archive in my mentioned text conversation with the moon i embedded all my youthful, naive thoughts and emotions from that time. this was in hungary in the early fifties under the rákosi dictatorship. after finishing that story i realized it couldn’t go on like this for long. there was a feeling of looming threat that made my stomach hurt. and now i just had to decide what profession to choose for further learning. both my mother and grandfather were physicians and it became obvious that i, too, would choose this apolitical, strictly fact-based profession, although it felt quite distant for me. flight my paternal grandfather, gyula garzuly, was a ship's captain, he also sailed around the world. later in bratislava (preßburg, pozsony), when it was still a hungarian city, he became the supervisor of the vessels of the hungarian river and shipping company. in 1916 he received a major award, the silver degree of the civil military cross of merit – ships on the danube also took part in the events of the first world war. with this, he earned to be imprisoned in ilava in 1918. he died shortly after his release, i didn’t even know him. my other grandfather, dr. rezső limbacher, the son of a blacksmith in an earldom, was such a skillful blacksmith „assistant” that the count taught him. graduated from university, he became a recognized obstetrician-gynecologist in bratislava (preßburg, pozsony). the first postmortem sectio cesarea in the city was performed by him, an article about this case appeared in the hungarian medical weekly. his son, rezső peéry, as a journalist and writer, was one of those who held the soul with his articles in the hungarians of bratislava after the treaty of trianon. fig. 2. my grandfather’s publication in the hungarian medical weekly, 1910 after the world war ii we had to flee from bratislava. every hungarian escaped who could. the future prime minister, edvard beneš , will announce it shortly: our people cannot live in a common country with the germans and the hungarians. dictatorship and university i was admitted to the medical university of pécs. at that time there were several internationally known professors at the university. one of these was jános szentágothai, the professor of anatomy, whose lectures resembled a grand performance. learning between his posters and drawings of anatomical figures was most interesting and exciting. another professor engraved himself in the hearts of the students with his directness, warm demeanor and his special approach to facts and events. that was györgy romhányi, the professor of pathology. the sentence pathology is the science of human suffering (1) would remain in the memories of his students for a lifetime. during my studies i was most impressed by neurology and psychiatry – at the time these two specializations were not separated yet. professor istván környey was the head of the university clinic in pécs, a well-known and prestigious scientist abroad. neurologist, psychiatrist, neurosurgeon and neuropathologist in one person, a wiry, strict and consistent man. he kept a kind of prussian discipline in the clinic and would not even be broken by the rákosi system. fig. 3. professor dr. istván környey (1901-1988) i decided to become a neurologist and psychiatrist myself. the field of psychiatry was a bit of an exit or detachment from the classical medical professions, although i could only get to know freud’s books under my blanket at that time. neurology, meanwhile, involved the precise mapping and careful evaluation of the patient complaints. particularly the physical examinations were much more complex, such as utilizing tools from internal medicine, palpation, percussion, auscultation and blood pressure measurements. i was ready to take that challenge. after graduation i was eligible to apply for three positions. it was evident that i could not stay at the university in pécs, considering that i did not actively participate in the kisz, the communist youth association. others were much smarter, they knew what needed to be done to stay in pécs and to become future professors. they weren't really friends of mine. i applied for positions of neurology and psychiatry in rural regions. since i did not have any patronage from the comrade secretariat of the university, i was not given the opportunity to do so. i had to choose from pathological positions, because there were usually fewer applicants for these and the vacancies had to be filled with people like me. it was intended as a punishment. this was in 1962 in the times after the failed revolution, not yet in the period of the soft goulash communism, but still in the hard dictatorship of the early sixties – there was no place for complaints. i felt that pathology was the medical field which was farthest from my interests and abilities. i remember my first autopsy practice at the university – when we entered the autopsy room and i saw a female corpse opened up with the wide layer of fat and intestines, i had to hold on to the wall so that i wouldn’t faint. still, i had no choice but to take on the appointed task. the mirror of sufferings i ended up at the hospital of szombathely, now called markusovszky teaching hospital of the university of pécs. the current building complex was built in 1929, and at that time it was the largest hospital in the western part of hungary. after the treaty of trianon, which had tragic consequences for hungary, the university of pozsony (today bratislava) was forced to move 1919 to hungary. szombathely was the only other city after pécs which was considered suitable for the new place of the university (1923). fig. 4. the hospital of county vas and szombathely, 1929 i started working in the pathology department in 1962. slowly i became accustomed to the silent dead and i started to warm up to the colorful histological preparates. i discovered the world that is our internal architecture and the macro-and microscopic war our bodies wage against our ferocious enemies – which, of course, have the same right to live just as we do. yes, it is true what professor romhányi said: pathology is the science of human sufferings and what i see during autopsy is the mirror of sufferings. today i stand at the same autopsy table where romhányi had also worked four years after the war. yes, pathology needs to be approached this way, i understand now. and i began to see the value of analysing medical treatments through the autopsies and also its worth for helping patients in the future. fig. 5. professor dr. györgy romhányi (1905-1991) despite these insights, i still wanted to be a neurologist and psychiatrist in the future. i even told this to the chief physician of pathology and he acknowledged my wishes in good faith. i still would have to wait two more years for this opportunity. looking back i am very grateful for this twist of fate and also to the party-member-packed secretariat of the university which had exiled me to szombathely, where i got to know pathology's world, methods and a point of view. black water, swirling time right at the start of my work at the department of pathology i discovered an old tied-up bundle of autopsy records on the shelves. the oldest ones could be dated back to 1920 and were still handwritten. from 1920 to 1929 autopsies were performed by the chief physician of the department of internal medicine and his doctors. the current complex of hospital buildings was built in 1929 and pathologists have been only working here ever since. fig. 6. a page of an autopsy report, 1923 the reports were interesting and instructive but also informative regarding the history of the pathology department itself and its doctors. many years later, in 2006 i looked back to examine this period. i reviewed the protocols from 1920 to 1956, written before and after world war ii till the hungarian revolution in 1956. they added up to a total of 6000 records. i wrote about it in a book with the title black water, swirling time –the message of autopsy reports (1920-56). fig. 7. black water, swirling time – the message of autopsy reports (1920-1956) during this period a lot of radical changes occurred. interestingly, sifting through the autopsy reports revealed that in the pre-war years every third death protocol was reported on tuberculosis. after the war the rate of infant mortality outstripped these deaths. the reports also included cases of diseases which have since been eradicated such as syphilis, tetanus, scarlet fever, whooping cough, diphtheria or poliomyelitis. they recount the typhus epidemic in the department of psychiatry and the epidemic of hepatitis in a child asylum. heart attacks or lymphoma barely occurred. curiously, patients under the age of 60 died three times as often in the hospital than those over the age of 60. in the meantime, political conditions changed drastically. to illustrate this in my book i have attached excerpts from the local newspaper vasvármegye. in 1940 for example, the chief physician of pathology, dénes görög, could have read news predicting a glorious future: the desertion of england has begun. the english population is in distress about a possible german landing… life in london is paralysed. the prices for a seat on one of the big ocean liners are horrendous... england will either surrender or be destroyed. after the war in 1948 chief physician romhányi might have been reading the local newspaper and be persuaded that the communist party had taken over the role of god in hungary: thank you for my recovery. i am a country woman with five acres. due to my lung problems i was lying in the pulmonology of the public hospital in szombathely. i was operated twice. i owe my recovery to the party and the doctors of the hospital. the events of the time shaped the human destinies severely. dénes görög was taken to auschwitz with his family in 1944. under mengele's supervision he performed autopsies with another hungarian doctor, miklós nyiszli. shortly before his death he said: miklós, with your willpower i am confident that you will make it home. i feel that i won´t survive. my wife and daughter died here in the gas chamber, i have proof that it happened. i hid a little boy with the monks of the monastery in kőszeg. this is my little son sándor, 12 years old. take him with you when you get home. i know for sure that i will die. this is my last wish. this wish was fulfilled and the little boy survived, became a chemist, a pharmaceutical researcher and a member of the hungarian academy of sciences. after his years in szombathely in 1951 györgy romhányi was appointed the head of the pathological institute of the university pécs. his experiences at the hospital in szombathely might have led him to formulate the sentence: the dead teach us. he may have also thought of the fate of dénes görög. neuropathology laboratory – without a boss during the two years i spent in pathology i performed the autopsy of those who died in the care of the department of psychiatry and neurology. i also studied the histological specimens and learned about their diseases. in 1963 this department was divided into two departments, one of them was headed by a new physician dr. lászló baltavári, from the környey-clinic in pécs. his wife worked in the laboratory of that clinic. the new chief physician managed to set up a neuropathology laboratory in the department within a year. at that time there was a vacant medical position in the department, which meant that i was now able to have a transition. since i already had a pathological past, so in addition to my daily medical work on patients, i also received the neuropathology laboratory as a gift. without a boss. i had to stand on my own two feet. i was already able to do the sections of the brains here and through it we were able to evaluate our medical work. i am still doing this work, although the laboratory (with me) has been relocated to the pathology department nearly twenty years ago. the number of brain cuts performed here is close to 6000. fig. 8. department of psychiatry and neurology, szombathely and its physicians, 1965 as chief physician of the department of neurology the departments were transformed into neurology (with 70 beds) and psychiatry (with 250 beds) in the 1980s. i stayed with the former. after twenty years dr. lászló baltavári died and for the next twenty years i became the head of the department. still, i couldn’t leave laboratory work for a minute, i would not give it up. the neuropathology laboratory strongly supported the medical care for patients. at that time there was no ct or mri. only the neuropathology gave us insights into the changes caused by the diseases which was teaching us many lessons even after the death of the patients. it also gave us histological diagnoses which radiological examinations still cannot provide today. because of my medical work both in our and in other departments of the hospital (for example pediatrics, infectiology, intensive care) i often encountered rare diseases. many of them were fatal and difficult to diagnose. i have increasingly used the capabilities of our laboratory to solve these mysterious cases. this was made possible by a number of contacts which i had established through the years, mainly with the institute of neurology (neurologisches institut) in vienna. deus ex machina i have to look back to 1966 when something extraordinary happened. by this time i was already working in the 2nd department of neurology and psychiatry. i received an invitation from professor franz seitelberger, head of the famous institute of neurology in vienna, for a three-month scholarship-funded study trip. beyond the iron curtain, to the land of the capitalist. i was absolutely surprised and only got to know later what happened. the first woman to graduate from the technical university of vienna was my father's sister, margarethe garzuly. she was admitted in 1919 after world war i and graduated in 1923. she moved from bratislava to vienna, became a staff member of the university, habilitated and in the meantime her husband became the head of the department of organic chemistry. when my aunt found out that i was working in neuropathology she called professor seitelberger. it was unbelievable. and it was even more incredible that i received a passport for the trip and could leave the country. at a time when hardly anyone could travel to the west. to this day i still wonder how it could have happened. maybe the party knew (as they knew everything) that i would always come back to my very good friend, a young, nice and very talented ceramic artist, maria geszler. fig. 9. maria geszler, a young and talented ceramic artist and a prince with big nose under the wings of pécs and budapest dr. lászló baltavári advised me not to go to professor seitelberger completely untrained. i should study in pécs for a month under professor környey before i would leave for vienna. shortly afterwards i found myself at the clinic in pécs. in the mornings professor környey was always sitting in a gigantic room behind a very wide baroque desk and about 20 doctors would be standing in front of him. if the floor creaked under one of them he looked up to see, who was that undisciplined one who couldn't stand still just for half an hour? there were three female assistants working in the laboratory and a young doctor, who vanished shortly after. every day i received histological preparates for study from professor környey accompanied by offprints, e. g. about tick-borne encephalitis. in the meantime (or later?) i became acquainted with ferenc gallyas, who was a young chemist. he began to investigate silver impregnations for glial cells at that time. later, this made him world-famous. on such specimens discovered mátyás papp the so called „papp-lantos bodies”. we became friends although i was very surprised that his lunch always consisted of three eggs. from a culinary point of view the everyday life at the clinic was interesting as well, for example lunch time you could choose between soup or bread. rom professor környey i received spielmeyer’s book on neuropathology which included many coloured hand drawn illustrations and i used it to prepare for the journey ahead. in later years környey became my supporter and our relationship deepened especially after he retired and moved to budapest. there he lived in one of the apartments in the park of the national institute of psychiatry and neurology (built in 1868 for mentally ill patients) courtesy of the director professor istván tariska who was also an excellent neuropathologist. the neuropathology laboratory at the national institute of psychiatry and neurology in use here was the leading laboratory in the country. one great advantage of the laboratory was that the well-trained neuropathologist katalin majtényi worked here full-time. i have been there many times with majtényi, tariska and környey to consult histological specimens. környey wrote his book on neuropathology here. to illustrate the chapter about the hallervorden-spatz disease he asked me for some photos. after i handed these to him, he told me that once in berlin hallervorden and spatz faced him on the sidewalk. hallervorden raised his hat and said, guten tag while spatz greeted him with a heil-hitler. after that i always thought about hallervorden as a decent person but later i was very disappointed when i found out about the circumstances of developing the hallervorden collection. in nazi germany, a large number of mentally disabled were killed. the majority of pediatric brains were transferred to the kaiser wilhelm institut für hirnforschung, led by hugo spatz, and was included in the hallervorden collection. i also often met lóránt leel-össy here who headed the neurology department in esztergom but he also had a laboratory in miskolc. he became the first president of the hungarian society of neuropathology. then the years passed, many things changed in the castle-like building, until in 2007 the huge building closed its gates. it is still empty and only owls are hooting in it, missing angry psychiatric patients and clever neuropathologists. fig. 10. the ruins of the national institute of psychiatry and neurology, 2017 source: hu.wikipedia.org a young man slips through the iron curtain – the neurologisches institut at hegyeshalom, on the hungarian-austrian border, i transferred to a train departing for vienna. it carried two wagons, surrounded by four border guards armed with machine guns. i successfully arrived in vienna. i have already mentioned my father’s elder sister, who had two more siblings – both sisters – who fled from bratislava to vienna after the war. they supported me during my stay here. the neurological institute – on the schwarzspanier street – was at the first floor of a huge building. i learned later that sigmund freud, whose writings on decoding of dreams were my favorite readings in those times, had lived on a sloping part of this street not so very long ago. i have found professor seitelberger in his room. he was very friendly with me, he talked a bit to me, and then i got a table in the large lecture hall in front of the benches. i saw him again and again most of the time as he presented the institute to his foreign acquaintances. they also looked into the lecture hall. i greeted everyone politely. there was a constant typewriter-knocking coming from the room next to the lecture hall. kurt jellinger worked here diligently. from the intensity of the knocks, one would think that the number of his publications will at least approach a thousand, if not more. i was very grateful to him, because he took me under his wings immediately: he provided me with tasks and instructions, and i received a case from him for analysis – syringomyelia combined with spinal haemangioblastoma. it was interesting, i was looking for similar disease combinations in the literature, in old and new books and journals in the large library, which always rang from emptiness like a cathedral. unfortunately, i always had to be afraid that someone would lock the iron door of the library from outside and i would have to say goodbye to my young life here, and so i can never see the nice and talented ceramic artist again. i gave a presentation later on the edifications from the analyses at the end of the study tour. since then, i feel bad to have left this area for the sake of rare diseases. but the latter was required by the daily practice. slideshows were not known at that time, so i had to draw my illustrations: the drawings and the related notes could be projected on a stretched canvas with a huge episcope. after the presentation, professor seitelberger invited me to a nearby restaurant for a farewell lunch and i also got a ticket from him to pablo casals’ performance at the musikverein. i last saw professor seitelberger many, many years later, in 1992, in szombathely. i invited him to give a lecture in our hospital library. he chose an interesting title for his performance, i was amazed: the neurobiology of aging – brain aging and cognition. i think he felt that this was a dangerous area for everyone. steps towards rare diseases my trip to vienna was the first initial step, on which further contacts, personal ties and consultations with the institute and its doctors were built. i visited the institute on several occasions for a longer or shorter period of time later, although it was initially difficult due to the iron curtain. but the evaluation, consultation of cases was – however – solvable by sending paraffin blocks too. fig. 11. congress of neuropathology in kyoto, 1990. on the left foto: franz seitelberger, his wife and hans lassmann left, right one of my friends, herbert budka. on the right foto: riki okeda (tokyo medical and dental university), my other friend and i i visited professor kurt jellinger and later his successor, professor herbert budka, and his doctors, first of all ellen gelpi, romana höftberger and later gábor g. kovács with special and rare cases: these meetings also resulted in publications (2-17). neuropathological diagnosis of diseases was a difficult task, especially for the deceased ones at the pediatric department. there has been almost no information or descriptions about the neuropathological abnormalities caused by these diseases, so it was worth dealing with them. we were able to diagnose some of these clinically – just like the family with the lowe-syndrome (2) – but e. g. the disease underlying symmetrical cerebral heterotopias and strange knee calcifications – the zellweger-syndrome (3) – was recognized by kurt jellinger. adult cases were also usually limited to rare diseases such as the foix-alajouanine disease (4), or the jakob-creutzfeldt disease (5). efforts to diagnose an undiagnosable disease of the many rare and interesting cases i have encountered, i would like to highlight only two. in the first, the story began when one of our female hospital assistants became disoriented for half an hour, later not remembering it. we found strange calcification along the sylvian-fissures on the ct scans. the total protein content of the csf was extremely high. the cause of the global transient amnesia was not found. later, it occurred to me that i had seen a similar ct scan from a female patient with suspected multiple sclerosis three years earlier. we have found this recording and it turned out, that this patient also had a very high csf total protein. and we discovered – quite by accident – that these two patients were related to each other, cousins. we mapped the whole family. many years ago, two men from this family passed away at our department: before that, they visited the department many times because of their central nervous system symptoms (migraine like headache, transient global amnesia, nystagmus, spastic paresis, ataxia, hearing loss, incipient dementia). we found the paraffin blocks of the deceased (but only blocks made from the brain). examination of the histological specimens revealed that amyloid was deposited in the central nervous system, primarily subpial. the area next to the deposited amyloid was calcified. it became apparent that these calcifications could be seen on the ct scans. none of the patients had any peripheral nervous system symptoms. however, neither the symptoms, nor the high protein content of the csf was consistent with any of the known central nervous system amyloidoses. and then, one of the two mentioned female patients passed away. after talking to the family, the deceased was brought to the hospital by ambulance and an autopsy was made. it turned out that although the patient had no symptoms of involvement according to the other organs – apart from central nervous system abnormalities – amyloid was found in small amounts in the kidneys, heart, and liver. so the autopsy referred to systemic amyloidosis. but our cases could not be classified here either. our cases were very similar to the very rare, so-called oculoleptomeningeal amyloidosis cases, but the patients had no visual impairment. consultations again and again (neurological institute, university of vienna, max-planck-institut für biochemie, martinsried, departments of pathology and neurology, new york university medical center), immunohistochemical and molecular biological studies revealed a completely unexpected result: the disease was caused by a transthyretin mutation. we summarized findings in our article (authored by vidal r, garzuly f, budka h, lalowski m, linke rp, brittig f, frangione b, wisniewski t) in the american journal of pathology in 1996 as follows: we describe a novel transthyretin mutation at codon 18 where asp is replaced by gly (d18g) in a hungarian kindred. this mutation is associated with meningocerebrovascular amyloidosis. fifty different transthyretin mutations are related to amyloid deposition, typically producing a peripheral neuropathy or cardiac dysfunction… with this report we establish that transthyretin amyloid deposition can also produce central nervous system dysfunction as the major clinical symptom (7). fig. 12. amyloid showing birefringence and the cover of am j pathol with an image from our case in the journal neurology, the patient's clinical symptoms were summarized, naming the disease hungarian type amyloidosis (8). in this form of familiar amyloidosis, the pathological mutant is produced in large amount in the plexus choroideus and – upon entry into the csf – immediately precipitates on the membranes, ependymes, unlike to forms that cause peripheral neuropathy, where mutants remain in the blood (9). now we know that the so-called oculoleptomeningeal amyloidoses are also caused by transthyretin mutations. unfortunately, these "cerebral" forms of amyloidoses are not yet curable, although our neurology department has just admitted the next sick member of our family. establishment of the hungarian society of clinical neurogenetics, szombathely, 1997 five years of efforts made clear to me, that underneath the layer of neuropathology stretches another layer, clinging to it: the neurogenetics of diseases. without any knowledge of this area, we cannot treat the hereditary diseases. without any molecular-biological tests, we can do essentially nothing for diagnosing or researching them, and the key to curing these diseases is also buried here. the recognition that neurogenetics will have a significant development and that the work in hungary should be coordinated, was also evident to other neurologists and neuropathologists. as a result of the joint efforts, we held the first scientific meeting on neurogenetics in hungary: with a significant number of foreign participants, it took place in szombathely on may 9–10, 1997. among the foreign professors of the gathering were george karpati, kurt jellinger, herbert budka, parviz mehraein, hans (hano) bernheimer, jean bénard, manuel b. graeber. the event had a lot of hungarian participants. professors ferenc mechler and mária judit molnár from the clinic of neurology in debrecen also participated at the meeting. they suggested the establishment of the hungarian society of clinical neurogenetics. fig. 13. a group of participants of the first scientific meeting on neurogenetics in hungary. standing row from right: gábor kiss, jean bénard and wife, parviz mehraein and wife, maria geszler, sámuel komoly, in front of them manuel b. graeber and i, george karpati and wife and miklós wenczl are sitting. to this day, the society diligently holds annual scientific meetings and looks to a prosperous future. at the tenth anniversary gathering of the society, professor ferenc mechler and i had the honor to become life members of the association. however, it took more than twenty years to wait for an excellent neurologist and researcher, professor bernadette kálmán – who has spent several decades in the united states – to establish a molecular pathology laboratory at the markusovszky hospital. autopsy at night the other interesting disease was a variant of the autosomal dominantly inherited fabry disease. a patient – who suddenly became unconscious – was brought to our department in the evening. angiography was performed, the images showed an extremely wide basilar artery – a so called megadolichobasilar artery – which was thrombosed. the next morning, during the visit, i lifted the patient's quilt. and then i saw on the skin of the abdomen the characteristic angiokeratomas of fabry disease. i recognized it immediately, because three years ago i saw a similar patient. it was later revealed that the patient seen three years ago was the same. the patient was expected to die within a few days. i called maria mázló, the head of the electron microscope laboratory, of the national institute of psychiatry and neurology in budapest. she said, the investigation would be carried out, but asked us to send fresh samples to the laboratory. i discussed the immediate autopsy with the head physician of our pathological department. the patient died at one o'clock at night. after the obligatory two-hour wait we were standing in the autopsy room, and the autopsy was done at three o'clock. this was the first autopsy in hungary by a patient who died of fabry disease, no previous reports were found. and we believe it was also the first autopsy at night in our country. fig. 14. electron microscopic image of the stored material of our patient with fabry’s disease three years later, a similar case occurred in a neighboring town's hospital: a thrombotized giant basilar artery caused death. we received the formalin-fixed material samples. the lesions were completely similar to our case. and within a short time it turned out that the two patients were related to each other. these cases were also discussed with the colleagues in vienna, meanwhile the molecular pathological examinations were completed at the university of debrecen. the cases were published in the brain with the following title: megadolichobasilar anomaly with thrombosis in a family with fabry's disease and a novel mutation in the alpha-galactosidase a gene (11). shortly after the publication, it became possible to treat fabry-patients, and some of the family members may already be happy with this opportunity. changing times – changing diseases n 2014, our neuropathology laboratory turned 50 years old. on the occasion of the anniversary we published an article in the medical journal orvosi hetilap (18). nearly 5500 autopsy examinations were carried out during the preceding 50 years. the documentation reflects on variations in the occurrence of diseases, and it draws attention to those disorders, which can be prevented or treated today, but may represent diagnostic challenges. measles-related subacute sclerosing panencephalitis caused death in 13 cases, the last occurred in 1991. the mandatory vaccination against the causative morbilli virus has eliminated this severe neurological complication. fourteen lives were lost due to herpes simplex encephalitis, including the last case that occurred in 1999. feasibility of early diagnosis and the availability of acyclovir therapy resulted in better outcome without fatality. tuberculous meningitis still occurred in most recent years, although only sporadically. recognition of this condition is not straightforward due to its rarity, considerations for this disease are often omitted from the routine differential diagnosis. the low mortality rates in tick-borne encephalitis dropped further after the introduction of vaccination, 8 cases were documented altogether. with our colleagues in vienna, we published two reports on tick-borne encephalitis (12, 13). the last fatal cases of neurolues were seen in the 1990s. however, syphilis itself has not disappeared, and the number of cases with newly acquired infection continues to rise. meningosis leukaemica caused the death of 20 patients, mainly children, between 1972 and 1987. it was a sad time for me to witness this as a neurologist. i wrote also my phd dissertation on neuropathological features of leukemias and lymphomas. introduction of intrathecal methotrexate and radiation therapy made the prevention and effective treatment of meningosis leukaemica possible (19, 20). however, a careful coordination of these treatment's modalities is important as nervous system complications may develop in the form of disseminated necrotizing (methotrexate) leukoencephalopathy. we had four such cases, we needed professor herbert budka’s help to diagnose the first one. among patients and colleagues – establishment of the west pannon neurological forum, szombathely, 1998 fifty years is a long time, many things have happened apart from the events in the neuropathology laboratory. i would like to highlight two of these. in the 1980s, the rehabilitation of neurological patients had an increasing emphasis. this was the beginning of the golden era of neurorehabilitation – not only of neuropathology as herbert budka referred to in his recently published reflections (21). in szentgotthárd – a town next to us – a large rehabilitation hospital was established on the site of a former pulmonology sanatorium. together with the hospital's director we visited similar institutes in austria. we contacted professor gerhard barolin, who was the main organizer of the neurological patients' rehabilitation in austria. my friend divided his rehabilitation hospital into three parts and reserved one for neurorehabilitation. in 1988, we organized a two-day scientific meeting on neurorehabilitation entitled current issues in neurorehabilitation, with guest participation of austria, switzerland and germany. in a similar form, the gathering was repeated in 1993. these were the first events in hungary dealing exclusively with neurorehabilitation. maria geszler's ceramic exhibition in the picture galery and the scientific meeting on neurorehabilitation opened at the same day, one after the another in 1993. fig. 15. ceramic statue of maria geszler: falling figure. maria geszler's ceramic exhibition and the scientific meeting on neurorehabilitation opened at the same day. szombathely is located on the western border of hungary, near vienna, but far from the universities of budapest and pécs. it became clear, that our county and the three neighboring counties need to work together. nearly one and a half million people live in this area. this way, we established the west pannon neurological forum with its first meeting in 1998 in szombathely (22), for neurologists, neurosurgeons, and neurorehabilitation physicians. neuropathology was represented consequently by our department. this one-day meeting is held every six months in one of the cities of the four counties. the gatherings are mainly based on case studies with an educational lecture. the event created dialogues among the doctors of these four counties and helps them to solve medical problems. the 50th anniversary meeting will take place next year. fig. 16. one of the west pannon neurological forum's program booklets with crests of the host cities spotlight on rare diseases i retired in 2002 and started to work full-time at the department of pathology. i took the neurohistology laboratory with me: the lab had a better, more spacious place here. sections were always performed in the presence of clinicians and then the evidence was always compared with the results of radiological examinations in the presence of radiologists. we also collaborated with the laboratory of molecular pathology, and several published works resulted from this cooperation (23-27). fig. 17. thrombosis of the basilar artery in a young woman. comparison of the pathological with the radiological findings. the department of pathology organized the clinico-pathological conferences since the early 1950s. these were implemented on a soviet model. professor pavel ivanovich sapochkov said at that time: i work at clinic no. i in moscow and our clinic is extremely restless (sic) when it comes to the patient’s autopsy. pathology examines the relationship between clinical and pathological diagnosis the most accurate way. every two weeks the doctors are called together and the diagnostic differences in each case are talked over, and it’s a shame (sic) for a doctor to be invited to such a hearing, where his fault is discussed. after moving on to the pathology, i was entrusted with organizing these conferences, every three months, with mandatory attendance for hospital physicians. it became clear that the handling of these cases – where an error has occurred – falls within the competence of other forums. we expanded the discussions of cases by talking not only about the deceased ones, but also the healed patients. the conferences no longer featured the doctors who worked poorly, but those who worked well. and we gradually set the focus of the conference on cases that were interesting in some way, predominantly discussing rare diseases. fig. 18. our books about rare diseases the pathology department offers a very good angle to observe to the medical activities of our very large hospital and it is easy to discover interesting cases. over the past nearly twenty years, we have written five books about rare diseases relying on these cases (the sixth one is also in progress) with lots of coloured illustrations. professor romhányi said: one case is not a case. but we relied on 260 cases to present the rare diseases – with the required information and notes – we have experienced during our work. and in addition, at the hospital's lecture hall was named after professor romhányi. fig. 19. the lecture hall of the hospital, named after györgy romhányi, the venue for conferences on rare diseases the music of the 21st century the nice and talented ceramic artist, maria geszler is the one for whom i came back from the bewitching western world. she is my wife now for many-many years. her great-grandmother sang so beautifully as a young girl, that after a concert she was kissed by franz liszt on the forehead. the grandmother, margit tessényi, a student of vianna da motta, was a well-known pianist, her father, györgy geszler a composer and pianist, who gave concerts also with béla bartók. it is no coincidence that her sisters are also musicians. our daughter anna endowed the talent as a gift. she studied in budapest, munich and new york, and became a flutist. she frequently participated in music competitions and was usually among the winners, so she could even give an evening at carnegie hall in new york. her swedish husband, henrik wahlgren is an excellent oboist. they were both members of the gewandhaus orchester in leipzig, but our daughter is currently a professor of chamber music at the musikhochschule in leipzig and a flute professor in weimar. she was a founding member of the quintessenz leipzig flute ensemble, which has now celebrated its 20th anniversary. all three of our grandchildren also play and sing, so music has become a constant companion of our lives. fig. 20. the quintessenz leipzig flute ensemble, in the middle anna garzuly wahlgren we shared a lot of things: good ones and sad ones too. but we are still working. i work in my hospital, as maria does in her studio, or having a lecture somewhere, exhibiting in europe, or elsewhere in the world. she is a ceramic artist well-known everywhere. she won a lot of prizes: last year she was awarded with the grand prix in vallauris, the internationally famous ceramics biennial. fig. 21. maria geszler: arctic meteorite, porcelain object one of her artworks can be seen on the june issue's cover of new ceramic – the most famous magazine for ceramic artists – and a long article about her in the journal. there, she writes following: our nerves are strained like wires, we live among electrical discharges of love and hatred, above our heads there is special buzz of cables, that is the music of the 21st century. we all dream to see something new, to live, to discover, to understand. this experience has pushed mankind since the stone age, even in the millennium of moon travel. who didn’t feel something like this? wandering on the banks of rivers, standing on the sandy shores of the seas, at the foot of the mountains, looking at the peaks? who didn’t feel the attraction of the unknown? to go as far as possible, to see the horizon, to discover the mountain peaks, to reach the skies. references 1. prof. dr. med josef 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f, brittig f, baltavári l, pócza k, istván l: incidence of various neuroleukemia types in a 5-year record of autopsy cases. orv hetil 1984;125:1685-90 (in hungarian) 20. garzuly f: pathological features of neuroleukemia. changes and lessons. orv hetil 1994;135:1291-5 (in hungarian) 21. budka h: the golden era of neuropathology. free neuropathology 2020;1:1-26 22. garzuly f, grubits j, nikl j: antecedents to the commencement and history of the west-pannonic neurological forum. ideggyogy sz/clin neurosci 2016;69:139-43 (in hungarian) 23. bobest m, tóth c, gyurcsó m, molnár mj, garzuly f: nonsense mutation 193c>t of neurofibromatosis type 2 – a neurosurgical challenge. ideggyogy sz/cli neursci 2007;60:41-5 (in hungarian) 24. kálmán b, szép e, garzuly f, post de: epidermal growth factor receptor as a therapeutic target in glioblastoma. neuromolecular med 2013;152:420-34. doi: 10.1007/s12017-013-8229-y 25. sinko g, garzuly f, kálmán b: striking pathology in leigh syndrome associated with the mtatp6 t8993g mutation. pediatr neurol 2014;51:585-6. doi: 10.1016/j.pediatrneurol.2014.07.015 26. nagy á, garzuly f, kálmán b: pathogenic alterations within the neurofibromin gene in various cancers. magy onkol 2017;61:327-336. (in hungarian) 27. nagy á, garzuly f, padányi g, szűcs i, feldmann á, murnyák b, hortobágyi t, kálmán b: molecular subgroups of glioblastoma – an assessment by immunohistochemical markers. pathol oncol res 2019;25:21-31. doi: 10.1007/s12253-017-0311-6 books on rare diseases (in hungarian) ferenc garzuly: in the labyrinth of diagnostics (2013) györgy pfliegler, ferenc garzuly: rare diseases – diagnostic challenges (2013) ferenc garzuly, csaba tóth, bernadette kálmán: rare diseases, struggle for life (2013) ferenc garzuly, csaba tóth, bernadette kálmán: rare diseases, special forms of diseases (2015) ferenc garzuly, csaba tóth, bernadette kálmán: rare diseases, diagnosis and therapy (2017) ferenc garzuly, csaba tóth, bernadette kálmán: rare and hiding diseases (2020, in progress) other books (in hungarian) garzuly: black water, swirling time – message of autopsy reports (1920-1956) (2006) garzuly: unknown season – irregular diary (2008) garzuly: my father is reading newspapers 1953-1957 (2011) garzuly: good morning, mr. head physician! (2019) copyright: © 2020 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. amygdala granular fuzzy astrocytes as lesions preceding development of argyrophilic grains: data from 239 autopsy cases feel free to add comments by clicking these icons on the sidebar free neuropathology 3:18 (2022) letter amygdala granular fuzzy astrocytes as lesions preceding development of argyrophilic grains: data from 239 autopsy cases osamu yokota1,2,3,4, tomoko miki1,2,3,4, chikako ikeda2,3, hideki ishizu3, takashi haraguchi4, akinori miyashita5, takeshi ikeuchi5, shintaro takenoshita6, seishi terada2 1 department of psychiatry, kinoko espoir hospital, kasaoka, japan 2 department of neuropsychiatry, okayama university graduate school of medicine, dentistry and pharmaceutical sciences, okayama, japan 3 department of laboratory medicine and pathology, zikei institute of psychiatry, okayama, japan 4 department of neurology, national hospital organization minami-okayama medical center, okayama, japan 5 department of molecular genetics, brain research institute, niigata university, niigata, japan 6 department of neuropsychiatry, okayama university hospital, okayama, japan corresponding author: osamu yokota · department of neuropsychiatry · okayama university graduate school of medicine · dentistry and pharmaceutical sciences · 2-5-1 shikata-cho · okayama 700-8558 · japan oyokota1@yahoo.co.jp submitted: 15 june 2022 accepted: 21 july 2022 copyedited by: irati bastero published: 27 july 2022 https://doi.org/10.17879/freeneuropathology-2022-4285 additional resources and electronic supplementary material: supplementary material keywords: amygdala, argyrophilic grain disease, artag, blood vessel, granular fuzzy astrocyte, tau cases of argyrophilic grain disease (agd) frequently have astrocytes containing non-argyrophilic tau-positive fine granules in the foot processes sprouting from cell bodies in the amygdala. they were first called bush-like astrocytes by botez et al. [1], and later they were classified as one of the age-related tau astrogliopathies and re-named granular fuzzy astrocytes (gfas) [2]. in our previous studies, the formation of gfas was not simply associated with age alone, and their distributions were influenced by underlying tauopathies [3,4]. especially in agd cases, gfas preferentially develop in the amygdala rather than the frontal cortex and striatum [3]. kovacs et al. previously noted the possibility that gfas in the amygdala might be a forerunner of argyrophilic grains [5]. however, there is no comprehensive pathological data regarding the relationship between the frequencies and severities of agd and gfas in the amygdala, and whether argyrophilic grains develop in the absence of amygdala gfas remains unclear. to address these issues, first, we analyzed the relationship between the presence or absence of agd and the severity of amygdala gfas in a case series. then, their spatial relationship was examined using double staining with the gallyas method and anti-phospho-tau (at8) immunohistochemistry. among a total of 1,166 autopsy cases registered in our database, 474 cases that were evaluated using modern standardized methods, including a panel of immunohistochemistry, modified bielschowsky silver stain, and gallyas-braak silver stain from 2001 to 2022, were selected (supplementary file 1). all cases died in psychiatric hospitals or neurological departments of general hospitals. of these cases, we extracted 263 cases for which semiquantitative data of the at8-positive gfas, as well as the saito agd stage [6] (the distribution of argyrophilic grains in the cerebrum) assessed using the gallyas method, are available. then, 24 cases having progressive supranuclear palsy, corticobasal degeneration, pick’s disease, globular glial tauopathies, post-encephalitic parkinsonism, and myotonic dystrophy were excluded because distinct tau-positive astrocytic lesions were noted. finally, 239 cases were included in the present study. in all of these cases, gfas were semiquantitatively assessed in the frontal cortex, caudate nucleus, putamen, and amygdala on at8-stained sections, using the following grading system (gfa stage): stage 0, no lesion in the anatomical region (i.e., the superior frontal gyrus, caudate nucleus, putamen, and amygdala); stage 1, more than one lesion in the anatomical region but less than one lesion per ×200 visual field; stage 2, one lesion per ×200 visual field; or stage 3, two or more lesions per ×200 visual field. among the 239 cases, 112 had at least one gfa in the amygdala, while 127 lacked amygdala gfas (amygdala gfa stage 0) (table 1). the age at death was significantly higher in the amygdala gfa stage 1, 2, and 3 groups than in the amygdala gfa stage 0 group (p<0.001, 0.0025, and 0.0023, kruskal-wallis and steel-dwass tests). however, the sex ratio, brain weight, braak stage, thal phase, proportions of lewy body disease (lbd) pathological subtypes, and proportions of tar dna-binding protein 43 (tdp-43) proteinopathy pathological subtypes did not significantly differ between the four amygdala gfa stage groups. table 1. demographic data in all subjects stratified by severity of gfas gfa: granular fuzzy astrocyte, agd: argyrophilic grain disease, n: number of cases, nft: neurofibrillary tangles, lbd: lewy body disease, late-nc: limbic-predominant age-related tdp-43 encephalopathy neuropathological change, als-tdp: amyotrophic lateral sclerosis with tdp-43-positive inclusions, ftld-tdp: frontotemporal lobar degeneration with tdp-43-positive inclusions. agd was noted only when a case had at least one gfa in the amygdala; that is, there was no case that lacked amygdala gfas but had agd (figure 1a). on the other hand, cases having amygdala gfas did not always have agd: of all 112 cases having amygdala gfas, 40 cases (35.7%) had argyrophilic grains in the amygdala (figure 1a). the frequency of cases with agd increased parallel to the amygdala gfa stage: 18.7% in gfa stage 1, 61.5% in gfa stage 2, and 75.0% in gfa stage 3 groups (figure 1b). the saito agd stage was significantly correlated with the amygdala gfa stage (ρ=0.5878, p<0.001, spearman rank-order correlation test). the binomial logistic regression analysis was used to evaluate whether the age at death, braak stage, thal phase, frontal lobe gfa stage, caudate nucleus gfa stage, putamen gfa stage, or amygdala gfa stage could be used as possible predictors of the development of agd, and it demonstrated that the amygdala gfa stage (the standardized partial regression coefficient (β), 1.79; odds ratio, 6.63; 95% confidence interval (ci), 3.57–12.32; p<0.001), the age at death (β, 1.34; odds ratio, 1.09; 95% ci, 1.02–1.16; p<0.001), the frontal lobe gfa stage (β, 0.62; odds ratio, 3.47; 95% ci, 1.06–11.33; p=0.0391), and the putamen gfa stage (β, 0.64; odds ratio, 3.10; 95% ci, 1.005–9.56; p=0.0490) were significant independent predictors of the occurrence of argyrophilic grains. figure 1. relationship between the formation of agd and amygdala gfas (a) the relationship between the occurrence of agd and the presence or absence of amygdala gfas. agd was noted only in cases having amygdala gfas. (b) the severity of agd by amygdala gfa stage. the frequency and severity of agd increase with the amygdala gfa stage in presence of amygdala gfas. (c-e) gfas in the amygdala. at8 immunohistochemistry. gfas are often in contact with vessel walls (asterisks). further, argyrophilic grains are scattered around gfas (arrows). (f-h) gallyas method. the amygdala. argyrophilic grains (arrows) are scattered at a distance around blood vessels (asterisks). (i-k) gallyas (black)-at8 (light blue) double staining. the amygdala. argyrophilic grains (black) are scattered around gfas (light blue) contacting the vessel walls (asterisks). all scale bars: 30 μm as previously reported [3,4], gfas with tau-positive astrocytic endfeet were often in contact with blood vessel walls on at8-immunostained sections of the amygdala (figures 1c-1e). in addition, a small number of at8-positive argyrophilic grains were often scattered around the gfas (figures 1c-1e). although gfas are not stained by the gallyas method, a potential association between argyrophilic grains and vessels was also noted on gallyas-stained sections: a few argyrophilic grains are scattered at a distance around vessels (figures 1f-1h). likewise, double staining with the gallyas method and at8 immunohistochemistry showed the proximity of argyrophilic grains, gfas, and blood vessels (figures 1i-1k). noteworthy, these phenomena could be observed only in regions where argyrophilic grains were relatively scarce rather than abundant. the findings presented here support the notion that amygdala gfas may develop prior to the formation of argyrophilic grains in the amygdala [5] and suggest that amygdala gfas may play some role in the occurrence of argyrophilic grains, which are preferably formed in the dendrospinal portion of neurons [7]. these findings also suggest that at least some cases having amygdala gfas but lacking argyrophilic grains may be in the earliest ‘pre-grain’ stage in the pathological process of agd (supplementary file 2). whether amygdala gfas affect cognitive function and psychiatric status, especially in elderly people, also needs to be explored. finally, table 1 shows that some cases had gfas in the frontal cortex, caudate nucleus, and/or putamen even when the case lacked amygdala gfas and argyrophilic grains (amygdala gfa stage 0). based on our previously reported findings [3], this fronto-striatal-predominant distribution pattern of gfas led us to consider the possibility that the case has an early pathological process of progressive supranuclear palsy (psp). for a better understanding of the relationship between argyrophilic grains, gfas, subcortical neuronal tau accumulation, and psp as the pathological disease entity, the sequence of the appearance of these lesions and pathological conditions needs to be further examined. declarations ethics approval autopsy was carried out after written informed consent was obtained from family members, and all experiments in this study were approved by the ethical committees of the okayama university graduate school of medicine, dentistry and pharmaceutical sciences, national hospital organization minami-okayama medical center, niigata university, and zikei hospital. competing interests oy is an editorial board member but was not involved in the editorial handling of this manuscript. the other authors declare that they have no conflicts of interest. funding this work was supported by grants from the strategic research program for brain sciences from the japan agency for medical research and development (amed, jp22wm0425019, jp22dk020704) and grants from the zikei institute of psychiatry. authors’ contributions oy, tm, sht, and set macroscopically and histopathologically assessed all subjects; ci, hi, and th conducted the autopsies; nh, am, and ti administered the brain bank; oy and tm drafted the manuscript. all authors reviewed the manuscript. acknowledgements we thank ms. y. matsuo for her technical assistance. references 1. botez g, probst a, ipsen s, et al.: astrocytes expressing hyperphosphorylated tau protein without glial fibrillary tangles in argyrophilic grain disease. acta neuropathol 1999;98:251-6. https://doi.org/10.1007/s004010051077. 2. kovacs gg, ferrer i, grinberg lt, et al.: aging-related tau astrogliopathy (artag): harmonized evaluation strategy. acta neuropathol 2016;131:87–102. https://doi.org/10.1007/s00401-015-1509-x. 3. miki t, yokota o, haraguchi t, et al.: factors associated with development and distribution of granular/fuzzy astrocytes in neurodegenerative diseases. brain pathol 2020;30:811-830. https://doi.org/10.1111/bpa.12843. 4. ikeda c, yokota o, nagao s, et al.: the relationship between development of neuronal and astrocytic tau pathologies in subcortical nuclei and progression of argyrophilic grain disease. brain pathol 2016;26:488-505. https://doi.org/10.1111/bpa.12319. 5. kovacs gg, xie sx, robinson jl, et al.: sequential stages and distribution patterns of aging-related tau astrogliopathy (artag) in the human brain. acta neuropathol commun 2018;6:50. https://doi.org/10.1186/s40478-018-0552-y. 6. saito y, ruberu nn, sawabe m, et al.: staging of argyrophilic grains: an age-associated tauopathy. j neuropathol exp neurol 2004;63:911-918. https://doi.org/10.1093/jnen/63.9.911. 7. ikeda k, akiyama h, kondo h, et al.: a study of dementia with argyrophilic grains. possible cytoskeletal abnormality in dendrospinal portion of neurons and oligodendroglia. acta neuropathol 1995;89:409-414. https://doi.org/10.1007/bf00307644. copyright: © 2022 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. an atypical teratoid/rhabdoid tumor (at/rt) with molecular features of pleomorphic xanthoastrocytoma (pxa) in a 62-year-old patient feel free to add comments by clicking these icons on the sidebar free neuropathology 2:31 (2021) case report an atypical teratoid/rhabdoid tumor (at/rt) with molecular features of pleomorphic xanthoastrocytoma (pxa) in a 62-year-old patient matthias dottermusch1,2, ali alomari3, nesrin uksul3, ulrich j. knappe3, julia e. neumann2,1 1 institute of neuropathology, university medical center hamburg-eppendorf, hamburg, germany 2 center for molecular neurobiology (zmnh), university medical center hamburg-eppendorf, hamburg, germany 3 department of neurosurgery, johannes wesling klinikum, university hospital of ruhruniversität bochum, minden, germany corresponding author: julia e. neumann · center for molecular neurobiology (zmnh) · university medical center hamburg-eppendorf · falkenried 94 · 20251 hamburg · germany ju.neumann@uke.de submitted: 20 october 2021 accepted: 09 november 2021 copyedited by: shino magaki published: 15 november 2021 https://doi.org/10.17879/freeneuropathology-2021-3640 additional resources and electronic supplementary material: supplementary material keywords: atypical teratoid/rhabdoid tumor, pleomorphic xanthoastrocytoma, dna methylation atypical teratoid/rhabdoid tumors (at/rt) are aggressively growing malignant embryonal neoplasms of the central nervous system (cns), which mainly affect young children. loss of smarcb1/ini1 (or smarca4/brg1 in rare cases) is recognized as the genetic hallmark of at/rts. furthermore, these tumors can be distinguished into three distinct dna-methylation based molecular subgroups (i.e. -myc, -shh and -tyr) (1–3). while most at/rts are considered to occur de novo, previous studies have recognized secondary smarcb1/ini1-deficient rhabdoid tumors arising from other low grade cns tumors in young patients (4–9). moreover, three at/rts, which harbored epigenetic and mutational characteristics of pleomorphic xanthoastrocytoma (pxa), while being entirely void of nuclear smarcb1/ini1 expression were recently described in older children (10). we here report the first case of an at/rt with molecular features of pxa in an older adult. a 62-year-old woman presented with diffuse headaches since several weeks. medical examination revealed no clinically significant neurological deficits. an mri showed a right-sided tumor in temporomesial location with dimensions of 3.8 x 3.7 x 4.1 cm. the tumor displayed contrast-enhancement, central necrosis und perifocal edema (figure 1a). additionally, a smaller lesion of the infundibulum was identified. preoperative lumbar puncture with examination of cerebrospinal fluid revealed extensive presence of tumor cells. the temporomesial tumor was subtotally resected. figure 1: radiology, histopathology and epigenetic analysis. a) representative axial mri demonstrating the preoperative finding of a large temporomesial tumor (large arrow). contrast enhancement was found partially within the tumor and additionally in the region of the infundibulum (small arrow). t1-weighted image plus contrast medium. b) histomorphology revealed geographical necrosis, prominent rhabdoid morphology and brisk mitotic activity of tumor cells (inset). c) the tumor showed a prominent network of reticular fibres, visible in the gomori silver impregnation. d) immunostaining for vimentin was vastly strongly positive. e) immunostaining for cytokeratins (ae1/3) was vastly strongly positive in the majority of tumor cells. f) immunostaining for gfap was negative in tumor cells while demarcating residual brain tissue. g) ki67 proliferative index amounted to about 40%. h) tumor cells exhibited loss of nuclear smarcb1/ini1 staining. retained nuclear staining was found in blood vessels and inflammatory cells (inset). scale bar in b – h is 150 μm. i) copy number profile of the tumor indicated a homozygous cdkn2a/b (short arrow) and smarcb1/ini1 (long arrow) deletion. j) tsne including a reference set of brain tumors (gse90496, (11)) showed affiliation of the tumor to the group of pxa. clicking the figure will lead you to the full virtual slide (h&e). upon histopathological examination, we saw a highly cellular, partially necrotic tumor, which was entirely composed of pleomorphic rhabdoid cells with eosinophilic cytoplasms (figure 1b). gomori-staining demonstrated a prominent reticulin network (figure 1c). immunohistochemical staining showed positivity for vimentin (figure 1d) and cytokeratin ae1/3 (panck, figure 1e). gfap was negative within the tumor cells (figure 1f). strong proliferation was demonstrated by brisk mitotic activity and a proliferative index of 40% in the ki67-staining (figure 1g). smarcb1/ini1 nuclear signal was consistently lost in tumor cells (figure 1h). further immunohistochemical stainings are displayed in supplementary figure 1. dna was extracted from the tumor and subjected to dna methylation profiling using the illumina epic beadchip array. using the brain tumor methylation classifier (v11b4 and v12.3) (11), the tumor did not match with a defined methylation class (no calibrated score was ≥ 0.3). the copy number profile revealed a homozygous smarcb1/ini1 and cdkn2a/b deletion, gains in chromosomes 2 and 7q as well as losses of chromosomes 9p, 14q, 16 and 22q (figure 1i). t-sne analysis displayed similarity of the case with pxa (figure 1j). dna panel sequencing confirmed a braf v600e mutation (table 1). taking all diagnostic layers together, we saw a malignant rhabdoid tumor, not elsewhere classified, with braf v600e mutation, homozygous smarcb1/ini1 and cdkn2a/b deletion, and a methylation profile with similarity to pxa. in correspondence with the provisional designation of thomas et al. (10), the case was signed out as at/rt with molecular features of pxa. postoperative clinical staging of the patient revealed a smaller lesion of uncertain dignity in the cervical myelon. no further malignancies outside of the cns were found. unfortunately, the patient developed postoperative hemiplegia, infarctions of the basal ganglia and displayed progressive worsening of the clinical status. palliative chemotherapy was administered with intrathecal application of methotrexate. the patient died in the 6th postoperative week. table 1: ngs dna panel results. as it is the case for the herein presented tumor, it remains unclear, if at/rts with molecular features of pxa correspond to secondary rhabdoid tumors, which have emerged from and entirely outgrown preexisting pxas. in contrast, such tumors might also occur independently from precursor lesions and therefore represent a distinct entity. of note, the cns who classification of 2016 has previously recognized high-grade rhabdoid components with loss of smarcb1/ini1 in anaplastic pxas and suggested the term “smarcb1-deficient anaplastic pxa” for such lesions (12). however, since the tumor presented here was entirely void of smarcb1/ini1 retained tumor cells and a preexisting lesion had not been described beforehand, the tumor’s origin is left to speculation. it is moreover important to note that the single genetic and epigenetic alterations we found are not pathognomonic for pxas (13) and the tumor did not match with a defined methylation class of the brain tumor classifier. however, the combination of all molecular findings – including the epigenetic resemblance to pxas via tsne analysis – clearly demonstrates similarity with molecular features of pxas. a complicating aspect of the current and soon to be updated cns who classification of 2016 is that smarcb1/ini1-deficient rhabdoid brain tumors tend to be vastly designated as at/rts. the herein presented case exemplarily demonstrates that especially without molecular investigations this may lead to the subsumption of malignancies with various epigenetic and mutational landscapes as well as different cellular origins. while the preceding summary of the new who classification of 2021 (14) has announced the recognition of epigenetic analyses in at/rts, it is currently still unclear how smarcb1/ini1-deficient brain tumors with rhabdoid morphology and lack of epigenetic features of at/rts are to be classified. future studies should evaluate if predictive and prognostic implications of these tumors call for a more concise classification and terminology. data availability the data that supports the findings of this study is available from the corresponding author upon request. a digital h&e stained slide is deposited here. conflict of interest the authors have no competing interests to declare. acknowledgements we thank celina soltwedel, carolina janko, karin gehlken, ulrike rumpf, tasja lempertz, nicole bernhardt, helena zinn and ulrich schüller (hamburg) for excellent technical support. we thank annika wefers for helpful discussions. we thank martin hasselblatt and christian thomas from the institute of neuropathology of the university hospital münster for confirming the diagnosis. j.n. was supported by the deutsche forschungsgemeinschaft (dfg, emmy noether programme). m.d. was supported by the erich und gertrud roggenbuck-stiftung. ethical statement study approval was obtained from the local ethics committee of the hamburg state chamber of physicians. the patient and/or guardian gave their informed consent for scientific use of the data. author contributions m.d. and j.n. conceived the study and drafted the manuscript. all authors acquired and analyzed data and approved the final version of the manuscript. references johann, p. d. et al. atypical teratoid/rhabdoid tumors are comprised of three epigenetic subgroups with distinct enhancer landscapes. cancer cell 29, 379–393 (2016). frühwald, m. c., biegel, j. a., bourdeaut, f., roberts, c. w. m. & chi, s. n. atypical teratoid/rhabdoid tumors current concepts, advances in biology, and potential future therapies. neuro-oncol. 18, 764–778 (2016). richardson, e. a., ho, b. & huang, a. atypical teratoid rhabdoid tumour: from tumours to therapies. j. korean neurosurg. soc. 61, 302–311 (2018). allen, j. c., judkins, a. r., rosenblum, m. k. & biegel, j. a. atypical teratoid/rhabdoid tumor evolving from an optic pathway ganglioglioma: case study. neuro-oncol. 8, 79 (2006). chacko, g., chacko, a. g., dunham, c. p., judkins, a. r., biegel, j. a. & perry, a. atypical teratoid/rhabdoid tumor arising in the setting of a pleomorphic xanthoastrocytoma. j. neurooncol. 84, 217–222 (2007). kleinschmidt-demasters, b. k., birks, d. k., aisner, d. l., hankinson, t. c. & rosenblum, m. k. atypical teratoid/rhabdoid tumor arising in a ganglioglioma: genetic characterization. am. j. surg. pathol. 35, 1894–1901 (2011). nadi, m., ahmad, t., huang, a., hawkins, c., bouffet, e. & kulkarni, a. v. atypical teratoid rhabdoid tumor diagnosis after partial resection of dysembryoplastic neuroepithelial tumor: case report and review of the literature. pediatr. neurosurg. 51, 191–198 (2016). bertrand, a. et al. rhabdoid component emerging as a subclonal evolution of paediatric glioneuronal tumours. neuropathol. appl. neurobiol. 44, 224–228 (2018). nobusawa, s. et al. secondary ini1-deficient rhabdoid tumors of the central nervous system: analysis of four cases and literature review. virchows arch. 476, 763–772 (2020). thomas, c. et al. atypical teratoid/rhabdoid tumor (at/rt) with molecular features of pleomorphic xanthoastrocytoma. am. j. surg. pathol. 45, 1228–1234 (2021). capper, d. et al. dna methylation-based classification of central nervous system tumours. nature 555, 469–474 (2018). louis, d. n. et al. who classification of tumours of the central nervous system. (international agency for research on cancer, 2016). vaubel, r. et al. biology and grading of pleomorphic xanthoastrocytoma-what have we learned about it? brain pathol. 31, 20–32 (2021). louis, d. n. et al. the 2021 who classification of tumors of the central nervous system: a summary. neuro-oncol. 23, 1231–1251 (2021). copyright: © 2021 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. neurodegeneration: 2022 update feel free to add comments by clicking these icons on the sidebar free neuropathology 3:13 (2022) review neurodegeneration: 2022 update john f. crary neuropathology brain bank & research core, department of pathology, nash family department of neuroscience, department of artificial intelligence & human health, ronald m. loeb center for alzheimer's disease, friedman brain institute, icahn school of medicine at mount sinai, new york, ny, usa corresponding author: john f. crary, md-phd · friedman brain institute · ronald m. loeb center for alzheimer’s disease · icahn school of medicine at mountsinai · 1 gustave l. levy place box 1194 · new york, ny 10029 · usa john.crary@mountsinai.org submitted: 18 april 2022 accepted: 05 may 2022 copyedited by: georg haase published: 10 may 2022 https://doi.org/10.17879/freeneuropathology-2022-3866 keywords: neurodegeneration, neuropathology, aging, microglia, alzheimer disease, tauopathy, huntington’s disease, α-synucleinopathy, tdp-43 proteinopathy, traumatic brain injury abstract here, we review a collection of recent manuscripts and research trends on the neuropathology of neurodegeneration that are considered by the author to be among the potentially most impactful. to the greatest extent possible, we chose to focus on histopathological studies that are most relevant to experimental and diagnostic neuropathology. while there has been an abundance of important recent discoveries and developments in neurodegenerative disease research, there was a deliberate effort here to provide balance to prevent disease categories and experimental approaches from overshadowing the others. the result is a diverse series of outstanding studies, together showing the landscape of progress across neurodegenerative disorders. one is a stereological study examining dystrophic microglia in aging. we highlight the first large genetic study of primary age-related tauopathy, showing convergence and divergence from classical alzheimer’s disease. there were further advances in the neuropathological criteria and staging of chronic traumatic encephalopathy. links suggesting a causal role for tmem106b in tdp-43 proteinopathy emerged. attempts to subtype alzheimer’s disease on the molecular level were made. evidence for a role for the vegf family in cognitive impairment was advanced. comparison of gene expression profiles from myeloid cells in peripheral blood and brain tissues from parkinson’s disease patients revealed pathways that may lead to new mechanistic insights and biomarkers. a large autopsy series identified an increased frequency of central nervous system developmental malformations in huntington’s disease. a robust and reliable system for assessing lewy body pathology was proposed. finally, we continue to be plagued by the covid-19 pandemic, with lingering concerns of a long-term link with neurodegeneration. 1. neurodegeneration-specific alterations in microglial morphology as the data from genetic, cellular and animal studies accumulates, the importance of microglial and neuroimmune function continues to grow. microglia take on a number of different morphologies, including ramified, hypertrophic and dystrophic, that are demonstrable using various histopathological techniques. the importance of the dystrophic morphology is currently unclear and has been implicated in disease and age-related cellular senescence. unfortunately, there has been a dearth of rigorous histopathological human studies looking at them across the aging spectrum. critically, our understanding of dystrophic microglia has been hindered by a lack of well-designed and implemented quantitative studies of these various microglial morphological patterns across the human lifespan and in disease. shahhidehpour et al. performed a stereological study, published in the neurobiology of aging, specifically investigating these microglial morphologies in human post-mortem brain tissues across the lifespan (shahidehpour et al., 2021). the authors set out to test the hypothesis that age causes microglial dystrophy. abnormalities in microglial function may be a strong contributor to age-related neurodegenerative disease, with cellular senescence being a critical hallmark of aging. the strength of the study was the rigorous stereological counts of microglial morphologies including hypertrophic and dystrophic types alongside total, across a collection of brains ranging in age in years from teenage to nineties (figure 1). surprisingly, while an age-associated increase in the total number of dystrophic microglia was found, this was not significant when compared to the total number of microglia which also increased with age. in contrast, the total number of dystrophic microglia was greater in subjects with a number of neurodegenerative diseases, including alzheimer's disease, dementia with lewy bodies, and limbic predominant age-related tdp-43 encephalopathy. the authors also provided evidence for a role of altered iron homeostasis in these dystrophic microglia using ferritin light chain as a marker of intracellular iron indicating a possible mechanism. this result pinpoints microglia dystrophy as a critical disease-associated signature in neurodegeneration. figure 1. examples of ramified (a, b), hypertrophic (c, d) and dystrophic microglia (e, f) reproduced with permission from shahidehpour et al. (shahidehpour et al., 2021). 2. pathogenetic study in part clarifies genetic overlap with ad and identifies jade1 in 2014, a group of neuropathologists, led by the author of this article, defined criteria for a new category of neurodegenerative change, termed primary age-related tauopathy (part) (crary et al., 2014). subsequently, there have been many clinical and neuropathological studies that have explored the contours and implications of this designation, both challenging and supporting its scientific validity as an independent age-associated neurodegenerative category. while the terminology has been broadly adopted, part remains controversial and poorly understood. one of the biggest gaps in knowledge is the extent to which part represents a distinct pathological process or shares mechanistic underpinnings with ad and perhaps the other tauopathies. this was addressed with the first genome-wide association study (gwas) in primary age-related tauopathy published online in november 2021 in acta neuropathologica (farrell et al., 2022). given that part is a ubiquitous neuropathological feature in essentially all aged human brains, farrell et al. conducted a case-only quantitative trait analysis using braak neurofibrillary stage as an endophenotype. while this was a relatively small gwas (n=647), it was noted that most similar studies rely on clinical phenotypes, which are noisy relative to autopsy-derived endophenotypes, such as the braak neurofibrillary tangle (nft) stage used in this study. looking at candidate genes implicated in alzheimer’s disease and tauopathies revealed some associations, inducing with the mapt h1 haplotype and other candidate loci, including slc24a4, ms4a6a, hs3st1 and eif2ak3. as with previous studies, there was no association between part and the most important and common risk allele for sporadic alzheimer’s disease, apoe. the study also identified a novel locus on chromosome 4 that met genome-wide significance. given that the locus contains a number of genes, the investigators used a computational approach, including examination of a single-cell tangle-containing rna-seq dataset, to nominate gene for apoptosis and differentiation in epithelia 1 (jade1) as the best candidate in the locus. the jade1 protein has some interesting neuropathological features, co-localizing by immunohistochemistry with nft in all tauopathies except pick disease, the prototype tauopathy with three microtubule-binding domain repeats (3r). this was intriguing given that jade1 protein biochemically interacts with only tau isoforms that contain 4 microtubule-binding domain repeats (4r). finally, the jade1 homolog in drosophila, rhinoceros (rno), was shown to be a potent regulator of cell death in the fly eye and brain. together, these findings suggest that jade1 might be a component of a pathway that shared by many tauopathies. 3. neuropathological criteria for chronic traumatic encephalopathy: consensus update as awareness and scientific interest in chronic traumatic encephalopathy (cte) continues to expand, the neuropathological criteria continue to be a focus area. concussions are extremely common, especially in military populations and contact sports athletes, and the long-term neurological sequelae remain under-investigated. previously, the nih convened a consensus panel of neuropathologists to establish preliminary consensus criteria for the neuropathological diagnosis of cte (mckee et al., 2016). this landmark study has been highly influential, but questions persisted. for example, it was unclear as to what is the minimum requirement for a cte diagnosis. further, it was important to know whether cte could be reliably differentiated from age-related tauopathies like primary age-related tauopathy (part) and aging-related tau astrogliopathy (artag). further, a four-tiered staging system has been proposed but has not been independently validated (mckee et al., 2013). to address this, the nih convened a second consensus panel meeting with eight neuropathologists expert in neurodegenerative tauopathies (bieniek et al., 2021). the panel blindly reviewed 27 cases (17 cte and 10 other tauopathies), applying the 2016 cte criteria. first, there was substantial agreement in the diagnosis across raters which validated the robustness of the approach. next, the panel turned to staging and developed a simplified and practical tool for the assessment of the cte stage (figure 2). the tool involves assessment of the 11 brain regions proposed by the first consensus meeting with a minimum threshold as a single pathognomonic lesion (with or without glial tangles). a point system was devised encompassing the numerous features of cte with a cutoff of five, dichotomizing “low" and “high" cte neuropathologic change. this simplified system had excellent correlation with the cte stage as previously described (mckee et al., 2013). numerous challenges and controversies in the field remain, but this represents a milestone in establishing rigor and reproducibility in cte autopsy studies and provides a foundation for further advances in the field. figure 2. flowchart for diagnosis of cte reproduced with permission from bieniek et al. (bieniek et al., 2021). 4. a causal role for tmem106b in tdp-43 pathology inclusions containing the tar dna-binding protein 43 (tdp-43) are the hallmark neuropathological feature of a number of neurodegenerative diseases with divergent neuroanatomical vulnerability and symptomatology including amyotrophic lateral sclerosis (als), frontotemporal lobar degeneration (ftld-tdp), and limbic age-related tdp-43 encephalopathy (late). the underlying reasons for these divergent pathoanatomical signatures are unclear and numerous factors likely contribute. while mutations in the tdp-43 gene are only rarely associated with tdp-43 proteinopathy, genetic variation in other genes including tmem106b and c9orf72 are much more frequent in this context. to explore this, mao et al. performed a rigorous pathoanatomical study published in acta neuropathologica examining the influence of genetic variation in tmem106b and c9orf72 on the burden and distribution of tdp-43 proteinopathy in 899 brains from the university of pennsylvania center for neurodegenerative disease research (cndr) brain bank, including cases with als, ftld-tdp, alzheimer’s disease and lewy body disease (mao et al., 2021). tmem106b risk alleles for ftld-tdp were demonstrated to modify tdp-43 burden in als (but curiously not in ftld-tdp). c9orf72 expansion was associated with tdp-43 burden in both als and ftld-tdp. the interaction between tdp-43 and tmem106b was confirmed in a cellular model. these data are intriguing because they suggest a causal role for tmem106b in some forms of tdp-43 pathology (figure 3). figure 3. schematic showing the effects of tmem106b on tdp-43 proteinopathy reproduced with permission from mao et al. (mao et al., 2021). 5. defining molecular subtypes for alzheimer’s disease according to a widely held dogma, alzheimer’s disease arises secondarily from amyloid-beta toxicity that in turn leads to neurofibrillary degeneration and cell death. however, there is increasing evidence that ad is more heterogeneous than recognized, manifesting not only in the ad mimics such as limbic age-related tdp-43 encephalopathy and comorbid dementias such as lewy body disease and cerebrovascular disease) but also within the group of subjects that meet the neuropathological criteria for ad. neuropathological subtypes of ad have been proposed (janocko et al., 2012; murray et al., 2011) and gene expression studies in ad have been previously performed, but defining a molecularly defined subtype has never been attempted. armed with a large rna-seq dataset of over 1543 transcriptomes derived from four brain regions from two independent brain bank series, neff et al. set out to define molecular subtypes of ad (neff et al., 2021). to accomplish this, they leveraged a novel clustering method which uncovered five stable subtypes in three classes: a, b1/2 and c1/2 (figure 4). one of these subtypes termed class c (“typical") which represented about one third of all cases, displayed expression changes similar to those previously reported in alzheimer’s disease (blalock et al., 2011) with increased immune response and decreased synaptic signaling. the class c cases also showed strong up-regulation of pathways involving amyloid-beta clearance, fiber formation and scavenger receptor activity. in contrast, expression changes in class a (“atypical") were generally opposite to class c (blalock) changes. class b (“intermediate") did not show changes in either direction. tau-related pathways were strongly upregulated in both a and b1/2 subtypes. certain pathway changes were subtype-specific illustrated by the up-regulation of protein degradation-related genes (e.g., ubiquitination) in class a or the up-regulation of organic acid-related genes in class b. of note, the different classes were associated with distinct clinical and neuropathological phenotypes. class c1 for instance showed a more frequent association with increased amyloid plaque burden than the tau-related class a and also had an increased apoe ε4 allele frequency whereas class a displayed increased mapt pathway activation and decreased nft association, thus potentially representing a resilient subtype. many questions remain, especially how these subtypes align with previously proposed ad subtypes. nevertheless, this study provides evidence that ad is likely not a single entity and paves the way towards additional studies addressing this critical barrier. figure 4. molecular subtypes of alzheimer’s disease defined by gene expression changes. changes in the mean expression of gene pathways are indicated for ad subtypes a, b1, b2, c1 and c2 in comparison to normal control samples. data from a previous study msigdb are indicated on the right. changes in gene expression are grouped by major areas of biological activity. reproduced with permission from neff et al. (neff et al., 2021). 6. cerebrovascular disease: vegf gene family expression in cognitive aging and alzheimer’s neuropathology vascular endothelial growth factor (vegf) is an angiogenic factor induced by hypoxia that plays an important role in angiogenesis. the vegf gene family has several members which interact with a complex set of receptors and co-receptors which trigger (figure 5) various signaling pathways highly relevant to neurodegeneration such as neurogenesis, neuroprotection, apoptosis, cell proliferation, and inflammation. likewise, previous studies have linked vegf signalling to various neurodegenerative and cerebrovascular diseases. figure 5. illustration of the vegf family of growth factors and their receptors. asterisks indicate genes that are differentially expressed between ad and controls. reproduced with permission from mahoney et al. (mahoney et al., 2021). a focused study published in molecular psychiatry by mahoney et al. found intriguing direct evidence that changes in vegf signaling are closely associated with the development of ad neuropathology and the progression of cognitive impairment (mahoney et al., 2021). the authors leveraged the religious orders study (ros) and the rush memory and aging project (map), using global composited cognitive scores from 17 neuropsychological tests and autopsy measurements of amyloid plaque and tau burden from a total of 531 subjects with normal cognition, mild cognitive impairment (mci) and ad-type dementia. the authors found that expression in the frontal cortex of four key components of vegf signaling, vegfb, flt4, flt1 and pgf, were associated with accelerated cognitive decline. all four of these genes were also associated with increased tau and amyloid burden. additional work is required to elucidate exactly how vegf signaling relates to ad neuropathology and cerebrovascular disease. this study, which requires additional confirmation and replication with molecular studies and cellular/animal modeling, spotlights the potential importance of vegf in dementia. 7. monocytes and microglia in parkinson’s disease evidence is accumulating that the immune system plays a critical role in parkinson’s disease (pd). investigating the pathogenic events occuring in critical immune cells especially in the pd brain remains of great interest. myeloid cells and microglia have been understudied. genetic studies have identified a large number of risk loci that alter the expression of nearby genes in easily accessible peripheral monocytes and there is enrichment of gene sets expressed in microglia. there are many functions of myeloid cells, including clearing of debris and maintaining brain homeostasis, that are highly relevant to pd pathogenesis. further, alpha-synuclein can promote microglial release of neurotoxic factors potentially leading to death of dopaminergic neurons. intriguingly, the possibility that alpha-synuclein pathology might be initiated in the enteric nervous system further highlights the potential importance of peripheral myeloid cells to influence pathogenesis. how myeloid cells are involved in pd is unclear and the extent to which specific immune cell types, in the periphery or in the central nervous system, participate remains a critical knowledge gap. in a paper published in nature aging by navarro et al., an international team of researchers conducted a study looking at monocytes in peripheral blood as well as monocytes and microglia in post mortem brains from pd patients (navarro et al., 2021). the authors sorted cd14+ peripheral monocytes and microglia from the blood and brains and performed a large-scale unbiased single cell transcriptomic study. the strength of this study are the direct comparisons between gene expression in the peripheral blood with the brain as most studies in pd have focused on dopaminergic neurons, fibroblasts, or whole blood. they identified genes and co-expression networks dysregulated in pd myeloid cells. they identified genes that are involved in mitochondrial and proteasomal function that are highly expressed in a proinflammatory intermediate subpopulation. they identified 17 variants that influence mrna expression and pd risk. this enabled the authors to show that there are actually opposite mitochondrial transcriptomic profiles between microglia and macrophages (down regulated) versus peripheral monocytes (upregulated). these results provide further support for a prominent role for immune dysfunction in the pathogenesis of pd. 8. neurodevelopmental alterations in huntington’s disease huntington’s disease (hd) is the prototype hyperkinetic movement disorder caused by polyq repeat expansion in the huntingtin gene (htt). while the neuropathology of hd has been extensively studied, accumulating evidence from cellular, animal and human studies pointed to the existence of neurodevelopmental abnormalities in hd. of note, barnat et al. have reported defects in neuronal migration in the brains of mutant htt-fetuses and linked them to alterations in the interkinetic nuclear migration of progenitor cells (barnat et al, science 2020). neuropathological evidence in adult human hd has however been lacking. in a paper published in acta neuropathologica, hickman et al. provided the first neuropathological evidence that malformations are present in adult human hd (hickman et al., 2021) through systematic evaluation of two large brain collections: a discovery cohort from the new york brain bank (nybb) at columbia university and a validation cohort from the harvard brain tissue resource center (hbtrc), total n=3918. of 130 hd brains examined from the nybb, eight (6.2%) had at least one malformation compared to 12 (0.75%) in the 1600 non-hd brains. periventricular nodular heterotopias (pnh) were the most frequent malformation (figure 6), confirming abnormalities in neuronal migration in hd. intriguingly, the neurons in the pnh contained p62-positive aggregates indicative of ongoing proteinopathy. other cerebral malformations included hamartomas, cerebellar nodular heterotopias, and one multinodular vacuolating tumor of the cerebellum (mvnt). the frequency of hd-associated cerebral malformations was also significantly increased in the hbtrc cohort but at a lower frequency than in the nybb cohort, which was likely explained by differences in sampling and assessment protocols. the asymmetric nature of the cerebral malformations in hd suggested that they were secondary to somatic mutation. their higher frequency in women may be related to sexual dimorphism. the possibility that hd brains contain additional developmental or neurodevelopmental abnormalities merits further investigation. nevertheless, this study provides concrete evidence that developmental abnormalities occur in human brains with hd and provide the foundation for a new avenue into studying htt-related mechanisms. figure 6. a periventricular nodular heterotopia (pnh) in a 41-year-old woman with hd. gross (a) and low power (lh&e stain) images (b) of a subependymal heterotopia containing non-dysmorphic neurons (c) that are immunopositive for htt and p62 (d, arrows). reproduced with permission from hickman et al. (hickman et al., 2021). 9. criteria for lewy body pathology: approaching consensus the spectrum of lewy body disease (lbd) pathology includes parkinson’s disease (pd), dementia with lewy bodies (dlb) and pd with mild cognitive impairment or dementia. currently, a neuropathological diagnosis of lewy body disease can be rendered using any of four different proposed systems (adler et al., 2019; braak et al., 2003; leverenz et al., 2008; mckeith et al., 2005). all use semi-quantitative assessment of the neuroanatomical distribution of lewy pathology in neurites and cell bodies, but each has specific strengths and weaknesses. each system suffers from issues related to interrater reliability and/or the inability to classify certain cases with lewy pathology (lp), for example cases with olfactory bulb-only or amygdala-only disease. this stems from the heterogenous pathoanatomical distribution of lp. in 2021, new neuropathological consensus criteria that aim to address the limitations of these systems, termed the lp consensus criteria (lpc), were developed and published in acta neuropathologica in order to improve interrater reliability and to unequivocally classify all possible cases with lp (attems et al., 2021). to improve inter-rater reliability, the authors implemented a dichotomized scoring system, with lp scored as either present or absent. the diagnostic categories were: olfactory-only, amygdala-predominant, brainstem, limbic, and neocortical lp. the 16 raters from 13 centers applied the lpc and each of the four existing systems to 34 cases with lp from the newcastle brain tissue resource and the university of pennsylvania brain bank. interrater reliability was good for the lpc, being comparable to the mckeith and leverenz system (krippendorff’s alpha ≈ 0.6), and considerably better than for braak and adler (beach). the lpc system was as applicable as the beach system and able to unequivocally classify essentially all cases, which was not the case for the leverenz, mckeith and braak systems which failed in 10%, 25% and nearly 30% of cases respectively. given the reliability and applicability of the lpc system, we expect that it will be adopted broadly in the post-mortem evaluation of cases with lewy body pathology. 10. long-covid intersecting with neurodegeneration during the covid-19 pandemic, a number of neuropathological studies addressed the changes in the human brain from patients succumbing to acute sars-cov-2 infection. microthrombi and strokes were among the most common findings together with indirect inflammatory and autoimmune changes but encephalitis was rare. after the peak of the pandemic, roughly one third of patients emerged with a clinical syndrom often termed post-acute sequelae of sars-cov-2 infection (pasc) and commonly referred to as “long-covid". cardiac and pulmonary symptoms are prominent in pasc but there are also many alarming non-specific neurological and psychiatric symptoms including fatigue, cognitive complaints (“brain fog"), sensorimotor defects, headaches, insomnia, depression, and post-traumatic stress disorder (taquet et al., 2021). a meta analysis confirmed decreased cognitive function seven months following covid-19 infection (crivelli et al., 2022). pasc has drawn attention in the field of neurodegeneration given the possibility that infection with the virus may trigger a long-term neurodegenerative process (lennon, 2020). the mechanisms whereby pasc may be linked to neurodegeneration remained however elusive. many investigators have raised the questions as to whether sars-cov-2 can cause neurodegeneration through its neurotropism or otherwise (krey et al., 2021). some investigators have proposed that sars-cov-2 enters the brain and may activate an ad-like program (shen et al., 2022). the ace2 covid-19 receptor was indeed shown to be upregulated in the hippocampal formation of ad patients (zhao et al., 2021), which may explain why medial temporal lobe structures might be vulnerable, potentially contributing to some post-infection memory impairment and neurodegeneration (ritchie et al., 2020). notably, signaling pathways leading to tau hyperphosphorylation in ad including gsk3β may be activated in post-mortem covid-19 brains (reiken et al., 2022). adand covid-19 signaling pathways may converge at the level of the nrlp3 inflammasome through dysregulation of amyloid clearance by microglia (cama et al., 2021, p. 3). alternatively, sars-cov-2 infection may worsen ad for various reasons (villa et al., 2022). while this constitutes perhaps the most urgent and potentially important research trend at this time, it remains to be seen whether there will be sustainable momentum. acknowledgement dr. crary receives research funding from the nih (r01ag054008, r01ns095252, r01ag062348, r01ns086736, u54ns115266, u54ns115322). we further acknowledge the rainwater charitable trust/tau consortium, david and elsie werber, alexander saint amand scholar award and karen strauss cook 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(2021). ubiquity of the sars-cov-2 receptor ace2 and upregulation in limbic regions of alzheimer’s disease brain. folia neuropathologica, 59(3), 232–238. https://doi.org/10.5114/fn.2021.109495 copyright: © 2022 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. post-mortem neuropathologic examination of a 6-case series of car t-cell treated patients feel free to add comments by clicking these icons on the sidebar free neuropathology 3:23 (2022) original paper post-mortem neuropathologic examination of a 6-case series of car t-cell treated patients nuria vidal-robau1*, gabriela caballero1*, ivan archilla1, andrea ladino2, sara fernández2, valentín ortiz-maldonado3, montserrat rovira3, marta gómez-hernando3, julio delgado3, maría suárez-lledó3, carlos fernández de larrea3,8, olga balagué1, gerard frigola1, abel muñoz1, estrella ortiz1, teresa ribalta1, miguel j. martinez5,6, maria angeles-marcos5, marta español-rego7, azucena gonzález7, daniel benitez-ribas7, eugenia martinez-hernandez4, pedro castro2,8, iban aldecoa1,9 1 pathology department, biomedical diagnostic centre (cdb), hospital clinic of barcelona – university of barcelona, barcelona, spain 2 medical intensive care unit, hospital clinic of barcelona – university of barcelona, barcelona, spain 3 haematology department, hospital clinic of barcelona – university of barcelona, barcelona, spain 4 neurology department, hospital clinic of barcelona – university of barcelona, barcelona, spain 5 microbiology department, biomedical diagnostic centre (cdb), hospital clinic of barcelona – university of barcelona, barcelona, spain 6 barcelona institute for global health (isglobal), hospital clinic of barcelona – university of barcelona, barcelona, spain 7 immunology department, biomedical diagnostic centre (cdb), hospital clinic of barcelona – university of barcelona, barcelona, spain 8 august pi sunyer biomedical research institute (idibaps) – university of barcelona, barcelona, spain 9 neurological tissue bank, biobank of hospital clinic of barcelona – idibaps, barcelona, spain   * these authors contributed equally corresponding author: iban aldecoa ansorregui · pathology department. 3rd stair, 5th floor · hospital clinic of barcelona · st. villaroel 170 · 08036 barcelona · spain ialdecoa@clinic.cat submitted: 04 august 2022 accepted: 20 october 2022 copyedited by: alessia sciortino published: 27 october 2022 https://doi.org/10.17879/freeneuropathology-2022-4365 keywords: hematologic malignancies, chimeric antigen receptor (car) t-cell, cytokine release syndrome (crs), neurotoxicity, neuropathology, immunohistochemical stains abstract introduction: chimeric antigen receptor (car) t-cell therapy is a promising immunotherapy for the treatment of refractory hematopoietic malignancies. adverse events are common, and neurotoxicity is one of the most important. however, the physiopathology is unknown and neuropathologic information is scarce. materials and methods: post-mortem examination of 6 brains from patients that underwent car t-cell therapy from 2017 to 2022. in all cases, polymerase chain reaction (pcr) in paraffin blocks for the detection of car t cells was performed. results: two patients died of hematologic progression, while the others died of cytokine release syndrome, lung infection, encephalomyelitis, and acute liver failure. two out of 6 presented neurological symptoms, one with extracranial malignancy progression and the other with encephalomyelitis. the neuropathology of the latter showed severe perivascular and interstitial lymphocytic infiltration, predominantly cd8+, together with a diffuse interstitial histiocytic infiltration, affecting mainly the spinal cord, midbrain, and hippocampus, and a diffuse gliosis of basal ganglia, hippocampus, and brainstem. microbiological studies were negative for neurotropic viruses, and pcr failed to detect car t -cells. another case without detectable neurological signs showed cortical and subcortical gliosis due to acute hypoxic-ischemic damage. the remaining 4 cases only showed a mild patchy gliosis and microglial activation, and car t cells were detected by pcr only in one of them. conclusions: in this series of patients that died after car t-cell therapy, we predominantly found non-specific or minimal neuropathological changes. car t-cell related toxicity may not be the only cause of neurological symptoms, and the autopsy could detect additional pathological findings. introduction chimeric antigen receptor (car) t-cell therapy is a promising immunotherapy for the treatment of refractory hematopoietic malignancies. normal and malignant b-cells express the cd19 protein in the cell membrane. lymphodepletion followed by transfer of autologous t-cells that have been genetically modified to express a car targeting cd19 have been effective in clinical trials, producing remission of refractory or relapsed acute lymphoblastic leukemia (all), chronic lymphocytic leukemia and non-hodgkin’s lymphoma (nhl)1–5. recently, bcma targeted cars have been used to treat multiple myeloma6. among toxic effects expected after lymphodepletion and anti-cd19 car t-cell infusion, cytokine release syndrome (crs) and neurotoxicity are the most relevant but, unlike crs, the physiopathology of neurological events is less known and neuropathologic information is scarce7. clinical presentation develops mostly as non-specific brain dysfunction without focal signs,most of them being mild and transient. nevertheless, focal symptoms such as seizures and fatal cerebral oedema have been described. neurotoxicity can occur in the context of severe crs, where disruption of the blood-brain barrier (bbb) by cytokine production and subsequent endothelial activation leads to an increase in proinflammatory cytokine levels in cerebrospinal fluid (csf)7–10. however, neurotoxicity is also associated with expansion and activation of car t-cells that lead to a direct parenchymal car t-cell infiltration. in addition to crs following car t-cell infusion, factors that favor car t-cell proliferation in blood, such as the addition of fludarabine to cyclophosphamide lymphodepletion chemotherapy9, are also associated with an increased risk of neurotoxicity. fludarabine is a antineoplastic agent that added to the conditioning regimens improves the expansion and persistence of car t-cells, as well as duration of remission and disease-free survival2,4,5. its contribution to neurotoxicity may be driven by different mechanisms, and is yet to be elucidated. however, fludarabine-associated neurotoxicity has a later onset, and cerebral oedema is not usually reported11. reports of neuropathological findings after car t-cell therapy are scarce, independent of the presence or absence of neurological symptoms prior to death. we only found 4 cases described in the literature, which showed variable and non-specific neuropathological changes7–10. here, we report detailed neuropathological findings of 6 patients who died after car t-cell therapy12. materials and methods six brains from patients that underwent car t-cell therapy from 2017 to 2022 in a tertiary university hospital were examined. we selected all cases with car t-cell therapy from requested medical autopsies at the pathology department during this period. patients received ari-0001 (anti-cd19) and ari-0002h (anti-bcma, for patients with multiple myeloma) based car t treatment. for ari-0001, the planned target cell dose varied depending on the patient's disease: typically, 1 × 106 ari-0001 cells/kg for patients with acute lymphoblastic leukemia and chronic lymphocytic leukemia; and 5 × 106 ari-0001 cells/kg for non-hodgkin lymphoma patients. full details of ari-0001 and ari-0002h cell production and a phenotypic characterization of these cells can be found elsewhere6,12–14. clinical data was retrospectively retrieved from the electronic medical records. formalin-fixed and paraffin-embedded tissue sections from the frontal cortex, visual cortex, cingulate, hippocampus, amygdala, thalamus, basal ganglia, white matter, midbrain, pons, medulla oblongata, spinal cord and cerebellum that had been stained with hematoxylin-eosin (he) were histologically assessed using light microscopy. in some of the cases the study was extended with immunohistochemistry (ihc). ihc was performed on 5-µm formalin-fixed, paraffin-embedded (ffpe) sections using the roche benchmark ultra platform with usual antigen retrieval protocols. the primary antibodies used were cd3 (2gv6, roche), cd20 (l26, roche), cd4 (sp35, roche), cd68 (kp-1, roche), glial fibrillary acidic protein (gfap) (ep672y, roche), terminal deoxynucleotidyl transferase (tdt) (roche), beta amyloid (de2b4, roche), tau (at8, thermo-fisher scientific), neurofilament (2f11, roche), herpes simplex virus (hsv)-1 (10a3, roche), hsv2 (dbm15.69, sanbio), human herpes virus (hhv)-6 (a & b, gp 60/110, millipore), hhv-8 (13b10, roche), cytomegalovirus (cmv) (8b1.2, 1g5.2, and 2d4.2, roche), epstein-barr virus (ebv) latent membrane protein (lmp)-1 (cs1-4, roche), epstein-barr virus (ebv)-encoded small rnas (eber) (roche), and polyomavirus (mrq-4, roche). ihc studies were performed in the initial diagnostic workup when clinical suspicion or neuropathological evidence of any pathologic process was observed. in addition, a systematic retrospective analysis was performed: cd3 and cd20 were screened on hippocampal and midbrain sections in all cases, on spinal cord sections in all cases except case 1, on frontal cortex in all cases except cases 2 and 3, and on basal ganglia only in cases 4 and 6. gfap ihc was performed on frontal sections in all cases, on basal ganglia in all cases except case 3, on hippocampus in all cases except case 1, and on midbrain only in case 4. polymerase chain reaction (pcr) in paraffin blocks was performed in case 5 for the detection of hhv-6, west nile virus, adenovirus, enterovirus, measles, mumps and lymphocytic choriomeningitis. samples of all cases from hippocampus, midbrain, and spinal cord (only in cases 2 to 6) were used for car t-cell detection by pcr. five micrometer sections were obtained from paraffin blocks, and dna was extracted using qia amp dna ffpe tissue kit (qiagen, hilden, germany). real-time pcr was used to measure number of copies/cell. genomic dna was amplified as previously described13. amplification reactions were performed in triplicate. an eight-point standard curve was generated using a sequence close to the cdkn1a gene (genebank: z86995) as comparator. the test was calibrated to detect a number of copies ranging from 5 to 106 per 500 ng of genomic dna. results see table 1 for a summary of the cases here presented and the other cases reported in the literature, and figure 1 for the comparison of gliosis and t-cell infiltrates of the cases. table 1. clinical data and histopathologic findings of our cases and previously described cases (modified from7) m: male; f: female; n/a: not applicable. ffpe: formalin fixed paraffin embedded. figure 1. figures co to f1: gfap immunohistochemistry of hippocampus (upper third ca1 sector -right sideand subiculum – left side-) and parahippocampal area (lower third, transenthorinal cortex) of a control (co) and cases 2 (b1), 3 (c1), 4 (d1), 5 (e1) and 6 (f1). the control was a 52-year-old male who died due to an acute bilateral adrenal hemorrhage with extensive clasmatodendrosis (insets, parahippocampal white matter, 400x). the cases showed variable gliosis, predominantly mild, higher in case 5 (e1) who had an encephalitis, and case 4 (d1) who died with an extensive acute pneumonia. both had mild and isolated clasmatodendritic changes in white matter (see insets). row 3 and 4 show t-cell infiltrates (cd3+) in parahippocampal cortex (left half) and substantia nigra pars compacta (right half), in cases 1 (a2), 2 (b2), 3 (c2), 4 (d2), 5 (e2) and 6 (f2). only case 5 (e2) presented marked infiltrates, while other cases has only scattered to isolated perivascular t-cells. case 2 (b2), which was positive for cart pcr in brain tissue, did not have significantly higher t-cell infiltrates (figures co, b1 to f1 20x scale bar 500 µm; insets 400x, scale bar 20 µm. figures a2 to f2 200x, scale bar 50 µm). case 1 a 44-year-old woman with acute b-cell lymphoblastic leukemia presented to an outside hospital with central nervous system (cns) involvement and a paravertebral mass (d8-d10) without spinal compression. at disease onset, she had lymph node involvement and leukocytosis and was refractory to five lines of treatment. at her first consultation in our center she did not present any neurological symptoms. basal brain magnetic resonance imaging (mri) showed a nonspecific lacunar lesion in the left frontal white matter and discrete homogeneous dural enhancement in cerebral convexity; the cerebrospinal fluid (csf) study was normal. after the conditioning with cyclophosphamide and fludarabine, she received car-cd19 treatment. three days after the infusion she developed fever, pulmonary interstitial infiltrates, and pleural effusion. the radiology was suggestive of non-cardiogenic oedema, with isolated colonies of staphylococcus haemolyticus in respiratory samples culture. she received antimicrobial therapy, depletive treatment, pleural drainage, and non-invasive mechanical ventilation, as well as tocilizumab, as a crs could not be ruled out. her clinical status improved, although she had persistent fever with negative microbiological culture. fourteen days after the infusion she progressed with fever, hemodynamic instability and multi organ failure, as well as car t-cells amplification in peripheral blood. with the diagnosis of crs, a second dose of tocilizumab was administered with only partial improvement. steroids were added, but the patient developed confusion, suggesting neurotoxicity. the electroencephalogram (eeg) showed generalized slowing, compatible with encephalopathy, and the brain mri showed two non-specific signal abnormalities/foci of signal alteration without mass effect or contrast uptake, lateral to the left ventricular atrium and in the right temporo-occipital area adjacent to the ventricular occipital horn. flow cytometry (fc) of the csf revealed the presence of mature t lymphocytes and isolated blasts, and the microbiological csf study was weakly positive for hhv-6. nineteen days after the infusion she presented rapidly progressive respiratory and hemodynamic deterioration requiring intubation and vasoactive treatment. the tracheal aspirate was positive for stenotrophomona maltophila and the diagnostic of septic shock due to nosocomial pneumonia was established. antibiotic treatment was escalated. however, she progressed with pulmonary hemorrhage and died 23 days after the infusion. neuropathologic gross examination revealed a 1280 g fixed brain with diffuse petechiae in the dura mater. coronal sections showed slight discoloration of right temporal white matter. histology showed mild arteriosclerosis and arteriolar hyalinosis, minimal lymphocytic perivascular infiltrate (t-cell predominant) and occasional intravascular infiltration by atypical cells with tdt positivity (figure 2), confirming the presence of leukemic cells, without remarkable parenchymal involvement. car t-cell pcr in the brain was negative and there were no signs suggestive of encephalitis associated to viral infection. full body autopsy demonstrated massive dissemination of the lymphoproliferative process affecting the lungs, liver, spleen, bone marrow, lymph nodes and mammary gland. the cause of death was attributed to disease progression. figure 2. remarkable histological features: case 1 presented scant intravascular infiltration of the leukemia (a), which was positive for tdt (a, inset). case 5 showed an encephalomyelitis, with predominant involvement of limbic areas (b, entorhinal cortex), basal ganglia (c, hypothalamus), brainstem (d, substantia nigra) and spinal cord (e, lumbar spinal cord). t-cell lymphocytic infiltrates were both cd8+ and cd4+, with predominance of the former (f and g, cd8+ and cd4+ infiltrates in lumbar spinal cord, which did not show marked tropism for motor neurons (arrowheads)) (a 400x, scale bar 20 µm, inset 600x, scale bar 20 µm; b to g, 200x, scale bar 50 µm). case 2 a 19-year-old woman presented with primary mediastinal diffuse large b-cell lymphoma with infiltration of soft tissues. she had a protracted course with persistent mediastinal disease, gastric relapse, superior vena cava syndrome and pericardial and pleural malignant effusions. she received multiple therapy lines including autologous stem cell transplant, local radiotherapy, and several chemotherapy lines comprising burkimab protocol and ibrutinib, before considering car t treatment. her initial mri showed mild global cerebral atrophy without other remarkable findings. on admission she was hypotensive and tachycardic and positron emission tomography (pet) – computed tomography (ct) scan showed pericardial infiltration in addition to supra and infradiaphragmatic lymphadenopathies, with soft tissue, muscle, bone, and pleural multifocal enhancement. the presence of bone marrow aplasia delayed the conditioning with fludarabine-cyclophosphamide. respiratory distress increased after conditioning along with the pleural effusion, and she was admitted to the intensive care unit (icu) to receive car-cd19 infusions. the following 48 hours, her respiratory status worsened, adding hemodynamic instability and fever. a nosocomial pneumonia was diagnosed by x-ray and ultrasound examination, without microbiological isolation, and she received antibiotics and fluconazole. despite treatment, her status worsened, requiring invasive mechanical ventilation and vasoactive treatment. subsequently, she presented superior vena cava syndrome and worsening of pericardial effusion with progression of hemodynamic instability. although she received corticosteroids and rituximab aiming to diminish the tumoral mass, she progressed to organic failure, and died 33 days after car t infusion. neurological symptoms were not detected in the course of the disease, although it was difficult to examine the patient due to sedation administered during invasive mechanical ventilation. neuropathologic gross examination was unavailable. histology revealed basal ganglia mild gliosis, and occasional perivascular hemosiderin deposits in parenchymal blood vessels of the white matter and basal ganglia. immunohistochemical studies showed scarce perivascular cd3+ t-cells with minimal intraparenchymal extension in the midbrain tegmentum, as well as in perithalamic white matter and cervical spinal cord. t-cells were not detected in the hippocampus, and there were almost no cd20+ b-cells. pcr for the detection of car t-cells in paraffin blocks of midbrain and spinal cord was positive, while it was negative in the hippocampus. full body autopsy revealed a large mediastinal mass with pericardial extension causing severe constrictive pericarditis, as well as extension to cervical region, diaphragm, rib wall and skin, and multiorgan tumor implants. no clear histological signs of acute neutrophilic pneumonia were found, although the immunosuppressive and neutropenic status may have had hindered the presence of histological signs. the cause of death was attributed to disease progression. case 3 a 19-year-old man with acute b-cell lymphoblastic leukemia was referred to our center one year after disease onset. he had relapsed disease despite induction protocol and six consolidation treatment cycles, as well as one cycle of inotuzumab-ozogamicine. he received cyclophosphamide and corticosteroid bolus as bridge therapy before the infusion of car-cd19. his initial brain mri showed multiple bilateral cortical and subcortical frontoparietal signal abnormalities suggesting chronic hemorrhages from multiple cerebral cavernomatosis, but no contraindication for the car t therapy was found. after conditioning with fludarabine and cyclophosphamide, antibiotics were started due to fever and isolation of enterococcus faecalis in urine culture. twelve hours after the first car t-cell infusion, he presented fever and hemodynamic instability refractory to fluids. he was transferred to the intensive care unit (icu) with the diagnosis of crs grade 3. tocilizumab, high doses of methylprednisolone and siltuximab, as well as support treatment with several vasopressors, mechanical ventilation, and empirical treatment with antifungal and antibacterial agents were administered. however, he developed refractory multiorgan failure, with negative microbiological analyses, and he died five days after car t-cell infusion. no neurological symptoms were noted, and he was clinically diagnosed as fatal crs (grade 5). neuropathologic gross examination revealed a 1255 g fixed brain. coronal sections showed mild congestive appearance of left amygdala, putamen, front-temporo-parieto-occipital white matter, and pontine nuclei, as well as mild greyish discoloration of white matter, midbrain tegmentum and pons. the brainstem showed moderate substantia nigra depigmentation. histology confirmed mild white matter oedema with mild gliosis, mainly in basal ganglia (ventral thalamus) and midbrain tegmentum/periaqueductal grey matter. blue dark neurons were observed in cortical areas, predominantly in layers ii and iii, suggestive of incipient mild hypoxic-ischemic changes. histology also revealed mild congestion and minimal arteriosclerosis of parenchymal blood vessels with occasional perivascular histiocytes with hematic component, without perivascular tissue damage. no evidence of venous dilations or vascular malformations were observed; the changes reported by mri study were attributed to hemodynamic fluctuations in life rather than to the presence of established vascular malformations. pcr for the detection of car t-cells in paraffin blocks was negative. full body autopsy revealed no significant findings. the cause of death was attributed to crs after car t-cell infusion. case 4 a 63-year-old male patient with a grade 3a, stage iv follicular lymphoma with abdominal extranodal and mesenteric involvement that had been treated with a first line of r-chop and had reached partial response. following maintenance with rituximab for 2 years, he remained stable one more year, until progression of the abdominal mass, new gastric infiltration, and pet hypermetabolism in the esophagus. after a third line with obinotuzumab and bendamustine, a failed attempt of autologous stem cell aphaeresis, and maintenance with obinutuzumab, a fourth line with idelalisib was started. however, the abdominal disease progressed with involvement of peripancreatic, renal and iliac lymph nodes, the patient was not eligible to allogeneic stem cell transplant, and he received car-cd19 therapy with prior fludarabine and cyclophosphamide conditioning. after the third day of consecutive infusion, the patient developed fever with mild hypotension that was diagnosed as grade 2 crs and had a good response to tocilizumab. he had an episode of toxic epidermolytic necrolysis (diagnosed by skin biopsy) with severe extensive rash and oral lesions. he also presented with severe aplasia, as well as a septic shock due to urinary tract infection that was treated in the icu. after initial improvement, he presented a pulmonary hemorrhage with colonization by candida glabrata, and developed kidney failure, hyperkaliemia, tracheobronchitis, more episodes of septic shock due to bacteremia of intestinal and skin origin (bacteroides fragilis, pseudomona aeruginosa, enterococcus faecium), and persistent candidemia as well as cmv reactivation. due to persistent skin lesions, an alternative diagnosis of car t-cell associated dermatosis was suspected. immunosuppression was restarted, improving the skin lesions, but it had to be stopped due to bone marrow aplasia and worsening neutropenia. of note, the refractory follicular lymphoma showed morpho-metabolic decrease in post car t infusion ct and pet scans, attributed to the presence of circulating car t-cells. eventually, the last episode of septic shock and ventilator associated pneumonia were refractory, which led to death after 4 months in the icu at 5 months of car t-cell therapy. he died without evidence of neurological symptoms in the course of the disease, and a brain computerized tomography (ct) done 45 days after the car t infusions showed no significant findings. neuropathologic gross examination showed a 1240 g fixed brain. coronal sections showed mild congestive changes in caudate, putamen, thalamus, and parieto-occipital white matter. histology revealed cortical gliosis in frontal and parahippocampal cortices, in possible relation to acute hypoxic-ischemic injury, as well as mild scattered foci of gliosis with minimal clasmatodendrosis in frontal and parahippocampal subcortical white matter, highlighted with gfap immunohistochemistry (see figure 1). mild parenchymal blood vessel arteriosclerosis and arteriolar hyalinosis, with occasional hemosiderin deposits, and isolated perivascular rarefaction in deep vessels was observed. histology also showed isolated diffuse cortical amyloid beta deposits, and minimal amygdala neurofibrillary pathology. pcr for the detection of car t-cells in paraffin blocks was negative. full body autopsy excluded residual neoplasia and evidenced a pneumonic process in context of the septic condition. the final cause of death was a respiratory failure. case 5 a 28-year-old man with b-cell acute lymphoblastic leukemia with disease progression with orbital infiltration. after diagnosis, induction, and consolidation treatment, he received haploidentical stem cell transplant (sct), which was previously conditioned with thiotepa, fludarabine, busulfan and prophylaxis of graft versus host disease (gvhd) with tacrolimus and cyclophosphamide. he developed hemorrhagic cystitis, cmv reactivation, escherichia coli orchitis and tacrolimus nephrotoxicity as complications, but never gvhd. he relapsed one year after sct with testicular infiltration, which required blinatumomab rescue treatment and lymphocyte donor infusion plus intrathecal prophylaxis, but ultimately led to radiotherapy and posterior bilateral orchiectomy. two years after diagnosis, he had progressive disease and was planned for car-cd19 therapy. his initial brain mri showed few nonspecific subcortical white matter hyperintense signal abnormalities, and tumor infiltration of the right orbit. after lymphodepletion with fludarabine and cyclophosphamide, he received a first car t-cell infusion with good tolerance, no signs of crs and good response observing non-neoplastic b-cells. due to loss of car t-cells in peripheral blood, he received two reinfusions in a 10-month period. the second was followed by an early loss of car t-cells due to antibodies, and the third infusion was made after plasma exchange to remove the antibodies. after the third infusion, he presented with fever without a clear origin that was treated with antibiotics, with low clinical suspicion of crs due to the absence of analytical alterations. almost one month after this last infusion, he presented with confusion, bradypsychia and generalized tremor. repeated, a brain mri showed no significant changes, and persistent tumor infiltration of the right orbit. eeg study showed diffuse slowing, and there were not significant findings in the csf study. the neurological symptoms were refractory, and he died 43 days after the third car t-cell infusion. neuropathologic gross examination showed a 1385 g fixed brain. coronal sections showed softening in both putamen. histology revealed diffuse gliosis predominant in basal ganglia, hippocampus, substantia nigra and brainstem. the most relevant finding was interstitial and perivascular lymphocytic infiltrates, together with histiocytic infiltrates, which were severe in anterior horns and grey matter of spinal cord, and to a lesser extent in the midbrain and hippocampus (figure 2). immunohistochemical studies revealed that lymphocytic infiltrates were cd3+ t-cells, predominantly cd8+, with practically no b-cells being observed, and were accompanied by severe cd68+ microglial activation with foamy histiocytes. neurofilament staining showed occasional phosphorylated somas as well as isolated axonal spheroids in cortical and hippocampal white matter, without axonal loss in spinal cord tracts. gfap staining showed mild and focal clasmatodendritic changes in the white matter. all the findings above were concordant with the diagnosis of a diffuse lymphocytic encephalomyelitis predominant in brainstem, spinal cord and limbic system, with milder disease in cortex and basal ganglia, and relative sparing of cerebellum. no viral inclusions were observed, and virus detection stains for hsv-1, hsv-2, hhv-6, hhv-8, cmv, ebv and polyomavirus, and pcr for hhv-6, west nile virus, adenovirus, enterovirus, measles, mumps and lymphocytic choriomeningitis were negative. pcr in paraffin blocks for the detection of car t-cells was negative. full body autopsy revealed extensive pulmonary hemorrhage with no apparent signs of infection and foci of extramedullary hematopoiesis in the liver. after all, the cause of death was attributed to an unspecified encephalomyelitis, suggesting primarily an infectious etiology but not ruling out immune causes. case 6 a 71-year-old woman with multiple myeloma iga kappa r-iss ii, diagnosed from a scalp extramedullary plasmacytoma. cytogenetic showed high risk (17p deletion) and it relapsed after four therapy lines. she received fludarabine and cyclophosphamide conditioning after bridging therapy. after one week of the first car-bcma infusion, she was admitted to the icu due to a grade 2 crs with fever and hypotension that were successfully treated with one dose of tocilizumab and corticosteroids. ten days later, she presented episodes of hypotension without fever or alterations in chest and brain ct or echocardiography. in the differential diagnosis, neurotoxicity related to treatment was considered, corticosteroid dose was increased, and antibiotics were added. she worsened with agitation, tachypnoea, and respiratory failure; and progressed to refractory multiorgan failure with ischemic hepatitis, renal failure, and hemorrhagic complications in the context of thrombocytopenia. a macrophagic activation syndrome was suspected due to ldh and ferritin increase, and treatment was attempted with corticosteroids, anakinra, siltuximab and cyclophosphamide. amid this situation, she presented a blood culture positive for escherichia coli without detectable septic focus. despite all measures, she remained in multiorgan failure with acidosis and hyperlactacidemia, and she died 19 days after car t-cell therapy start. additional neurotoxicity signs were not evident. neuropathologic gross examination showed a 1045 g fixed brain with mild hippocampal and amygdalar atrophy. neuropathologic evaluation revealed mild arteriosclerosis (with focal mural calcification in globus pallidus arteries) and diffuse minimal gliosis. histology revealed scattered perivascular t-cells (cd3+) in basal nuclei, frontal cortex, and hippocampus. minimal microglial activation highlighted with cd68 accompanied these findings. she also had a definite primary age related tauopathy or part (braak stage i/vi, thal 0) and mild periamygdaline age related tau astrogliopathy or artag. pcr for the detection of car t-cells in paraffin blocks was negative. the most remarkable systemic finding was a submassive acute hepatic necrosis. the prominent center lobular location, the lack of cytopathic changes and negative immunohistochemical stains for hsv-1, hsv-2, hvv-8, cmv, ebv lmp1 and eber oriented to a hemodynamic–hypovolemic etiology. there were additional acute hemorrhagic foci, like in myocardium and intestines, but the lack of tissue ischemia or inflammatory reaction surrounding them suggested they were acute premortem ischemic changes. remarkably, there was hypercellular bone marrow with t-cell expansion and hemophagocytosis. immunohistochemistry for cd68 in different organs showed moderate histiocytic activation in the spleen and mild activation in the heart, liver, intestine and brain. in the latter, it did not seem to induce secondary tissue alterations, hence its impact may had been limited. a lithic focus in the cranium was constituted mainly by fibrotic reactive tissue without evidence of plasma cell disease. after all, the cause of death was attributed to submassive liver necrosis of probable ischemic cause. discussion here we present the neuropathological findings of 6 deceased patients, 3 women and 3 men, who received a cd19(5) and bcma(1) targeted car t-cell therapy. three of them had a b-cell acute lymphoblastic leukemia, one a follicular lymphoma 3a grade, one a primary mediastinal diffuse large b-cell lymphoma, and the other a multiple myeloma with plasmocytoma. only 2 of them presented explicit neurological symptoms in the course of the disease. brain gross examination did not show significant alterations in any of the patients. histological findings in one of the patients with neurological symptoms were mild arteriosclerosis and arteriolar hyalinosis, minimal lymphocytic perivascular infiltrate (mainly cd5+) and leukemic cells, in line with the systemic progression of the hematologic malignancy as the cause of death. the histological examination of the other patient with neurological symptoms showed a severe perivascular and interstitial lymphocytic infiltration, predominantly cd8+, admixed with a diffuse interstitial histiocytic infiltration, affecting mainly the spinal cord, midbrain, and hippocampus, along with a diffuse gliosis of basal ganglia, hippocampus, and brainstem. microbiologic studies were negative for neurotropic viruses, and pcr failed to detect car t-cells. finally, the cause of death was attributed to a lymphocytic encephalomyelitis of unknown etiology, infectious versus toxic, without being able to demonstrate viral infection nor car t-cells in brain tissue. one of the cases without neurological signs showed mild clasmatodendrosis in context of terminal hypoxic-ischemic damage. the rest showed a mild patchy gliosis and microglial activation, with no other remarkable histologic changes. car t-cells were detected by pcr only in one of them, without histological particularities. reports of neuropathological findings after car t-cell therapy are scarce. neuropathological findings in autopsies of different types of patients vary, but there are common changes suggesting astrocyte dysfunction, blood-brain barrier disruption with endothelial damage and activation, and a subsequent increase in vascular permeability. we found four cases described in the literature, all of them with neurological involvement that led to death. the autopsy of a patient who died from fulminant cerebral oedema showed signs of blood-brain-barrier disruption, clasmatodendrosis and microglial activation8. the findings in the autopsy of another patient who died from progressive neurologic deterioration were diffuse gliosis with severe neuronal loss and degeneration of white matter, dense infiltration of macrophages with numerous microglial cells, as well as a cd8+ infiltrate. it is worth noting that before the hematologic malignancy, he had the diagnosis of optic atrophy, which could alter the findings10. the autopsy findings of another 2 patients were vascular in nature, with multifocal microhemorrhages, perivascular infiltration of cd8+ cells, intravascular von willebrand factor (vwf) binding, and platelet microthrombi, along with more severe vascular findings, such as karyorrhexis and fibrinoid vessel wall necrosis. reactive microglia was also noted, the percentage of t-cells in the parenchyma mirrored the percentage of those cells in the csf9. it is worth highlighting that the clinical setting of patients undergoing car t-cell therapy makes it difficult to determine the cause of the observed neurological symptoms. while crs is widely studied, neurological toxicity itself remains less understood. neurological damage due to the underlying hematological disease, opportunistic infections in a setting of severe immunosuppression, and pharmacological toxicity of accompanying chemotherapy drugs are conditions that should be taken into consideration when evaluating the possible effects of car t-cell therapy. fludarabine, a drug used for the conditioning with cyclophosphamide in all our cases, seems to promote a discernible form of neurotoxicity, with unspecific but distinctive gliosis and white matter involvement11. basic neuropathologic assessment allows reliably detecting or discarding malignant cell infiltration in the central nervous system. it also enables to demonstrate the presence of inflammation with or without infectious agents in neurological tissue, although their absence does not completely rule out infection as in our fifth case. alterations of the bbb cannot be reliably studied in he alone and specially without a prior technical background. nevertheless, without clear evidence of brain weight gain, oedema, and white matter changes, nor minimal vascular damage and perivascular cellular reactions, bbb disruption may be at best limited in cases without neurological involvement in life. on the other hand, astrocyte injury can be highlighted with ihc showing gfap aberrant beading and fragmentation, a phenomenon called clasmatodendrosis, that relates to blood barrier dysfunction8,15. we reexamined our cases with gfap, and only saw scattered foci of clasmatodendrosis in 2 cases, one with hypoxic-ischemic changes in the context of nosocomial pneumonia and one with encephalomyelitis. clasmatodendrosis has been related to acute metabolic disturbances in astrocytes (hypoxia, ischemia, hypoglycemia…)15, and as such may not be pathognomonic of crs brain pathology or fludarabine neurotoxicity, for example, especially when is mild and focal and may have alternative causes. the relative lack of clasmatodendrosis as well as overall gliosis in most of our cases would support the notion that bbb dysfunction is not a systematic feature in car t-cell treated critical patients when they do not have specific and severe neurological symptomatology. we present 6 patients who died after car t-cell therapy, 2 of whom displayed neurologic clinical manifestations with only one showing significant neuropathological findings. this case had an encephalomyelitis of immune-toxic versus infectious origin, being the findings in the rest of the patients unspecific and minimal, with mild chronic vascular disease and changes suggestive of terminal hypoxic-ischemic etiology. interestingly, the encephalomyelitis changes could not be related to the presence of car t-cells in brain tissue, while we were able to demonstrate car t-cells in brain tissue of one patient without relevant histological changes nor neurological symptoms. in conclusion, patients that die after car t-cell therapy show non-specific or minimal neuropathological changes, especially when they lack evident neurological symptoms and these do not contribute in patient death. car t-cell related direct toxicity may not be the only cause for neurological symptoms, and autopsy may detect other pathological processes, such as disease progression. car t-cell therapy is a new therapeutic approach that needs further research to elucidate its potential adverse effects and the underlying pathological mechanisms behind them. conflicts of interest the authors have no conflicts of interest that relate to the content of this article. acknowledgments we are thankful to the pathology technicians in the immunohistochemistry and molecular sections of the pathology department for the performed studies. funding the authors have not received specific funding for this study. cfl thanks the spanish institute of health carlos iii (projects pi19/00669 and ici19/00025; co-funded by the european union). references 1. kochenderfer, j. n. et al. chemotherapy-refractory diffuse large b-cell lymphoma and indolent b-cell malignancies can be effectively treated with autologous t cells expressing an anti-cd19 chimeric antigen receptor. j. clin. oncol. 33, 540–549 (2015). pmid: 25154820. https://doi.org/10.1200/jco.2014.56.2025 2. turtle, c. j. et al. cd19 car-t cells of defined cd4+:cd8+ composition in adult b cell all patients. j. clin. invest. 126, 2123–2138 (2016). pmid: 27111235. pmcid: pmc4887159. https://doi.org/10.1172/jci85309 3. maude, s. l. et al. chimeric antigen receptor t cells for sustained remissions in leukemia. n. engl. j. med. 371, 1507–1517 (2014). pmid: 25317870. pmcid: pmc4267531. https://doi.org/10.1056/nejmoa1407222 4. turtle, c. j., riddell, s. r. & maloney, d. g. cd19-targeted chimeric antigen receptor-modified t-cell immunotherapy for b-cell malignancies. clin. pharmacol. ther. 100, 252–258 (2016). pmid: 27170467. https://doi.org/10.1002/cpt.392 5. turtle, c. j. et al. immunotherapy of non-hodgkin’s lymphoma with a defined ratio of cd8+ and cd4+ cd19-specific chimeric antigen receptor-modified t cells. sci. transl. med. 8, 355ra116 (2016). pmid: 27605551. pmcid: pmc5045301. https://doi.org/10.1126/scitranslmed.aaf8621 6. oliver-caldes, a. et al. first report of cart treatment in al amyloidosis and relapsed/refractory multiple myeloma. j. immunother. cancer 9, e003783 (2021). pmid: 34876408. pmcid: pmc8655576. https://doi.org/10.1136/jitc-2021-003783 7. hunter, b. d. & jacobson, c. a. car t-cell associated neurotoxicity: mechanisms, clinicopathologic correlates, and future directions. j. natl. cancer inst. 111, 646–654 (2019). pmid: 30753567. https://doi.org/10.1093/jnci/djz017 8. torre, m. et al. neuropathology of a case with fatal car t-cell-associated cerebral edema. j. neuropathol. exp. neurol. 77, 877–882 (2018). pmid: 30060228. https://doi.org/10.1093/jnen/nly064 9. gust, j. et al. endothelial activation and blood–brain barrier disruption in neurotoxicity after adoptive immunotherapy with cd19 car-t cells. cancer discov. 7, 1404–1419 (2017). pmcid: pmc5718945. https://doi.org/10.1158/2159-8290.cd-17-0698 10. schuster, s. j. et al. chimeric antigen 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experience from an academic phase i clinical trial. front. immunol. 11, 482 (2020). pmid: 32528460. pmcid: pmc7259426. https://doi.org/10.3389/fimmu.2020.00482 15. balaban, d., miyawaki, e. k., bhattacharyya, s. & torre, m. the phenomenon of clasmatodendrosis. heliyon 7, e07605 (2021). pmid: 34368479. pmcid: pmc8326353. https://doi.org/10.1016/j.heliyon.2021.e07605 copyright: © 2022 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. 63rd meeting of the french society of neuropathology meeting abstracts , december 3rd. 2021 feel free to add comments by clicking these icons on the sidebar free neuropathology 2:32 (2021) meeting abstracts 63rd meeting of the french society of neuropathology meeting abstracts december 3rd, 2021   the meeting will take place online at: https://us02web.zoom.us/s/87471519403 the french society of neuropathology was created in 1989, succeeding the french club of neuropathology set up in 1965.   submitted: 19 november 2021 accepted: 19 november 2021 published: 23 november 2021 https://doi.org/10.17879/freeneuropathology-2021-3682 keywords: french society of neuropathology, sfnp, meeting abstracts, 63rd meeting dec. 2021   9:00 introduction       short communications 9:00 deciphering the genetic and epigenetic landscape of pediatric bithalamic tumors 9:15 hedgehog-activated anterior skull base meningiomas overexpress gab1 9:30 1p deletion and fgfr1 alterations as specific diagnostic tools to discriminate dlgnt from pa in medullary location: an integrative radiological and histomolecular series of 28 cases 9:45 classification of pituitary neuroendocrine tumors: genomic data on cell lineage 10:00 large brain vessel vasculitis in immunocompromised patients 10:15 cerebellar ataxia with neuropathy and vestibular areflexia syndrome (canvas): a case report 10:30 neuropathological and amyloid peptide differences between down syndrome and familial alzheimer’s disease with duplications and missense mutations in app gene 10:45 characterization of the endolysosomal compartment of noradrenergic neurons of the locus cœruleus in neurodegenerative diseases 11:00 neuropathology analysis as a precious tool to ascertain the pathogenicity of nearsplice / intronic variants in amyotrophic lateral sclerosis: example of a sod1 nearsplice / intronic mutation       conferences 11:15 implication of microglial cells and peripheral macrophages in amyotrophic lateral sclerosis (als) 14:15 the neuro-ceb brainbank: a brief history (15 years! ) of neuropathology 14:30 human brain imaging with stochastic optical reconstruction microscopy (storm) 14:45 ai applied in neuropathology: automated detection of intraepidermal nerve fibers for the diagnosis of small-fiber neuropathies 15:00 histological description of myopathy related to chronic graft versus host disease and characterization of inflammatory infiltrate by imaging mass cytometry 15:15 highlighting autophagy in a fatal case of pompe’s disease 15:30 diagnosis and classification of peripheral nerve vasculitis: attempt at simplification bases on a retrospective study at limoges university hospital between 2014 and 2020 15:45 experimental evaluation of myosuppressive effects of ifn-gamma       general assembly (members only) short communications:   free neuropathol 2:32:3 deciphering the genetic and epigenetic landscape of pediatric bithalamic tumors arnault tauziède-espariat1, marie-anne debily2,3, david castel2,4, jacques grill2,4, stéphanie puget5, alexandre roux6, raphaël saffroy7, guillaume gauchotte8, ellen wahler1, lauren hasty1, fabrice chrétien1, emmanuèle lechapt1, volodia dangouloff-ros9, nathalie boddaert9, philipp sievers10,11#, pascale varlet1# 1 department of neuropathology, ghu paris-psychiatrie et neurosciences, sainte-anne hospital, 75014 paris, france 2 u981, molecular predictors and new targets in oncology, inserm, gustave roussy, université paris-saclay, 94805 villejuif, france 3 université evry, université paris-saclay, 91000 evry, france 4 department of pediatric oncology, gustave roussy, université paris-saclay, 94805 villejuif, france 5 department of pediatric neurosurgery, necker hospital, aphp, université paris descartes, sorbonne paris cite, 75015 paris, france 6 department of neurosurgery, ghu paris-psychiatrie et neurosciences, sainte-anne hospital, 75014 paris, france 7 department of biochemistry and oncogenetic, paul brousse hospital, 94804 villejuif, france 8 department of pathology, chru, nancy, france 9 pediatric radiology department, hôpital necker enfants malades, ap-hp, university de paris, france 10 department of neuropathology, institute of pathology, university hospital heidelberg, heidelberg, germany 11 clinical cooperation unit neuropathology, german consortium for translational cancer research (dktk), german cancer research center dkfz, heidelberg, germany # these authors contributed equally to this work. over the past several years, based on the results of the literature: 1) diffuse midline gliomas (dmg) were defined as a new tumoral type in the 2016 who classification, and 2) four different subtypes are now defined depending on their molecular characteristics, and/or locations: dmg h3.3 k27–mutant, dmg h3.1 or h3.2 k27–mutant, dmg h3-wildtype with ezhip overexpression, and dmg egfr-altered. however, in this rapidly evolving field, a more comprehensive analysis of pediatric bithalamic gliomas is needed. we investigated retrospectively data from 19 pediatric bithalamic gliomas, confirmed by a central radiological review. we also performed a comprehensive clinical, histopathological and molecular evaluation, as well as dna methylation profiling.     free neuropathol 2:32:4 hedgehog-activated anterior skull base meningiomas overexpress gab1 julien boetto1,2, julie lerond2,3, matthieu peyre2,4,5, suzanne tran5,6, pauline marijon2,4, michel kalamarides2,4,5, franck bielle2,3,5,6,7 1 department of neurosurgery, gui de chauliac hospital, montpellier university hospital center, montpellier, france 2 icm inserm u1127 cnrs umr 7225, paris brain institute, paris, france 3 siric curamus (cancer united research associating medicine, university & society) site de recherche intégrée sur le cancer iuc aphp.6 sorbonne université, paris, france 4 department of neurosurgery, ap-hp, hôpital pitié-salpêtrière, paris, france 5 sorbonne université, upmc université paris 06, paris, france 6 department of neuropathology, ap-hp, hôpital pitié salpêtrière, paris, france 7 onconeurotek, ap-hp, hôpital pitié-salpêtrière, paris, france anterior skull base meningiomas were previously showed to have mutations activating the hedgehog (hh) signalling pathway. however, identification of hh-activated tumours is hampered by the lack of a reliable immunohistochemical marker. we report gab1 as a potential immunohistochemical marker of hh-activated meningiomas. gab1 immunolabeling was compared to smo mutation detection with sanger and ngs techniques as well as hh pathway activation study through mrna expression level analyses in a discovery set of 110 anterior skull base meningiomas. we showed that a cut-off score of 250 for the gab1 expression score (from 0 to 400) lead to excellent detection of hh pathway mutations (sensitivity 100%, specificity 86%). the prospective validation set of 21 meningiomas confirmed the excellent negative predictive value of gab1 expression score. gab1 immunohistochemistry is a fast and cost-efficient tool to screen anterior skull base meningiomas and to facilitate the identification of candidate tumours for targeted therapy.     free neuropathol 2:32:5 1p deletion and fgfr1 alterations as specific diagnostic tools to discriminate dlgnt from pa in medullary location: an integrative radiological and histomolecular series of 28 cases alice métais1, arnault tauziède-espariat1, wael yacoub2, thomas kergrohan3, raphael saffroy4, dominique figarella-branger5, emmanuele uro-coste6, annick sevely7, delphine larrieu-cirron8,9, alexandre roux10, sandro benichi11, lila saidoun3, yassine bouchoucha12, françois doz12, volodia dangoulof-ros2, jacques grill3, pascale varlet1 1 department of neuropathology, ghu paris-psychiatrie and neurosciences, sainte-anne hospital, paris, france 2 department of pediatric radiology, hôpital necker-enfants malades, paris, france 3 département de cancérologie de l'enfant et de l'adolescent, institut gustave roussy, université paris-sud, villejuif, france 4 department of biochemistry, paul-brousse hospital, villejuif, france 5 department of pathology and neuropathology, timone hospital, marseille, france 6 département d'anatomie et cytologie pathologiques, chu de toulouse, iuct-oncopole, toulouse, france 7 department of radiology, purpan university hospital, toulouse, france 8 department of neurology, toulouse university hospital, toulouse, france 9 department of medical oncology, iuct-oncopole, toulouse, france 10 department of neurosurgery, ghu paris-psychiatrie et neurosciences sainte-anne hospital, paris, france 11 department of pediatric neurosurgery, hôpital necker-enfants malades, paris, france 12 siredo center (care, innovation, research in, children, adolescent and young adults oncology), institut curie, paris, france new tumor types such as diffuse leptomeningeal glioneuronal tumors (dlgnt) have been added to the 2016 who. their distinction from others intramedullary gliomas is poorly defined. we retrospectively studied a cohort of 28 children with low-grade intramedullary tumors. the radiological, histopathological and molecular portrait including methylation profiling was established as well oncological treatments data. we observed two main tumor types: dlgnt and pilocytic astrocytomas (pa) that were hardly distinguishable by neuroradiology or histopathology alone. the major criteria to segregate these two tumor types presenting a mapkinase alteration was the 1p deletion. we observed that these tumors interested different populations (infants for pa, school children for dlgnt). these tumors evolved insidiously during a long period of time, remission was rarely achieved (5/28). two cases had a deadly evolution. we recommend to systematically assess 1p deletion in intramedullary low-grade glioma. improving the management of these diseases remains a major challenge.     free neuropathol 2:32:6 classification of pituitary neuroendocrine tumors: genomic data on cell lineage villa c.1,2, armignacco r.2, jouinot a.2, perlemoine k.2, baussart b.3, bertherat j.4,2, gaillard s.3, assié g. 4,2 1 department of neuropathology, hôpital de la pitié-salpêtrière aphp sorbonne université, 47-83 bd de l’hôpital 75651 paris, france 2 inserm u1016, institut cochin, 75014 paris, france; cnrs umr 8104, 75014 paris, france; université paris descartes-université de paris, 75006 paris, france 3 department of neurosurgery, hôpital de la pitié-salpêtrière aphp sorbonne université, 76561 paris, france 4 department of endocrinology, center for rare adrenal diseases, assistance publique-hôpitaux de paris, hôpital cochin, 75014 paris, france the 2017 world health organization (who) classification of pituitary adenomas is based on cell lineage and transcription factors (tfs). transcriptome of 134 pitnets (rna sequencing) was used to determine tfs expression at mrna level, and to provide a canonical transcriptome signature for each cell-types. pathological study of the present serie of pitnets included the histological examination and the immunohistochemical tests for all pituitary hormones, proliferation markers and tfs including gata3. pit1 lineage : based on transcriptome classification, accurate thresholds of immunoexpression for gh, prl and tsh were established in order to define the different pit-1 subtypes. t-pit lineage : t-pit mrna showed the expected expression in corticotroph pitnets (35/35), lower in silent ones (wilcoxon p<10-5). gonadotroph lineage : sf1 mrna expression showed the expected high expression in gonadotroph pitnets (29/29), but also in a subset of somatotroph pitnets (9/21). sf1 immunopositivity was confirmed in this somatotroph subgroup.     free neuropathol 2:32:7 large brain vessel vasculitis in immunocompromised patients isabelle plu1, elie haddad2, arnaud fekkar3, sophie bonnin4, natalia shor5, valérie touitou4, véronique leblond6, nicolas weiss7, eric caumes2, sophie demeret7, valérie pourcher2, danielle seilhean1 sorbonne université, ap-hp, hôpitaux universitaires pitié-salpêtrière charles foix, 75013 paris, france 1 département de neuropathologie 2 service de maladies infectieuses et tropicales 3 service de parasitologie-mycologie 4 service d’ophtalmologie 5 département de neuroradiology 6 service d’hématologie 7 service de neurologie inflammation of the large arteries of the brain is a relatively rare condition whose best known mechanism is hyperimmunity with granulomatous reaction or circulating immune complexes. some vasculitides are secondary to a systemic hyperimmune disease or to an infectious disease (purulent or tuberculous meningitis). we describe here the occurrence of cerebral infarcts in three immunocompromised patients with neutrophilic aseptic meningitis. the mri appearance was vasculitis of the anterior cerebral arteries (one case) or basilar arteries (two cases). pcr found aspergillus fumigatus in the csf two days before a positive cavum biopsy in one case. in the other two cases, aspergillus fumigatus was found postmortem. the neuropathologic appearance was that of filament-rich necrotizing arteritis. there was no other organ involvement. these observations highlight a probably underestimated cause of central nervous system arteritis whose curability relies on early diagnosis based on repeated and extensive mycological testing in csf.     free neuropathol 2:32:8 cerebellar ataxia with neuropathy and vestibular areflexia syndrome (canvas): a case report susana boluda1, david grabli2, vincent huin3, giulia coarelli4, alexandra durr4, danielle seilhean1 1 sorbonne université, institut du cerveau paris brain institute icm, inserm, cnrs, aphp, laboratoire neuropathologie raymond escourolle, hôpital de la pitié salpêtrière, paris, france 2 ap-hp, pitié salpêtrière university hospital, department of neurology, sorbonne university, paris, france 3 sorbonne université, institut du cerveau paris brain instituteicm, inserm, cnrs; university of lille, univ. lille, inserm, chu lille, u1172 lilncog (jparc) lille neuroscience & cognition, lille, france 4 sorbonne université, institut du cerveau paris brain institute icm, inserm, cnrs, aphp, genetics department, hôpital de la pitié salpêtrière, paris, france canvas is a rare disease characterized by an intronic biallelic aaggg expansion in rfc1. we report the case of a 74-year-old man who presented with cramps, mixed sensory and cerebellar ataxia, vestibular areflexia and neuropathic pain in addition to parkinsonian symptoms. macroscopic examination showed atrophy of the cerebellar vermis and pallor and atrophy of the posterior columns. microscopic examination revealed, in addition to diffuse lewy body pathology, axonal loss in the dorsal columns and a moderate loss of purkinje cells in the cerebellum. there was marked astrocytic gliosis in contact with the dendrites in the spinal cord and molecular layer of the cerebellum as well as disorganization of the radial glia in the cerebellum. we report the first complete neuropathological examination of the brain and spinal cord of an rfc1 patient and describe astrocytic abnormalities that would explain the severe clinical phenotype in the absence of severe neuronal loss.     free neuropathol 2:32:9 neuropathological and amyloid peptide differences between down syndrome and familial alzheimer’s disease with duplications and missense mutations in app gene amal kasri1, léa durix1, susana boluda1,2, lev stimmer1, eleni. gkanatsiou3, gunnar brinkmalm3, yannick vermeiren4,5, sarah e. pape6, peter p. de deyn4,5, charles duyckaerts1,2, henrik zetterberg3,7, andre strydom6, marie-claude potier1 1 paris brain institute, icm, cnrs umr7225 inserm u1127 – upmc, paris, france 2 laboratoire de neuropathologie r escourolle, hôpital de la pitié-salpêtrière, ap-hp, paris, france 3 institute of neuroscience and physiology, the sahlgrenska academy at the university of gothenburg, gothenburg, sweden 4 department of biomedical sciences, neurochemistry and behavior, institute born-bunge, university of antwerp, antwerp, belgium 5 department of neurology and alzheimer center, university of groningen, university medical center groningen (umcg), groningen, netherlands 6 institute of psychology and neuroscience, king’s college london, 16 de crespigny park, london, united kingdom 7 sahlgrenska university hospital, clinical neurochemistry laboratory, mölndal, sweden cerebral amyloid angiopathy (caa) is present in 80% of alzheimer's disease (ad) patients. however, caa is more prominent in familial cases with app mutations or duplications (dupapp) and in individuals with down syndrome (ds). in order to explain these differences, we investigated the alterations in the endo-lysosomal pathway, aβ species and caa grading in post-mortem human brain tissues. using immunohistochemistry, we identified increased rab5 puncta size in all cases except those with app mutations compared to controls. moreover, we found elevated levels of aβ40 but not aβ42 in dupapp and in ds correlating with caa grading. altogether, these results suggest that caa arises from specific accumulation of aβ40 species or, alternatively, that aβ40 is the principal aβ species produced in dupapp and ds and is more prone to aggregation in blood vessels. the ongoing aβ and caa grading analysis aim to unravel pathophysiological mechanisms involved in specific aβ production and deposits.     free neuropathol 2:32:10 characterization of the endolysosomal compartment of noradrenergic neurons of the locus cœruleus in neurodegenerative diseases marta fructuoso1, susana boluda1,2, lev stimmer1, yannick vermeiren3, peter p. de deyn3, debby van dam3, andre strydom4, charles duyckaerts1,2, marie-claude potier1 1 icm institut du cerveau et de la moelle épinière, cnrs umr7225, inserm u1127, upmc, hôpital de la pitié-salpêtrière, 47 bd de l’hôpital, paris, france 2 laboratoire de neuropathologie r escourolle, hôpital de la pitié-salpêtrière, ap-hp, 75013 paris, france 3 department of neurology and alzheimer center, university medical center groningen, university of groningen, groningen, the netherlands. laboratory of neurochemistry and behavior, department of biomedical sciences, institute born-bunge, university of antwerp, antwerp, belgium 4 department of forensic and neurodevelopmental sciences, institute of psychiatry, psychology & neuroscience, king's college london, london, uk degeneration of the locus coeruleus (lc) is a common feature in parkinson's disease (pd), alzheimer's disease (ad), and down syndrome (ds). lc is the main source of noradrenaline in the brain, and neuronal loss in this nucleus is associated with dementia. endolysosomal abnormalities may contribute to the accumulation of toxic proteins. we hypothesize that this could be one mechanism of lc neurons vulnerability (jpnd-heroes, www.heroes-jpnd.eu). using post-mortem paraffin fixed lc of control and pathological cases, we confirm the presence of aβ plaques and p-tau in ad and ds brains, and lewy bodies in pd samples. analysis of confocal microscopy images of immunofluorescence revealed that area of the rab5 positive puncta of endosomes was increased in ad whereas the area cathepsin b positive puncta of lysosomes was reduced in pd. our results suggest that endolysosomal alterations could be a mechanism implicated in the degeneration of noradrenergic neurons, and might be disorder-specific.     free neuropathol 2:32:11-12 neuropathology analysis as a precious tool to ascertain the pathogenicity of nearsplice / intronic variants in amyotrophic lateral sclerosis: example of a sod1 nearsplice / intronic mutation françois muratet1, elisa teyssou1, aude chiot1, séverine boillée1, christian s. lobsiger1, delphine bohl1, beata gyorgy1, justine guegan1, yannick marie1, maria-del-mar amador1,2, françois salachas1,2, vincent meininger3, emilien bernard4,5, jean-christophe antoine6, jean-philippe camdessanché6, william camu7, cécile cazeneuve8, anne-laure fauret-amsellem8, eric leguern1,8, kevin mouzat9, claire guissart9, serge lumbroso9, philippe corcia10,11, patrick vourc’h11,12, aude-marie grapperon13, shahram attarian13, annie verschueren13, danielle seilhean1,14* and stéphanie millecamps1* 1 cm, institut du cerveau, inserm u1127, cnrs umr7225, sorbonne université, paris, france 2 ap-hp, département de neurologie, centre de référence sla ile de france, hôpital de la pitié-salpêtrière, paris, france 3 hôpital des peupliers, ramsay générale de santé, paris, france 4 centre de référence sla, hôpital neurologique pierre wertheimer, hospices civils de lyon, université de lyon, bron, france 5 institut neuromyogène, cnrs umr5310, inserm u1217, faculté de médecine rockefeller, université claude bernard lyon i, lyon, france 6 service de neurologie, centre de ressource et de compétence sla, hôpital nord, chu de saint-etienne, saint-etienne, france 7 centre de référence sla, hôpital gui de chauliac, chu et université de montpellier, montpellier, france 8 département de génétique et cytogénétique, unité fonctionnelle de neurogénétique moléculaire et cellulaire, aphp, hôpital pitié-salpêtrière, paris, france 9 laboratoire de biochimie et biologie moleculaire, chu nimes, nîmes, motoneuron disease: pathophysiology and therapy, inm, inserm, univ. montpellier, montpellier, france 10 centre sla, chu tours, tours, france 11 umr 1253, université de tours, inserm, tours, france 12 service de biochimie et biologie moléculaire, chu tours, tours, france 13 centre de référence pour les maladies neuromusculaire et la sla, chu de marseille, hôpital de la timone, marseille, france 14 ap-hp, département de neuropathologie, hôpital pitié-salpêtrière, paris, france * equal contribution among etiologic factors assumed to be responsible of amyotrophic lateral sclerosis (als), several plausible causative genes have emerged. it becomes crucial to determine the pathogenicity of any genetic variant identified through whole exome/genome sequencing analysis, including those located in non-coding regions. we describe a c.358-10t>g nearsplice/intronic variant in the sod1 gene, encoding superoxide dismutase 1, as the second prominent mutation among the sod1 related-french als families. this variant leads to the addition of three amino acids in the protein sequence and impairs the protein secondary structure. biochemical and neuropathological analyses performed on patient tissue revealed massive cytoplasmic sod1 and neurofilament accumulation in spinal motor neurons, similar to those observed in spinal cord of patients with d83g or g93d sod1 mutations. these neuropathology findings ascertain this variant pathogenicity, which is a crucial information in the context of patient enrolment in ongoing clinical trials targeting sod1 by antisense oligonucleotides     conferences:   free neuropathol 2:32:13 implication of microglial cells and peripheral macrophages in amyotrophic lateral sclerosis (als) aude chiot1,2 , félix berriat f1, matthieu ribon1, sakina zaïdi1, charlène iltis1,3, michel mallat1, delphine bohl1, stéphanie millecamps1, danielle seilhean1,4, christian s lobsiger1, séverine boillée1 1 sorbonne université, institut du cerveau – paris brain institute icm, inserm, cnrs ap-hp, hôpital de la pitié-salpêtrière, paris, france 2 current address: department of molecular microbiology and immunology, oregon health and science university, portland, or 97239, usa 3 current address: cancer biology & genetics program, memorial sloan kettering cancer center, new york, ny 10065, usa 4 département de neuropathologie, assistance publique hôpitaux de paris (ap-hp), hôpital pitié-salpêtrière, paris, france in mouse models of amyotrophic lateral sclerosis (als), the most common motor neuron (mn) disease of the adult, microglial cells/ macrophages have been shown to participate to disease progression. however, the respective contributions of microglial cells (mg) and peripheral macrophages (pm) were not documented. we have now shown that pm were present around mn axons of als patients, both in motor roots and peripheral nerves. in als mice, pm were progressively activated and their infiltration into the spinal cord was very limited and disease-length dependent. transcriptomics analysis showed that mg and pm reacted differently to mn degeneration. replacing pm by macrophages more neurotrophic, at disease onset, increased als mouse survival and downregulated not only pm activation in peripheral nerves but also suppressed proinflammatory responses of mg in the spinal cord, with a switch toward neuronal support. thus, targeting pm, directly at the periphery, could be of therapeutic value in als.     free neuropathol 2:32:14 the neuro-ceb brainbank: a brief history (15 years! ) of neuropathology marie-claire artaud-botté1, sabrina leclère-turbant2, charles duyckaerts2, marie laure martin-négrier3, franck letournel4, neuro-ceb neuropathologist’ network5, isabelle plu6, susana boluda2,6, danielle seilhean2,6 1 association neuro-ceb (paris, france): arsla, association france dft, association france parkinson, csc, fondation arsep, fondation vaincre alzheimer 2 icm-paris brain institute cnrs umr7225 inserm u1127 – sorbonne university gh pitié-salpêtrière, paris france 3 gh pellegrin, bordeaux, france 4 chu d’angers, france 5 neuro-ceb neuropathologist’ network: susana boluda (gh pitié-salpêtrière, paris, france), jean boutonnat (hôpital michalon, grenoble, france), fanny burel-vandenbos (hôpital pasteur, nice, france), françoise chapon (hôpital côte de nacre, caen, france), dan-christian chiforeanu (hôpital pontchaillou, rennes, france), vincent deramecourt (chu salengro, lille, france), charles duyckaerts (gh pitié-salpêtrière, paris, france), maxime faisant (hôpital côte de nacre, caen, france), catherine godfraind (chu gabriel montpied, clermont-ferrand, france), béatrice lannes (hôpital de hautepierre, strasbourg, france), annie laquerrière (chu charles nicolle, rouen, france), franck letournel (chu d’angers, france), benoit lhermitte (hôpital de hautepierre, strasbourg, france), florent marguet (chu charles nicolle, rouen, france), marie-laure martin-négrier (gh pellegrin, bordeaux, france), andré maues de paula (chu timone 2, marseille, france), claude-alain maurage (chu salengro, lille, france), david meyronet (hôpital pierre wertheimer, lyon, france), isabelle plu (gh pitié-salpêtrière, paris, france), valérie rigau (hôpital gui de chauliac, (montpellier, france) 6 laboratoire de neuropathologie, gh pitié-salpêtrière, ap-hp-sorbonne université, paris, france the neuro-ceb national brainbank was founded in 2006 at the initiative of patients’ associations, to support a post mortem brain donation program. the missions are to collect, sample, prepare and store nervous tissues (brain and spinal cord), taken post mortem from "control" subjects (not suffering from neurological diseases) and from patients suffering from alzheimer or parkinson disease, multiple sclerosis, ataxia, amyotrophic lateral sclerosis, frontotemporal lobar degeneration, or cadasil. the biological ressources are provided to research teams presenting a suitable project reviewed by a scientific committee. the organization relies on a network of neuropathologists from 14 hospitals. they are in charge of brain sampling and neuropathological diagnosis. the expertise of these neuropathologists ensures a high quality of the samples. their involvement over the past 15 years alongside research teams, has led to the publication of more than 100 scientific articles. this illustrates the impact of the neuro-ceb biobank at an international level.     free neuropathol 2:32:15 human brain imaging with stochastic optical reconstruction microscopy (storm) philippe codron1,2,3, franck letournel1,2, guy lenaers1,3, arnaud chevrollier3 1 service de neurologie, centre hospitalier universitaire d’angers, angers, france 2 service de neurobiologie et neuropathologie, centre hospitalier universitaire d’angers, angers, france 3 équipe mitolab, institut mitovasc, inserm u1083, cnrs 6015, université d'angers, angers, france the recent development of stochastic optical reconstruction microscopy (storm) has contributed to major advances in neuroscience. however this technique is restricted to cultured cells and rodent brain, and no experiment on human samples has been reported so far. to this end, we combined cellular microscopy protocols with neuropathology tissue preparation techniques to characterize physiological and pathological structures in brain samples with 2d-, 3dand two-color storm. this approach proved to be particularly effective at visualizing the organization of dense protein inclusions in samples from patients affected with neurodegenerative diseases. these very first results open further gates to a more comprehensive understanding of the human brain organization and revelations about the underlying mechanisms responsible for neurodegenerative disorders.     free neuropathol 2:32:16 ai applied in neuropathology: automated detection of intraepidermal nerve fibers for the diagnosis of small-fiber neuropathies labeyrie c1,2,3, adam c.2,3, lorenzo a.4, brunel n.4, blot v.4, sevillia h.4, hervault d.4, trassard o.3, morassi o.1, adams d.1,4, guettier c2,3 1 service de neurologie adulte, crmr neuropathie amyloïde familiale et autres neuropathies périphériques rares (nnerf), chu bicêtre, 78 rue du gal leclerc, 94275 le kremlin-bicêre, aphp, france 2 service d’anatomopathologie, chu bicêtre, 78 rue du gal leclerc, 94275 le kremlin-bicêre, aphp, france 3 institut biomédical de bicêtre, ums32, chu bicêtre, 78 rue du gal leclerc, 94275 le kremlin-bicêre, aphp, france 4 quantmetry, 52 rue d’anjou, 75008 paris, france 5 umr1195, université paris sud, faculté de médecine bicêtre, 80 rue du gal leclerc, 94276 le kremlin-bicêre, aphp, france the measurement of intra-epidermal nerve fiber density (ienfd) in skin biopsy is the gold standard for the diagnosis of small fiber neuropathy (sfn). this recently described entity causes neuropathic pain and dysautonomia. numerous etiologies are described, although the majority of them remain idiopathic. the classic dfnie reading method, derived from a 3mm punch biopsy and 50 micron sections, is based on the anti-pgp9.5 labelling with a visual count related to the analyzed length, a long process thus slowing down its development. thanks to the digitization of the double-labeled slides in immunofluorescence anti-pgp9.5 and anti-coll iv since 2012 allowing the computer labeling of the traversing fibers, we were able to train an automated reading algorithm which obtains an accuracy of 71% and a recall of 77%.     free neuropathol 2:32:17 histological description of myopathy related to chronic graft versus host disease and characterization of inflammatory infiltrate by imaging mass cytometry amélie bourhis1,2, baptiste hervier3,4, arnaud uguen1,2, pascale marcorelles1, patrice hemon2, sarah léonard-louis5,6 1 pathology department, hospital morvan, brest, france 2 lbai, umr1227, univ brest, inserm, labex igo, brest, france 3 internal medicine and clinical immunology department, french referral centre for rare neuromuscular disorders, hôpital pitié-salpêtrière, aphp, paris, france 4 inserm umr-s 1135, cimi-paris, upmc & sorbonne université, paris, france 5 neuropathology department, aphp, pitié salpétrière, sorbonne university, paris, france 6 institute of myology, neuromuscular reference center nord/est/ile de france, ap-hp, pitié-salpêtrière hospital, sorbonne university, paris, france chronic graft versus host disease (cgvhd) is a long-term side effect of allogenic hematopoietic cells transplantation, affecting muscles in 3,4 to 7,7% of patients. this study proposes a complete histological and immunohistological description of myopathy in the context of cgvhd and a characterization of the inflammatory infiltrate by imaging mass cytometry technology (imc). histological description was performed on 19 patients and divided patients into three groups: 10 patients with no fiber necrosis and no or very low inflammatory infiltrate, 6 patients with fiber necrosis and high inflammatory infiltrate and 3 patients with fasciitis. all patients had variations in fiber size, diffuse mch-i and perifascicular mch-ii immunostaining. 7 patients were analyzed by imc. the main inflammatory population was macrophage cells, but eosinophils represented more than 5% of inflammatory cells in 5 cases and seemed to be specific of cgvhd induced myopathy as they were already described in other cgvhd locations.     free neuropathol 2:32:18 highlighting autophagy in a fatal case of pompe’s disease julien gouju1, philippe codron1,2, caroline savary1, aleksandra nadaj-pakleza3, marco spinazzi1,2, franck letournel1 1 département de pathologie – chu angers, angers, france 2 département de neurologie – chu angers, angers, france 3 centre de référence des maladies neuromusculaires nord-est-ile de france, service de neurologie, hôpitaux universitaires de strasbourg, strasbourg, france pompe’s disease is a rare autosomal recessive disorder caused by mutations in the acid α-glucosidase (gaa) gene. alpha-glucosidase deficit leads to lysosomal and non-lysosomal accumulation of glycogen with different forms of the disease, ranging from early-onset to late-onset form (lof). we report the case of a lof treated by enzyme replacement therapy (ert), who died of myocardial infarct. we describe expression of several lysosomal and autophagy markers in several tissues. we observed a glycogen-accumulation in brain, associated to increased expression of p62, lc3a/b and lamp-2 localized in the neocortex and hippocampus. both neurons and glial cells were affected, however cerebral vessels were normal. similar results were observed in muscle, heart and liver. our data suggest that autophagy impairment could be an important pathogenic mechanism in the muscles and the nervous system of patients with pompe’s disease     free neuropathol 2:32:19 diagnosis and classification of peripheral nerve vasculitis: attempt at simplification bases on a retrospective study at limoges university hospital between 2014 and 2020 camille guibert1, laurent magy2, mathilde duchesne3 1 service d’anatomie pathologique, chu de dijon, france 2 service de neurologie, chu de limoges, france 3 service d’anatomie pathologique et laboratoire de neurologie, chu de limoges, france peripheral nerve vasculitis remain rare and challenging to diagnose. we tried to simplify criteria of the pns 2010 by searching for those strongly associated with certain and probable vasculitis, and to propose a management of the biopsy. this retrospective study included 46 patients with definite and probable vasculitis according to the criteria of the pns 2010 who underwent neuromuscular biopsy and 21 controls (neurolymphomatosis and cidp). clinical, biological and pathological features were collected. criteria discriminating vasculitis from controls were asymmetry, axonal involvement, biological inflammation, t cells in vessel wall, and nerve haemosiderin deposits. two criteria distinguish definite and probable vasculitis: ovoid and endoneurial haemosiderin deposits. the techniques useful for pathological diagnosis are serial cuts, typing of inflammatory elements and perls staining. we proposed a slightly simplified classification of vasculitis according to the pns 2010 and a management of neuromuscular biopsy in this indication.     free neuropathol 2:32:20 experimental evaluation of myosuppressive effects of ifn-gamma cyrielle hou1; baptiste périou1, marianne gervais1, juliette berthier1, yasmine baba-amer1, sarah souvannanorath1,2, edoardo malfatti1,2, frédéric relaix1,2, françois jérôme authier1,2 1 inserm u955-eq. relaix, faculté de santé, université paris est-créteil; créteil, france 2 hu henri mondor (aphp), centre de référence des maladies neuromusculaires, département de pathologie, créteil, france dysimmune and inflammatory myopathies (dims) are acquired idiopathic myopathy associated with immune response dysregulation. inclusion body myositis (ibm), the most common dims, is characterized by endomysial infiltrates of cytotoxic t lymphocytes cd8, muscle type ii-interferon (ifnγ) signature, and by the lack of response to immunomodulatory therapies. we showed that ibm differs from other myopathies by the presence of chronic degenerative myopathic features including the altered functions of skeletal muscle stem cells. here, we demonstrated that, in vitro, protracted ifnγ treatment inhibits the activation, proliferation, migration, differentiation, and fusion of myogenic progenitor cells and promotes their senescence through jak-stat-dependent activation. jak-stat inhibitor, ruxolitinib abrogates the deleterious effects of ifnγ, in conclusion, our results indicate that ifnγ could impair muscle regeneration in the context of inflammatory myopathies and that jak inhibitors could represent interesting therapies for immune myopathies with ifnγ signature.   copyright: © 2021 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. 61st annual meeting of the canadian association of neuropathologists association canadienne des neuropathologistes (canp-acnp) meeting abstracts feel free to add comments by clicking these icons on the sidebar free neuropathology 2:29 (2021) meeting abstracts 61st annual meeting of the canadian association of neuropathologists association canadienne des neuropathologistes (canp-acnp) meeting abstracts october 14–16, 2021 submitted: 26 october 2021 accepted: 27 october 2021 published: 29 october 2021   the canadian association of neuropathologist – association canadienne des neuropathologistes (canp-acnp) held their 61st annual meeting via zoom from october 14th to 16th, 2021, under the leadership of dr. peter gould, president of the canp-acnp, dr. peter schutz, secretary treasurer of the canp-acnp, and with technical support from canp administrators heather dow and colleen fifield. the academic program comprised 17 scientific abstracts, 8 unknown cases, the presidential symposium on epilepsy and neoplasms, and a neuropathology in practice session on the implementation of the new who classification of cns neoplasms in canada. digital pathology images from the 8 unknown cases are available for viewing online (www.canp.ca). the unknown case sessions were moderated by dr. andrew gao. the presidential symposium 2021 on epilepsy and neoplasms featured the david robertson lecture given by dr. eleonora aronica entitled evolving classification of epilepsy-associated tumors: understanding epileptogenesis, the gordon mathieson lecture delivered by dr. harvey sarnat on why are some cerebral malformations more epileptogenic than others? clues from developmental neuropathology, and the jerzy olszewsky lecture presented by dr. maria thom on neuropathology in sudden and unexpected death in epilepsy and future directions. the program was completed dr. george ibrahim’s lecture on epilepsy as a network disorder. the mary tom award for best clinical science presentation by a trainee went to dr. suzy kosteniuk (supervisor dr. s. das), and the morrison h. finlayson award for best basic science presentation by a trainee was won by dr. nicole schwab (supervisor dr. l.n. hazrati). the following abstracts were presented at the 61st annual meeting of the canadian association of neuropathologists – association candienne des neuropathologistes (canp-acnp) in october 2021. https://doi.org/10.17879/freeneuropathology-2021-3671 keywords: canadian association of neuropathologists, canp, meeting abstracts, 61st meeting oct. 2021 contents abstract 1: histologic and clinical spectrum of brain tumours harbouring fgfr3-tacc3 fusions abstract 2: ganglion cell maturational markers in peripheral neuroblastic tumours of children abstract 3: long term disease-free survival after surgical resection only: malignant idh-mutated astrocytoma versus ganglioglioma abstract 4: diagnostic challenges in unusual high-grade gliomas abstract 5: regional variability of α-synuclein inclusion size in multiple system atrophy abstract 6: cognitive resilience in individuals with severe alzheimer’s disease neuropathology abstract 7: linking iron homeostasis to vulnerability patterns in progressive supranuclear palsy abstract 8: friedreich cardiomyopathy is a secondary desminopathy abstract 9: glial senescence (not tau) is the driver of post-concussive symptoms abstract 10: hail to astrogliosis! the unsung hero of the cns tissue reaction to the spinal cord injury abstract 11: ischemic-like pathology in aberrant white matter tracts of fetal holoprosencephaly: a case series abstract 12: brain pathology in patients with congenital heart disease abstract 13: characterizing the hippocampal dentate gyrus involvement in temporal lobe epilepsy abstract 14: deep learning approaches to deciphering intra-tumoural heterogeneity in glioblastoma abstract 15: integrating morphologic and molecular histopathological features through whole slide image registration and deep learning abstract 16: neuropathology of eight cases of the new brunswick cluster of neurological syndrome of unknown cause (nsuc) abstract 17: covid-19 pandemic impact on surgical neuropathology services at london health sciences centre     abstract 1 free neuropathol 2:29:4 histologic and clinical spectrum of brain tumours harbouring fgfr3-tacc3 fusions francesca gianno1, jennifer a. chan2, phedias diamandis3,4, john a. maguire5, qi zhang6, cynthia hawkins1, lili-naz hazrati1 1 department of laboratory medicine and pathobiology, university of toronto, toronto, ontario, canada; department of pathology, the hospital for sick children, toronto, ontario, canada 2 university of calgary, calgary, alberta, canada 3 princess margaret cancer centre/laboratory medicine program, university health network, toronto, ontario, canada 4 departments of laboratory medicine and pathobiology and medical biophysics, university of toronto, toronto, ontario, canada 5 department of pathology & laboratory medicine, costal health-vancouver general hospital, canada 6 department of pathology, london health sciences centre, london, canada fibroblast growth factor receptor (fgfr) gene family alterations, including point mutations and fusions, have been described in brain tumours as oncogenic drivers (ng 2013; 45(6): 602–612). fgfr3-tacc3 fusions were initially described in idh wildtype glioblastomas (science 2012; 337:1231–1235) and are most frequently seen in morphologic or molecular gbm although occasional who grade 2 astrocytomas may harbour this alteration. the significance of this alteration as a single driver in low-grade glioma is unclear. in order to better understand the clinical implications of finding this alteration, a series of low-grade gliomas underwent molecular testing at sickkids. the series includes six samples of diffuse low-grade gliomas, with a mean age of 24.8 years (range 1 to 44 years), three males and three females. five out of six tumours are localized in the supratentorial region, and one in the spinal cord. three patients presented with seizures, one was asymptomatic. morphologic appearance showed different aspects, from cells with oligo-like features in a neuropil-rich stroma to spindle cells with fibrillary cytoplasm in a loose microcystic background. single nucleotide polymorphism (snp) array was performed on two samples and one of them shows the gain of chromosome 7 and loss of chromosome 10, indicating a molecular profile of high grade glioma. follow-up was available for four patients, all are alive without progressive disease with follow-up ranging from six months to two years. longer follow-up will be required to determine the significance of this alteration in low grade glioma.   abstract 2 free neuropathol 2:29:5 ganglion cell maturational markers in peripheral neuroblastic tumours of children harvey b. sarnat1 1 university of calgary & alberta children's hospital, calgary, alberta, canada peripheral neuroblastic tumours of neural crest origin are the most frequent solid neoplasms outside the cns in children. neuroblastoma/ganglioneuroblastoma have a natural evolution of histological differentiation over time. together with mitosis-karyorrhexis index and patient age (international neuroblastoma pathology classification criteria), ganglion cell maturation determines grading and prognosis. maturation presently is assessed only by h&e histology. methods: immunocytochemical markers of neuroblast maturation in fetal cns were applied to peripheral neuroblastic tumours arising in adrenal medulla or sympathetic chain. resected tumours of 4 toddlers were examined using antibodies demonstrating neuronal identity and maturation: map2; synaptophysin; chromogranin-a; neun; keratan sulfate (ks); glutamate receptor antibody (glur2). others: ki67; s-100β protein; vimentin; nestin; α-b-crystallin; neuroblastoma marker phox2b. results: degrees of neuroblastic maturation were demonstrated by map2, chromogranin, synaptophysin, ks and glur2; neun was negative consistent with sympathetic neural crest lineage. ks was sparsely distributed within the tumours adherent to somata and proximal neuritic trunks. phox2b did not distinguish maturational stages. ki67 was expressed in scattered primitive cells that also expressed vimentin and nestin, but not in differentiated neoplastic neurons. s-100β protein and α-b-crystallin labeled schwann cells, especially schwannian ganglioneuroma. conclusions: immunocytochemical markers of neuroblast maturation in fetal brain also are useful in peripheral neuroblastic tumours, providing greater precision than histology alone. the most practical are map2 and synaptophysin. prognosis and choice of treatment including chemotherapy might be influenced.   abstract 3 free neuropathol 2:29:6 long term disease-free survival after surgical resection only: malignant idh-mutated astrocytoma versus ganglioglioma roland n. auer1,5, egiroh omene2, sarah e. edwards1, kotoo meguro1, daryl r. fourney1, jose f. tellez-zenteno2, gary r. w. hunter2, kyle moulton3, vijayananda kundapur4 1 department of surgery (neurosurgery) , university of saskatchewan, saskatoon, saskatchewan, canada 2 department of neurology, university of saskatchewan, saskatoon, saskatchewan, canada 3 department of radiology, university of saskatchewan, saskatoon, saskatchewan, canada 4 department of oncology, university of saskatchewan, saskatoon, saskatchewan, canada 5 department of pathology and laboratory medicine, university of saskatchewan, saskatoon, saskatchewan, canada gangliogliomas are important to distinguish from anaplastic astrocytoma and glioblastoma multiforme, to avoid potentially damaging radiation and for accurate prognostication. two patients with a seizure presentation at age 31 and 30 had large, well demarcated brain tumors each 4.3 cm with no suggestion of diffuse glioma on imaging. both patients requested tumor resection. the first patient intra-operatively was found to have the tumor in close proximity to the motor cortex, and resection was thus incomplete, leaving residual tumor in situ. the second patient had a gross total resection. an initial diagnosis of ganglioglioma, was changed to malignant astrocytoma based on idh1 mutation, loss of atrx, abundant mitoses and a high ki67 labelling index. conventional histology revealed glial, neuronal and intermediate forms in both tumors. both patients declined treatment following neurosurgery and remain without tumor growth on neuroimaging at 6 and 3 years later, respectively. the findings illustrate the conundrum of tumors diagnosed as malignant astrocytomas based on molecular profile, but showing no growth after no treatment, as well as the superiority of resection over needle biopsy, as the first tumor was called a diffuse astrocytoma on a needle biopsy prior to resection. without diffuse astrocytoma appearing on long-term follow-up in the absence of radiochemotherapy, a diagnosis of diffuse, malignant astrocytoma is increasingly untenable as years pass. both patients and their clinicians question the malignant diagnosis. gangliogliomas have been reported with idh mutation (brain pathol 2011; 21: 564-574). ganglioglioma versus diffuse astrocytoma remains a difficult differential diagnosis in neuropathology.   abstract 4 free neuropathol 2:29:7 diagnostic challenges in unusual high-grade gliomas namita sinha1, arie perry2 1 department of pathology, health sciences center, university of manitoba, winnipeg, manitoba, canada 2 department of pathology, university of california, san francisco, california, usa diagnosis of most of adult high-grade gliomas is largely based on morphology and immunohistochemistry, with an increasing role of molecular studies to aid proper classification (idh wildtype and idh-mutant gliomas, h3k27m mutant diffuse midline glioma). on occasions, despite all the work-up, the results do not allow for a definite diagnosis that are established in the most recent who classification. some glial tumor shows dedifferentiation and looses gfap expression. in these cases, methylation profiling is a power tool in establishing the diagnosis. therefore, it is very important that the tissue should be utilized optimally and carefully especially when the biopsies are very small and extensive work may be required. on rare occasions, definitive diagnosis can be challenging despite complete histological and molecular characterization. some variants of glioblastoma may show epithelial or primitive neuronal differentiation and some variants may have increased predilection for csf metastasis and therefore it is important to subclassify these variants. these challenges will be discussed with some examples of high-grade gliomas that we received in our institution at health sciences center, university of manitoba.   abstract 5 free neuropathol 2:29:8 regional variability of α-synuclein inclusion size in multiple system atrophy ain kim1,2,3, ivan martinez-valbuena2,3, gabor g. kovacs1,2,3* 1 department of laboratory medicine & pathobiology, university of toronto, ontario, canada 2 tanz centre for research in neurodegenerative disease, university of toronto, ontario, canada 3 krembil research institute, university health network, ontario, canada relevance: multiple system atrophy (msa) patients show variable duration of illness and distinct constellation of clinical symptoms suggestive of different pathological α-synuclein strains. objective: based on this, our aim is to investigate whether the seeding and aggregation process of α-synuclein are different in msa brains, leading to variability of size and detectability of oligodendrocytic inclusions using different anti-α-synuclein antibodies. methods: the putamen and cerebellum of 6 msa cases were immunostained using nitrated, truncated, phosphorylated and disease-specific α-synuclein (clone 5g4) antibodies. sections were scanned using tissuescopetm and images were processed using photoshop (v.21.0.3). 5g4-immunoreactive oligodendrocytes with visible nucleus were optically dissected and inclusion sizes were evaluated using algorithms that we developed using the software image j. results: the size of 5g4-reactive inclusions were significantly larger in the cerebellum than in the putamen in 4 out of 6 cases. in one case, the inclusion size was significantly larger in the putamen than in the cerebellum, while a single case showed similar sizes. in addition, case-wise comparison revealed significant variability between the size of inclusions in both regions. in addition, substantial differences were noted between the number of inclusions stained by the different α-synuclein antibodies. conclusions: we developed a novel computer-based method to measure the size and number of α-synuclein inclusions in msa. our observations on the variability of size and number of inclusions, detected by different α-synuclein epitopes between brain regions and cases, support the notion of distinct subtypes of disease. our study further provides a first step to developing ai-based evaluation strategies for large scale comparative studies.   abstract 6 free neuropathol 2:29:9 cognitive resilience in individuals with severe alzheimer’s disease neuropathology narges ahangari1, corinne e. fischer2,3,4, tom a. schweizer2,4,5, david g. munoz1,2,6 1 division of pathology, st. michael’s hospital, toronto, ontario, canada 2 keenan research centre for biomedical research, the li ka shing knowledge institute, st.michael’s hospital, toronto, ontario, canada 3 department of psychiatry, faculty of medicine, university of toronto, ontario, canada 4 institute of medical sciences, university of toronto, toronto, ontario, canada 5 division of neurosurgery, faculty of medicine, university of toronto, ontario, canada 6 department of laboratory medicine and pathobiology, university of toronto, ontario, canada we sought to identify demographic, clinical, genetic, and neuropathological features associated with cognitive resilience in subjects with severe alzheimer’s disease (ad) neuropathology. data for this study was obtained from a national alzheimer’s coordinating centre (nacc) dataset. study inclusion criteria are as follows: severe ad pathology, i.e., frequent neuritic plaques and braak & braak stage v/vi pathology, interval between last visit and death ≤ 2 years, and absence of other primary neuropathology diagnoses. cognition was assessed by the mini-mental status examination (mmse) score. a total of 654 cases met our criteria. of these, 59 (9%) persons had mmse scores ≥24 at their last visit and were categorized as cognitively resilient. first, bivariate analysis was done to compare resilient with non-resilient groups. then, variables with significant results were entered into the multivariable model. based on our binary logistic regression model, resilient subjects were older (odds ratio [or]=1.03; 95% confidence interval [ci]=1–1.07), had more years of education (or=1.16; 95% ci=1.04-1.29), had lower bmi (or=0.91; 95% ci=0.85-0.99), were more likely to be a smoker (or=2.78; 95% ci=1.45-5.34), and were more likely to use an anticoagulant/antiplatelet at last visit compared with subjects with impaired cognition (or=1.87; 95% ci=1.01-3.48). in addition to expected protective factors such as higher education and lower bmi, our results showed that more smoking and frequent use of an anticoagulant/antiplatelet at last visit could also be possibly associated with resilience to clinical expression of ad severe pathology. pharmacological approaches that mimic the effects of nicotine may be useful in amelioration of ad symptoms.   abstract 7 free neuropathol 2:29:10 linking iron homeostasis to vulnerability patterns in progressive supranuclear palsy seojin lee1, ivan martinez-valbuena1, suganthini ilaalagan1,3, naomi p. visanji2,4, gabor g. kovacs1,2,3,4 1 tanz centre for research in neurodegenerative disease, university of toronto, toronto, ontario, canada 2 department of laboratory medicine and pathobiology and department of medicine, university of toronto, toronto, ontario, canada 3 laboratory medicine program & krembil brain institute, university health network, toronto, ontario, canada 4 edmund j. safra program in parkinson’s disease and rossy program in progressive supranuclear palsy, toronto western hospital, toronto, ontario, canada progressive supranuclear palsy (psp) is a 4-repeat tauopathy in which pathology starts in select subcortical areas including the globus pallidus (gp) and the substantia nigra (sn), regions also associated with age-related iron accumulation. toxic iron burden in these regions of psp brains have been examined but given the differences in cellular iron homeostasis across cell types and the heterogeneity in psp tau cytopathology, we aim to examine the possible role of iron accumulation on the cellular selective vulnerability of tau pathology in the vulnerable anatomical regions of psp brains. using human post-mortem brain tissue of the early-affected psp brain regions (gp, sn, and putamen), we visualized iron deposition in the neurons, astroglia, oligodendrocytes, and microglia using a combination of dab-enhanced perl’s (ferric) and turnbull (ferrous) iron staining with immunohistochemistry of cell type-specific markers. iron deposition was also examined in relation to their tau cytopathologies using at8-immunohistochemistry. in all three regions, astrocytes and microglia were seen to predominantly accumulate both species of iron. moreover, tau-positive astrocytes showed the highest frequency of cellular iron deposition compared to neurofibrillary tangles and oligodendroglial coiled bodies. on the other hand, association of iron burden with different cellular tau pathologies was species-specific in the same regions, suggesting iron functions in psp tau cytopathology may be distinct by species. our mapping of cellular iron burden in relation to pathology in psp brains suggests a selective cellular vulnerability to iron deposition in diseased brains, and further supports the role of pathological iron in the early pathogenesis of psp.   abstract 8 free neuropathol 2:29:11 friedreich cardiomyopathy is a secondary desminopathy arnulf h. koeppen1,6, rahman f. rafique1, joseph e. mazurkiewicz2, steven pelech3,4, catherine sutter3, qishan lin5 , jiang qian6 1 research service, veterans affairs medical center, albany, new york, usa 2 department of neuroscience and experimental therapeutics, albany medical college, albany, new york, usa 3 kinexus bioinformatics corporation, vancouver, british columbia, canada 4 division of neurology, department of medicine, university of british columbia, vancouver, british columbia, canada 5 rna epitranscriptomics & proteomics resource, university at albany, albany, new york, usa 6 department of pathology, albany medical college, albany, new york, usa hypertrophic cardiomyopathy with or without arrhythmia is the predominant cause of death in friedreich ataxia (fa). the clinical and pathological phenotypes of fa are diverse. the pathogenesis of the fa-related lesions in the central and peripheral nervous systems, heart, skeleton, and endocrine pancreas is incompletely understood. the hypothesis in this research is that frataxin deficiency affects the cellular proteome in downstream mechanisms. antibody microarrays of pooled fa heart lysates showed upregulation in several proteins, including alpha b crystallin, a desmin chaperone. western blots of individual and pooled heart lysates revealed a prominent extra desmin band at 47 kda that was absent or under-expressed in control samples. mass spectrometry confirmed the origin of this band from canonical desmin (53 kda). co-immunoprecipitation of fa heart lysates with anti-desmin, recovery of the antigen-antibody complex with protein a-sepharose, sodium dodecylsulfate polyacrylamide gel electrophoresis, and western blotting confirmed the interaction of desmin and αb crystallin. slide techniques, including immunohistochemistry and double-label immunofluorescence, disclosed desmin and alpha b crystallin aggregation near intercalated discs and z-discs. congo red fluorescence microscopy did not confirm the formation of amyloid. we suggest that accumulation of a truncated desmin and aggregation with αb crystallin cause accelerated heart disease in fa.   abstract 9 free neuropathol 2:29:12 glial senescence (not tau) is the driver of post-concussive symptoms lili-naz hazrati1,2, nicole schwab1,2 1 the hospital for sick children, toronto, ontario, canada 2 department of laboratory medicine and pathobiology, university of toronto, toronto, ontario, canada mild traumatic brain injury (mtbi) leads to chronic symptoms in some patients. pathologically, mtbi is associated with chronic traumatic encephalopathy (cte), a neurodegenerative disease characterized by hyperphosphorylated tau (p-tau) in neurons and astrocytes at the depths of cortical sulci. cte has been proposed as the driver of these symptoms, despite many patients presenting with severe symptoms yet minimal pathology. it is known that low levels of p-tau are present in cognitively intact individuals, including ageing-related tau astrogliopathy (artag), and that p-tau accumulates prior to a diagnosis of alzheimer’s disease. we propose that examining changes beyond p-tau is critical to understand mtbi pathology. the objective of this study is to identify novel pathological markers of mtbi which may explain clinical symptoms and allow for the development of therapeutic targets. using a brain bank of former professional athletes with mtbi history we used immunohistochemistry and gene expression analysis to show cellular senescence as a mechanism driving brain dysfunction after mtbi. compared to controls, mtbi brains show widespread dna damage (γh2ax) and cellular senescence in ependymal cells, astrocytes, and oligodendrocytes even in cases with no neuropathology. gene expression analysis revealed upregulation of the senescence-associated secretory phenotype (sasp), a form of chronic low-level inflammation which has been proposed to drive cognitive dysfunction and proteinopathy. in a mouse model of mtbi, we have recapitulated these results and shown that eliminating senescent cells with senolytic therapy is beneficial. mtbi brains are characterized by early ageing through cellular senescence, which may drive clinical symptoms and p-tau pathology.   abstract 10 free neuropathol 2:29:13 hail to astrogliosis! the unsung hero of the cns tissue reaction to the spinal cord injury. jacek m. kwiecien1 1 department of pathology and molecular medicine, faculty of health sciences, mcmaster university, hamilton, ontario, canada spinal trauma results in a localized spinal cord injury (sci) with an area of hemorrhage and necrosis surrounded by edema in acute phase that lasts 2 days followed by an inflammatory phase beginning on day 3 with infiltration of necrosis by inflammatory cd68+/cd163macrophages whose numbers rapidly increase and persist at high levels for 4 weeks and then decline but are still present in low numbers at 16 weeks. this destructive inflammation is fuelled by myelin-rich necrotic debris and the macrophage activation causes ongoing damage to the surrounding spinal cord and further damaged myelin to sustain a mechanism of vicious cycle. the severity of post-sci inflammation becomes inhibited and eliminated by a spinal cord tissue response in the 3rd phase, resolution. astrogliosis is a hallmark of reaction to sci. in the 1st week astrocytes surrounding the lesion hypertrophy and define a cavity of injury (coi) containing macrophage-rich inflammation and accumulation of water. progression of severity of astrogliosis around the coi coincides with the reduction of numbers of macrophages and leads to formation of quiescent syrinx. while the inflammation in the coi leads to damage of blood vessel in the surrounding spinal cord resulting in vasogenic edema beyond 16 weeks post-sci, astrocytes equipped with aquaporin-4 pass excess edema water to 4 extra-spinal spaces; (1) the sub-arachnoid, (2) the central canal, (3) the blood vessels, and (4) the coi, a novel mechanism. astrogliosis is a beneficial element in neuroplasticity following the sci and should not be inhibited in anti-inflammatory treatments effecting neuroprotection.   abstract 11 free neuropathol 2:29:14 ischemic-like pathology in aberrant white matter tracts of fetal holoprosencephaly: a case series sumit das1, jessica saunders2, sarah nikkel3, lindsay brown4, christopher dunham2 1 department of pathology and lab medicine, university hospital, edmonton, alberta, canada 2 division of anatomical pathology, bc children’s hospital, vancouver, british columbia, canada 3 department of medical genetics, bc children’s hospital, vancouver, british columbia, canada 4 division of genome diagnostics, bc children’s hospital, vancouver, british columbia, canada holoprosencephaly (hpe) is due to defective early forebrain induction resulting in a “lack of cleavage” of the primitive prosencephalon into paired cerebral hemispheres. hpe is generally considered sporadic, although clear environmental and genetic factors have been recognized. from a genetic perspective, an autosomal dominant inheritance pattern is most commonly noted. chromosomal abnormalities are common in hpe, seen in 25-50% of affected individuals, with trisomy 13 being the most frequent. a number of structural chromosomal abnormalities (e.g., 21q22.3 deletion) and pathogenic copy number variations have also been described, as have syndromic associations. pertinent biologic pathways include shh, nodal and bmp; in turn, several single gene mutations related to these pathways have been uncovered, including shh, zic2, and six3. although not frequently emphasized in neuropathologic descriptions of hpe, abnormal white matter has been reported, mainly in the form of aberrant tracts (e.g., absence of the corpus callosum and hypoplasia of the corticospinal pathway). in this study, we detail the clinicopathologic features of 8 fetal bcch cases bearing a seemingly unique dual form of white pathology. all 8 cases demonstrated ischemic-like pathology, with 7 of 8 exhibiting such pathology in a similar location – ventral to the fused deep grey nuclei. diffusion tensor imaging (dti) studies suggest that the neuroanatomic substrate of this aberrant white matter is fused superior and inferior occipito-frontal fasciculi, with the resultant fibers crossing the midline. while the etiology of this ischemic-like pathology is unclear, the literature raises the possibility of a role for the shh pathway.   abstract 12 free neuropathol 2:29:15 brain pathology in patients with congenital heart disease murad a. alturkustani1,2,3, linda j. szymanski2 1 king abdulaziz university, jeddah, saudi arabia 2 children’s hospital los angeles, los angeles, california, usa 3 western university, london, ontario, canada brain pathology in patients with congenital heart disease (chd) are associated with neurodevelopmental delay. imaging studies support vascular etiology for both white and gray matter lesions. in this retrospective study, we describe the pathological changes in the brains of patients with chd. the last 20 autopsy cases in patients with chd at our institution were retrieved and the clinical and the autopsy report were reviewed. the available h&e, special, and immunostains were evaluated and at least one section from each case was stained with gfap, app, and hla-dr. five control cases included telencephalic leukoencephalopathy (3) and no significant pathological changes (2). the following histological features were assessed: necrotic cells in cortex, hippocampus and the cerebellum; app and gfap staining pattern, and presence of focal lesions and amphophilic globules. twenty patients (10 males, 10 females) were identified with age range of 2 weeks – 19 years. the pathological findings are as follows: 8 cases had changes consistent with acute global hypoperfusion, 5 cases showed features consistent with chronic global hypoperfusion, 3 cases showed focal white matter lesions (1 secondary, to septic emboli), 4 cases with only widespread gliosis, one case with only old focal neuronal loss in dentate gyrus, and one with no significant changes. subarachnoid hemorrhages were present in 5 cases and germinal matrix hemorrhage in 3 cases. in conclusion, most of the pathological changes could be explained by cerebral hypoperfusion and better techniques to improve the cerebral perfusion are warranted in the management of these patients.   abstract 13 free neuropathol 2:29:16 characterizing the hippocampal dentate gyrus involvement in temporal lobe epilepsy carolyn twible1, qi zhang1,2 1 department of pathology and laboratory medicine, schulich school of medicine and dentistry, western university, london, ontario, canada 2 department of pathology and laboratory medicine, london health sciences centre, london, ontario, canada hippocampal sclerosis (hs) is the most common pathology finding for drug resistant temporal lobe epilepsy (tle) and is characterized by neuronal loss and gliotic cornu ammonis. nearly 20% of surgical specimens from drug-resistant tle surgery contain “normal” populations of neurons, termed no-hs, yet still benefit from surgical resection. this observation suggests a neuropathological explanation for the epileptogenic focus in the resected tissue that is not detected through standard diagnostic practices. the goals of this project are to increase the neuropathological understanding of drug resistant tle, and to elucidate the structural changes of hs and no-hs, focusing on the granule cell layer (gcl). in this study, 21 tle surgical resection cases were examined, including 14 hs and 7 no-hs cases, to investigate gcl morphometry. information on histopathological diagnosis and post-operative outcome were collected. the digital image analysis software qupath was used to perform cell detection analysis on the gcl. measures including delaunay mean cell density were analyzed. hs patients show a significant increase in granule cell spacing and decrease in granule cell density within the gcl compared to no-hs patients. regardless of the histological diagnosis, patients who achieved seizure freedom post-operatively demonstrated an increase in granule cell spacing and decrease in granule cell density in comparison to those who did not achieve seizure freedom post-operatively. hs and no-hs diagnosis groups have diseaseand post-operative outcome dependent morphometry differences. the post-operative outcome dependent morphometry observed in hs and no-hs patients presents a potential additional method to evaluate post-operative prognosis for tle surgical-resection patients.   abstract 14 free neuropathol 2:29:17 deep learning approaches to deciphering intra-tumoural heterogeneity in glioblastoma anglin dent1, brian lam1, kevin faust1, alberto j. leon3, phedias diamandis1 1 princess margaret cancer centre, university health network, toronto, ontario, canada background: emerging evidence strongly implicates intra-tumoral heterogeneous biology in treatment resistance and disease progression across many cancer types (1). using glioblastoma (gbm) as a prototype, i have aimed to leverage the computational power of artificial intelligence (ai) and deep learning (2) to develop an autonomous workflow for the objective definition and quantification of biologically distinct tumour subpopulations. objectives: i hypothesize that ai-defined tumoral clusters predict spatially distinct molecular profiles and therapeutic targets. methods: i apply our developed image clustering workflows (3) to quantify ai-defined subregions within a clinical cohort of 10 gbm patient tumors and use laser capture microdissection and mass spectrometry-based proteomics to address if ai-defined subregions show intra-tumoral molecular variation. further, i leverage existing pharmacogenomic databases (4) and carry out drug sensitivity and transcriptional clustering to define biomarkers and validate their intra-tumoral expression in my clinical gbm cohort. results: preliminary data shows that region-to-region heterogeneity can be found in idh wild-type gbm using our unbiased omics approach, in addition to predicting different pharmacogenomic sensitivities. conclusions: this project aims to develop the first ai-driven tool to guide the routine and systematic molecular analysis of spatial morphogenomic heterogeneity. further, this tool may have the potential to provide novel approaches for personalized care by selecting drug combinations that target a larger fraction of a tumor’s true biology.   abstract 15 free neuropathol 2:29:18 integrating morphologic and molecular histopathological features through whole slide image registration and deep learning michael k. lee1, kevin faust2,3, anglin dent1, clare fiala3, alessia portante1, madhu rabindranath1, noor alsafwani3,4, andrew gao1,3, ugljesa djuric5, phedias diamandis1,3,5,6 1 department of laboratory medicine and pathobiology, university of toronto, toronto, ontario, canada 2 department of computer science, university of toronto, toronto, ontario, canada 3 laboratory medicine program, department of pathology, university health network, toronto, ontario, canada 4 department of pathology, college of medicine, imam abdulrahman bin faisal university, dammam, saudi arabia 5 princess margaret cancer centre, toronto, ontario, canada 6 department of medical biophysics, university of toronto, toronto, ontario, canada background and objective: despite recent innovations in deep learning, integrating histomorphologic and molecular information found on respective h&e and ihc-stained tissue sections still remains a challenge. while human observers can easily align these different tissue sections, routine computational approaches for image registration of giga-byte sized wsis are still needed. here, we aim to address this issue by incorporating another computer vision tool, scale-invariant feature transform (sift), to align h&e-stained sections with accompanying ihc studies for automated subclassification of gliomas. method: to test the workflow, we first trained a vgg19 convolutional neural network (cnn) using pathologist-annotated h&e wsis to recognize histological patterns of 16 common tissue and lesion classes. afterwards, we optimized a different set of cnns to recognize various ihc markers, such as idh1-r132h and atrx, which are relevant for molecular subclassification of gliomas. for the integrated analysis, we employed sift to find features for image matching which were used to align lesional regions of h&e and ihc slides. results: the histomorphologic classifier (cnnh&e) excelled at classification with accuracies of 100% for glioma, meningioma and metastatic carcinoma, and 93% for schwannoma (n = 125). using the newly developed cnnh&e and sift-based alignment, the quantitative analysis by the ihc classifiers significantly improved for atrx retained and idh1-r132h positive wsis when compared to that of unaligned wsis. conclusion: sift can work in concert with deep learning tools to provide a pathologist-inspired workflow to help automate advanced immunohistochemically diagnostic tasks, such as subclassification of glioma.   abstract 16 free neuropathol 2:29:19 neuropathology of eight cases of the new brunswick cluster of neurological syndrome of unknown cause (nsuc) gerard h. jansen1, sidney e. croul2, angela k. miller3, alexander s. easton2, john m. j. woulfe1 1 department of pathology and laboratory medicine, university of ottawa, ottawa, ontario, canada 2 department of pathology, qeii health science centre, halifax, nova scotia, canada 3 department of pathology, health sciences centre, winnipeg, manitoba, canada (previously department of pathology, moncton hospital, moncton, new brunswick, canada) on march 17, 2021 a press conference aired featuring a moncton neurologist and new brunswick’s chief medical health officer, regarding a cluster of patients in new brunswick who had symptoms reminiscent of cjd, and were claimed to have a novel neurological syndrome. the onset of the disease was between 2015 and 2021. all patients in that cluster had at that time been reported by one neurologist, although subsequently a few incidental cases were reported by other neurologists. the size of this cluster has been reported at around 50 cases. further news publications have suggested that an environmental factor is the causative factor for this cluster. this news has significantly disturbed the medical community. since 2019 eight patients have died that belonged to this cluster. we report their neuropathological findings and hope this will bring some clarity. there was one case of metastatic carcinoma, one case of ftld-tdp43, one case of neocortical lewy body pathology, one case of neocortical lewy body pathology and ad, 2 cases of ad with vascular pathology, one case of mainly vascular pathology, and one case without significant pathology (consistent with the patient’s previous history). in these 8 patients no evidence for a prion disease was found, nor novel pathology. we suggest that these 8 patients represent a group of misclassified clinical diagnoses. since june 3, 2021 the oversight committee was established in new brunswick reviewing the clinical and epidemiological data of the patients in this cluster. we hope that our findings are useful to them.   abstract 17 free neuropathol 2:29:20 covid-19 pandemic impact on surgical neuropathology services at london health sciences centre shervin pejhan1, chris tran1, david driman1, robert hammond1,2, lee cyn ang1,2, qi zhang1,2 1 department of pathology & lab medicine, london health sciences centre, london, ontario, canada 2 department of clinical neurological sciences, london health sciences centre, london, ontario, canada the covid-19 pandemic has had a significant impact on medical services. many jurisdictions postponed non-urgent procedures to balance individual patient care with public health precautions. surgical backlogs caused by the covid-19 pandemic have been evaluated by different groups. however, the impact of this pandemic on pathology and specifically neuropathology (np) services has received limited attention. in this study, we reviewed all surgical np reports of our centre from march 2018 (two years before the pandemic declaration) through july 2021. patient demographic information and pathological variables were collected. for tumours, site, type, and who grade were analyzed. within the period under study, the total number of np samples was lowest in april 2020, corresponding to the first ontario provincial lockdown. in comparison to the dramatic decrease in other surgical pathology subspecialties (camj 2021;193(10): e343), the np surgical specimen initially had a minimal volume reduction, with a rapid return to baseline. among the different types of np surgical specimens, muscle biopsy and epilepsy-related specimens showed a more significant reduction. there was a slight increase in higher-grade tumours. interestingly, a gradual increase of brain biopsies for inflammatory conditions was noticed. our results show that the neuropathology service volume reduction due to the covid-19 pandemic has not been as significant as other pathology subspecialties. studying the variations in histopathological diagnoses in pandemic years could be helpful for future planning in both clinical and pathological sectors, especially when the data is strengthened by the experiences of other medical centers.   copyright: © 2021 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. top ten discoveries of the year: neurodegeneration free neuropathology 1:12 (2020) review top ten discoveries of the year: neurodegeneration john f. crary neuropathology brain bank & research core, department of pathology, nash family department of neuroscience, ronald m. loeb center for alzheimer's disease, friedman brain institute, icahn school of medicine at mount sinai, new york, ny, usa corresponding author: john f. crary, md-phd · friedman brain institute · ronald m. loeb center for alzheimer’s disease · icahn school of medicine at mount sinai · 1 gustave l. levy place box 1194 · new york, ny 10029 · usa john.crary@mountsinai.org submitted: 26 january 2020 accepted: 02 april 2020 copyedited by: nima sharifai published: 08 april 2020 https://doi.org/10.17879/freeneuropathology-2020-2634 keywords: neurodegeneration, neuropathology, alzheimer’s disease, limbic-predominant age-related tdp-43 encephalopathy, amyotrophic lateral sclerosis, parkinson’s disease, huntington’s disease abstract as we embark on a new year of scientific inquiry in neurodegenerative disease research, it is helpful to take a look back and consider the contributions in the field with the potential to be the most impactful. the purpose of this review is to highlight recent advances in 2019 which have the potential to be transformative in the field of neurodegenerative neuropathology. substantive scientific progress rarely occurs as a “eureka moment”, and when possible, we opted to highlight collaborative efforts and research trends. we also included groundbreaking methodologies and tools. the generous increases in federal funding in the united states and elsewhere have massively expanded the total number of active programs researching alzheimer’s disease. this exacerbates an imbalance, and an effort was made to highlight innovations across disease categories, and not to permit dementia to crowd out movement disorders, motor neuron disease, ataxias, etc. thus, our overall goal was to highlight some of the most important discoveries, tools or methods that we feel will most likely directly enhance our ability to understand and diagnose neurodegenerative brain diseases. given space limitations and the targeted readership of this journal, we selected ten topics most relevant to neuropathologists and clinical neuroscientists: 1. a new neurodegenerative disease category, 2. a new approach to probing gene expression on the single cell level, 3. a new approach merging histology and gene expression profiling, 4. a new computational approach using deep machine learning and computer vision, 5. a neuropathological substrate for sleep disturbance in alzheimer’s disease, 6. a candidate pathogenic agent for alzheimer’s disease, 7. a comprehensive approach to morphometric analysis of cerebellar neurodegeneration, 8. the strongest evidence yet linking neurodegeneration to contact sports, 9. mounting evidence for gut to central nervous system transmission in parkinson’s disease, and 10. a spotlight on glia in huntington’s disease. 1. late, a new old neurodegenerative disease as the population ages, researchers are gleaning fundamental new insights from the oldest-old population (i.e., those over the age of 80 years) whose vulnerability to neuropathology diverges from the younger-old. in a paper published in brain, a large group of neuropathologists led by dr. peter nelson at the university of kentucky introduced a new diagnostic category, limbic-predominant age-related tdp-43 encephalopathy (late), describing a type of neurodegeneration that mostly strikes elderly individuals over 80 years old (nelson et al., 2019). this work was the culmination of many years of study and a consensus group report, the goal of which was to address how to deal with the surprisingly large number of patients that variably develop an amnestic dementia that is very similar to that seen in alzheimer’s disease (ad), but with abnormalities in the transactive response dna binding protein of 43 kda (tdp-43). historically, the first study to recognize that there was something different occurring in these patients did so by identifying 13 autopsied subjects with dementia who had marked neuronal loss with reactive gliosis in the medial temporal lobe, but had no alzheimer’s neuropathological changes; this was termed “hippocampal sclerosis” (dickson et al., 1994). this neuropathological signature is not specific, and it was not until tdp-43 was discovered in amyotrophic lateral sclerosis (als) and frontotemporal lobar degeneration (ftld) patients that investigators thought to also look in hippocampal sclerosis patients and ultimately link the two. it was also recognized that subjects with tdp-43 proteinopathy only variably present with a pattern histologically considered hippocampal sclerosis on hematoxylin and eosin (h&e) stain. neuropathologically, subjects with late have the tell-tale abnormal translocation of tdp-43 from the nucleus to the cytoplasm, where it becomes phosphorylated (figure 1). in late, tdp-43 pathology can be detected in numerous brain regions, some of which overlap with ftld, but the amygdala is most severely affected, followed by the hippocampal formation. nelson et al. provided diagnostic criteria and a proposed hierarchical staging system for assessing patients for late neuropathologic change: tdp-43 immunohistochemistry is essential (hippocampal sclerosis pathology only is not sufficient) and should be performed in the amygdala (stage 1), mid-level hippocampus (stage 2) and middle frontal gyrus (stage 3).   figure 1. limbic-predominant age-related tdp-43 encephalopathy (late). (a) late neuropathological change in an 83-year-old woman with dementia and the clinical diagnosis of ad. marked atrophy and neuronal loss in the hippocampal formation, but with minimal ad neuropathological change. (b) tdp-43 proteinopathy is shown in the dentate gyrus. image courtesy of dr. peter nelson (nih grant p30 ag028383). there is a great deal that we have to learn by studying late. while a few genes have been implicated as risk factors (i.e., apoe, grn, tmem106b, abcc9, kcnmb2), larger genetic studies have the potential to better delineate mechanisms and causal pathways that could serve as biomarkers and therapeutic targets. further, we have very little understanding of the extent to which late affects non-caucasian populations; studying more diverse populations has the potential to expand on risk factors, genetic and otherwise. biochemical and molecular studies of tdp-43, including secondary modifications such as phosphorylation, have the potential to lead to biomarkers that could be used pre-mortem. finally, co-morbidities are nearly universal in the oldest-old population, and the extent to which they interact with late pathology will be important for understanding its clinical significance. 2. single-cell transcriptomics nothing has plagued molecular profiling of post-mortem neurodegenerative disease brain tissue more than the problem of normalization in the context of variable and often massive neuronal loss with commensurate reactive gliosis. numerous approaches have been developed to address this problem (e.g., deconvolution of bulk rna sequencing or expression profiling of pooled cells isolated by laser capture microdissection), but they are laborious and results often fall short. recent advances in high-throughput single cell sorting using microfluidics coupled with next-generation sequencing have enabled a paradigm shift in how gene expression profiles can be investigated. the first papers deploying single-cell rna sequencing (scrna-seq) in human post-mortem neurodegenerative disease tissues have emerged in 2019 and we expect this to transform transcriptomic studies (del-aguila et al., 2019; grubman et al., 2019; mathys et al., 2019). one of the major barriers that investigators encountered in applying single cell technology to human post-mortem brain is that it is not possible to isolate single cells from frozen tissue given the membrane breakdown. thus, scrna-seq is currently best done from cells isolated from fresh brain tissue, and precludes using archival frozen banked brains. previous work in animal models, however, has shown that single nuclei can withstand the freezing process and yield nuclear rna profiles that can serve as a proxy for those generated from cytoplasmic rna. because of this, groups are focusing on this variant approach termed single nuclei rna-seq (snrna-seq, or just nuc-seq). three studies were published in 2019 using nuc-seq in post-mortem human brain tissue from patients with neurodegenerative disease. in the first, published online in august by del-aguila et al, investigators at washington university in saint louis used nuc-seq to profile a patient with the psen1 p.a79v mutation and compared it to two related family members with sporadic ad (non-mutational), and proposed a collection of best practices (del-aguila et al., 2019). all of these studies have made the data available online (figure 2). in november, grubman et al., from monash university (australia) and duke-national university of singapore medical school, examined the entorhinal cortex from 6 sporadic patients and 6 controls. their group found that ad is associated with downregulation of apoe in oligodendrocyte precursors and astrocytes, and with upregulation of apoe in a subpopulation of microglia. they also explored transcription factor regulatory modules (grubman et al., 2019). finally, in the largest study published in june by mathys et al., investigators at the broad institute of mit/harvard profiled the prefrontal cortex from 48 individuals from the rush rosmap cohort and identified sex differences in disease-associated subpopulations of cells, and found interesting recurrent differences in myelination that might play a role in ad (mathys et al., 2019). together, these results are the first step towards providing a highly detailed cell-type specific molecular atlas of neurodegenerative diseases.   figure 2. single-nuclei rna-seq (snrna-seq) brain expression browser. cell type specific expression profiles can be shown using a publicly available interactive web-based resource (http://ngi.pub/snuclrna-seq). 3. spatial transcriptomics essentially all approaches to measuring gene expression are limited by the fact that isolation of nucleic acids from tissue by necessity disrupts the spatial relationship of the cells of interest. losing the cellular architecture is a major barrier to understanding the context of genome-wide changes in rna expression. this year we saw the first use of spatial transcriptomics, a method first introduced in 2016 that bridges rna-sequencing and histopathology by allowing researchers to generate thousands of gene expression profiles from a single histological section using a slide printed with an array of barcoded specialized mrna-capturing probes (ståhl et al., 2016). in a study led by hemali phatnani at the new york genome center and published in science (maniatis et al., 2019), spatial transcriptomics was applied to post-mortem human amyotrophic lateral sclerosis tissue from seven patients with either lumbaror bulbar-onset sporadic als (figure 3). they also profiled sod1-g93a transgenic mice. together, they quantitated the spatial distribution of 11,138 mouse and 9624 human genes in spinal cord sections and confirmed the alteration in expression of some known als genes. they were also able to identify an abnormal microglial signature that preceded astroglial dysfunction. they chose to follow up on a mechanism involving trem2 and its partner tyrobp, revealing a spatiotemporal ordering with tyrobp expression upregulated pre-symptomatically before trem2. changes in other genes that are part of this mechanism (e.g., apoe, cx3cr1, lpl, b2m and cx3cr1) occurred later. these results demonstrate how this technology can lead to novel insights by being deployed as part of an interdisciplinary approach to neurodegenerative disease.   figure 3. identification of expression programs using spatially resolved transcriptomics. (a) example data for a single gene from spatial transcriptomics profiling of postmortem lumbar and cervical spinal cord sections from a sporadic als patient presenting with bulbar symptom onset. (b) data from many spatial transcriptomics arrays were aligned to a common coordinate system, and expression levels were hex binned spatially. this procedure enables reconstruction of larger regions than can be profiled with a single array and reveals expected spatial expression patterns for genes expressed by cell types present in specific tissue domains. (c) spatially resolved coexpression analysis reveals gene sets with tissue domain specific expression patterns, a subset of which also exhibit differences with respect to proximity to the site of symptom onset. figure courtesy of drs. hemali phatnani and silas maniatis. 4. artificial intelligence and computer vision since alois alzheimer first described the disease that bears his name in 1906, revealing striking morphological changes using ammoniacal silver stains, there has been very little progress in the way these cellular changes are interpreted. visual microscopic examination remains state-of-the-art, but it is slow, imprecise, marginally reproducible and requires the experience of a highly trained neuropathologist, the numbers of which are dwindling. the task is highly repetitive and fatigue inducing, which increases the likelihood of error. the whole process would be best suited to automation, but the concept of a machine that could substitute for a neuropathologist seemed implausible until recent developments in the field of artificial intelligence. increases in computational power combined with improved algorithms for training artificial neural networks have placed us at the precipice of a transformation moment in neuropathology. the most common machine learning approach to analyzing visual imagery is the convolutional neural network (cnn). these are a type of deep learning, a class of artificial neural networks with multiple layers that progressively extract more complex features from the input. cnns are based on the architecture of the organization of the animal visual cortex, encompassing simulations of overlapping receptive fields what were described by hubel and wiesel in the cat and monkey in the 1950s and 1960s. this year we saw the first two papers deploying deep learning to analyze digital whole slide images from patients with neurodegenerative diseases to identify both amyloid plaques and neurofibrillary tangles. one paper, published in nature communications and led by dr. brittany dugger (university of california davis), reported a deep learning pipeline that identifies amyloid plaques and cerebral amyloid angiopathy in immunohistochemically stained slides (tang et al., 2019). to build this, they annotated over 70,000 plaques that served as ground truth to train and evaluate the cnn achieving 0.993 receiver operating characteristic (roc) curve and 0.743 precision recall curve. in another paper, investigators from the center for computational and systems pathology at the icahn school of medicine at mount sinai, led by dr. john f. crary (author of this review) and published in laboratory investigation, focused on neurofibrillary tangle pathology (signaevsky et al., 2019). this study used 3177 images derived from patients with primary age-related tauopathy, chronic traumatic encephalopathy, progressive supranuclear palsy and alzheimer’s disease to train and test the network, achieving recall, precision and f1 scores of 0.91, 0.80 and 0.85 respectively. we expect that this approach will be transformative in the near future with the development of sophisticated neural networks capable of rapidly and reproducibly analyzing slides alongside, or perhaps instead of, the neuropathologist. in the long term, this will certainly transform the skillset of the neuropathologist, who will perhaps be less adept at picking up subtle morphological features by eye, but hopefully more able to deploy computational and other modern tools. 5. a neuropathological substrate for sleep-wake disruption in alzheimer’s disease sleep-wake disruption is common in aging and associated with cognitive decline and decreased quality of life. sleep disturbance is seen in a number of neurodegenerative diseases and is amongst the earliest symptoms of ad. this includes reduced total, slow wave, and rapid eye movement (rem) sleep time. there are arousal deficiencies, including daytime sleepiness, that can precede cognitive impairment. competition between subcortical populations of both wake promoting neurons (wpns) and sleep promoting neurons (spns) provide a switch that coordinates sleep behaviors. among these populations, wpns in the locus coeruleus, orexin/hypocretin-producing neurons in the lateral hypothalamic area, and histaminergic neurons in the tuberomammillary nucleus (tmn) play critical roles in stimulating the cerebral cortex and contributing to arousal. the neuropathological substrate for sleep disturbances in ad remains unclear, but attention has been focused on amyloid-beta (aβ) as sleep promotes its clearance. accumulation of abnormal hyperphosphorylated tau (p-tau), however, is a stronger correlate of neurodegeneration and functional decline, and accumulation of p-tau in wpns is among the first ad neuropathological changes to appear in the brain. in a powerful study published in alzheimer’s & dementia, a team led by dr. lea grinberg (ucsf) directly addressed the question of p-tau pathology in brainstem wpns in ad by performing rigorous quantitative study, using stereological measures of wake-promoting cells in the locus coeruleus, lateral hypothalamic area and tuberomammillary nucleus in ad, compared to two amyloid-independent primary tauopathies (progressive supranuclear palsy and corticobasal degeneration) and healthy controls (oh et al., 2019). they found that while all three of these diseases displayed the presence of p-tau in wpns, only the ad patients exhibited clinically relevant cell loss (figure 4). the implications are that loss of these cells are an important component of sleep disturbance in ad, but also that amyloid-independent tauopathies may benefit from alternative clinical approaches (e.g., suppression of the arousal system).   figure 4. tau pathology in the locus coeruleus in alzheimer’s disease compared to progressive supranuclear palsy, corticobasal degeneration and controls. double-label immunohistochemistry showing colocalization of tau pathology (brown) and norepinephrine (red). figure courtesy of dr. lea grinberg. 6. periodontal disease as a risk factor for alzheimer’s disease neuroinflammation is a well-recognized feature of ad, with activation of microglia, inflammasomes, and complement. over the years, a number of infectious agents have been postulated to trigger this inflammation and participate in disease pathogenesis. the aβ peptide itself has even been demonstrated to have antimicrobial properties, supporting this hypothesis. one candidate pathogenic agent that has emerged is porphyromonas gingivalis, a pathogen that plays a critical role as a keystone pathogen in chronic periodontitis (cp). previously, among other supportive data, p. gingivalis was shown to be associated with cognitive decline in ad patients compared to controls without cp. furthermore, oral infection of apoe-/transgenic mice resulted in brain infection and complement activation. in a paper published in science advances, researchers led by drs. jan potempa (university of louisville) and stephen s. dominy (cortexyme) have provided the strongest evidence yet that infiltration of the brain with p. gingivalis might be causal and that their toxicity is through their virulence factors termed gingipains (dominy et al., 2019). gingipains are secreted cysteine proteases that play critical roles related to host colonization, inactivation of host defenses, iron/nutrient acquisition, and destruction of tissue. these researchers demonstrated the presence of gingipains in post-mortem ad brain tissue alongside the presence of p. gingivalis dna. remarkably, treatment with a small molecule gingipain inhibitor blocks gingipain-induced neurodegeneration in an animal model with an associated decrease in host aβ response to p. gingivalis. this orally available gingipain inhibitor is currently being tested in human clinical studies. more work needs to be done, and it is anticipated that this work might trigger a new research trend around periodontal disease in ad. 7. deconstructing cerebellar degeneration using “patholog-omics” essential tremor (et) is a common condition that can be associated with significant morbidity. for many years, the neuropathological features underlying et had been a complete mystery, with autopsy studies being limited and qualitative. recently, investigators have been building large cohorts of et patients and a series of et-related neuropathological changes have been reported, but many had been observed in other neurodegenerative diseases affecting the cerebellum, raising questions of specificity. how these features and the other cerebellar degenerations are related had never been formally or systematically studied. in what could be considered a tour de force, investigators led by drs. elan louis (yale university) and phyllis faust (columbia university) published as landmark paper in acta neuropathologica applying what they termed a “patholog-omics” approach using 37 quantitative morphological metrics (louis et al., 2019). they compared 156 brains from healthy controls and patients with a spectrum of cerebellar diseases, including essential tremor, spinocerebellar ataxia, multiple system atrophy, parkinson’s disease, and dystonia, mapping unique cellular patterns of neurodegeneration. measures included purkinje cell loss, axonal changes (e.g., torpedoes), basket cell hypertrophy, climbing fiber synaptic changes and others. they then used principal component analysis to derive distinctive and overlapping signatures marking each of these disorders. this study, which is the first of its kind to attempt to apply “omics” approaches to histomorphology, paves the way towards applying the approach to other neurodegenerative diseases. 8. chronic traumatic encephalopathy and american football: a dose-response relationship while the fact that repetitive mild traumatic brain injury leads to devastating long-term sequelae in professional boxers has been recognized for close to a century without controversy, chronic traumatic encephalopathy (cte) only recently began to achieve substantial scientific scrutiny and public interest when it was identified in american football players. while there is strong evidence that repetitive head injuries are causal for cte, skeptics remain. among the criticisms is that while clinicopathological studies are powerful, they are association analyses that ostensibly cannot prove a causal relationship on their own. this is not the whole story. in 1965, sir austin bradford hill, an english statistician, proposed nine criteria that, when met, provide epidemiologic evidence of causality. in terms of cte, eight of these criteria had been met, including strength (effect size), reproducibility, specificity, temporality, plausibility, coherence, experimentation and analogy. the missing criterion, until now, was evidence of a biological gradient: the presence of a dose-response relationship showing that increased exposure leads to a greater incidence/magnitude of the effect. in a report published online in november 2019 in the annals of neurology, researchers led by drs. jesse mez and ann c. mckee (boston university) measured exposure to contact sports in the largest autopsy series of american football players ever assembled and calculated cte risk. they found that the odds of cte doubled with every 2.6 years of american football played (mez et al., 2020). this paper firmly establishes that there is a dose-response relationship between exposure to american football and cte, which the strongest indicator of causality obtainable. this study also provides hard data for athletes and families trying to determine how much exposure might be considered safe. 9. parkinson’s disease: the gut-brain connection the pathological spread of abnormal protein aggregates through “prion-like” templated misfolding is strongly implicated in a host of neurodegenerative diseases. parkinson’s disease is no exception, with rigorous neuroanatomical studies by heiko braak and colleagues describing a hierarchical spread of α-synuclein pathology caudally to rostrally in the central nervous system (braak et al., 2003). parkinson’s disease is notable in that it is not restricted to the central nervous system, with α-synuclein aggregates present throughout the body including the gastrointestinal tract. this, together with the fact that the dorsal motor nucleus of the vagus nerve, which heavily innervates the gut, is among the first regions to show α-synuclein pathology, has fueled investigation around whether parkinson’s disease may be initiated in the gut. this hypothesis is plausible given that a number of cohort studies of human subjects have a reduction in parkinson’s disease risk following truncal vagotomy. further, injection of various forms of human or recombinant α-synuclein fibrils into the gut at various sites leads to central pathology, but this pathology was minimal and transient (holmqvist et al., 2014; manfredsson et al., 2018; uemura et al., 2018). investigators at the johns hopkins university school of medicine published a manuscript in neuron showing results using a new experimental paradigm, which demonstrated severe and widespread α-synuclein pathology in the brains of transgenic mice following injection of preformed fibrils in the gut (kim et al., 2019). they injected α-synuclein fibrils directly into the muscularis layer of the duodenum and pylorus and tracked the progression of pathological aggregate generation to the dorsal motor nucleus, caudal hindbrain, and onward to the substantia nigra with accompanying cell loss and functional impairment. truncal vagotomy blocked this spread, confirming the direct spread through this nerve, confirming what was seen previously. further, knockout of α-synuclein prevented the spread as well, indicating that the propagation is dependent on the molecule. this new animal model provides a robust experimental system, and together with the previous studies adds to the growing trend focusing on gut to brain transmission in parkinson’s disease. 10. huntington’s disease: a spotlight on astrocytes astrocytes play a critical role in a broad spectrum of nervous system functions, through direct interactions with neurons, blood vessels and other glial cell populations. thus, it is not surprising that they have been implicated in a number of neurodegenerative disorders. glial pathology is well-documented in tauopathies (e.g., tufted astrocytes of psp) and synucleinopathies (e.g., papp-lantos bodies in oligodendrocytes in msa). huntington’s disease (hd) principally affects the striatum and cortico-striatal-thalamic circuitry with medium spiny neurons being most vulnerable. intriguingly, the huntingtin gene (htt) gene is expressed in all cell types, but abnormalities in astrocytes have been demonstrated in post-mortem hd brains tissue. astrocytes express mutant htt (mhtt) with increased gfap expression and reactive changes. reactive astrocytosis is a constant feature of neurodegeneration that accompanies neuronal loss, but how these changes contribute to disease and the extent to which they are secondary changes or contribute causally is unclear. emerging evidence from transcriptomic studies has begun to paint a picture suggesting that astrocytes may not be passive bystanders, but may play an active role in some diseases, including hd. these studies have revealed two broad categories of astrocytes, termed a1 and a2, with a1 being neurotoxic (liddelow et al., 2017; zamanian et al., 2012). these and other studies prompted a group of investigators at ucla, led by dr. baljit s. khakh, to take a closer look at astrocytes in hd. their study, published in october 2019 in science translational medicine, used transcriptomic approaches in post-mortem human brain tissue from 36 huntington’s disease patients, vonsattel stage 0-3, alongside two transgenic mouse models to probe astrocyte pathology in hd (diaz-castro et al., 2019). in their 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(2012). genomic analysis of reactive astrogliosis. the journal of neuroscience: the official journal of the society for neuroscience, 32(18), 6391–6410. https://doi.org/10.1523/jneurosci.6221-11.2012 copyright: © 2020 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. neuromuscular disease: 2022 update feel free to add comments by clicking these icons on the sidebar free neuropathology 3:5 (2022) review neuromuscular disease: 2022 update marta margeta1 1 department of pathology, university of california, san francisco, ca, usa address for correspondence: marta margeta · ucsf pathology, box 0511 · 513 parnassus ave., hsw-514 · san francisco, ca 94143 · usa marta.margeta@ucsf.edu submitted: 16 february 2022 accepted: 02 march 2022 copyedited by: henry robbert published: 04 march 2022 https://doi.org/10.17879/freeneuropathology-2022-3805 keywords: covid-19, guillain-barré syndrome, mlip, vwa1, optic neuropathy, autophagy, peripheral neuropathy, systemic sclerosis, microvessels, idiopathic inflammatory myopathy, gene expression profiling, pompe disease abstract this review highlights ten important advances in the neuromuscular disease field that were reported in 2021. as with prior updates in this article series, the overarching topics include (i) advances in understanding of fundamental neuromuscular biology; (ii) new / emerging diseases; (iii) advances in understanding of disease etiology and pathogenesis; (iii) diagnostic advances; and (iv) therapeutic advances. within this general framework, the individual disease entities that are discussed in more detail include neuromuscular complications of covid-19 (another look at the topic first covered in the 2021 review), autosomal recessive myopathy caused by mlip mutations, autosomal recessive neuromuscular disease caused by vwa1 mutations, leber’s hereditary optic neuropathy, myopathies with autophagic defects, trna synthetase-associated charcot-marie-tooth disease, systemic sclerosis-associated myopathy, humoral immune endoneurial microvasculopathy, and late-onset pompe disease. in addition, the review highlights a few other advances (including new insights into mechanisms of muscle and nerve regeneration and the use of gene expression profiling to better characterize different subtypes of immune-mediated myopathies) that will be of significant interest for clinicians and researchers who specialize in neuromuscular disease. abbreviations ars aminoacyl-trna synthetase, ass anti-synthetase syndrome, assm anti-synthetase syndrome-associated myositis, avm autophagic vacuolar myopathy, casa chaperone-assisted selective autophagy, covid-19 coronavirus disease 2019, ck creatine kinase, cmt charcot-marie-tooth disease, dm dermatomyositis, ert enzyme replacement therapy, gaa acid α-glucosidase, gbs guillain-barré syndrome, hiem humoral immune endoneurial microvasculopathy, hmgcr 3-hydroxy-3-methylglutaryl-coa reductase, iim idiopathic inflammatory myopathy, imnm immune-mediated necrotizing myopathy, isr integrated stress response, lhon leber’s hereditary optic neuropathy, lopd late-onset pompe disease, m6p mannose-6-phosphate, mct1 monocarboxylate transporter 1, mlip muscular a-type lamin-interacting protein, mmcp minimal myositis with capillary pathology, msa myositis specific antibody, msp multiple system proteinopathy, musc muscle stem cells, mxa myxovirus (influenza virus) resistance 1, interferon-inducible protein p78, nampta nicotinamide phosphoribosyl transferase a, nmd neuromuscular disease, sibm sporadic inclusion body myositis, ssc systemic sclerosis, vwa1 von willebrand factor a containing 1 protein.   in this annual update, i will briefly describe ten neuromuscular field advances from last year that i consider to be most important and/or interesting; as in the earlier updates (margeta, 2020b, 2021), these advances will be grouped into different “discovery clusters” and listed in no particular order. advances in fundamental neuromuscular biology with implications for neuromuscular disease 1. revisiting the role of macrophages in nerve and muscle regeneration in the last year’s review (margeta, 2021), i highlighted the 2020 discovery of a crosstalk between macrophages and satellite cells in skeletal muscle repair. excitingly, two studies published in 2021 extended that work by further dissecting the molecular mechanisms by which macrophages promote regeneration not only in skeletal muscle (ratnayake et al., 2021) but also in peripheral nerves (jha et al., 2021). together with other recent work in the field of regenerative medicine, these two studies provide evidence that macrophages – which are readily detectable within necrotic muscle fibers (fig. 1a-b) and adjacent to degenerating peripheral nerve axons (fig. 1c-d) – play a major role in the pns repair, extending well beyond the long-recognized phagocytic clearance of necrotic cellular debris at injury sites. figure 1. macrophages home on degenerating muscle and nerve fibers. a representative field from an h&e-stained muscle section (a) shows muscle fibers in varying stages of necrosis and repair, starting with early coagulative necrosis, progressing through myophagocytosis, and ending with regeneration mediated by macrophage-activated satellite cells; in this case, the fiber injury was immune-mediated, but similar changes are seen in all necrotizing myopathies regardless of the underlying etiology. the cd68 stain of the same area (b) highlights macrophages within necrotic muscle fiber segments (myophagocytosis). a representative field from an h&e-stained peripheral nerve section (c) shows a marked loss of large myelinated axons and abundant digestion chambers (fragments of degenerating axons) in a case of acute axonal neuropathy. the cd68 stain of the same microscopic field (d) highlights endoneurial macrophages associated with degenerating axon fragments. the stains were performed on formalin-fixed, paraffin-embedded tissue; scale bar, 50 μm. ratnayake and colleagues used sophisticated genetic and pharmacologic approaches to delineate the macrophage-stem cell interactions that are required for muscle wound repair in both zebrafish and mice (ratnayake et al., 2021). using the zebrafish laser-ablation injury model, they showed that a subset of tissue-resident macrophages that are recruited to the wound following injury remained at the injury site, creating a “dwelling” macrophage population that had spherical morphology (in contrast to transient macrophages, which had a stellate appearance); these dwelling macrophages formed intimate connections with muscle stem cells (musc) that were reminiscent of dendritic cell-t cell immunological synapses, forming a transient pro-proliferative stem cell niche that persisted until musc underwent cell division. based on their gene expression profiles, dwelling macrophages were shown to be a heterogenous cell population; however, the authors identified a single “mature” subpopulation (defined by expression of markers associated with the anti-inflammatory macrophage phenotype, such as arginase 2 and matrix metalloproteinase-9) that mediated muscle repair by stimulating musc proliferation. to promote musc mitogenesis, these mature dwelling macrophages (which constitute ~70% of all dwelling macrophages) secreted nicotinamide phosphoribosyl transferase a (nampta), which bound to the chemokine receptor ccr5 expressed by musc; interestingly, this mitogenic effect of nampta was independent of its enzymatic activity. the zebrafish findings were replicated in mice using the volumetric muscle loss model, which simulates irrecoverable muscle injury that occurs following trauma, tumor excision, or infarction (quarta et al., 2017; sicari et al., 2012): delivery of human recombinant nampt (the human analogue of zebrafish nampta) into the excisional muscle defect using a fibrin hydrogel led to a complete restoration of muscle architecture, with an increase in the number of proliferating satellite cells and centrally nucleated, regenerating muscle fibers relative to the control condition (fibrin hydrogel without nampt). while these results are very promising from the tissue engineering standpoint, it remains to be seen whether a similar approach can be used to promote muscle fiber regeneration in muscle dystrophies and other muscle diseases that show a significant loss of muscle volume. the study by jha and colleagues (jha et al., 2021) focused on the role of macrophage metabolism in the peripheral nerve repair following mechanical injury, and their nerve regeneration findings in many ways echo the muscle regeneration findings made by ratnayake et al. using the murine sciatic nerve crush model of wallerian degeneration, jha et al. showed that the activity of monocarboxylate transporter 1 (mct1, encoded by slc16a1 gene) in macrophages, but not in dorsal root ganglion neurons, schwann cells, or perineurial cells, plays a key role in axon regeneration. selective deletion of mct1 from macrophages did not affect macrophage recruitment to the portion of the nerve distal to the site of injury; instead, it impaired the ability of recruited macrophages to phagocytose axon and myelin debris. in addition, mct1 deletion led to a decrease in the expression of macrophage transcription factor atf3, thereby attenuating macrophage reprogramming into a pro-regenerative, anti-inflammatory phenotype characterized by high expression of arginase 1 and chitinase-like 3. excitingly, the authors also showed that i.v. treatment with bone marrow-derived wt macrophages fully restored peripheral nerve regeneration in mice with macrophage-selective mct1 deletion, while macrophage-specific mct1 upregulation led to improved nerve regeneration. taken together, these findings suggest that adoptive transfer of patient-derived macrophages engineered to upregulate their mct1 activity could be used to treat severe peripheral nerve injuries. however, this study has one very significant caveat: while both anti-regenerative and pro-regenerative effects of various experimental manipulations could be demonstrated on the tissue level (by quantifying regenerating axon clusters, relative thickness of axon myelin sheaths, and the number and distribution of neuromuscular junctions in the gastrocnemius muscle), they were not accompanied by parallel measures of behavioral recovery. one possible explanation for this discrepancy is that behavioral recovery actually preceded tissue recovery in this mouse model, suggesting that alternate recovery / behavioral compensation pathways are important, at least in rodents; nonetheless, this animal model discrepancy needs to be resolved before exploring the efficacy of macrophage-based therapies for human axonal neuropathies. newly defined / emerging neuromuscular diseases 2. neuromuscular complications of covid-19: what have we learned after one more year of the pandemic? covid-19, the novel infectious disease caused by sars-cov-2, primarily targets the respiratory system but can also affect many other tissues and organs, including the pns. in the last year’s review (margeta, 2021), i summarized what was known about the neuromuscular complications of covid-19 at the end of 2020; given the preliminary nature of those investigations, i decided to revisit this topic in the current review, providing an update on what has been learned since then. one area of intense interest within the neuromuscular field has been the possible link between covid-19 and guillain-barré syndrome (gbs), an acute immune-mediated polyneuropathy characterized by ascending weakness, mild-moderate sensory abnormalities, and pain. most gbs cases are triggered by a bacterial or viral infection that precedes neurologic symptoms by 1-3 weeks; the strongest association is seen with campylobacter jejuni bacterium (~100 gbs cases / 100 000 c. jejuni infections) and with zika virus (~50 gbs cases / 100 000 zika virus infections). while small early studies from italy and spain suggested that gbs could also be triggered by a preceding sars-cov-2 infection (filosto et al., 2021; fragiel et al., 2021), no such link was found in a more comprehensive epidemiologic study performed in the uk (keddie et al., 2021) [for a more detailed discussion of the 2020 data, see the last year’s review (margeta, 2021)]. discrepancies between these initial studies prompted animated discussion among the gbs experts, but also led to additional investigations undertaken by several different groups; taken as a whole, this new evidence provides further support for a lack of a strong link between covid-19 and gbs. for example, the data originally reported by keddie at al. reflected the first uk covid-19 surge (in march and april 2020); extension of the same study until the end of 2020 showed essentially the same findings, with a large increase in the number of covid-19 cases in the fall of 2020 that was accompanied by a decrease, rather than an increase, in the number of gbs cases during the same time period [(lunn et al., 2021) and fig. 2]. moreover, as reported by the same group of investigators in a very recently published medrχiv preprint (keh et al., 2022), this disassociation between covid-19 and gbs numbers has persisted into 2021, with a clear uptick in gbs cases observed in march and april of 2021 (secondary to covid-19 vaccination; further discussed below), but no similar increase was seen in august and september of 2021, during the delta variant-associated covid-19 surge. (no data is yet available for the most recent covid-19 surge caused by the omicron variant of sars-cov-2.) a similar lack of association between covid-19 and gbs was observed by a team of investigators in singapore (umapathi et al., 2021). in contrast, a weak association between sars-cov-2 infection and gbs (1.5 excess gbs cases per 100 000 sars-cov-2-positive tests) was reported by a different group of uk investigators (patone et al., 2021); however, that study was based on the hospital coding data, which are not always entirely accurate, and included gbs cases that were diagnosed on the same day as covid-19 (suggesting a coincidental rather than causative relationship). finally, no excess gbs cases were observed by researchers of the international gbs outcome study consortium, which includes gbs experts from china and japan in addition to several european countries. while this study was not designed for systematic gbs surveillance, a significant increase in the gbs case incidence should have led to an observable increase in the study inclusion rate; instead, the inclusion rate in the first 5 months of 2020 was comparable to inclusion rates in 2017, 2018 and 2019 (luijten et al., 2021). taken together, the preponderance of data available to date suggests that a preceding sars-cov-2 infection is not a major risk factor for development of gbs; however, it remains possible that rare gbs cases could be triggered by infection with this virus. figure 2. daily uk infections with covid-19 by pcr (blue bars) and monthly cases of gbs. 2020 = red dotted line, 2019 = green dashed line, secondary y-axis. the graphs demonstrate no visible increase in gbs in the last quarter of 2020 with a rise in case numbers between 30% and 1000% more than in march/april. note significant alterations in testing occurred in the uk in april 2020 resulting in the subsequent enhanced detection of most symptomatic cases. hospital admissions were 30% higher on 1 january 2021 than in april 2020. sources: covid cases (left axis, blue bars) https://coronavirus.data.gov.uk/details/cases; nhse national immunoglobulin database courtesy mdsas, manchester, uk. [with publisher’s permission, this figure and its legend are reproduced from (lunn et al., 2021).] in contrast to a lack of definite association between gbs and covid-19, there is a clear link between gbs and the first dose of the chadox1 ncov-19 (astrazeneca) adenoviral covid-19 vaccine, with an excess risk of ~0.4-0.6 gbs cases for each 100 000 administered vaccine doses (keh et al., 2022; patone et al., 2021). interestingly, a similar excess gbs risk was seen in the us during the 1976-77 flu vaccination campaign, with 0.5-0.6 gbs cases observed for each 100 000 doses of the “swine flu” vaccine given that year; the risk attributable to later influenza vaccines has been significantly lower (~0.1 gbs cases for each 100 000 vaccines), and no increase in the gbs risk has been observed with the mrna-based covid vaccines, bnt162b2/tozinameran (pfizer) and mrna-1273 (moderna). given that all these covid-19 vaccines target the spike protein of sars-cov-2, the differences among them suggest that the excess gbs risk is attributable to the adenoviral vector rather than the sars-cov-2 spike protein antigen; supporting this hypothesis, a warning about a link between the adenovirus-based ad26.cov2.s (j&j) covid-19 vaccine and gbs was announced by the us food and drug administration in july 2021 (https://fda.gov/news-events/press-announcements/coronavirus-covid-19-update-july-13-2021), although no formal scientific study about this association has been published to date. what about covid-19-associated myopathy? although initial studies were extremely limited, they suggested that weakness and high creatine kinase (ck) levels seen in a significant subset of covid-19 patients were immune-mediated rather than due to direct viral infection of skeletal muscle [reviewed in (margeta, 2021)]. based on the two large autopsy case-control studies published since then (aschman et al., 2021; suh et al., 2021), this initial impression was essentially correct. aschman and colleagues examined deltoid and quadriceps muscles from 43 patients with covid-19 and compared them to the same muscles sampled from 11 severely ill patients negative for sars-cov-2. they found that ~60% of patients who died with severe covid-19 showed evidence of immune-mediated muscle pathology including some combination of mild-severe inflammation (mostly consisting of cd8+ t cells and macrophages), degenerating muscle fibers, and mhc-i and mhc-ii upregulation in muscle fiber sarcolemma (the latter mostly seen in chronic cases). three samples showed perifascicular upregulation of mhc-i and mhc-ii; however, no sarcoplasmic upregulation of dermatomyositis (dm) marker mxa [myxovirus (influenza virus) resistance 1, interferon-inducible protein p78; also known as mx1] was seen in any of the specimens. (interestingly, some covid-19 samples showed increased mxa expression in the endomysial capillaries, possibly reflecting a nonspecific response to systemic viral infection.) in addition, a few covid-19 samples showed evidence of small and medium-vessel angiitis. although sars-cov-2 rna was detectable by pcr in some muscle homogenates, immunohistochemistry against sars-cov-2 spike protein was negative in all cases and no viral particles were demonstrated on electron microscopy (aschman et al., 2021). very similar findings were reported by suh et al., who studied psoas muscles from 35 patients who died following covid-19 infection and 10 severely ill patients who were covid-19-negative. in addition to diffuse type 2 fiber atrophy (which was present in almost all cases and controls), these authors found evidence of immune-mediated muscle pathology in ~70% of covid-19 cases, with necrotizing myopathy in 9 patients, t celland macrophage-mediated myositis in 8 patients, and isolated diffuse or multifocal mhc-i upregulation in additional 8 patients. like aschman et al., suh et al. observed perifascicular pattern of mhc-i upregulation without concurrent sarcolemmal mxa upregulation in one covid-19 case, while mxa expression restricted to endomysial capillaries was seen in 8 covid-19 cases. immunohistochemistry against sars-cov-2 nucleocapsid protein was negative in all 35 covid-19 cases. in contrast to the findings in these autopsy studies, direct viral infection of muscle fibers was recently documented by both immunohistochemistry and electron microscopy in muscle biopsies from two of three covid-19 patients with critical illness myopathy and persistent sars-cov-2 positivity on pcr testing (dodig et al., 2022). it is currently unclear what accounts for the discrepancy between these three studies; one possibility is that the difference reflects the sample type used (muscle biopsy vs. autopsy), although that does not seem likely given (1) that sars-cov-2 viral infection can be demonstrated by both immunohistochemistry and electron microscopy in the lungs of autopsied covid-19 patients and (2) that no sars-cov-2 was definitively detected in other muscle biopsies from covid-19 patients reported to date. more likely, this difference can be attributed to the effects of a long-standing and persistent sars-cov-2 infection, which was documented in the patients described by dodig et al. but is not a common feature of covid-19. importantly, and regardless of the specific reason for the rarity of this finding, the study by dodig and coauthors demonstrates that direct sars-cov-2 infection of skeletal muscle can occur, and raises the possibility that such infection is present in the early stages of covid-19 in many patients, acting as a trigger for the subsequent immune-mediated muscle pathology that develops in some instances. 3. autosomal recessive myopathy caused by truncating mutations of mlip (muscular a-type lamin-interacting protein) unexplained persistent elevation of serum ck levels points to a chronic myopathic process that is likely genetic in origin, but identifying the specific underlying etiology can be challenging if no other symptoms and signs are present to narrow the differential diagnosis. when chronic hyperckemia is associated with exercise intolerance and exercise-induced rhabdomyolysis, the underlying problem is usually metabolic; if, on the other hand, chronic ck elevation is accompanied by muscle weakness and wasting, the patient likely has a muscular dystrophy. two interesting papers published in 2021 (lopes abath neto et al., 2021; salzer-sheelo et al., 2021) uncovered a novel autosomal recessive myopathy that is caused by loss-of-function mutations in mlip (muscular a-type lamin-interacting protein; also known as muscular lmna-interacting protein) and is clinically characterized by persistent basal hyperckemia. among the twelve affected individuals identified to date, eight developed symptoms in childhood (with symptom onset between 8 months and 7 years of age); their clinical findings included exertional myalgia, episodes of rhabdomyolysis (occasionally exercise-induced, but without clear trigger in most cases), and/or mild progressive proximal muscle weakness in addition to chronically elevated ck levels. the remaining four individuals were identified based on genetic screening of the subject cohort enrolled in the university of maryland study of old order amish; based on the evaluation of banked serum specimens (available for 3 of 4 individuals), those patients, who currently range from 24 to 67 years in age, also have chronic hyperckemia, but no other known neuromuscular symptoms. muscle biopsies were performed for 7 of 8 clinically affected patients and showed a wide spectrum of histopathologic findings, ranging from mild nonspecific features to necrotizing and – less frequently – dystrophic changes. thus, it is not yet entirely clear whether to classify this new disease as a metabolic myopathy or a muscular dystrophy; while muscular dystrophy is more likely, the final classification will require careful clinical follow-up and repeat muscle biopsies of the known symptomatic individuals (who currently range from 5-19 years of age), as well as identification of additional patients by genetic testing of individuals with otherwise unexplained chronic hyperckemia. mlip is a ubiquitously expressed, alternatively spliced protein (23-57 kda) that directly interacts with lamins a/c (two intermediate filaments, both derived from the lmna gene, that are the main constituents of the nuclear envelope); its expression is highest in the heart, skeletal muscle, and brain, but its function remains poorly understood (ahmady et al., 2011). in a murine model, mlip deletion in the heart led to metabolic abnormalities, abnormal adaptation to stress, and accelerated progression from compensated cardiac hypertrophy to decompensated heart failure (cattin et al., 2015; huang et al., 2015). in contrast, mlip deletion in murine skeletal muscle led to increased central nucleation but no obvious neuromuscular phenotype (liu et al., 2020). in humans, variants in the mlip genetic locus [a single coding missense variant (nm_138569, c.475g>a, p.val159ile) and several noncoding variants] have been shown to increase the risk of dilated cardiomyopathy (esslinger et al., 2017); however, prior to studies by lopes abath neto et al. and salzer-sheelo et al., it was not known whether mlip mutations can directly cause human disease. twelve affected individuals from 8 unrelated families that are described in these two studies all carry either homozygous or heterozygous biallelic truncating mutations of mlip gene, with mutation hotspots in exons 4 and exons 9; while the molecular consequences of these mutations still need to be fully elucidated, initial work suggests that they lead to nonsense-mediated mrna decay and presumed loss of protein expression [although the latter is likely specific for individual splice variants and has therefore been difficult to establish (lopes abath neto et al., 2021)]. interestingly, 1 of 8 affected children and all 4 affected adults with biallelic mlip mutations showed evidence of mild cardiac involvement, raising the possibility that mlip deficiency causes cardiac abnormalities in humans as well as mice; this is particularly intriguing given the broad spectrum of human disease associated with lmna mutations, which includes cardiomyopathy in addition to muscular dystrophy, peripheral neuropathy, lipodystrophy, and premature aging (ho and hegele, 2019). future work will be required to fully define the nature and scope of skeletal and cardiac muscle phenotypes caused by mlip deficiency and to elucidate whether mlip interaction with lamins a/c plays an important role in the pathogenesis of this new human disease. 4. autosomal recessive neuromuscular disease caused by mutations in vwa1 (von willebrand factor a containing 1 protein) in the past, a disease was typically defined based on its unique clinicopathologic features, with the underlying etiology and pathogenesis established later. in the era of comprehensive genetic testing, this traditional sequence is being inverted: new genetic diseases are identified and defined based on their etiology, and future work is required to fully define their clinicopathologic features. as with the mlip-associated recessive myopathy described in discovery #2, two different research groups (deschauer et al., 2021; pagnamenta et al., 2021) used a genetic approach to identify a novel autosomal recessive neuromyopathy caused by mutations in vwa1 (von willebrand factor a containing 1 protein), also known as warp (von willebrand factor a domain-related protein). deschauer et al. started their study in a more “conventional” manner, by performing exome sequencing of 10 individuals with presumed genetic neuromuscular disease but without genetic diagnosis; 2 of these 10 patients turned out to carry biallelic truncating mutations in vwa1, a gene not previously associated with human disease but with high expression in peripheral nerve and skeletal muscle tissues. follow-up analyses of the patients’ families and additional “unsolved” patient cohorts identified 13 additional individuals with biallelic vwa1 mutations; 5 of 6 identified mutations are predicted to result in protein truncation, and all 6 are predicted to result in a loss of protein function. pagnamanta et al. used a slightly different approach: they identified vwa1 as a potentially interesting gene based on its high expression in the pns and searched the data from the 100k genome project (turnbull et al., 2018) for individuals carrying biallelic vwa1 variants. this analysis ultimately yielded 10 unrelated patients that fit the inclusion criteria, with 7 additional individuals identified through follow-up studies of other patient cohorts. interestingly, 15 of these 17 patients (from 14 of 15 separate families) carry the same truncating vwa1 mutation (10-bp insertion, nm_022834, c.62_71dup; pgly25argfster74) in either homozygous or heterozygous state; this mutation was also identified in 3 of 6 families described by deschauer et al., and is estimated by pagnamanta et al. to have arisen in europe ~8000 years ago. interestingly, the same 10-bp tandem repeat is deleted (rather than duplicated) in other affected individuals, suggesting that it is prone to de novo alterations. what is the clinicopathologic phenotype of the vwa1-associated neuromuscular disease? the affected individuals typically develop slowly progressive non-length dependent axonal motor neuropathy that presents with distal weakness and foot deformities, often in early childhood; however, proximal weakness with scapular winging and mildly elevated ck level is sometimes also present, and it is not entirely clear whether this myopathic involvement represents a separate phenotype of vwa1 deficiency, or whether myopathic changes are secondary to the primary neuropathic process. muscle biopsies were evaluated for a subset of cases (7 of 32 patients across both studies had biopsies that were available for review), showing a similarly complex picture: well-developed chronic neurogenic changes were accompanied by some myopathic features (including fiber size variation, internal nucleation, endomysial fibrosis, fiber lobulation and/or whirling, and mitochondrial alterations) but no basement membrane abnormalities. vwa1 is a 445 aa extracellular matrix protein that contains the n-terminal von willebrand factor a domain followed by two fibronectin type iii repeats; it is expressed in the cartilage and in the basement membranes of peripheral nerves (endoneurium), skeletal muscle (endomysium), and the cns vasculature, where it interacts with collagen vi and perlecan (two proteins that are also associated with genetic nmd) (allen et al., 2009). vwa1-null mice show a delayed response to painful stimuli and impairment of fine motor coordination; histologically, their peripheral nerves show aberrant fusion of schwann cell basement membranes, while their skeletal muscles and other vwa1-expressing tissues show no obvious abnormalities (allen et al., 2009). knockdown of zebrafish vwa1 gene (zebrafish vwa1 analogue) leads to dose-dependent abnormalities of motor neuron development, with reduced axon branching of primary motor neurons, axon truncation of secondary motor neurons, and reduced number of neuromuscular junctions, as well as some muscle fiber disorganization (pagnamenta et al., 2021). taking together the human and animal model data, it is clear that vwa1-deficiency affects motor neuron axons and leads to neuropathic changes in the affected musculature; however, it remains to be established whether these abnormalities represent a developmental or degenerative disease, and whether there is a separate but concurrent myopathic phenotype. in addition, it needs to be elucidated how the subtle basement membrane abnormalities lead to these neuromuscular phenotypes. advances in understanding of etiology and pathogenesis of neuromuscular diseases 5. impaired repair of mitochondrial respiratory chain complex 1 causes autosomal recessive variant of leber’s hereditary optic neuropathy leber’s hereditary optic neuropathy (lhon), first defined as a clinical entity in 1871, is a degenerative disease of retinal ganglion cells and their axons that presents with subacute bilateral painless loss of central vision. most lhon cases are maternally inherited; three point mutations in mitochondrial dna (mtdna), each of which alters the coding sequence of a different subunit of mitochondrial respiratory chain complex i, account for ~90% of the maternally inherited cases, with other mtdna mutations accounting for additional ~5%. however, approximately 5% of lhon patients carries no pathogenic mtdna mutations; in their groundbreaking work published last year, stenton et al. showed that many of these previously unresolved lhon cases carry biallelic missense mutations in the gene encoding dnajc30 chaperone (stenton et al., 2021). among 33 individuals with dnajc30-mutated, autosomal recessive form of lhon (arlhon) characterized by stenton and colleagues, 29 carry the same missense mutation (nm-032317, c152a>g, ptyr51cys; estimated to have arisen in eastern europe ~85 generations / ~2100 years ago); 2 additional missense dnajc30 mutations were identified in the remaining 4 patients. clinically, arlhon cases are indistinguishable from mtlhon cases, although arlhon has a significantly earlier age of onset (19.9+/-7.9 vs. 30.7+/-15.0 years). like mtlhon, arlhon is characterized by incomplete disease penetrance, with some homozygous mutation carriers not expressing the clinical phenotype. interestingly, mutation penetrance is sex-dependent in both mitochondrial and autosomal recessive forms of lhon, with disease prevalence 5-10 times higher in male compared to female mutation carriers; both incomplete penetrance and male predominance are unusual for an autosomal recessive disease, and the mechanism(s) underlying these phenomena are yet to be elucidated. importantly, arlhon patients have a better response to treatment with idebenone [a coenzyme q10 analogue that bypasses complex i and is currently approved for lhon treatment in europe (amore et al., 2021)] than mtlhon patients; this difference in the treatment response highlights the clinical significance of early molecular diagnosis of arlhon cases. notably, these genetic and clinical findings were replicated by another very recent study (kieninger et al., 2022), which also identified two additional dnajc30 pathogenic variants. how do dnajc30 mutations cause lhon? all three initially identified variants are located in the j domain, a conserved portion of dnajc30 with homology to the heat shock family of proteins. muscle biopsies from arlhon patients showed a small but significant defect in complex i activity (but no change in complex iv and complex v activities), and this complex i-specific defect was recapitulated in patient-derived fibroblast cell lines and dnajc30-knockout hek293 cells. interestingly, the functional defect of complex i was accompanied by a small but consistent increase (rather than decrease) in the expression level of complex i subunits, with no corresponding increase in the level of mrnas that encode these subunits. the increase in the complex i subunit expression level was accompanied by a decrease in the turnover of subunits that comprise the n-module, which normally have the highest turnover rate due to a high degree of oxidative damage (szczepanowska et al., 2020). taken together, these data indicate that dnajc30 maintains structure and function of complex i by facilitating exchange of oxidatively damaged subunits comprising its n-module; impairment of this repair mechanism leads to accumulation of damaged type i complexes, functional complex i defect, and lhon clinical phenotype. interestingly, dnajc30 was also shown to interact with several subunits of complex v / atp-synthase (tebbenkamp et al., 2018); however, clinical significance of that finding is unclear given that muscle biopsies from patients with dnajc30 mutations show no changes in complex v abundance and no functional complex v deficits. it is worth noting that complex i dysfunction is thought to play an important role in the pathogenesis of parkinson disease, cancer, and diabetes; a long-term clinical follow up of arlhon patients will be necessary to determine whether they are at increased risk for these common age-associated disorders. 6. autophagy defects in skeletal myopathies: the etiologic spectrum widens degradative macroautophagy (hereafter referred to as autophagy) is a conserved catabolic pathway required for cellular adaptation to nutrient deprivation and for degradation of damaged intracellular organelles and insoluble protein aggregates. autophagy is a highly regulated cellular process that involves multiple steps (starting with autophagy induction and ending with degradation of sequestered cargo by lysosomal hydrolases), disruption of each can lead to human disease [reviewed in detail in (margeta, 2020a)]. from the muscle pathology perspective, the most recognizable autophagy defect involves inhibition of the bulk autophagic flux, which results in the autophagic vacuolar myopathy (avm) phenotype; however, defects of autophagy induction and cargo sequestration can also result in muscle disease (fig. 3). several new diseases with autophagic impairment were identified in 2021, expanding the spectrum of etiologies that cause skeletal myopathy through dysregulation of autophagy; interestingly, however, all of them map on one of the four previously identified pathogenetic pathways illustrated in fig. 3. figure 3. the role of autophagic defects in the pathogenesis of skeletal myopathies. in myopathies with autophagy induction defects, the accumulation of damaged cellular organelles activates signaling cascades that contribute to fiber atrophy and apoptosis; however, there is no accumulation of autophagosomes. in contrast, autophagic vacuolar myopathies are caused by defects in the general autophagic flux that lead to a massive accumulation of autophagosomes and undigested cargo, ultimately resulting in a reduction of the mechanical force generated during muscle contraction (i.e., muscle weakness) and a failure of muscle regeneration. finally, cargo recognition defects lead to two different, defect-specific disease phenotypes: a granulophagy defect results in rimmed vacuole formation and inclusion body myopathy, while a casa defect causes myofibrillar myopathy by impairing z-disc stability. the electron micrographs illustrate (a) subsarcolemmal accumulation of damaged mitochondria that show marked variation in their size and shape; (b) an autophagic vacuole with an accumulation of undigested, electron-dense autophagic cargo; (c) a rimmed vacuole with a rim of autophagic material and a center that consists of aggregated proteins; and (d) myofibrillar disorganization with streaming of the z-disc material. [this figure and its legend are reproduced from (margeta, 2020a) with permission from the annual review of pathology: mechanisms of disease, volume 15 © 2020 by annual reviews, http://www.annualreviews.org/.] when there is a block of autophagy induction, autophagosomes do not normally form and therefore cannot significantly accumulate; however, there is accumulation of damaged and degenerating intracellular organelles, which ultimately leads to fiber atrophy and apoptosis. the autophagy induction defect plays an important role in the pathogenesis of at least some muscular dystrophies and congenital myopathies; however, the complete block of autophagy induction is embryonically or perinatally lethal in animal models, and no human disease has previously been attributed to mutations in one of the core autophagy genes (margeta, 2020a). that changed in 2021, when collier at al. identified five independent families with autosomal recessive mutations in atg7 (a core autophagy-related protein that is required for lipidation of lc3, the key step in induction of degradative autophagy); the affected individuals have a complex developmental disorder that commonly includes cerebellar hypoplasia, corpus callosum abnormalities, facial dysmorphism, and mild proximal myopathy (collier et al., 2021). muscle biopsies were performed on a few affected individuals and showed mild myopathic changes mimicking those seen in the autophagy-deficient murine muscle, with moderate mitochondrial abnormalities, lipofuscin accumulation, subsarcolemmal accumulation of autophagic cargo receptor p62/sqstm1 (encoded by sqstm1 gene), and diminished levels of lc3-ii (the lipidated, autophagosome-bound form of lc3); while some autophagosomes were present, there was no accumulation of autophagic vacuoles. intriguingly, another potential myopathy with impaired autophagy induction was reported last year by napolitano and co-authors (napolitano et al., 2021): while these authors classified their patient 6 as having an avm, his muscle biopsy showed high p62 and low lc3-ii (as would be expected with the autophagy initiation defect) rather than high levels of both p62 and lc3-ii (as would be expected in a true avm caused by the autophagic flux defect). interestingly, that individual was found to carry a potentially deleterious variant in tbc1d5 gene, which has not previously been associated with human disease but was shown to regulate autophagy initiation (popovic and dikic, 2014). specific classification of tbc1d5-associated myopathy aside, napolitano et al. showed that the avm pathology mimicking late-onset pompe disease (lopd) can be associated with mutations in genes not previously associated with this clinicopathologic phenotype (such as titin, dysferlin, plectin, and perilipin 3, among others); this highlights the importance of genetic diagnosis for lopd, which can be treated by enzyme replacement therapy (ert; further discussed in advance #10) – a treatment that is unlikely to be effective if the underlying molecular defect is not acid α-glucosidase (gaa) deficiency. cargo recognition defects, which result in selective (rather than global) autophagy dysfunction, can also cause muscle disease: impairment of granulophagy (autophagy of stress granules, which are ribonucleoprotein foci that form under cellular stress and, if persistent, need to be degraded via autophagy) leads to an inclusion body myopathy phenotype, while impairment of chaperone-assisted selective autophagy (casa), which is critical for the z-disc maintenance, results in myofibrillar myopathy (fig. 3). each of these phenotypes has been associated with mutations in many different genes; in 2021, two new genes were added to this ever-growing list. in the first study, leoni et al. showed that autosomal dominant missense variants in annexin a11, a 505 aa calcium-dependent phospholipid-binding protein, lead to hereditary inclusion body myopathy, amyotrophic lateral sclerosis, and frontotemporal lobar degeneration (leoni et al., 2021); given that other mutations in anxa11 (annexin a11-encoding gene) were previously shown to cause amyotrophic lateral sclerosis and frontotemporal degeneration (zhang et al., 2018), anxa11 is now the sixth gene known to cause multiple system proteinopathy (msp type 6). the other 5 genes associated with the msp phenotype encode either rna-binding proteins that form stress granules (hnrnpa2b1, hnrnpa1, and matr3 genes) or autophagy-associated regulatory proteins (vcp and sqstm1 genes); while the precise function of annexin 11 still needs to be elucidated, it most likely falls in the latter category given that other annexin a proteins play a role in autophagy regulation (xi et al., 2020). interestingly, rimmed vacuoles in msp-6 patients are positive for annexin 11 in addition to tdp-43, while rimmed vacuoles in sporadic inclusion body myositis (sibm) and other inclusion body myopathies are annexin 11-negative (leoni et al., 2021). in the second study, johari and colleagues showed that autosomal dominant missense mutations in smpx (small muscle protein x-linked) cause distal myopathy with protein inclusions (johari et al., 2021). smpx is a small (88 aa), proline-rich protein that is highly expressed in slow muscle fibers and cardiomyocytes, where it localizes to costameres and intermyofibrillar spaces; its function is currently unknown, but loss-of-function smpx mutations cause non-syndromic hearing loss without muscle weakness. muscle biopsies from patients with smpx-related distal myopathy, who do not have hearing loss, showed lc3-positive rimmed vacuoles, abundant smpx-and p62-positive sarcoplasmic inclusions, as well as smpx-negative inclusions that were positive for αb-crystallin and myotilin in addition to casa chaperones bag3 and hspb8; however, there was no significant myofibrillar disarray on electron microscopy. thus, it is not yet clear whether this new disease should be classified as a rimmed vacuolar (inclusion body) myopathy or as a myofibrillar myopathy; while a lot of additional work is needed to fully elucidate its pathogenesis, initial experiments suggest that smpx may affect stress granule dynamics (johari et al., 2021). thus, it will be interesting to see whether smpx mutations can also cause other diseases in the msp spectrum, either alone or in combination with mutations in other msp-associated genes. 7. activation of the integrated stress response plays a key role in the pathogenesis of trna synthetase-associated subtypes of axonal charcot-marie-tooth disease non-syndromic inherited sensory-motor peripheral neuropathies, also known as charcot-marie-tooth (cmt) diseases, are broadly classified into demyelinating, axonal, and intermediate subtypes, each of which is associated with a large number of causative genes and with more than one mode of inheritance (stavrou et al., 2021; zhang et al., 2021). within the axonal cmt category, an interesting and relatively recently recognized disease subset is caused by mutations in aminoacyl-trna synthetases (arss), which are ubiquitously expressed enzymes that charge amino acids onto their cognate trnas during protein synthesis. of 20 arss, 5 have been associated with autosomal dominant forms of axonal cmt disease [yars (tyrosyl-rs; cmtdic), gars (glycyl-rs; cmt2d), aars (alanyl-rs; cmt2n), hars (histidyl-rs; cmt2w), and mars (methionyl-rs; cmt2u)], while two (wars / tryptophanyl-rs and kars / lysyl-rs) have been associated with hereditary motor neuropathy type 9 (hmn9) and autosomal recessive intermediate cmt disease cmtrib, respectively. (of note, gars mutations can cause hmn5a in addition to cmt2d, while hars mutations can cause hmn2 in addition to cmt2w.) not much was known about pathogenesis of ars-associated forms of cmt until last year, when two fascinating studies were published back-to-back in science, together providing important mechanistic insights and therapeutic promise for this subcategory of inherited neuropathies (spaulding et al., 2021; zuko et al., 2021). spaulding et al. used three different mouse models of gars-cmt (cmt2d) and one mouse model of yars-cmt (cmtdic) to investigate molecular pathways that are affected by mutant gene expression (spaulding et al., 2021). in all four investigated mouse models, they found that the integrated stress response (isr) was selectively activated in alpha motor neurons and a subset of sensory neurons; the isr activation preceded neurologic symptom onset, was associated with increased expression of genes regulated by transcription factor atf4, and required sensor kinase gcn2. [the isr is a conserved cellular signaling pathway that alters gene expression program in response to various cellular stressors, including protein homeostasis defects, nutrient deprivation, viral infection, and oxidative stress (costa-mattioli and walter, 2020). these stresses are sensed by four specialized kinases (perk, gcn2, pkr and hri), activation of which ultimately leads to phosphorylation of the eukaryotic translation initiation factor eif2 and general reduction in protein synthesis along with an increase in translation of specific mrnas, such as atf4. if these adaptive responses fail to mitigate cellular stress, the isr triggers programmed cell death.] genetic deletion of gcn2 completely rescued the neuropathy phenotype in the gars mutant mice, with beneficial effects of deletion observed behaviorally, physiologically (as improved sciatic nerve conduction velocity), and pathologically (as ameliorated axon loss, increased axon diameter, and decreased muscle denervation). while only one of three gars-cmt mouse models was tested in these genetic studies, similar results were observed in a different gars-cmt model following pharmacologic inhibition of gcn2, with treatment that was initiated at disease onset (2 weeks of age); interestingly, the effect of pharmacologic gcn2 inhibition was greater in male than female mice, but the reason for this sex difference is currently unclear. in a parallel study, zuko et al. investigated mechanisms underlying inhibition of global protein synthesis that was previously documented in 6 different gars-cmt and yars-cmt mouse lines and could not be rescued by overexpression of wt gars or yars. while manipulation of translation initiation and upstream regulatory pathways had no significant effect, overexpression of trnagly rescued both the protein synthesis deficit and neuropathy phenotype in two drosophila models of gars-cmt. these findings were replicated in two mouse models of gars-cmt, with trnagly overexpression resulting in an essentially complete behavioral, physiologic, and pathologic rescue of peripheral neuropathy (zuko et al., 2021). in addition, zuko and colleagues showed that inhibition of global protein synthesis in this mouse model was due to trnagly sequestration by mutant gars, which led to ribosome stalling at gly codons; as a result, the neuropathy in gars-cmt mice was worsened by concurrent deletion of the ribosome rescue factor gtpbp2, which by itself does not lead to nerve damage. finally, connecting the dots between the two studies, zuko et al. showed that trnagly overexpression abrogated activation of the isr in the gars-cmt mouse model, indicating that depletion of the normal trnagly pool and consequent ribosome stalling are the upstream signals that lead to the isr activation. taken together, these two studies suggest that ars mutations cause autosomal dominant axonal cmt disease through a toxic gain-of-function mechanism: by binding more tightly to their cognate trnas, they deplete the free trna pool, ultimately leading to ribosome stalling, aberrant isr activation, and axonal degeneration. despite the very comprehensive nature of this work, additional questions need to be answered. for example, it remains to be shown whether the same (or similar) molecular mechanism plays a role in the pathogenesis of the aars-, hars-, and mars-associated cmt, and in axonal cmt more broadly. in addition, it is not clear which downstream effect of the isr activation (protein synthesis inhibition or the atf4 pathway activation) leads to axonal degeneration. nonetheless, and very excitingly, this work opens a new direction for the cmt treatment development: manipulation of the isr is emerging as a promising therapeutic avenue for several age-associated diseases, including neurodegeneration and cancer (costa-mattioli and walter, 2020), and at least some subtypes of axonal cmt can now be added to that list. [as an aside, ars enzymes also play an important role in anti-synthetase syndrome (ass), where they are targeted by disease-causing autoantibodies (histidyl-rs is targeted by anti-jo-1 antibodies, alanyl-rs by anti-pl12 antibodies, glycyl-rs by anti-ej antibodies, lysyl-rs by anti-sc antibodies, and tyrosyl-rs by anti-yrs antibodies; witt et al., 2016). ass and cmt disease have entirely different clinical manifestations, so the underlying pathogenetic mechanisms are likely also different despite this common link; while there is some evidence that protein translation is affected in ass – see advance #9 – it remains to be established whether the isr activation and/or protein synthesis inhibition play a role in the ass pathogenesis.] advances in neuromuscular disease diagnostics 8. microvascular pathology in immune-mediated neuromuscular disease major vascular pathology (such as vasculitis or amyloid vasculopathy) has long been recognized as an important cause of neuromuscular disease, and vessel evaluation on routine stains is a standard component of diagnostic neuromuscular pathology workup. the role of microvascular pathology is less well understood, but some microvascular abnormalities [such as deposition of c5b9 (complement membrane attack complex) in the endomysial capillaries] are included in the diagnostic criteria for several idiopathic inflammatory myopathies (iims) including dm, anti-synthetase syndrome-associated myositis (assm), and immune-mediated necrotizing myopathy (imnm). two studies published last year (siegert et al., 2021; trikamji and pestronk, 2021) expanded the spectrum of microvascular abnormalities seen in immune-mediated neuromuscular disorders, highlighting that careful evaluation of endomysial and endoneurial microvessels (arterioles and capillaries) can increase the diagnostic yield of muscle and nerve biopsies. systemic sclerosis (ssc) is a systemic autoimmune disease characterized by fibrosis of skin and internal organs, vasculopathy, and evidence of immune system dysregulation; skeletal muscle involvement is variable, with some patients developing a superimposed specific iim or less specific “overlap myositis”, while others (~20%) have a poorly understood muscle disease specific to ssc. to better define the latter disease entity, siegert et al. performed detailed retrospective evaluation of muscle biopsies from 18 ssc patients with muscle symptoms (derived from a larger cohort of 367 ssc patients) using routine light microscopy as well as semi-quantitative large scale electron microscopy [described as advance #9 in last year’s review (margeta, 2021)]. two thirds of evaluated ssc biopsies (12 of 18) showed a novel morphologic pattern (which the authors termed “minimal myositis with capillary pathology”; mmcp) characterized by sparse endomysial t cells, rare necrotic and regenerating muscle fibers, diffuse mhc-i and focal mhc-ii upregulation, and enlarged capillaries with prominent pericytes but no capillary c5b9 deposition; ultrastructurally, mmcp cases showed well-developed but mild capillary pathology, including thickening and reduplication of basement membranes, reactive endothelial changes, and capillary ensheathment by pericyte processes (fig. 4). similar but more severe capillary alterations were seen in other 6 ssc muscle biopsies, which also showed additional capillary abnormalities (such as tubuloreticular inclusions) and more prominent inflammatory changes; therefore, capillary pathology is a uniform feature of mmcp cases but is not pathognomonic for this condition, and must be interpreted in the context of other muscle biopsy findings. interestingly, mmcp ssc cases were clinically less severe than non-mmcp ssc cases, with higher frequency of localized (versus diffuse) skin sclerosis, less prominent internal organ involvement, and lower ck levels; however, these muscle abnormalities developed early in the disease course, with mean disease duration of 3 years at the time of biopsy (<1 year in 6 of 12 cases). figure 4. ultrastructural characterization of skeletal muscle biopsies with histological minimal myositis with capillary pathology (mmcp) phenotype. entire ultrathin sections were recorded by large-scale digitization at 7.3 nm pixel size; digitally magnified regions of interests (roi) of patients 3 (a, d, g–l), 8 (b, m), 6 (e), 2 (f), 18 (n; severe necrotizing myositis with capillary pathology and fibrosis), 1 (o) and non-diseased control (c). a markedly thickened capillaries can be clearly identified at low magnification (arrows). b capillary with pronounced thickening and reduplication (arrow) of the basement membrane (bm) as well as ensheathment by pericyte processes (white asterisk), mildly activated endothelium (black asterisk), lumen (lu), adjacent muscle fiber (mf). c healthy capillary with thin bm (arrow): note that no reduplication is apparent, endothelium with no signs of activation (asterisk), lumen with erythrocyte (er). different types of bm (bm) thickening were detected (d–f): distinct reduplication (d; arrows), endothelium (asterisk), fibroblast (fi); fuzzy appearance with less pronounced reduplication (e): note that the bm (arrow) directly underneath the endothelium (asterisk) is clearly visible, fibrous long spacing collagen (arrowhead); homogeneous thickened bm (f), capillary lumen (lu), neighboring muscle fiber (mf) with basal lamina (white arrow). different types of endothelial activation (g–i): increased size and number of endothelial cells (g; asterisk, lu lumen), note the granular appearance of the cytoplasm, probably mostly due to ribosomes; prominent endothelial membrane organelles (h; black asterisk, er erythrocyte in lumen), note the prominent and mostly small pericyte processes demonstrating mild ensheathment (white asterisks); degraded capillary (i), showing some remaining membrane structures, probably of endothelial or pericyte origin (arrowheads), almost empty bm “sack” with mild reduplication (arrows). [this figure and its legend were adopted from figure 3 in (siegert et al., 2021); this use is permitted under the creative commons attribution 4.0 international license.] while at least some aspects of microvascular pathology are routinely evaluated in muscle biopsies, the same is not true for nerve biopsies; the 2021 study by trikamji and pestronk suggests that this approach to nerve biopsy processing should probably change. these authors have retrospectively analyzed a cohort of 16 patients with a motor-sensory axonal polyneuropathy and complement deposition in endoneurial microvessels, detected by c5b9 immunohistochemistry performed on nerve cryosections. this patient cohort showed unique clinicopathologic features that could reflect a new / previously unrecognized autoimmune neuropathy, which the authors termed “humoral immune endoneurial microvasculopathy” (hiem). clinically, hiem patients showed progressive asymmetric distal weakness that was more prominent in lower extremities; there was no evidence of a systemic inflammatory process. pathologically, there was a patchy / asymmetric axon loss that preferentially affected myelinated axons, with rare digestion chambers and rare regenerating axon clusters but no significant inflammation; strong c5b9 staining was seen surrounding the endothelium in all or nearly all endoneurial microvessels, and was also detectable in endomysial capillaries in the majority of paired muscle biopsies (trikamji and pestronk, 2021). a potentially confounding factor is that similar capillary changes were previously documented in nerve and muscle biopsies from diabetic patients (yell et al., 2018); 56% of the patients in the hiem cohort also had diabetes, and the mean age of onset was lower in diabetic than non-diabetic patients. however, and very compellingly, 15 of 16 hiem patients showed a measurable strength improvement following a short course of corticosteroid therapy; this would not be expected if the observed nerve pathology was mainly due to chronic (and likely irreversible) diabetic neuropathy. while very intriguing, these findings need to be replicated in other patient cohorts, with corticosteroid treatment effectiveness confirmed by double-blind clinical trials. in addition, both nerve and muscle biopsies from hiem patients should be evaluated by electron microscopy: it is possible that ultrastructural evaluation will yield additional useful clues, either from pathogenetic or diagnostic perspective. nonetheless, trikamji and pestronk’s findings suggest that a portion of each nerve biopsy should be frozen for specialized immunohistochemical studies, which is currently not a standard operating protocol in the majority of neuromuscular pathology laboratories: in addition to enabling identification of potentially treatable hiem patients, such processing would also facilitate detection of immunoglobulin deposits that are important for accurate classification of amyloid and vasculitic neuropathies. 9. gene expression profiling in idiopathicinflammatory myopathies based on the current clinicoseropathologic criteria, iims are divided into four distinct subcategories [dm, assm, imnm, and sibm; for a brief overview of the diagnostic criteria and other recent advances in the iim field, see the 2020 update in this article series (margeta, 2020b)]. an interesting study published last year (amici et al., 2021) has used a gene expression profiling approach to take a deeper look into these disorders; while the current data is mainly interesting from the standpoint of disease pathogenesis, this study also provides a glimpse into the use of transcriptomic profiles for disease diagnostics. the authors used rna-sequencing data from 119 iim muscle biopsies [39 dm cases, 18 assm cases, 49 imnm cases, and 13 sibm cases; all assm cases were positive for anti-jo-1 antibodies, while the dm and imnm groups were heterogenous with respect to the patient’s serologic status, with 5-40 cases per myositis-specific antibody (msa) subgroup] and 20 normal muscle biopsies to generate transcriptome maps and identify gene expression modules that are dynamically regulated in iims. some of the results were not surprising; for example, dm biopsies showed activation of the type 1 interferon-inducible gene module, which was not significantly upregulated in other iim subtypes, while sibm cases were unique in showing consistent overexpression of t cell genes. other findings, however, were novel and somewhat surprising. for example, two gene modules – the stress response acute phase response module and the neutrophil degranulation module – were upregulated across all iim subtypes (including imnm cases, which generally lack significant inflammation), as was the cytoskeleton remodeling module, which includes tgf-β and could be related to endomysial fibrosis seen in many long-standing iim cases; in contrast, the large muscle function, redox metabolism, and ubiquitination gene module was downregulated across all iim subtypes. expression changes in some gene modules were more specific and characteristic for individual iim subtypes: for example, the previously mentioned upregulation of the type 1 interferon-induced gene module in dm cases was strongly correlated with downregulation of the titin transcript, while the assm cases showed upregulation of the rna binding, splicing, and translation genes. in addition to studying gene expression changes across different iim subtypes, the authors of this study converted individual trascriptomic profiles into images suitable for computational analysis by a convolutional neural network. unsupervised hierarchical clustering by neural network-derived features led to an interesting hierarchy of different patient subgroups, with the severe dm cases clustering closer to the assm cases and severe imnm cases than to the mild dm and imnm cases; one severe anti-mi-2 dm case was even misclassified within the assm group. this finding is congruent with the experience on a diagnostic muscle pathology service, where one can occasionally see a significant histopathologic overlap between anti-mi-2 dm, assm, and severe imnm (fig. 5). interestingly, in the group of 119 iim cases studied by amici at al., disease severity did not correlate with a specific msa subtype; instead, both mild and severe cases were seen in each msa-defined subgroup. if validated by future studies and found to correlate with the response to therapy and/or patient outcomes, this finding suggests that transcriptomic analysis might be able to extract clinically useful information not captured by serotype / disease subtype, and that gene expression profiling therefore may at some point become a routine component of diagnostic muscle biopsy work-up. excitingly, the authors of this study have shared the source code and model weights on their website (https://github.com/mendillolab), and this tool can be used by other researchers who perform muscle transcriptomics to further test and improve their algorithm. figure 5. histologic overlap between assm (a-c), anti-mi-2 dm (d-f), and a severe case of anti-hmgcr imnm (g-i). a typical assm case shows perifascicular fiber atrophy and necrosis (a) along with marked perimysial abnormalities [edema, fragmentation, macrophage-rich inflammation (best visualized on acid phosphatase stain; b) and a linear pattern of alkaline phosphatase positivity (c)]. anti-mi-2-associated dm often leads to a more severe muscle involvement than other dm subtypes and can histologically mimic assm; in this example (d-f), there was a prominent perimysial involvement including macrophage-rich inflammation (e) and strong alkaline phosphatase staining (f). markers of type 1 interferon response (such as mxa) can help differentiate dm from assm, but in the ucsf experience mxa upregulation is often weak in anti-mi-2 dm cases. typical imnm cases show little to no inflammation and are generally easily distinguished from other autoimmune myopathies; however, severe cases (such as the one shown in g-i) can be diagnostically challenging. this anti-hmgcr imnm case showed abundant macrophage-rich inflammatory infiltrate in the perimysium (h) along with well-developed perimysial alkaline phosphatase positivity (i); the only clue to the correct diagnosis was dense punctate p62 staining of muscle fibers (inset in g), which is not typically seen in either dm or assm. representative cryosection images are shown for all three cases; scale bars, 100 μm. advances in neuromuscular disease treatment 10. clinical trials: late-onset pompe disease revisited pompe disease is an autosomal recessive metabolic disorder caused by loss-of-function mutations in gaa, the lysosomal glycogen-catabolizing enzyme; depending on the level of residual gaa activity, the disease can present in infancy, childhood, or adulthood. while early onset pompe disease is a systemic disorder that affects multiple organs systems, lopd primarily affects skeletal musculature and is characterized by progressive weakness of the axial, limb-girdle, and respiratory muscles; pathologically, vacuolated muscle fibers show an accumulation of membrane-bound as well as free glycogen and a build-up of autophagic debris (fig. 6), so this disease is currently classified as an avm (fig. 6). ert with alglucosidase alfa, recombinant form of human gaa, was approved in 2006, constituting a breakthrough in pompe disease management; however, most lopd patients on this therapy experience a short-term improvement followed by progressive decline. the failure of ert in lopd is thought to primarily reflect inefficient delivery of alglucosidase alfa to skeletal muscle lysosomes: efficient lysosomal targeting is based on binding of mannose 6-phosphate (m6p)-glycans and bis-m6p-glycans within the recombinant protein to the cation-independent m6p receptor in the target tissue, and only a minor fraction of originally developed recombinant gaa contains bis-m6p ligands. figure 6. muscle biopsy findings in lopd. a representative h&e-stained cryosection (a) shows largely unremarkable skeletal muscle with a single highly vacuolated muscle fiber in this microscopic field (black arrowhead). the same microscopic field from a pas (periodic acid-schiff)-stained cryosection (b) shows two vacuolated muscle fibers with mild glycogen accumulation (black arrowheads); the less vacuolated fiber at the bottom of the image shows no obvious changes on the corresponding h&e-stained section (a). electron microscopy shows an increase in the lysosomal (membrane-bound) glycogen (white arrowheads in c) as well as accumulation of free sarcoplasmic glycogen accompanied by electron dense autophagic debris (d). the focal nature of these pathologic findings is typical for lopd, although the degree of muscle fiber involvement varies from case to case. scale bars: a and b, 60 μm; c and d, 2 μm. to try circumventing this problem, two new versions of recombinant gaa were developed and tested against the current standard of care (alglucosidase alfa), with the results of both clinical trials published last year in lancet neurology (diaz-manera et al., 2021; schoser et al., 2021). to generate the first investigational drug, avalglucosidase alfa, synthetic bis-m6p-glycane-containing oligosaccharides were chemically conjugated to recombinant human gaa; the safety and efficacy of this drug was evaluated by the comet study, a phase 3, randomized, double-blind clinical trial involving 55 sites in 20 countries (diaz-manera et al., 2021). the second investigational drug, cipaglucosidase alfa, is a novel recombinant human gaa with a high bis-m6p glycan content that is co-administered with a pharmacologic chaperone (miglustat) to increase its bioavailability; the safety and efficacy of this drug combination was evaluated by the propel study, also a phase 3, randomized, double-blind clinical trial that involved 62 sites in 24 countries (schoser et al., 2021). the comet trial enrolled 100 ert-naïve lopd patients who were randomized into equally sized avalglucosidase alfa and alglucosidase alfa groups; the propel trial, on the other hand, had a parallel group design (28 ert-naïve participants and 95 participants previously treated with alglucosidase alfa), with two thirds of participants in each subgroup receiving cipaglucosidase alfa + miglustat and one third receiving alglucosidase alfa + placebo. while the details of the study design differed slightly between the two trials, they had similar duration (49 weeks for comet and 52 weeks for propel) and their outcomes were quite similar: (1) the safety of both investigational drugs was high, with no new safety signals reported; (2) neither investigational drug was inferior to alglucosidase alfa, the current standard of care; and (3) while there was a trend toward increased efficacy for both investigational drugs, neither reached a statistically significant primary end-point. given the very promising results of prior preclinical (mouse model) studies and initial phase 1/2 trials (meena et al., 2020; puertollano and raben, 2021), these results are somewhat disappointing. intriguingly, however, the cipaglucosidase alfa + miglustat treatment was significantly more effective than the alglucosidase alfa + placebo treatment in the ert-experienced subgroup of the propel study, which is arguably most representative of the segment of the lopd patient population with the greatest need for new and more effective lopd therapies. both trials have now moved into their last phase (open label extended treatment period), the aim of which is to assess the long-term safety and efficacy of these new drugs; it will be interesting to see whether the difference in the efficacy profile between either investigational drug and alglucosidase alfa becomes greater over time or whether they will show a similar loss of efficacy over time. in addition, it will be interesting to directly compare the two new lopd drugs and determine whether one of them is superior to the other with respect to the size of initial improvement, effect duration, or both. most importantly, however, the modest results of these large and expensive clinical trials indicate that the cure for lopd remains elusive and highlight the need for a novel, ideally more effective and durable, lopd treatment approach going forward. disclosure statement the author receives research support from astellas gene therapies (formerly known as audentes therapeutics, inc) as a member of the muscle biopsy review committee for the aspiro (nct03199469) and fortis (nct04174105) clinical trials, which are evaluating the safety and efficacy of gene transfer therapy for x-linked myotubular myopathy (aspiro) and lopd (fortis). acknowledgements i am grateful to drs. nigel g. laing, gina ravenscroft, and benedikt schoser for helpful input during the conceptualization stage of this review. in addition, i would like to thank ms. christine lin for assistance with figure preparation. references ahmady, e., deeke, s.a., rabaa, s., kouri, l., kenney, l., stewart, a.f., and burgon, p.g. 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(2021). trna overexpression rescues peripheral neuropathy caused by mutations in trna synthetase. science 373, 1161-1166. copyright: © 2022 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. considering the myelin-centric hypothesis: insights from budka's historical adrenomyeloneuropathy case report feel free to add comments by clicking these icons on the sidebar free neuropathology 4:23 (2023) letter considering the myelin-centric hypothesis: insights from budka's historical adrenomyeloneuropathy case report ettore salsano1, chiara benzoni1 1 unit of rare neurological diseases, fondazione irccs istituto neurologico c. besta, milano, italy corresponding author: ettore salsano · unit of rare neurological diseases · fondazione irccs istituto neurologico c. besta · milano · italy ettore.salsano@istituto-besta.it submitted: 22 november 2023 accepted: 17 december 2023 copyedited by: georg haase published: 21 december 2023 https://doi.org/10.17879/freeneuropathology-2023-5218 keywords: adrenoleukodystrophy (ald), adrenomyeloneuropathy (amn), demyelinating, neuropathology we are writing in light of dr. budka's recent flashback paper on his seminal article on adrenomyeloneuropathy (amn) [1]. our letter aims to underscore a critical aspect that we believe dr. budka himself may have overlooked when revisiting his work after nearly fifty years: the likely myelin-centric nature of amn in its early stages. the original study by dr. budka et al. [2] contributed significantly to our understanding of amn. in his flashback paper dr. budka provided virtual microscopy of original histological slides and discussed the historical context. yet, there seems to be an under-appreciation of one of his most striking findings: the occurrence of demyelination independent of axonal degeneration in an early-stage case of amn. in the original study [2], dr. budka et al. conducted a pathological examination on an amn patient who exhibited the shortest duration of neurological symptoms prior to death, at the young age of 24, succumbing to acute adrenal failure only two years after the onset of neurological symptoms. this case stands in clear contrast to the average neurological history of 11.8 years (range 2-21) and the average age at death of 38.1 years (range 24-52) among the ten reported autopsied amn males [3]. remarkably, the index patient presented with a pattern of incomplete, symmetrical demyelination predominantly in the pyramidal tracts from the cerebral peduncles to the lumbar cord, with a pronounced involvement in the medulla oblongata's pyramids. similar, demyelination was also noted in the medial and lateral lemnisci, brachium conjunctivum, spinocerebellar tracts, and the medial portion of the posterior columns, although less extensive than in the pyramidal tracts. notably, the integrity of axons within these regions was maintained [2]. the pathological changes observed in the study are visually documented in figure 2a of the original paper [2], and correspondingly in figure 5 of the flashback paper [1]. in our opinion, this observation is not just a mere detail but a pivotal insight, suggesting that demyelination can precede and possibly initiate axonal degeneration, an aspect corroborated in humans by the work of castellano et al., who used in vivo quantitative mri techniques that align with the hypothesis of a primarily myelopathic process [4]. furthermore, in the human brain, the amn-causing protein abcd1 appears to be predominantly expressed in glial cells, namely oligodendrocytes, microglia, and astrocytes, as well as in endothelial cells, but not in most neurons [5]. additionally, the expression of human abcd1 in the oligodendrocytes of a zebrafish model of adrenoleukodystrophy (ald) has been shown to be sufficient to rescue mutant phenotypes, providing additional support for this hypothesis[6]. this aspect is critical, as it shifts our understanding of amn pathogenesis toward a focus on myelin pathology, particularly in the initial phases of the disease. the common co-occurrence of myelin pathology and axonal degeneration in more advanced cases and the data from abcd1-deficient mice may have blurred this perspective, leading to ambiguity regarding the primary pathological event [3,7]. to clarify this issue, we would highly encourage the study of a conditional knockout mouse model with the abcd1 gene selectively inactivated in glial cells such as oligodendrocytes or astrocytes. this approach would provide definitive evidence on whether primary damage to glial cells leads to the axonal degeneration seen in abcd1-deficient mice [7], thereby reinforcing the hypothesis of a myelin-centric nature of early-stage amn. this hypothesis, strongly suggested by dr. budka et al.'s study, is noteworthy. it further acknowledges the original contribution of their work and should guide future research towards a more targeted understanding of amn pathology. references 1. h. budka, a historical look using virtual microscopy: the first case report of adrenomyeloneuropathy (amn), free neuropathol. 4 (2023) 4–18. https://doi.org/10.17879/freeneuropathology-2023-5115 2. h. budka, e. sluga, w.d. heiss, spastic paraplegia associated with addison’s disease: adult variant of adreno-leukodystrophy, j neurol. 213 (1976) 237–250. https://doi.org/10.1007/bf00312873 3. j.m. powers, d.p. deciero, m. ito, a.b. moser, h.w. moser, adrenomyeloneuropathy: a neuropathologic review featuring its noninflammatory myelopathy, j neuropathol exp neurol. 59 (2000) 89–102. https://doi.org/10.1093/jnen/59.2.89 4. a. castellano, n. papinutto, m. cadioli, g. brugnara, a. iadanza, g. scigliuolo, d. pareyson, g. uziel, w. köhler, p. aubourg, a. falini, r.g. henry, l.s. politi, e. salsano, quantitative mri of the spinal cord and brain in adrenomyeloneuropathy: in vivo assessment of structural changes, brain. 139 (2016) 1735–1746. https://doi.org/10.1093/brain/aww068 5. j. berger, f. dorninger, s. forss-petter, m. kunze, peroxisomes in brain development and function, biochimica et biophysica acta (bba) molecular cell research. 1863 (2016) 934–955. https://doi.org/10.1016/j.bbamcr.2015.12.005 6. l.r. strachan, t.j. stevenson, b. freshner, m.d. keefe, d. miranda bowles, j.l. bonkowsky, a zebrafish model of x-linked adrenoleukodystrophy recapitulates key disease features and demonstrates a developmental requirement for abcd1 in oligodendrocyte patterning and myelination, human molecular genetics. 26 (2017) 3600–3614. https://doi.org/10.1093/hmg/ddx249 7. a. pujol, c. hindelang, n. callizot, u. bartsch, m. schachner, j.l. mandel, late onset neurological phenotype of the x-ald gene inactivation in mice: a mouse model for adrenomyeloneuropathy, hum mol genet. 11 (2002) 499–505. https://doi.org/10.1093/hmg/11.5.499 copyright: © 2023 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. top ten discoveries of the year: neuromuscular disease feel free to add comments by clicking these icons on the sidebar >free neuropathology 1:4 (2020) review top ten discoveries of the year: neuromuscular disease marta margeta department of pathology, university of california, san francisco, ca, usa corresponding author: marta margeta · ucsf pathology, box 0511 · 513 parnassus ave., hsw-514 · san francisco, ca 94143 · usa marta.margeta@ucsf.edu submitted: 15 january 2020 accepted: 21 january 2020 published: 23 january 2020 https://doi.org/10.17879/freeneuropathology-2020-2627 keywords: anterior horn disease, congenital myopathy, inflammatory myopathy, muscular dystrophy, neuropathy, radiculopathy, rna-seq, gene therapy abstract this review highlights ten important advances in the neuromuscular disease field that either were first reported in 2019, or have reached a broad consensus during that year. the overarching topics include (i) new / emerging diseases; (ii) advances in understanding of disease etiology and pathogenesis; (iii) diagnostic advances; and (iv) therapeutic advances. within this broad framework, the individual disease entities that are discussed in more detail include myoglobinopathy, popdc3-mutated limb-girdle muscular dystrophy, neuromuscular adverse events associated with the immune checkpoint inhibition therapy, neuroglial stem cell-derived inflammatory pseudotumor of the spinal cord and spinal cord roots, acute flaccid myelitis, congenital myopathies, idiopathic inflammatory myopathies (with particular emphasis on immune-mediated necrotizing myopathies and sporadic inclusion body myositis), spinal muscular atrophy, and duchenne muscular dystrophy. in addition, the review highlights several diagnostic advances (such as diagnostic rna sequencing and development of digital diagnostic tools) that will likely have a significant impact on the overall neuromuscular disease field going forward. abbreviations aav, adeno-associated virus; afm, acute flaccid myelitis; ai, artificial intelligence; asm, antisynthetase syndrome-associated myositis; aso, antisense oligonucleotide; casa, chaperone-assisted selective autophagy; dm, dermatomyositis; dmd, duchenne muscular dystrophy; ema, european medicines agency; ev, enterovirus; fda, federal drug administration; hmgcr, 3-hydroxy-3-methylglutaryl-coa reductase; ifn, interferon; iim, idiopathic inflammatory myopathy; imnm, immune-mediated necrotizing myopathy; irae, immune-related adverse event secondary to immune checkpoint inhibition; irmg, myasthenia gravis secondary to immune checkpoint inhibition; irmyositis, inflammatory myopathy secondary to immune checkpoint inhibition; irneuropathy, immune-mediated neuropathy secondary to immune checkpoint inhibition; mhc, major histocompatibility complex; mngs, metagenomic next generation sequencing; msa, myositis-specific antibodies; mxa, myxovirus resistance protein a; nm, nemaline myopathy; nmd, neuromuscular disease; n-scipt, neuroglial stem cell-derived inflammatory pseudotumor; pm, polymyositis; rna-seq, transcriptome analysis (rna sequencing); sibm, sporadic inclusion body myositis; sma, spinal muscular atrophy; srp, signal recognition particle; temra cells, terminally differentiated effector memory t cells; wes, whole exome sequencing introduction 2019 was an exciting year for the neuromuscular disease (nmd) field, with important developments on multiple fronts and in many different disease categories. in this review paper, i will summarize ten 2019 nmd discoveries that i consider to be most important and/or interesting; they are grouped in four different “discovery clusters” and listed in no particular order. (like most classifications, this one is not perfect – a few discoveries included in the “etiology/pathogenesis cluster” have direct implications for diagnostics and/or treatment of nmds – but i hope it will improve the overall readability of the review.) newly defined / emerging neuromuscular diseases 1. newly defined genetic diseases even as our understanding of the nmd genetics inches closer to completion, new genetic nmds continue to be defined; in this review, i will summarize key features of two particularly interesting entities that were first characterized in 2019. myoglobinopathy is caused by the c.292c>t, p.his98tyr mutation in myoglobin, a small cytoplasmic hemoprotein that buffers intracellular o2 concentration and regulates the intracellular redox potential in cardiomyocytes and type 1 muscle fibers (olive et al., 2019). through an effort that involved two research teams on two different continents, this mutation was detected in 14 individuals from six different families, all of whom were initially identified based on the unique clinicopathologic features of their disease. clinically, myoglobinopathy presents with a weakness of the proximal lower limb and axial muscles; the symptoms start in the fourth to fifth decade and are slowly progressive, ultimately involving distal lower limb, upper limb, and respiratory muscles. creatine kinase levels are usually normal, while cardiac involvement is clinically apparent in approximately half of the affected individuals. pathologically, all muscle biopsies (as well as the hearts from two individuals who underwent autopsy) showed characteristic sarcoplasmic bodies that are brown on h&e stain, red on modified gomori trichrome stain, and highly electron dense on electron microscopy (fig. 1); interestingly, these sarcoplasmic inclusions rarely contain myoglobin and seem to be a product of increased lipid oxidation. (late stage biopsies also show chronic myopathic changes and autophagic impairment including rimmed vacuoles.) although additional work will be required to fully elucidate the molecular mechanisms underlying this newly defined disease, the initial biochemical experiments suggest that mutant myoglobin is more prone to the loss of the heme moiety and is associated with elevated intracellular superoxide levels, suggesting that impaired redox balance plays a role in the pathogenesis of this disease. figure 1. ultrastructural characterization of sarcoplasmic bodies, the morphological hallmark of myoglobinopathy: electron micrographs (a–d) show collections of highly electron-dense bodies with some less dense material at their periphery. the sarcoplasmic bodies are seen under the sarcolemma (a), often next to the nuclei. some sarcoplasmic bodies are surrounded by a membrane (b). sarcoplasmic bodies of different electron densities are seen near several vesicular structures (c) and were observed in the cardiac muscle obtained post mortem from one of the affected individuals (d). electron micrographs (e–g) and the corresponding nanoscale secondary ion mass spectrometry images (h–j). blue indicates sulfur (32s), red phosphorus (31p), and green iron (56fe), respectively. sarcoplasmic bodies are seen interspersed between the myofibrils (e), next to nuclei (f), or inside an autophagic vacuole (g). note the high-sulfur signal in the sarcoplasmic bodies (h–j), and the iron signal (green dots within the sarcoplasmic bodies in i, j). scale bar in a = 2 µm, b = 5 µm, c = 0.5 µm, d = 1 µm, h–j = 4 µm. [this figure and its legend were adopted from figure 5 in (olive et al., 2019); this use is permitted under the creative commons attribution 4.0 international license.] a different, purely genetic approach was used to define a new autosomal recessive form of limb-girdle muscular dystrophy that is caused by biallelic mutations in popdc3 gene: 5 affected individuals from 3 different families were identified via exome sequencing of a cohort of 1500 patients with presumed genetic limb-girdle weakness and elevated creatine kinase levels; their muscle biopsies showed dystrophic features of variable severity (vissing et al., 2019). popdc3 (popeye domain containing 3) gene encodes a membrane protein that regulates camp signaling, is highly expressed in skeletal and cardiac muscles, and has not yet been associated with any human disease. interestingly, patients with popdc3 mutations do not show a cardiac phenotype, while mutations in two other members of the same gene family (popdc1 and popdc2) cause a cardiac phenotype (generally a conduction disorder) accompanied by a variable skeletal muscle involvement (de ridder et al., 2019; vissing et al., 2019). the pathogenesis of this new disorder remains to be elucidated; however, initial studies suggest a loss-of-function mechanism that leads to altered camp modulation. 2. emerging therapy-related diseases introduction of new therapies has led to the emergence of unusual or completely novel nmd phenotypes; in 2019, two new nmd categories, previously described only in individual case reports or small case series, have been more thoroughly defined. immune-checkpoint inhibitor therapy has revolutionized treatment of patients with advanced cancers, but is often associated with immune-related adverse events (iraes) that most commonly affect the skin, gi tract, and endocrine glands, as well as the pns. neuromuscular iraes can be severe and include myasthenia gravis (irmg), myopathy (irmyositis), and peripheral neuropathy (irneuropathy; most commonly acute demyelinating polyradiculoneuropathy) (johansen et al., 2019; mohn et al., 2019; psimaras et al., 2019). while these neuromuscular iraes share some commonalties with their sporadic counterparts, several unique features have emerged. for example, up to 25% of the patients show more than one neuromuscular irae, with the combination of irmg and irmyositis encountered most often (johansen et al., 2019). furthermore, irmg is more likely to be negative for achr antibodies than idiopathic mg, while irmyositis is more likely to involve oculomotor and bulbar muscles than any of idiopathic inflammatory myopathies (iims); these features make clinical distinction between irmg and irmyositis difficult in many cases (johansen et al., 2019; mohn et al., 2019). in addition, irmyositis often involves axial muscles, while iims do not (touat et al., 2018). finally, a significant fraction of patients with irmyositis shows cardiac involvement, which is not typically seen in iim patients and has a poor prognosis (anquetil et al., 2018; johansen et al., 2019; mohn et al., 2019). interestingly, while the rates of irneuropathies (as well as the overall irae rates) are higher in patients treated with ipilimubab (the anti-ctla-4 antibody), irmg and irmyositis are particularly common in patients treated with nivolumab or pembrolizumab (the pd-1 blocking antibodies) (mohn et al., 2019). the pathogenesis of neuromuscular iraes is incompletely understood, but is thought to involve both type iv (t cell-mediated) and type ii (antibody-mediated) immune mechanisms (psimaras et al., 2019). on a tissue level, irmyositis can take multiple forms but the most commonly observed pattern [perimysial foci of cd8+ t cells and cd68+ macrophages that are associated with necrosis of adjacent muscle fibers; touat et al. (2018) and fig. 2] is distinct from the patterns seen in iims. interestingly, in some cases identical clonal cd8+ cells have been detected within the cancer tissue, skeletal muscle, and myocardium, suggesting that cross-reactivity between cancer cells and myocytes plays at least some role in the pathogenesis of irmyositis (psimaras et al., 2019). figure 2. myositis secondary to therapy with immune checkpoint inhibitors (irmyositis): a representative h&e-stained cryosection (a) shows patches of dense perimysial inflammation that focally extend into the endomysium and abut clusters of degenerating/regenerating muscle fibers; away from the inflammatory foci, muscle fibers have a relatively normal appearance. immunohistochemistry for cd8 (b) and histochemistry for acid phosphatase (c) show that the inflammatory infiltrate largely consists of cytotoxic t cells and macrophages, respectively. the patient was treated with pembrolizumab and presented with evidence of myocarditis as well as limb and bulbar weakness approximately 6 weeks following the last antibody dose. scale bars: a, 100 µm; b-c, 50 µm. in recent years, stem cell treatments have also risen in popularity, although they have no documented health benefits and are – at least for now – completely unregulated. in 2019, it was recognized that one relatively common form of stem cell treatment, intrathecal injection of allogeneic stem cells, can lead to development of distinctive lesions of the lumbosacral spinal cord and spinal cord nerve roots. these lesions, named neuroglial stem cell-derived inflammatory pseudotumors [n-scipts; (sloan et al., 2019)], can clinically mimic inflammatory and neoplastic disease processes and usually consist of a haphazardly organized neuroglial tissue that resembles a low-grade neuroglial tumor (fig. 3); typically, there is at least focal inflammatory infiltrate, which likely represents a host immune response to foreign antigens. while it seems likely that direct injection of allogeneic stem cells into other parts of the body could lead to development of stem-cell derived pseudotumors with non-neuroglial differentiation, only neuroglial scipts have been reported to date (sloan et al., 2019). from a diagnostic perspective, it is critical to be aware of the n-scipt existence, so that these lesions (which at the moment seem to be best handled through watchful waiting) are distinguished from the spinal cord tumors that would generally warrant a more aggressive treatment. advances in understanding of etiology and pathogenesis of neuromuscular diseases 3. acute flaccid myelitis: mounting evidence for causation by non-polio enteroviruses acute flaccid myelitis (afm) is a clinical syndrome characterized by flaccid limb weakness that develops acutely and has a lower motor neuron pattern; the current diagnostic criteria also include spinal cord imaging findings suggestive of anterior myelitis (messacar et al., 2018). the most common form of afm used to be poliomyelitis, which is caused by poliovirus [a member of the en terovirus (ev) genus]; as a result, afm became rare worldwide after introduction of the anti-polio vaccine in the 1950s. a small number of afm cases in the period from 1970-90 was attributed to infection with ev-a71 and a few other viruses (dyda et al., 2018; messacar et al., 2018). since 2012, however, afm has started to increase in incidence. the current form of this disease, first documented in california, is characterized by limb paralysis that occurs 5-7 days after a flu-like respiratory illness; it primarily affects young children (3-9 years of age) and peaks in incidence every other year during the summer/fall season. the largest number of confirmed cases (>500 to date) has been reported in the us, with sporadic cases confirmed throughout the world (schubert et al., 2019). in 2014, the peak of afm incidence has shown a temporal and geographic overlap with the outbreak of ev-d68 infection, raising the possibility that ev-d68 (which rarely caused human disease prior to 2008) might be the cause of recent afm outbreaks (dyda et al., 2018). indeed, ev-d68 infection meets many of the bradford-hill criteria (nine principles applied to examine causality) that are required to establish it as a cause of afm (dyda et al., 2018; messacar et al., 2018). in particular, afm-like disease can be recapitulated in a mouse model through infection with a 2014 strain (but not the originally isolated 1962 strain) of ev-d68, suggesting that neurotropism might have emerged only recently in ev-d68 evolution (hixon et al., 2017). however, 2016 afm cases have not shown a clear association with an ev-d68 outbreak, raising doubts about the causal association between ev-d68 and afm (dyda et al., 2018). moreover, ev has not been detected in the respiratory or gi specimens from more than half of afm children, and ev rna is very rarely detected in the csf of afm patients (schubert et al., 2019); thus, concerns have persisted that recent afm outbreaks might be caused by a still unidentified pathogen or an autoimmune mechanism. the 2019 study by schubert et al. has used a multi-pronged approach [metagenomic ultra-deep next generation sequencing (mngs) of the csf to detect the pathogen-derived nucleic acids, and antiviral antibody detection in the csf using virscan phage display library and ev elisa) to analyze 42 afm cases (covering all outbreaks from 2012-2018) and 58 neurologic disease controls. except for one already known ev-d68-positive case, mngs failed to discover any csf pathogens in either group. however, virscan and ev elisa revealed significantly higher levels of anti-ev antibodies in cases compared to controls, providing a strong support for evs as causative agents of all recent afm outbreaks. interestingly, csf from afm cases commonly contained antibodies targeting more than one ev; thus, viral detection in the spinal cord tissue will likely be required to definitively establish which ev species can cause afm. in addition, further work will be required to determine the sensitivity and specificity of csf ev serology in afm diagnosis (schubert et al., 2019). figure 3. neuroglial stem cell-derived inflammatory pseudotumor (n-scipt): a representative h&e-stained paraffin section (a) shows a spinal cord nerve root surrounded by haphazardly organized but otherwise mature-appearing neuroglial tissue. neurofilament immunohistochemistry (b) highlights intensely stained but disorganized nerve cell processes (arrowheads) that surround the centrally located nerve root (arrow); the tissue at the periphery of the specimen shows glial differentiation and is neurofilament-negative. chronic inflammatory infiltrate was apparent on other sections; for additional images and clinical history details, see the original publication (sloan et al., 2019). scale bar, 100 µm. 4. congenital myopathies: new pathogenetic mechanisms congenital myopathies are a group of genetically and clinically heterogeneous disorders that typically present early in life with weakness / hypotonia and delayed motor development, and are subsequently characterized by a stable or slowly progressive disease course. histopathologic findings are variable and include type 1 fiber predominance and atrophy, cores and/or minicores, mislocalization of intracellular organelles (including nuclei), and various sarcoplasmic inclusions (such as nemaline rods, caps, and cytoplasmic bodies). the well-established pathogenetic mechanisms include impairments of calcium homeostasis, excitation-contraction coupling, sarcomeric filament assembly, force generation, and membrane remodeling. excitingly, this mechanistic spectrum has been significantly widened by the work published in 2019. some of the newly discovered mechanisms include (i) cell cycle acceleration accompanied by reduced cell growth (villar-quiles et al., 2019), (ii) impaired redox balance (lornage et al., 2019), (iii) altered muscle kinetics due to impaired sarcomere relaxation (de winter et al., 2020), and (iv) nuclear dysfunction secondary to impaired contractility (ross et al., 2019); these last two studies are particularly groundbreaking and will therefore be described in a greater detail. nemaline myopathy (nm) can be caused by mutations in at least 13 genes, the great majority of which encode proteins that either form thin filaments or are associated with these filaments. a recent tour-de-force study by de winter et al. (2020; advanced electronic publication in 2019) has established that kbtbd13 (kelch repeat and btb domain containing 13), the mutations in which cause nem6 subtype of nm, is also an actin-binding protein; when mutated, kbtbd13 impairs sarcomere relaxation by altering the structure of thin filaments. specifically, thin filaments are more tightly wound and stiffer than normal when they are bound to mutant kbtbd13r408c; this change in the thin filament compliance disrupts the positive feedback loop that is required for thin-thick filament detachment, resulting in slowing of muscle relaxation that clinically characterizes nem6. given the 8:1 stoichiometry of kbtbd13-actin binding, which is close to the 7:1 stoichiometry between troponin / tropomyosin and actin, it is possible that kbtbd13 acts in concert with those well-known sarcomeric regulatory proteins to fine-tune muscle contraction kinetics. interestingly, deletion of kbtbd13 in mice does not lead to slower muscle relaxation, suggesting that kbtbd13r408c is a gain-of-function mutation. it remains to be shown whether other mutations in this gene lead to similar functional deficits and to elucidate how defects in muscle relaxation ultimately lead to production of nemaline rods (which are recapitulated in the mouse kbtbd13r408c knock-in model). in 2019, we have also learned that impaired contractility and abnormal cytoskeletal organization produce secondary nuclear deficits, which structurally – and possibly functionally – mimic nuclear abnormalities seen in patients with mutations in the nuclear envelope proteins, such as lamins, nesprins, and emerin (ross et al., 2019). abnormal spacing of myonuclei, nuclear envelope defects, and chromatin alterations were present not only in the skeletal muscle from patients with two different genetic forms of nm and nm model mice, but also in skeletal muscle from patients with acquired (immune-mediated) forms of nm, underscoring that these nuclear abnormalities are secondary to impaired contractility rather than a direct consequence of nm mutations. in addition, muscle fibers from both nm patients and nm model mice showed extensive cytoskeletal disruption (disorganized microtubules, mislocalized intermediate filaments composed of desmin, and altered cortical microfilaments composed of actin); given that treatment with microtubule-disrupting agents was shown to alter nuclear shape in vitro, these cytoskeletal alterations likely also contribute to nuclear deficits seen in the nm muscle. indeed, we have recently observed very similar nuclear defects in a patient with a desmin mutation (fig. 4); thus, secondary nuclear defects (and consequent gene expression changes) may be an important but overlooked cause of muscle dysfunction that is more widespread than currently recognized. figure 4. nuclear abnormalities in a case of myofibrillar myopathy caused by a desmin mutation: representative electron micrographs show marked nuclear abnormalities (a-e) including invaginations of the nuclear membrane (a and b, arrowheads), abnormally dense chromatin (the nucleus highlighted by the arrow in b, as well as nuclei in c and d), and nuclear fragmentation (c-e). granulofilamentous material (f), a clue to the correct diagnosis, was seen only focally. subsequent genetic testing revealed a heterozygous des gene mutation [c.1237_1239delgag (p.e413del)]. scale bars: a and e, 1 µm; b-d, 2 µm; f, 4 µm. 5. sporadic inclusion body myositis: the role of terminally differentiated cytotoxic t cells sporadic inclusion body myositis (sibm) is the most common skeletal myopathy affecting people over the age of 50; it typically presents with a weakness of the quadriceps and deep finger flexor muscles, has a slowly progressive course resulting in eventual loss of mobility, and is currently without effective therapy. pathologically, sibm shows both inflammatory and myodegenerative features (fig. 5a); together with a lack of response to immunosuppressive treatment, this “dual” nature of sibm pathology has led to a long-standing disagreement about the role of inflammation in sibm pathogenesis.three studies published in 2019 (dzangue-tchoupou et al., 2019; greenberg et al., 2019; knauss et al., 2019) will likely break this impasse by providing a strong support for the autoimmune nature of sibm: starting with different questions and using different experimental approaches, these studies have demonstrated that terminally differentiated effector memory t cells (so-called temra cells) are enriched in sibm muscle biopsies (greenberg et al., 2019; knauss et al., 2019) as well as peripheral blood of sibm patients (dzangue-tchoupou et al., 2019), and that the presence of these cells is fairly unique for sibm when compared to other iims [although similar cells were also seen in biopsies from two patients with severe, treatment-resistant irmyositis (knauss et al., 2019)]. temra cells are positive for cd8, t-bet (a transcription factor that drives differentiation of naïve t cells into cytotoxic t lymphocytes), cd57 (a marker of replicative senescence), and klrg1 (killer cell lectin-like receptor g1; a marker of t cell exhaustion). they contain high concentration of cytotoxic enzymes (granzymes and perforin) that likely directly cause muscle fiber damage, are known to increase in peripheral blood with age, and are resistant to corticosteroids and drugs that target actively dividing cells (such as methotrexate); the latter two characteristics likely provide at least partial explanation for the age dependence and resistance to immunosuppression that are characteristic of sibm. figure 5. distinct patterns of lc3 and p62 immunostaining in sporadic inclusion body myositis (sibm) and immune-mediated necrotizing myopathy (imnm): a typical sibm case (a-c) shows inflammatory and myodegenerative changes on h&e staining (a) and dense, largely subsarcolemmal protein aggregates and vacuoles on lc3 (b) and p62 (c) immunostaining. in contrast, an imnm case (d-f) shows randomly distributed degenerating/regenerating fibers and the absence of significant inflammation on h&e (d) and multiple fibers with diffuse, finely punctate staining on lc3 (e) and p62 (f) immunostaining. for both cases, h&e stain was performed on cryosections, while lc3 and p62 immunohistochemistries were performed on formalin-fixed, paraffin-embedded tissue sections. scale bars, 50 µm. in addition to providing insight into sibm pathogenesis, these studies have important implications for the sibm diagnosis and treatment. of particular interest, the study by greenberg at al. (2019) included 10 samples from patients diagnosed with polymyositis (pm), an iim that was historically thought to share the sibm inflammatory profile but lack its myodegenerative phenotype and its refractoriness to therapy. the existence of pm as a distinct iim subtype has recently been called into question, given that most patients diagnosed with pm based on the 1975 bohan & peter criteria can be reclassified as one of the other iims [early stage sibm, immune-mediated necrotizing myopathy (imnm), antisynthetase syndrome-associated myositis (asm), or dermatomyositis (dm)] using the current clinicoseropathologic criteria (allenbach et al., 2017; tanboon and nishino, 2019). however, greenberg and co-authors have demonstrated an important difference between their “pm” and sibm samples: while cd8+ lymphocytes invading muscle fibers in sibm biopsies were abundant and cd57-positive, the cd8+ t lymphocytes invading muscle fibers in “pm” biopsies were sparse and cd57-negative. additional work will be required to establish whether pathologically diagnosed pm is a distinct (albeit rare) disease entity or whether it corresponds to an early stage of sibm that lacks senescent, cd57+klrg1+ temra cells and thus remains responsive to immunosuppressive therapy. either way, these findings raise a possibility that the presence of cd8+cd57+klrg1+ t cells in an iim biopsy could serve as a prognostic marker of resistance to the currently available immunosuppressive therapies, a clinically important application that warrants future study. in addition, expression of klrg1 by sibm temra cells may have therapeutic implications: given that this protein is not expressed by central memory t cells and regulatory t cells, it could be used as a basis for development of a future sibm therapeutic (greenberg et al., 2019). 6. immune-mediated necrotizing myopathies: mechanisms of cell injury mmune-mediated necrotizing myopathies (imnms) are a unique class of iims for which the consensus clinicoseropathologic criteria were defined at a workshop held in 2016 (allenbach et al., 2018). imnms have been linked to autoantibodies against signal recognition particle (srp) and 3-hydroxy-3-methylglutaryl-coa reductase (hmgcr), but approximately one third of cases is seronegative and requires histopathologic diagnosis (bergua et al., 2019). regardless of the serologic subtype, pathologic features of imnm include randomly distributed necrotic and regenerating muscle fibers (fig. 5d), variable mhc-1 upregulation, sarcolemmal complement deposition, and a scant to absent lymphocytic infiltrate. previous work has shown that in vitro treatment with either anti-srp or anti-hmgcr antibodies leads to impaired myoblast fusion and muscle fiber atrophy (arouche-delaperche et al., 2017), suggesting that these autoantibodies play a causative role in imnm pathogenesis. a study from the same group published in 2019 has taken the field a step further by demonstrating the causative effect of these two antibodies in vivo: mice treated with either anti-srp+ or anti-hmgcr+ patient-derived plasma develop weakness and necrotizing myopathy that closely resembles human imnm (bergua et al., 2019). in a further agreement with human disease, muscle deficiency is worse following treatment with anti-srp than with anti-hmgcr antibodies, but both antisera require functional complement pathway to produce the disease phenotype. taken together, these studies provide a conclusive evidence for the key role of type ii (antibodyand complement-mediated) immune mechanisms in imnm pathogenesis. interestingly, target antigens of the two known imnm autoantibodies (the signal peptide-binding 54 kda subunit of the srp ribonucleoprotein complex and hgmcr, the rate-limiting enzyme in the cholesterol biosynthetic pathway) are both expressed in the er of all cell types (bergua et al., 2019). given the ubiquitous expression of these antigens, it remains unclear why anti-srp and anti-hmgcr antibodies selectively target skeletal muscle in both humans and mice. however, the er localization of both autoantibody targets may be significant for imnm pathogenesis: a 2019 study has shown that human imnm muscle biopsies of any serotype show increased er stress, activation of the unfolded protein response, and upregulation of the chaperone-assisted selective autophagy (casa) pathway (fischer et al., 2019). notably, activation of the casa pathway in imnms is associated with a unique pattern of immunostaining for autophagy-associated proteins lc3 and p62/sqstm1: instead of the coarse lc3and p62-positive vacuoles and protein aggregates seen in autophagic vacuolar myopathies and sibm [figs. 5b and 5c; also reviewed in (margeta, 2019)], imnm biopsies show diffusely punctate and often quite strong staining of a variable subset of non-necrotic muscle fibers (figs. 5e and 5f). while it remains to be shown how antibody-mediated cell injury leads to activation of the casa pathway [the impairment of which plays a role in the pathogenesis of myofibrillar myopathies; reviewed in (margeta, 2019)], this unique lc3and p62-staining pattern is likely to prove useful in diagnostically challenging imnm cases; however, additional work will be required to establish its sensitivity and specificity for imnms relative to other conditions in the histopathologic differential diagnosis (toxic necrotizing myopathies and other iims). advances in neuromuscular disease diagnostics 7. idiopathic inflammatory myopathies: myositis-specific antibodies and interferon activation signatures as already mentioned, iims are currently divided into four distinct classes dm, asm, imnm, and sibm based on the most up-to-date clinicoseropathologic criteria [first comprehensively outlined in a review by allenbach at al. (2017) and further updated in a recent review by tanboon and nishino (2019)]. this classification has been developed based on advances made over the last 5-10 years and is largely (although not entirely) based on the association of individual iim classes with different myositis-specific antibodies (msa); while these autoantibodies sometimes directly mediate muscle injury (as already discussed for imnms in discovery #6), more often they represent a diagnostically useful epiphenomenon / disease marker. the discovery of msa has revolutionized iim diagnostics because of their high specificity, but the sensitivity remains relatively low. for example, two retrospective studies of large european iim cohorts published in 2019 found that msa are negative in 40-60% of iim patients (betteridge et al., 2019; montagnese et al., 2019); in these msa-negative cases (as well as in rare cases with more than one msa present or in cases for which msa information is lacking), muscle biopsy remains the cornerstone of iim diagnosis. figure 6. dermatomyositis (dm) variants: (a) a “classic” dm case shows marked perifascicular fiber atrophy but no significant inflammation in the perimysium or endomysium; the patient’s antibody status is not known, but the overall clinicopathologic findings fit best with the anti-tif1γ-associated dm. (b) a typical case of anti-mi-2-associated dm also shows perifascicular atrophy, but could be confused with anti-synthetase syndrome-associated myositis given the presence of perimysial and focally endomysial inflammation, perimysial fragmentation, and patchy necrosis of perifascicular muscle fibers. (c) a case of anti-mda5-associated dm shows moderate processing artifacts but no significant pathologic changes; the only significant abnormality was mild upregulation of mhc-i complex in all muscle fibers (not shown). for all three cases, images show representative h&e-stained cryosections. scale bar, 100 µm. histopathologically, most cases of dm, imnm, asm, and sibm are easily distinguishable [for a review of up-to-date pathologic criteria, see tanboon and nishino (2019)]; however, a few entities can create diagnostic difficulties. for example, anti-mda5-associated subtype of dm generally lacks perifascicular atrophy (a key diagnostic feature of dm; fig. 6a) and can therefore mimic imnm or even normal muscle (fig. 6c), while anti-mi-2-associated dm shows prominent perimysial inflammation and perifascicular fiber necrosis that can mimic asm (fig. 6b). the interferon-induced gene expression signatures associated with different iim classes, which were defined in two separate studies published in 2019 (pinal-fernandez et al., 2019; rigolet et al., 2019), confirm the validity of the current iim classification and identify immunohistochemical markers of these interferon responses, which will likely prove diagnostically useful. in particular, both studies have shown that all subtypes of dm (including the histologically atypical cases associated with anti-mda5 and anti-mi-2 antibodies) show activation of the type i interferon (infα/β) pathway and prominent upregulation of ifn1-inducible genes such as mxa (myxovirus resistance protein a), which can be used as an immunohistochemical marker of the inf1 response. in dm, mxa generally shows sarcolemmal staining in perifascicular fibers; the exception is anti-mda5-associated dm, where mxa staining is scattered or diffuse but not perifascicular (tanboon and nishino, 2019). in contrast, both asm cases (regardless of the nature of the associated anti-synthetase antibody) and sibm cases show activation of the interferon type ii pathway (i.e. upregulation of infγ-inducible genes); mhc-ii, an immunohistochemical marker of infγ activation, can be used to monitor this pathway in muscle biopsies (rigolet et al., 2019) and could therefore be useful for distinguishing asm from anti-mi-2-dm in challenging cases. [of note, dm muscle biopsies do show a weak activation of infγ pathway (pinal-fernandez et al., 2019; rigolet et al., 2019); however, at least for mhc-ii proteins that activation is several orders of magnitude lower than in asm and sibm biopsies. thus, it should be possible to optimize mhc-ii staining to effectively differentiate between dm and asm cases.] finally, both studies found that imnm biopsies do not exhibit either type i or type ii interferon signature, further validating their separation into a separate iim category (pinal-fernandez et al., 2019; rigolet et al., 2019). thus, neuromuscular pathology laboratories that see a lot of iim cases should consider adding mxa and mhc-ii immunostains to their stain repertoire, so that these markers can be evaluated in diagnostically challenging iim cases. what are the sources of infα/β and infγ that drive these differential responses? that remains to be definitively established; however, the most likely candidate for the infα/β source are plasmacytoid dendritic cells, which are increased in dm muscle, while the most likely candidate for the infγ source are cd8+ t cells, which were observed in a close vicinity of mhc-ii-expressing fibers in both asm and sibm (rigolet et al., 2019). 8. emerging diagnostic techniques the reduced cost and widespread availability of advanced genetic testing techniques [such as disease gene panels and whole exome sequencing (wes)] have led to a dramatic diagnostic improvement for many inherited nmds. however, even with the aid of these advanced molecular techniques, 50-75% of presumed genetic nmd cases currently remains undiagnosed (cummings et al., 2017); the underlying reasons include challenges involved in interpretation of the variants of unknown significance and of variants in the genes previously not linked to human disease, the inability of these techniques to detect certain types of genetic alterations (such as structural re-arrangements and copy number variants), and the lack of coverage of intronic and regulatory regions (gonorazky et al., 2019). in such diagnostically challenging cases, transcriptome analysis (rna-seq) will likely prove beneficial. indeed, a recent study has shown that rna-seq resolved 36% of their paneland/or wes-negative nmd cases (gonorazky et al., 2019); an essentially identical diagnostic yield (35%) was reported in a 2017 study that used a different nmd patient cohort (cummings et al., 2017). importantly, gene expression is tissue-dependent; thus, the effectiveness of rna-seq for diagnostic testing hinges on the use of disease-relevant source material. in the context of genetic muscle disease, biopsied muscle represents the ideal specimen, while peripheral blood is inadequate; unmodified skin-derived fibroblasts can be useful for some diseases (such as congenital muscular dystrophies), but the diagnostic yield is substantially enhanced if fibroblasts are transdifferentiated into myotubes in vitro (gonorazky et al., 2019). importantly, expression of the muscle-specific transcripts was significantly lower in transdifferentiated myotubes than in the matched muscle biopsy samples for most genes; therefore, muscle biopsies will likely remain the gold standard for diagnostic transcriptomics. however, the transdifferentiation approach may prove useful when muscle biopsy cannot be performed or when accurate diagnosis requires correlation of gene expression patterns between the proband and other family members (gonorazky et al., 2019). another approach for improving muscle disease diagnostics is based on the use of automated image-analysis methods and artificial intelligence (ai) tools during muscle biopsy evaluations. to this end, a french group has recently developed a fully automated macro script for image j that performs muscle fiber measurements (such as total fiber number, type 1 and type 2 fiber proportion, and muscle fiber diameter) that are routinely assessed during muscle biopsy evaluation, but are typically estimated and reported in a semi-quantitative fashion (reyes-fernandez et al., 2019). in addition to validating the macro script (which analyzes images from immunofluorescence-stained sections and is available for download as the manuscript supplementary material), this study has used quantitative morphometry to definitively demonstrate that the size of type 2 fibers decreases with age but increases with obesity, an observation that has relevance for routine muscle biopsy interpretation (reyes-fernandez et al., 2019). notably, the authors are in the process of adopting their macro script for use on immunoperoxidase-stained sections, which will make it more routinely applicable to most pathology practices. more provocatively, a study presented at the 2019 world muscle society congress (kabeya et al., 2019) has shown that a deep neural network model (“ai-based muscle histopathologist”) can use images from h&e-stained slides to differentiate several major muscular dystrophies subtypes better than physicians (although the study abstract does not indicate whether those physicians were expert muscle pathologists). while intriguing, the validity of these results needs to be replicated by future studies. in particular, nmd diagnosis generally requires interpretation of many different stains and integration of clinical, laboratory, and histopathologic findings; thus, it is fairly unlikely that the diagnostic proficiency of an “ai-based muscle histopathologist” will generalize to a broader set of nmds. at the same time, it is likely that the best diagnostic yield will ultimately be achieved through the combined efforts of ai and human pathologists, as was previously demonstrated for the other fields of medicine that are based on image interpretation (such as radiology). advances in neuromuscular disease treatment development of new disease therapies takes decades of work, with clinical trials that span years and report their results thorough multiple papers published over time; as such, it is not entirely appropriate to designate any of the recently approved genetic treatments as a 2019 discovery. at the same time, these new treatments were among of the “hottest” nmd advances in 2019, and therefore had to be included in this review. 9. a success story: genetic therapies for spinal muscular atrophy spinal muscular atrophy (sma) is an autosomal recessive neurodegenerative disease that affects the alpha motor neurons in the anterior horn of the spinal cord, resulting in neurogenic muscle atrophy and weakness; the onset of disease varies from the first 6 months of age for the most severe infantile form (sma1) to the second or third decade for the mildest form (sma4). sma is caused by loss-of-function mutations in both copies of smn1 (survival motor neuron 1) gene, but clinical phenotype is modified by the presence of the paralog gene, smn2, which yields only a small number of full length transcripts (due to a mutation in the exon 7 that alters gene splicing) and is present in a variable number of copies in different individuals. the first genetic therapy for sma, an intrathecally delivered antisense oligonucleotide (aso) called nusinersen, was approved in the us in 2016 and in europe in 2017 based on the results of clinical trials done with sma1 and sma2 infants; additional clinical trials for other sma subgroups are still ongoing. [for a succinct but comprehensive review of all treatment approaches summarized in this section, see (vita et al., 2019).] nusinersen binds to the smn2 pre-mrna downstream of exon 7, resulting in an increase in the expression of smn protein and a significant improvement in motor function and patient survival. risdiplam, an orally available small molecule therapeutic that also acts as a splicing modifier of smn2 mrna, is still in clinical trials; however, based on the promising early results, it has been placed on an accelerated pathway for the approval by ema (european medicines agency). an alternate approach for sma treatment is to directly replace the defective smn1 gene; this approach has led to development of aav9-smn (also called avxs-101 or zolgensma), an aav9 (adeno-associated virus 9) vector carrying human smn transgene that was approved for sma treatment by fda (federal drug administration) in may 2019 (al-zaidy and mendell, 2019). the availability of multiple treatment options raises many interesting questions that will need to be addressed going forward. are some of these treatments superior to others? (an indirect comparison between nusinersen and avxs-101 has been done and suggests that avxs-101 is more effective (dabbous et al., 2019); however, that study had significant limitations. thus, a direct comparison of these two drugs is needed, but will be challenging to perform given the associated cost and ethical considerations.) if two treatments are based on different genetic strategies, would a combination therapy be more effective than either treatment alone? how will treatment efficacy differ depending on the sma subtype and the age at which the treatment is started? as expected for a neurodegenerative disease, the treatment is more effective if it is initiated before significant neuron loss has occurred (de vivo et al., 2019); depending on the sma subtype, this may require perinatal or even prenatal diagnosis. indeed, a large autopsy study published in 2019 has shown that smn protein levels in the human spinal cord decline rapidly during development, with a critical window that spans from 3 months before to 3 months after birth (ramos et al., 2019); this finding highlights the need for development of newborn sma screening programs, which are already being piloted in several different countries (dangouloff et al., 2019; vill et al., 2019). 10. on the horizon: genetic therapies for inherited myopathies aside from sma, the only currently approved genetic therapies for neuromuscular disease are three new drugs for duchenne muscular dystrophy (dmd), which is a severe progressive x-linked muscle disorder caused by the lack of membrane-associated dystrophin protein; however, based on their rather modest effects to date and on the conditional and inconsistent nature of the approvals that were rendered by fda and ema, these treatments have yet to prove their therapeutic utility and are best still considered as being “under development” (vita et al., 2019). ataluren is an orally bioavailable drug that binds ribosomal rna and interferes with recognition of premature stop codons present in 10-15% of dmd patients; it received a conditional approval from ema in 2014, but the evaluation of long-term outcomes is still ongoing. the other two conditionally approved treatments for dmd are based on the exon-skipping aso technology; the goal of this therapy is to “trick” the cellular machinery to restore the reading frame by skipping over the mutant exon, resulting in an internally truncated but functional protein. eteplirsen, which targets the splice-donor region of exon 51 (mutated in 13% of dmd patients), was conditionally approved by fda in 2016 but failed to receive an approval from ema (aartsma-rus and goemans, 2019); a phase 3 clinical trial to evaluate its efficacy is still ongoing. golodirsen (exon 53; 8% of dmd patients) has been approved by fda in december 2019 (https://www.drugs.com/newdrugs/fda-approves-vyondys-53-golodirsen-duchenne-muscular-dystrophy-dmd-patients-amenable-skipping-exon-5119.html; accessed on 12/26/2019), while clinical trials for casimirsen (exon 45; 8% of dmd patients) and suvodirsen (exon 51) are still ongoing (vita et al., 2019). based on the animal model studies, similar exon skipping treatments may also prove valuable for limb-girdle and congenital muscular dystrophies (hwang and yokota, 2019). excitingly, new treatments for other genetic myopathies are also under active development; several different therapeutic strategies are being evaluated through ongoing or planned clinical trials and include aav vector-based gene replacement therapies (for x-linked myotubular myopathy and pompe disease), aso-based therapy aimed at reduction of toxic rna production (for myotonic dystrophy type 1), chaperone therapy (for pompe disease), and the second generation enzyme-replacement therapy (also for pompe disease) (vita et al., 2019). initial results that are available to date are promising; thus, with some luck, at least some of these new treatments will prove beneficial enough to be covered in a future update. at the same time, all these new gene therapies are likely to be highly expensive; for example, the list price of nusinersen treatment in the us is $750,000 in the first year and $375,000 / year after that. thus, an increase in the availability of genetic therapies raises important socio-economic and ethical questions that will have to be addressed before their full benefits can be realized. disclosure statement the author is a consultant for audentes therapeutics; she serves as a member of the muscle biopsy review committee for the aspiro clinical trial (nct03199469), which is evaluating the safety and efficacy of gene transfer in x-linked myotubular myopathy. acknowledgements i am grateful to drs. michael lawlor, nigel g. laing, and benedikt schoser for helpful input during the conceptualization stage of this review, to dr. emily sloan for n-scipt images, and to ms. christine lin for assistance with figure preparation. this work was supported by the muscular dystrophy association grant mda514303. references aartsma-rus, a., and goemans, n. 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(2019). genetic neuromuscular disorders: living the era of a therapeutic revolution. part 2: diseases of motor neuron and skeletal muscle. neurol sci 40, 671-681. copyright: © 2020 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. desmoplastic myxoid tumor of pineal region, smarcb1-mutant, in young adult feel free to add comments by clicking these icons on the sidebar free neuropathology 2:14 (2021) case report desmoplastic myxoid tumor of pineal region, smarcb1-mutant, in young adult branavan manoranjan1, yves p. starreveld1, robert a. nordal2, christopher dunham3, susanne bens4, christian thomas5, martin hasselblatt5, jeffrey t. joseph6 1 department of clinical neurosciences, division of neurosurgery, university of calgary, canada 2 department of radiation oncology, foothills medical centre, canada; department or diagnostic imaging, university of calgary, canada 3 department of pathology and laboratory medicine, university of british columbia, canada 4 institute of human genetics, ulm university & ulm university medical center, ulm, germany 5 institute of neuropathology, university hospital münster, germany 6 department of pathology and laboratory medicine, university of calgary, canada corresponding author: jeffrey t. joseph · department of pathology, mccaig tower 7539 · foothills medical centre and university of calgary · 1403 29 st nw, calgary, ab, t2n 2t9 · canada jtjoseph@ucalgary.ca submitted: 21 april 2021 accepted: 23 may 2021 copyedited by: bert m. verheijen published: 01 june 2021 https://doi.org/10.17879/freeneuropathology-2021-3340 additional resources and electronic supplementary material: supplementary material keywords: desmoplastic myxoid tumor, smarcb1-mutant, atypical teratoid/rhabdoid tumor, pineal, csf dissemination abstract we present a young adult woman who developed a myxoid tumor of the pineal region having a smarcb1 mutation, which was phenotypically similar to the recently described desmoplastic myxoid, smarcb1-mutant tumor of the pineal region (dmt-smarcb1). the 24-year-old woman presented with headaches, nausea, and emesis. neuroimaging identified a hypodense lesion in ct scans that was t1-hypointense, hyperintense in both t2-weighted and flair mri scans, and displayed gadolinium enhancement. the resected tumor had an abundant, alcian-blue positive myxoid matrix with interspersed, non-neoplastic neuropil-glial-vascular elements. it immunoreacted with cd34 and individual cells for ema. immunohistochemistry revealed loss of nuclear ini1 expression by the myxoid component but its retention in the vascular elements. molecular analyses identified a smarcb1 deletion and dna methylation studies showed that this tumor grouped together with the recently described dmt-smarcb1. a cerebrospinal fluid cytologic preparation had several cells morphologically similar to those in routine and electron microscopy. we briefly discuss the correlation of the pathology with the radiology and how this tumor compares with other smarcb1-mutant tumors of the nervous system. case presentation the patient is a 24-year-old woman who presented following 2-weeks of progressive headache, nausea, emesis, and diplopia. her medical history was significant for right sided hearing loss that began with tinnitus in 2014. neurological examination revealed bilateral grade i papilledema but no other focal motor or sensory deficits. preoperative non-contrast head computed tomography (ct) revealed a hypodense pineal region lesion extending into the posterior third ventricle and causing obstructive hydrocephalus (figure 1a). magnetic resonance imaging (mri) demonstrated a 3.0 x 3.4 x 2.6 cm mass in the pineal region (see below). beta-human chorionic gonadotropin (beta-hcg) and alpha-fetoprotein (afp) from serum and cerebrospinal fluid (csf) were within normal limits. a spinal mri did not identify visible leptomeningeal disease; however, csf cytology had atypical cells (see below). figure 1: preoperative neuroimaging. the tumor in the pineal region was hypodense on non-contrast ct head (a), hypointense in t1-weighted (b) and hyperintense in t2-weighted (c) mri sequences. it was strongly hyperintense in flair sequences (d), showed heterogeneous gadolinium enhancement (e), and did not demonstrate restricted diffusion (f). she underwent a successful endoscopic third ventriculostomy (etv) and biopsy of the pineal lesion on post-admit day 3. the initial biopsy did not identify a germ cell tumor or a lymphoma, so the patient underwent tumor resection via a suboccipital craniotomy. grossly, the tumor was opaque, grey-white in color, firm, and moderately vascular. the tumor was debulked, however it could not be completely resected where its superior edge was adherent to the vein of galen and the basal vein of rosenthal, and where it was adherent to the pulvinar. post-operative mri showed nodular enhancement along the right lateral margin of the surgical cavity. she was discharged home on postoperative day 8 but subsequently developed a pseudomeningocele that was treated with a programmable ventriculoperitoneal shunt. she remains well on follow-up at 5 months post-shunt insertion. because of the csf cytologic results (see below), she received craniospinal irradiation. curative intent craniospinal irradiation with a radiation boost to the primary tumor region was prescribed. 36 gy was delivered to the neuraxis in 1.8 gy fractions, and an additional 19.8 gy was given to the primary tumor volume. at last follow-up, five months after surgery, the patient remained well. neuroradiology preoperative non-contrast computed tomography (ct) revealed a hypodense pineal region tumor extending into the posterior third ventricle and causing obstructive hydrocephalus (figure 1a). magnetic resonance imaging (mri) showed that this mass was hypointense in t1-weighted images (figure 1b), hyperintense in t2-weighted (figure 1c) and flair images (figure 1d). the mass heterogeneously enhanced with gadolinium (figure 1e) and was in close proximity to the deep cerebral veins, including the vein of galen. it did not show diffusion restriction (figure 1f; adc map not shown). no leptomeningeal disease or drop metastases were demonstrable on spinal mri (data not shown). these indicated that the tumor was likely sparsely cellular, its matrix contained abundant but not free water, and it had vessels that lacked a blood-brain barrier. csf cytology the patient's initial csf cytology (figure 2), which was obtained from a lumbar puncture before surgical intervention, included several small clusters of atypical cells that had a high nuclear-to-cytoplasmic ratio, prominent nucleoli (white arrows in 2a), and variable amounts of cytoplasm. some cells had small zones of clear cytoplasm (black arrows in 2b) that were interpreted as vacuoles, while others lacked this feature (cell cluster in 2a). these cells were morphologically similar to those in the routine sections (figure 3f) and in the electron microscopy from the later tumor resection (see figure 6). although these cells had some features of chondrocytes (see arya, 2019; chen, 1990; takeda, 1981; bigner, 1981; and the chapter on csf cytology by cibas, 2014), they showed increased pleomorphism and displayed considerably less cytoplasm than previously illustrated chondrocytes in csf and lacked other debris that was illustrated in arya, 2019. figure 2: csf cytology. these cells from the preoperative csf, imaged under oil at 100x magnification, were stained with toluidine blue. they had large nuclei with prominent nucleoli (a, white arrows). some appeared multinucleated (a and b). their cytoplasm showed moderate staining cytoplasm (a), although several cells had partial cytoplasmic clearing or vacuoles (b, black arrows). tumor histology the tumor had two distinct features: a major myxoid component (figure 3) and a minor, intermixed, vascular component that was associated with neuropil and astrocytes (figure 5, see below). the former had sparsely (figure 3, black arrow in a and d) to moderately cellular regions (white arrow in 3a) containing occasional microcysts (3c, black arrow) that were delineated by a single layer of cells. although fragmented, the tumor had regions displaying a clear collagenous capsule (3b, black arrows). because this capsule was not adherent to other tissues, it likely indicated that the tumour grew into a fluid-filled space, either within the third ventricle or into a meningeal cistern. in more myxoid, less cellular areas, the tumor cells formed small clusters or chains of cells (3d, black arrow) that were embedded in the loose, myxoid stroma (3d, white arrow). fine eosinophilic strands meandered through the background stroma (3, d and e). although these regions were histologically reminiscent of chordoma, the tumor did not express brachyury (data not shown). tumor cells displayed a minor predilection for the perivascular region (3e, black arrow), although they did not form ependymal pseudorosettes or pilomyxoid rosettes. they often grew along barely visible eosinophilic strands (swirling pattern in 3e, chains of nuclei in 3d). at high magnification, the cells had small amounts of often eccentric, eosinophilic cytoplasm (3f, black arrow) and large, hyperchromatic nuclei. many cells appeared binucleated or multinucleated (3f, white arrow) these were morphologically similar to the few available cells in the csf cytology (compare with cells in figure 2). mitoses were present (3f, red arrow) but infrequent (less than one per ten high-power fields) and the ki67 proliferation index was 9% (see here). figure 3: routine histology. all panels stained with hematoxylin-eosin. the resection (a; 4x) had hypocellular (black arrow) and moderately cellular (white arrows) areas. in some fragments (b; 10x), the tumor was delimited by an outer collagenous layer (black arrows). more densely cellular regions (c; 10x) had microcysts (black arrow) lined by single cells. a loose, hypocellular area (d; 20x) displayed chain-like growth (black arrow), with cells set in a myxoid matrix having fine, eosinophilic strands. tumor cells showed a weak predilection for the perivascular region (e, white arrows; 20x) around thin-walled vessels (black arrow). at high magnification in f (oil immersion, 100x), the tumor cells had scant amount of often eccentric, eosinophilic cytoplasm (black arrow) and occurred in scattered small groups (white arrow). the red arrow highlights a mitotic figure. clicking the picture will lead you to the full virtual slide (h&e). the tumor's myxoid matrix and its scattered microcysts (figure 4a) diffusely and strongly stained with alcian blue (figure 4b), which indicates that the tumor elaborated a ground substance containing carbohydrate moieties. this myxoid matrix was pervasive throughout the tumor and was only absent in non-tumor vascular elements (see pink-staining vessels and islands in 4b). unlike similar tumors, this neoplasm did not display an overt desmoplasia in the surrounding parenchyma (see figure 3). however, the tumor did produce an extensive network of fine, eosinophilic strands (see figure 3d and 4c), which stained strongly blue in masson trichrome stains (figure 4d). this finding indicates that the tumor created an intrinsic desmoplastic collagenous stroma and likely explains why this myxoid tumor felt firm during surgery. figure 4: myxoid matrix and desmoplasia. matched panels a – b (10x) and c – d (20x) are from the separate histologic regions. the hematoxylin-eosin section in panel a includes several microcysts (black arrow), which stain with alcian blue (b, black arrow). in addition, the loose, slightly basophilic myxoid matrix in a and c also stains strongly for alcian blue (b). throughout the tumor, cells elaborated fine eosinophilic strands (c), which stained strongly blue in the masson trichrome (d). clicking the picture will lead you to the full virtual slide (elastica-van gieson). tumor immunophenotype immunohistochemistry was performed using the dako omnis. the overall results are presented in supplemental table 1. many areas of the tumor strongly expressed cd34 (figure 5a), which was positive in the perinuclear cytoplasm and to a lesser extent in the long processes extending from the cells. in contrast, only a minor population tumor cells expressed ema and only in a perinuclear location (panel 5b). however, this ema expression occurred throughout the tumor. gliovascular islands, composed of vessels encased within an astrocytic (glial fibrillary acidic protein, gfap; figure 5c) and synaptophysin-positive matrix (data not shown) were dispersed throughout the tumor. the myxoid component showed no expression of either of these markers, indicating it did not derive from primary brain parenchyma. the most informative stain was ini1, whose expression was lost in the tumor (figure 5d) but retained in the reactive gliovascular islands (black arrow in 5d). these findings indicated that the tumor had 1) a defect in the smarcb1 gene or its expression and 2) incorporated adjacent brain tissue. while individual tumor cells had cytological features reminiscent of rhabdoid cells, they did not express myogenin or desmin. see supplemental table 1. figure 5: immunohistochemistry. the tumor strongly immunoreacted for cd34 in many regions (panel a; 20x), in both perinuclear regions and cytoplasmic processes. although widespread, the tumor showed only perinuclear reactivity for ema, and only in a small subset of tumor cells (panel b; 40x). many regions of tumor had islands of neuropil and reactive astrocytes that stained for glial fibrillary acidic protein (gfap, panel c, black arrow; 20x). these islands retained strong reactivity for ini1 (panel d, from same region as panel c, black arrow; 20x), however, the encasing tumor had lost expression of this molecular marker. clicking the picture will lead you to the full virtual slide (smarcb1/ini1). electron microscopy toluidine-stained plastic 1-micron sections again showed a relatively hypocellular neoplasm have cells widely disbursed in the loose matrix (figure 6a). vessels and some surrounding cells represented portions of the gliovascular element (6a, red arrows). ultrastructurally, tumor cells had scant cytoplasm and large nuclei with prominent nucleoli (figure 6b, red arrowhead). although by both routine histology and in cytological preparation, the tumor had frequent cells that appeared to have several nuclei, in ultrastructure, at least some of these cells were separated by a plasmalemma (6c, red arrowheads). the ultrastructure of these tumor cells was similar to the cells in the csf cytology and resected tumor routine stains (compare figure 6 with figures 2 and 3f). the cytoplasm typically contained small vacuoles (6b, red arrows) and intermediate filaments (6d, red arrow). figure 6: electron microscopy. the toluidine-stained 1-micron plastic section (a, 40x) demonstrated a hypocellular tumor having cells embedded in a loose matrix, with embedded gliovascular elements (red arrows). in ultrastructural examination using a hitachi h-7650 electron microscope at 75 kv, the tumor cells have large nuclei with prominent nucleoli (b, arrowhead, and c), scant cytoplasm containing both vacuoles (b, red arrows) and intermediate filaments (d, red arrows). closely apposed cells were separated by plasmalemma (c, red arrowheads). molecular results on dna-methylation profiling (illumina methylation epic beadchip) using the heidelberg cns tumor classifier [capper, 2018], the neoplasm was not classifiable (calibrated score <0.3). copy number alterations derived from dna-methylation intensity values (figure 7a) revealed gains of chromosome 11q and 17q, losses of chromosomes 1q and 4p as well as heterozygous loss affecting the smarcb1 region on chromosome 22q11.23 (black arrow). to confirm the specific loss, smarcb1 fish (figure 7b) was performed as described previously [frühwald, 2020]. this demonstrated a heterozygous loss of smarcb1 locus. figure 7: copy number variation. copy number alterations (a) derived from dna-methylation intensity values revealed chromosomal gains (green values) and losses (red values), including losses affecting the smarcb1 region on chromosome 22q11.23 (arrow). fluorescence in situ hybridization (fish) analysis using a probe consisting of the two clones rp11-71g19 (spectrum orange) and rp11-911f12 (spectrum green) (b) showed one colocalized signal per nucleus (orange arrows) representing the intact smarcb1 locus and a heterozygous loss of the deleted smarcb1 allele (green arrows) containing the clone rp11-71g19 (loss of red signal) and parts of clone rp11-911f12 (diminished intensity of remaining single green signal). next, t-distributed stochastic neighbor embedding (t-sne) analysis was performed in comparison with dna methylation profiles of 2.801 previously published samples comprising 82 molecularly distinct cns tumor entities [capper, 2018] as well as dna methylation profiles of six previously published dmt cases [thomas, 2020]. here, the dna methylation profile of the present case clearly grouped with that of dmt (figure 8, right, gray oval). this grouping was close to but distinct from three different groupings of smarcb1-mutant atypical teratoid-rhabdoid tumors (atrt) (figure 8, right, blue ovals). figure 8: dna methylation studies and smarcb1 gene analysis. on t-distributed stochastic neighbor embedding (t-sne) analysis, the dna methylation profile of 82 distinct tumor entities (left), this patient's tumor grouped with that of dmt (right). discussion the tumour presented here is histologically and immunophenotypically similar to cases presented in thomas, 2020, matsumura, 2021, wang, 2021. as demonstrated in the dna methylation studies (figure 8), this tumour also falls into the same cluster as the desmoplastic myxoid tumour, smarcb1-mutant tumor described in thomas, 2020. available demographic data from all prior cases indicate no gender differences (f: 6; m: 4) and an average age of onset of 37 years (range 15-61 years old). of note, all prior cases have been in the pineal region. the smarcb1 gene, which is also known as ini1 (integrase interactor 1) and hsnf5 (snf5 homolog), encodes the swi/snf-related, matrix-associated actin-dependent regulator of chromatin, subfamily b, member 1 protein, which is a core component of the atp-dependent swi/snf chromatin-remodeling complex [phelan, 1999] and acts as a tumor suppressor [versteege, 2002]. biallelic inactivation of smarcb1 has been associated with the growth of benign and malignant tumors in children and adults [hollmann, 2011]. intracranial smarcb1-deficient tumors span a range of malignancies and include childhood atypical teratoid/rhabdoid tumor (atrt) [biegel, 1999], cribriform neuroepithelial tumor [hasselblatt, 2009], poorly differentiated chordoma [mobley, 2010], meningeal tumors having challenging histology [dadone, 2017], a young adult superficial (not pineal) rhabdoid tumour [bodi, 2018], poorly differentiated sinonasal carcinoma [agaimy, 2017], and the desmoplastic myxoid tumor, smarcb1-mutant, of the pineal region (dmt) that we present here [thomas, 2020; matsumura, 2021; wang, 2021]. two previous reports discussed dmt radiologic features. unlike the tumour described here, which was hypodense in ct and hypointense in t1-weighted mri (figure 1a and 1b), the tumor described in wang, 2021 was hyperdense on ct scans and slightly hyperintense on t1-weighted mri scans. it is likely that the more prominent desmoplasia illustrated in wang, 2021, compared to our tumor, correlated with their increased ct signal density. in our patient's tumor, the alcian blue myxoid component (see figure 5b) was predominant, which correlates with our increased t2 and flair signal (see figure 1c and 1d) and is dissimilar to the isointense t2-weighted signal described in matsumura, 2021. similar to the image illustrated in matsumura, 2021, gadolinium enhancement was heterogeneous in our patient's tumour. this tumor had several features not yet described in dmts. it had a pervasive myxoid matrix and had little of the overt and possibly reactive desmoplasia illustrated in thomas, 2020, wang, 2021, or matsumura, 2021. it did, however, diffusely elaborate fine, eosinophilic strands similar to those illustrated in thomas, 2020 and wang 2021 (see figure 4c and 3d), which stain like collagen (figure 4d). we feel that this represents intrinsic tumor desmoplasia, rather than the reactive desmoplasia illustrated in the other cases and common to many systemic cancers. the dmt also had non-neoplastic elements, including gliovascular islands (figure 5c) that strongly expressed gfap but retained ini1 expression (figure 5d). these islands likely originated from the underlying pineal gland or surrounding brain. ultrastructural studies demonstrated the tumour cells had large nuclei with prominent nucleoli. the cytoplasm was scant and had vacuoles and intermediate filaments (figure 6). unlike the prior reports, our patient had atypical cells in her cerebrospinal fluid (figure 2). because of the scant available material in the csf cytology, we have not been able to verify these cells as neoplastic; however, we feel their cytologic features were morphologically similar to those in light (figure 3f) and electron microscopy (figure 6) from the tumor resection and considered them neoplastic rather than chondrocytes. our tumor cells lacked desmin or myogenin expression. as in our tumor, most cases expressed cd34 (figure 5a). ema expression has been variable, with strong positivity in 5 of 6 cases in thomas, 2020, focally positive in matsumura, 2020, and absent in wang, 2021. in our patient, the ema expression was subtle but distinct (figure 5b). given these data, we feel the salient pathological features of this tumor are: regions having a pale or slightly basophilic, alcian-blue positive myxoid matrix areas demonstrating either frank desmoplasia or intrinsic, fine eosinophilic collagenous bands, which stain strongly blue in masson trichrome cytologic features include small to medium size cells having an increased nuclear-to-cytoplasmic ratio, a large nucleus having a prominent nucleolus, and variable amounts of eosinophilic cytoplasm, which can sometimes be scant or can elaborate fine eosinophilic filaments and small vacuoles variable expression of cd34 and ema; neoplastic cells lack expression of specific muscle, glial, or neuronal proteins loss of ini1 protein expression or demonstration of smarcb1 mutation the advent of integrated genomics has revolutionized our understanding of atrt through the identification of three molecular subgroups (atrt-shh, atrt-tyr, and atrt-myc), which differ in their demographics, location, prognosis, and smarcb1 mutational profile [johann, 2016; torchia, 2016]. comparative methylation profiling of dmt with other intracranial smarcb1-mutant tumors has shown dmt, smarcb1-mutant tumors most closely resemble atrt-myc and poorly differentiated chordomas [thomas, 2020]. by contrast, the superficial cd34+ smarcb1-deficient tumor described in bodi, 2018, clustered with the atrt-myc, using the heidelberg brain tumor classifier, and not with the dmt, smarcb1-mutant group [thomas, 2020]. the patient we present here also has a methylation profile that clusters together with the other described smarcb1-mutant myxoid pineal tumours (see figure 8). while it is interesting to speculate about the origins or common features of smarcb1-mutant tumours, and although it has some features that overlap with atrt, the neoplasm described here is histologically distinct from other smarcb1-mutant tumours. references agaimy a, hartmann a, antonescu cr, chiosea si, el-mofty sk, geddert h, iro h, lewis js jr, märkl b, mills se, riener mo, robertson t, sandison a, semrau s, simpson rh, stelow e, westra wh, bishop ja. smarcb1 (ini-1)-deficient sinonasal carcinoma: a series of 39 cases expanding the morphologic and clinicopathologic spectrum of a recently described entity. am j surg pathol. 2017 apr;41(4):458-471. arya a. cartilage cells a potential mimicker of malignant cells in cerebrospinal fluid and a diagnostic pitfall. j cytol. 2019 oct-dec;36(4):218-219. biegel ja, zhou jy, rorke lb, stenstrom c, wainwright lm, fogelgren b. germ-line and acquired mutations of ini1 in atypical teratoid and rhabdoid 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a, coutts m, michiels jf, pedeutour f, burel-vandenbos f, renop. meningeal swi/snf related, matrix-associated, actin-dependent regulator of chromatin, subfamily b member 1 (smarcb1)-deficient tumours: an emerging group of meningeal tumours. neuropathol appl neurobiol. 2017 aug;43(5):433-449. frühwald mc, hasselblatt m, nemes k, bens s, steinbügl m, johann pd, kerl k, hauser p, quiroga e, solano-paez p, biassoni v, gil-da-costa mj, perek-polnik m, van de wetering m, sumerauer d, pears j, stabell n, holm s, hengartner h, gerber nu, grotzer m, boos j, ebinger m, tippelt s, paulus w, furtwängler r, hernáiz-driever p, reinhard h, rutkowski s, schlegel p-g, schmid i, kortmann r-d, timmermann b, warmuth-metz m, kordes u, gerss j, nysom k, schneppenheim r, siebert r, kool m, graf n. age and dna methylation subgroup as potential independent risk factors for treatment stratification in children with atypical teratoid/rhabdoid tumors. neuro oncol. 2020 jul;22(7):1006-1017. hasselblatt m, oyen f, 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hk, lu jq, mehta v, albrecht s, pimentel j, chan ja, somers gr, faria cc, roque l, fouladi m, hoffman lm, moore as, wang y, choi sa, hansford jr, catchpoole d, birks dk, foreman nk, strother d, klekner a, bognár l, garami m, hauser p, hortobágyi t, wilson b, hukin j, carret as, van meter te, hwang ei, gajjar a, chiou sh, nakamura h, toledano h, fried i, fults d, wataya t, fryer c, eisenstat dd, scheinemann k, fleming aj, johnston dl, michaud j, zelcer s, hammond r, afzal s, ramsay da, sirachainan n, hongeng s, larbcharoensub n, grundy rg, lulla rr, fangusaro jr, druker h, bartels u, grant r, malkin d, mcglade cj, nicolaides t, tihan t, phillips j, majewski j, montpetit a, bourque g, bader gd, reddy at, gillespie gy, warmuth-metz m, rutkowski s, tabori u, lupien m, brudno m, schüller u, pietsch t, judkins ar, hawkins ce, bouffet e, kim sk, dirks pb, taylor md, erdreich-epstein a, arrowsmith ch, de carvalho dd, rutka jt, jabado n, huang a. integrated (epi)-genomic analyses identify subgroup-specific therapeutic targets in cns rhabdoid tumors. cancer cell. 2016 dec;30(6):891-908. versteege i, medjkane s, rouillard d, delattre o. a key role of the hsnf5/ini1 tumour suppressor in the control of the g1-s transition of the cell cycle. oncogene. 2002 sep;21(42):6403-6412. wang ye, chen jj, wang w, zhang al, zhou w, wu hb. a case of desmoplastic myxoid tumor, smarcb1 mutant, in the pineal region. neuropathology. 2021 feb;41(1):37-41. wilson bg, roberts cw. swi/snf nucleosome remodellers and cancer. nat rev cancer. 2011 jun;11(7):481-492. copyright: © 2021 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. the neurovascular unit in diffuse intrinsic pontine gliomas feel free to add comments by clicking these icons on the sidebar free neuropathology 2:17 (2021) original paper the neurovascular unit in diffuse intrinsic pontine gliomas fatma e. el-khouly1,2*, rianne haumann1,2*, marjolein breur3, sophie e.m. veldhuijzen van zanten1,2, gertjan j.l. kaspers1,2, n. harry hendrikse4,5, esther hulleman1,2, dannis g. van vuurden1,2#, marianna bugiani3# 1 emma children’s hospital, amsterdam umc – location vumc, department of pediatric oncology, cancer center amsterdam, de boelelaan 1117, amsterdam, the netherlands 2 princess máxima center for pediatric oncology, utrecht, the netherlands 3 amsterdam umc – location vumc, department of pathology, de boelelaan 1117, amsterdam, the netherlands 4 amsterdam umc – location vumc, department of clinical pharmacology & pharmacy, de boelelaan 1117, amsterdam, the netherlands 5 amsterdam umc – location vumc, department of radiology & nuclear medicine, de boelelaan 1117, amsterdam, the netherlands * co-first authorship # co-last authorship corresponding author: fatma e. el-khouly · amsterdam umc, location vumc · department of pediatric oncology/hematology · de boelelaan 1117 · 1081 hv amsterdam · the netherlands f.el-khouly@amsterdamumc.nl submitted: 24 april 2021 accepted: 27 june 2021 copyedited by: bert m. verheijen published: 5 july 2021 https://doi.org/10.17879/freeneuropathology-2021-3341 additional resources and electronic supplementary material: supplementary material keywords: diffuse intrinsic pontine glioma (dipg), neurovascular unit (nvu), blood-brain barrier (bbb), tight junctions, brainstem, pons abstract aims: diffuse intrinsic pontine glioma (dipg) is a childhood brainstem tumor with a median overall survival of eleven months. lack of chemotherapy efficacy may be related to an intact blood-brain barrier (bbb). in this study we aim to investigate the neurovascular unit (nvu) in dipg patients. methods: dipg biopsy (n = 4) and autopsy samples (n = 6) and age-matched healthy pons samples (n = 20) were immunohistochemically investigated for plasma protein extravasation, and the expression of tight junction proteins claudin-5 and zonula occludens-1 (zo-1), basement membrane component laminin, pericyte marker pdgfr-β, and efflux transporters p-gp and bcrp. the mean vascular density and diameter were also assessed. results: dipgs show a heterogeneity in cell morphology and evidence of bbb leakage. both in tumor biopsy and autopsy samples, expression of claudin-5, zo-1, laminin, pdgfr-β and p-gp was reduced compared to healthy pontine tissues. in dipg autopsy samples, vascular density was lower compared to healthy pons. the density of small vessels (<10 µm) was significantly lower (p<0.001), whereas the density of large vessels (≥10 µm) did not differ between groups (p = 0.404). the median vascular diameter was not significantly different: 6.21 µm in dipg autopsy samples (range 2.25-94.85 µm), and 6.26 µm in controls (range 1.17-264.77 µm). conclusion: our study demonstrates evidence of structural changes in the nvu in dipg patients, both in biopsy and autopsy samples, as well as a reduced vascular density in end-stage disease. adding such a biological perspective may help to better direct future treatment choices for dipg patients. introduction diffuse intrinsic pontine gliomas (dipgs) are rare and aggressive childhood malignancies of the brainstem. these tumors are characterized by a diffuse growth pattern closely interwoven within white matter tracts and grey matter structures, and an intrinsic nature, uttered by hypertrophy of the brainstem often encasing the basilar artery 1,2. with a median overall survival of eleven months, and a two-year survival rate of 10%, dipgs are the leading cause of brain tumor-related deaths in children 3-7. in the recent world health organization (who) classification of tumors of the central nervous system (cns), dipgs were reclassified as h3k27m mutated diffuse midline gliomas (dmg h3k27m) 8. though much research has been dedicated to dipg, its poor outcome has remained unchanged for the past 40 years 9. to date, radiotherapy remains the only (temporarily) effective, albeit palliative treatment, and no chemotherapy regimens prolonging survival have been identified yet. since in vitro and in vivo drug testing on patient-derived tumor cells has shown sensitivity to conventional cytotoxic agents and novel drugs, the lack of efficacy in patients is hypothesized to be related to ineffective drug delivery due to an intact blood-brain barrier (bbb) 10-12. the bbb is formed by endothelial cells interconnected and sealed by tight junctions. the abluminal surface of the endothelium is covered by a basement membrane in which pericytes are embedded. pericytes control the cerebral blood flow by regulating capillary diameter and vessel stability. the basement membrane is enclosed by astrocyte end-feet, also important for brain homeostasis. together, pericytes and astrocyte end-feet induce and maintain the integrity of the bbb 13,14. the bbb regulates transport of essential nutrients to the brain through active transport mechanisms, such as glucose transporters of the glut-family. the efflux of waste products and exogenous compounds is mediated through efflux transporters of the atp-binding cassette family (e.g., p-gp, bcrp, mrp-1) 14,15. additionally, the paracellular barrier capacities of the tight junctions limit transport of circulating monoamines and drugs across the bbb 14-16. the intimate contact and interaction of the bbb complex, formed by endothelial cells, tight junctions, pericytes and astrocyte end-feet, with neurons and perivascular microglia form a dynamic functional unit, called the neurovascular unit (nvu) 13,14,16. some studies report different expression of tight junction proteins throughout the brain, suggestive of regional heterogeneity in bbb permeability 17,18. however, little research has been done on the bbb and nvu in the brainstem and particularly in the pons. yet, better insight into the bbb and the nvu at these sites is needed to develop new treatment strategies for pediatric brainstem tumors. this especially holds true for dipg, where the bbb is thought to be a major contributor to therapeutic inefficacy. in this study, we aim at determining and comparing the histological and immunohistochemical characteristics of the nvu of the pons in children with dipg and age-matched controls. figure 1: characteristics of diffuse intrinsic pontine glioma (dipg). a: t2-weighted mri-image of a dipg patient showing an expanded tumor at the basis of the pons. the arrow indicates the biopsy sampling area; b: gross axial section of the pons and cerebellum showing the presence of a diffuse infiltrating tumor in the pons, reaching the middle cerebellar peduncles. the box indicates the sampling location of autopsy tissue at the non-necrotic tumor site; c-e: hematoxylin and eosin (h&e) staining of the vital tumor bulk showing morphologic heterogeneity compatible with who grade iv tumors (c and d) and grade i tumors (e); f-h: stains against intravascular plasma proteins pre-albumin (f), fibrinogen (g) and igg (h) showing extravasation of these protein into the dipg tumor parenchyma; i-k: hypoxia inducible factor-1α (hif-1α) staining demonstrating a high expression of hif-1α. (scale bar: 5 µm) patients and methods patients and samples dipg pre-treatment biopsy samples (n = 4) were obtained from the biobank of the princess máxima center for pediatric oncology, utrecht, the netherlands, and processed as formalin-fixed paraffin-embedded tissue. end-stage disease dipg autopsy samples (n = 6) were obtained from the ‘vumc brain autopsy in children with dipg’ study 19. this study was approved by the institutional review board of amsterdam umc, location vumc (metc vumc, study number: vumc2009/237) and the scientific committee of the dutch childhood oncology group (dcog). in this study, brain tissue was obtained within a post-mortem interval of less than six hours for dutch patients and less than nine hours for patients from abroad, and was processed as formalin-fixed paraffin-embedded tissue or snap frozen. biopsy samples were mri-guided and taken from the tumor area displaying the highest hyper-intensity on t2-weighted image (figure 1a). autopsy samples were obtained from the non-necrotic tumor core in the pons (figure 1b). age-matched, healthy pontine tissue samples (n = 20) where obtained from the nih neurobiobank, maryland, united states. samples were selected based on (i) brain region (pons), (ii) clinical brain diagnosis (unaffected control/sudden deaths), (iii) post-mortem interval (<17 hours), and (iv) presence of formalin-fixed tissue and frozen tissue. table 1 shows patient and treatment characteristics of the dipg patients. median age at diagnosis was 7.7 years (range 1.3-17.0 years). all patients had a h3k27m mutated dipg. all autopsy patients, except for the youngest, received radiotherapy at diagnosis. of these, at disease progression, three out of six received different chemotherapy regimens and two patients did not proceed to further treatment. median overall survival of patients that were autopsied was 19.5 months (range 5.5-24.0 months). supplementary table 1 shows the characteristics of the control group. median age was 7.0 years (range 1.0-19.0 years). all controls were healthy and had an accidental death. immunohistochemistry air-dried five-μm-thick cryosections were fixed in 2% formaldehyde for 10 min at room temperature (rt). aldehyde groups were blocked in 0.1 g glycine in 100 ml distilled water for 10 min at rt. sections were incubated overnight at rt with a primary antibody: (i) tight junction protein claudin-5 (1:50, invitrogen, carlsbad, ca, usa); (ii) tight junction protein zo-1 (1:50, invitrogen, carlsbad, ca, usa); (iii) basement membrane component laminin (1:500, novus biologicals, abingdon uk); or (iv) pericyte marker pdgfr-β (1:500, abcam, cambridge, uk). the sections were co-stained with glial fibrillary acidic protein (gfap; 1:1000, merck, darmstadt, germany). the following day, the sections were incubated with alexa fluor®-labelled secondary antibodies, background was quenched with 0.1% sudan black b, and sections were mounted in mounting medium (vectashield with 4',6-diamidino-2-fenylindool (dapi); vector laboratories inc., burlingame, ca, usa). five-μm-thick formalin‐fixed paraffin-embedded tissue sections were routinely stained with hematoxylin & eosin (h&e). a gross axial section through the pons and cerebellum was stained with luxol fast blue-periodic acid-schiff. for immunohistochemistry, sections were deparaffinized using xylene, and rehydrated through descending alcohol concentrations. endogenous peroxidase activity was blocked by incubating the slides for 30 min in phosphate buffered saline (pbs) containing 0.3% h2o2. heat-induced antigen retrieval was performed in 0.01 m citrate buffer (ph 6.0). after washing in pbs, the slides were incubated overnight at rt with primary antibodies against p-gp (1:20; millipore, ca, usa), bcrp (1:40; abcam, cambridge, uk), pre-albumin (1:50,000, dako, glostrup, denmark), fibrinogen (1:1,600, dako, glostrup, denmark), igg (1:800, dako, glostrup, denmark) and hypoxia-inducible factor 1α (hif-1α; 1:40, cayman chemical, michigan, usa). the next day, slides were incubated with ready-to-use envision™-hrp (dako, glostrup, denmark) for 1 hour at rt and visualized with 3,3'diaminobenzidine (dab+ dako; 1:50, glostrup, denmark) for 10 min. the slides were counterstained with hematoxylin for 1 min and mounted with quick-d mounting medium (klinipath, duiven, the netherlands). data analysis sections were imaged using a leica dm6000b microscope (400x magnification; leica microsystems bv, rijswijk, the netherlands). from each tissue slide, ten images were made. a semi-quantitative analysis of the bbb staining, comparing dipg samples with control samples, was done by two independent reviewers (fe and rh) using the leica application suite x: las x version 3.1.5.16308. the vascular density was assessed on claudin-5-stained tissue sections by counting the number of blood vessels per mm2. the luminal diameter of the blood vessels was measured with the leica application suite x: las x version 3.1.5.16308. statistics data were analyzed using an independent samples t-test (p-value = 0.05) using ibm spss statistics version 26. the levene’s test of equality of variance was used to first test the assumption of homogeneity or variance between the groups (p-value = 0.05). when equal variances were assumed, pooled estimates were used for the independent t-test statistics. when equal variances were not assumed, unpooled data and an adjustment to the degree of freedom (df) were used for the independent t-test statistics. results immunohistochemistry figure 1 shows typical dipg mri features with enlargement of the pons and contrast enhancement. gross inspection confirms the presence of a partly necrotic and hemorrhagic tumor center in the pons. microscopic examination shows variability of tumor cell morphology, ranging from grade i to grade iv according to the 2016 who classification of cns tumors. tumor areas were recognized based on cell density, the presence of (atypical) mitotic figures and features of high-grade glioma, including necrosis and microvascular proliferation. in these tumors, the integrity of the bbb is compromised, as demonstrated by extravasation of pre-albumin, fibrinogen and igg. this corresponds with expression of hif-1α, indicating tumor hypoxia. notably, as expected for a heterogeneous tumor such as dipg, the density of gfap-expressing astrocytes varied throughout the tumor 20. additionally, tumor cells were differentiated from pre-existing astrocytes by their higher expression levels of gfap, conceivably also related to their less differentiated state 20. figure 2: expression of tight junction proteins claudin-5 and zonula occludens-1 (zo-1) in dipg pre-treatment biopsy and post-mortem autopsy samples. in controls, claudin-5 and zo-1 are sharply defined and have a segmented pattern (a and d). claudin-5 and zo-1 show reduced expression in dipg samples (b, c, e, f). please note the non-activated state of gfap-expressing astrocytes in control tissue. (blue: nuclei; green: astrocytes; red: claudin-5 or zo-1; scale bar: 5 µm) figure 3: expression of basement membrane component laminin in dipg pre-treatment biopsy and post-mortem autopsy samples. in controls (a-c), laminin shows a continuous pattern. laminin expression was reduced at the glial basement membrane in both dipg biopsy (d-f) and autopsy samples (g-i). this was also observed in neovascularization in autopsy samples (j-l). of note: neovascular proliferation was not detected in biopsy samples. (blue: nuclei; green: astrocytes; red: laminin; scale bar: 5 µm) immunohistochemical staining of claudin-5, zo-1, laminin, pdgfr-β, p-gp and bcrp were evaluable in all samples. expression of tight junction proteins claudin-5 and zo-1 was lower at inspection in all dipg biopsy and autopsy samples compared to control samples (figure 2). the expression of basement membrane protein laminin was lower at the glial basement membrane in dipg biopsy and autopsy samples. interestingly, this was observed in both pre-existent vessels within the tumor cells and in neovascular proliferation (figure 3). expression of pericyte marker pdgfr-β was also reduced in both dipg biopsy and autopsy samples (figure 4). efflux transporter p-gp expression was lower in dipg biopsy and autopsy samples, whereas the expression of bcrp was not different in dipg compared to controls (figure 5). figure 4: expression of pericyte marker pdgfr-β in dipg pre-treatment biopsy and post-mortem autopsy samples. in controls (a-c), pdgfr-β shows a continuous pattern. expression of pdgfr-β was reduced in both dipg biopsy (d-f) and autopsy samples (g-i). (blue: nuclei; green: astrocytes; red: pdgfr-β; scale bar: 5 µm). figure 5: expression of efflux transporters p-gp and bcrp in dipg pre-treatment biopsy and post-mortem autopsy samples. p-gp and bcrp are sharply defined and have a segmented pattern in controls (a and d). expression of p-gp was reduced in both dipg samples (b and c). bcrp expression was unchanged in dipg samples (e and f). (blue: nuclei; green: astrocytes; red: p-gp or bcrp; scale bar: 5 µm) vascular density vascular density per mm2 was measured in non-necrotic biopsy and autopsy tissue. it was significantly reduced in dipg autopsy samples compared to controls (1.5±1.2/mm2 versus 17.5±9.5/mm2, respectively; t113,890 = 6.831, p-value <0.001; figure 6a). notably, the density of small blood vessels (<10 µm) was significantly lower in dipg autopsy samples than in controls (t180,609 = -4.303, p-value <0.001), whereas the density of large blood vessels (≥10 µm) did not differ between groups (t597 = -0.835, p-value = 0.404). most blood vessels in dipg autopsy and control samples had a diameter smaller than 10 µm. the median vascular diameter was 6.21 µm in dipg autopsy samples (range 2.25-94.85 µm), versus 6.26 µm in controls (range 1.17-264.77 µm; figure 6b). due to the very small size of the biopsy samples, it was not possible to statistically analyze the vascular density and diameter in these tissue samples. visual inspection of three patients, however, showed a mean vascular density of 7.5 vessels per mm2, and a median vascular diameter of 8.23 µm (data not shown). figure 6: vascular density and diameter in dipg post-mortem autopsy and healthy control samples. a: vascular density in dipg post-mortem autopsy samples and healthy control samples. mean vascular density was 1.5±1.2/mm2 in dipg versus 17.5±9.5/mm2 in controls (red line). b: vascular size distribution in dipg post-mortem samples and healthy control samples. discussion little research has been done to identify the nvu in dipg, while it is hypothesized that treatment failure is caused by an intact bbb. as summarized by figure 7, our study demonstrates structural changes in the nvu of dipg patients that are already present at diagnosis, suggesting these to be tumor-related and not only due to treatment. all studied dipg patients harbored a h3k27m mutation, thus fulfilling the diagnosis of dmg h3k27m according to the revised who classification 8. up to 85% of dipg patients harbor this mutation 21,22. since three out of six dipg autopsy patients were long-term survivors, the median overall survival of the patients in this group was longer than known from literature, 19.5 months versus 11 months, respectively 7. whether neuropathological grading (who ii-iv), tumor location or the presence of a h3k27m mutation have an impact on survival is still not clear 22-24. figure 7: graphical overview of the structural capillary changes observed in dipg pre-treatment biopsy and post-mortem autopsy samples: lower expression of tight junction proteins claudin-5 and zo-1, basement membrane component laminin, pericyte marker pdgfr-β and efflux transporter p-gp in dipg biopsy and autopsy samples; unchanged expression of efflux transporter bcrp-1 in dipg biopsy and autopsy samples. the barrier properties of the nvu strongly depend on the complex interaction between endothelial cells and their tight junctions, pericytes, basement membranes and astrocytes. in physiological conditions, tight junctions are formed by inter-endothelial connections between transmembrane proteins of the claudin-family (claudin-1, 3, 5, and 12), which regulate the function of these tight junctions25,26. claudins are anchored into the endothelial cells by proteins from the zonula occludens-family (zo-1, 2, and 3) that regulate adherens junctions and influence cytoskeletal organization, angiogenic potential and cell migration 27. moreover, zo-1 is responsible for the spatial organization of claudin-5 by linking it to the actin cytoskeleton 25. downregulation of zo-1 can lead to tight junction disruption and a larger intercellular distance between endothelial cells and thus pathologically increased paracellular transport 27. claudin-5 is most abundant in brain vessels (600-times higher expression than other claudins), where it has a heterogeneous distribution 26. the highest claudin-5 expression is seen in capillaries and small post-capillary venules 25,28. in a claudin-5 knockout mouse model, an increased leakage of molecules up to 800 da was observed 29, whereas the permeability of normal bbb only allows passage of molecules up to 500 da 30,31. when additional tight junction proteins are downregulated, a size-dependent increase in paracellular transport is seen of molecules with a size up to 10,000 da 29,32. in our study, a reduced expression of claudin-5 and zo-1 was observed in dipg patients both pre-treatment and post-mortem, indicating a barrier defect, and increasing paracellular transport across the bbb 27. nevertheless, there are more tight junction proteins of the zonula occludens and claudin-family expressed by brain endothelial cells. whether possible downregulation of claudin-5 and zo-1 is compensated by overexpression of other tight junction proteins remains unknown. endothelial cells are surrounded by a basement membrane that contains laminin produced by pericytes and astrocytes 33,34. laminin is essential for basement membrane assembly and maintenance of nvu integrity 35. in our study, employing a panlaminin antibody, we found that expression of laminin was lower at the glial basement membrane in both dipg biopsy and autopsy samples. this was observed in pre-existent vessels amongst the tumor cells and in neovascular proliferation. our results suggest a pathological involvement of pericytes and astrocytes in dipg that could have consequences on the behavior of the endothelial cells, thus also disrupting the integrity of the nvu 34. immunohistochemistry showed also a lower expression of pdgfr-β in dipg biopsy and autopsy samples, suggesting a reduction in pericytic coverage in dipg nvu. besides contributing to secretion of basement membrane components 33,34, pericytes are essential for regulating capillary diameter and vessel stability 13. the possible reduction of pericytic coverage observed in our study may explain the possible downregulation of laminin at the glial basement membrane in dipg patients. under physiological conditions, p-gp and bcrp are the most dominantly expressed efflux transporters in de bbb 36,37. our study shows a decreased p-gp expression and unchanged bcrp expression in dipg pre-treatment and post-mortem samples. this is in line with previous work showing a “moderate expression” of p-gp and intense staining of bcrp in dipg tumor vasculature 10. overall, our results show alterations of the nvu in dipg patients, which could result in or reflect a more leaky nvu. this hypothesis of a leaky nvu is supported by the demonstrated extravasation of some intravascular proteins, such as pre-albumin, fibrinogen and igg. theoretically, this might positively influence influx of chemotherapeutic agents into the tumor, based on passive diffusion. clinical data, however, do not support this possibility 38,39. a possible explanation for this discrepancy might be the markedly reduced vascular density in dipg that could overrule the effects of a leaky nvu. whether the reduction of vascular density is also present at diagnosis remains to be investigated. lack of therapy efficacy in dipg has been linked to an intact bbb. here, we demonstrated structural changes of the nvu together with a lower vascular density in these tumors. these findings have consequences for drug administration, since coverage of the whole tumor, including the migrating/diffusely growing tumor cells, is essential. our findings suggest that drug administration techniques that mostly rely on vascular density for drug distribution, including conventional systemic administration and microbubble mediated focused ultrasound, might show limited efficacy in dipg 12. in contrast, convection-enhanced delivery might be a more suitable technique, in which drug distribution across the tumor relies on a positive pressure gradient instead of passive diffusion 12,40,41. adding such a biological nvu perspective may help to better direct treatment choices for dipg patients in the future. acknowledgement we thank the employees of the expertise center for post-mortem diagnostics of the amsterdam university medical centers, location vumc, for assisting with the autopsies of the dipg patients. we also thank the nih neurobiobank for providing the control tissue. we would also like to thank the semmy foundation (stichting semmy) for financially supporting dipg research in the amsterdam university medical centers, location vumc. ethical approval the study was approved by the medical ethical committee of the amsterdam university medical center, location vumc (metc vumc, study number vumc2009/237). this study was conducted in accordance to the declaration of helsinki. data sharing and data accessibility the data that support the findings of this study are available from the corresponding author upon reasonable request.  references 1. epstein f, mccleary el. intrinsic brain-stem tumors of childhood: surgical indications. j neurosurg 1986; 64(1): 11-5. 2. yoshimura 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port for intermittent convection-enhanced delivery of carboplatin for recurrent glioblastoma. drug deliv 2016; 23(1): 167-73. 41. lewis o, woolley m, johnson d, et al. chronic, intermittent convection-enhanced delivery devices. j neurosci methods 2016; 259: 47-56. copyright: © 2021 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. neurovascular disease: 2022 update feel free to add comments by clicking these icons on the sidebar free neuropathology 3:15 (2022) review neurovascular disease: 2022 update louise d. mccullough department of neurology, mcgovern medical school, uthealth houston and memorial hermann hospital, houston, texas, usa corresponding author: louise d. mccullough · department of neurology · mcgovern medical school · uthealth houston and memorial hermann hospital · houston, texas · usa louise.d.mccullough@uth.tmc.edu submitted: 25 april 2022 accepted: 07 june 2022 copyedited by: félicia jeannelle published: 14 june 2022 https://doi.org/10.17879/freeneuropathology-2022-3910 keywords: stroke, vascular malformations, microglia, t cells, neuroinflammation abstract in this update we present a series of papers focused on topics that have emerged in vascular disease over the prior year. the first two papers focus on the pathogenesis of vascular malformations, the first on brain arteriovenous malformations, and the second on cerebral cavernous malformations. these disorders can lead to significant brain injuries from intracerebral hemorrhage (if they rupture) or other neurological complications, including seizures. the next set of papers reflects work that has advanced our understanding of how the brain and the immune system “communicate” after brain injury, including stroke (papers 3-6). the first of these shows that t cells are involved in white matter repair after ischemic injury, an effect dependent on microglia, demonstrating the important cross-talk between innate and adaptive immunity. the next two papers focus on b cells, which have been relatively understudied in the context of brain injury. the contribution of antigen-experienced b cells from the meninges and skull bone marrow, rather than blood-derived b cells in neuroinflammation opens up a very novel area of investigation. the possibility that antibody secreting b cells may contribute to vascular dementia will certainly be an active area for future investigations. similarly, in paper 6, investigators found that cns-infiltrating myeloid cells can originate from brain borders tissues. these cells have unique transcriptional signatures that are distinct from their blood-derived counterparts, and likely contribute to myeloid cell infiltration from bone-marrow niches in close proximity to the brain. the contribution of microglia, the primary innate immune cell of the brain, to amyloid deposition and propagation is then discussed, followed by work on how perivascular aβ is potentially cleared along the cerebral vessels in patients with cerebral amyloid angiopathy. the final two papers focus on the contribution of senescent endothelial cells and pericytes. the first used a model of accelerated senescence (hutchinson-gilford progeria syndrome; hgps) and shows the translational potential of an approach to reduce telomere shortening to slow aging. the final paper demonstrates how capillary pericytes contribute to basal blood flow resistance and slow modulation of blood flow throughout the brain. interestingly, several of the papers identified therapeutic strategies that could be potentially translated into clinical populations. introduction this “neurovascular disease: 2022 update” presents topics that were selected as the top 10 papers and discoveries published in the field of neurovascular disorders in 2021. these papers were selected based on their broad appeal, those with new methods or discoveries, and those that have a direct link to neurovascular inflammation, vascular abnormalities and neuroimmunology. it is important to note that these top ten papers were subjectively selected by many individuals when queried regarding what papers they felt were most impactful in 2021 (individual contributors are listed in the acknowledgement section). thus, there is certainly considerable “selection bias”. these papers were chosen by a large group of neurosurgery and neurology faculty, and are not ranked in any specific order or by number of citations. overall, the theme of the selected papers reflects our growing understanding of how immunological events, both in the brain and in the periphery, alter the response to vascular and degenerative diseases. the field of neuroimmunology has seen tremendous growth over the past decade, and this is reflected in the papers selected. we apologize to all the authors we could not highlight due to space limitations. topic 1: selective endothelial hyperactivation of oncogenic kras induces brain arteriovenous malformations in mice brain arteriovenous malformations (bavms) are a challenging cerebrovascular disease, characterized by tangled/enlarged vessels occurred by direct connections between arteries and veins without intervening capillary bed. bavms are a major cause of the most debilitating spontaneous intracerebral hemorrhage in children and young adults. however, the pathogenesis of the disease is largely unknown, and the treatment options for bavm patients are severely limited mostly relying on risky surgical methods. recently, a clinical study has reported that somatic activating kras mutations were detected in the endothelium of ~60% of human sporadic bavms (1). in addition, mosaic variants have also been found in the braf and map2k1/mek in intracranial or extracranial avms, especially in high-flow vascular malformations (2). these observations suggest the critical role of kras signaling, especially raf/mek/erk kinase cascade in avm pathogenesis. based on this evidence, the authors tested the role of kras mutation in bavm development using an adeno-associated virus (aav)-br1 specifically targeting brain vascular endothelial cells. strikingly, the systemic administration of aav-br1 encoding human krasg12v gene (aav-br1-krasg12v) induced vascular malformations in the mouse brains that recapitulates salient features of human bavms including the tangled nidus connected with feeding arteries and draining veins, multi-focal spontaneous intracerebral hemorrhages, ambiguous arterial/venous identity and irregular/incomplete mural cell coverage, and enhanced proliferating endothelial cells and angiogenic factors. the accumulation of activated microglia/macrophages, enhanced inflammatory response, and neuronal death were observed around bavms, and mice with bavm showed impaired behavioral functions. the study found the erk activation, but not pakt and p38, in the bavms suggesting that mek/erk signaling is the key mediator for the mutant kras-induced bavm development, and finally revealed that trametinib (an fda-approved mek inhibitor for cancer) attenuates the growth of krasg12v-induced bavms (figure 1). the novel approach by park et al. (3) clearly confirmed the causative role of kras mutations in bavm development, and generated a novel/clinically-relevant mouse model of bavms that will be useful to define the underlying pathomechanism in bavms. this is the first preclinical study showing the efficacy of pharmacological intervention in sporadic bavms associated with kras mutation providing a potential therapeutic strategy for bavm patients by repurposing a cancer drug. figure 1. the characteristics of mouse bavms induced by brain endothelial krasg12v overexpression. systemic injection of aav-br1 carrying krasg12v (aav-br1-krasg12v)-induced abnormal vasculatures that recapitulate the salient features of human bavms including tangled nidus, irregular mural cell coverages, and spontaneous hemorrhages. enhanced endogenous vascular endothelial growth factor (vegf) signaling and endothelial cell proliferation were observed in the bavms. accumulating activated microglia were seen around bavms and accompanied with increased expression of inflammatory factors (e.g., cytokines, matrix metalloproteinases) and apoptotic neurons. mice with bavm showed significantly impaired behavioral function. prolonged treatment with trametinib, an fda-approved mek inhibitor (for six weeks from one day after the aav-br1-krasg12v) inhibited bavm growth suggesting the mek/erk signaling is critical in bavm development induced by kras mutation (park et al., 2021). topic 2: pik3ca and ccm mutations fuel cavernomas through a cancer-like mechanism another interesting work, also on the topic of brain vascular malformations, comes from the laboratory of dr. khan (4). in this work the authors focused on cerebral cavernous malformations (ccms) rather than the avms explored in the prior manuscript. ccms develop due to inactivation of the endothelial ccm protein complex, which is needed to decrease the activity of the kinase mekk3. these malformations can lead to intracerebral hemorrhages and seizures, and often affect younger individuals. the clinical spectrum of disease presentation, including growth and rupture, varies between individuals. some ccms are clinically silent, whereas others grow rapidly and are at risk for repeated hemorrhages. identifying which subset of ccms are more likely to become symptomatic is important, as surgical resection may be warranted. however, surgery has its own risks, and patients often have multiple lesions. genetic studies have implicated a monogenic basis for ccm disease associated with biallelic loss of function mutations in three genes, krit1, ccm2, and pdcd10, that encode the components of a heterotrimeric ccm protein complex, as described by others. in this work, the authors demonstrate that the growth of ccms requires increased signaling through the phosphatidylinositol-3-kinase (pi3k)-mtor pathway as well as loss of function of the ccm complex. this work identified that symptomatic ccm disease arises through a cancer-like program, with the accumulation of multiple somatic mutations in the same cell. this results in both the loss of a vascular malformation suppressor gene (i.e., the ccm gene) and the gain of vascular malformation growth gene (i.e. pik3ca). the authors found somatic gain-of-function mutations in pik3ca and loss-of-function mutations in the ccm complex in the same cells in the majority of human ccms. they then moved to mouse models and found that the growth of ccms requires both pi3k gain of function (gof) and ccm loss of function (lof) in endothelial cells, as both were required for ccm development. both ccm loss of function and increased expression of the transcription factor klf4 (a downstream effector of mekk3) increased mtor signaling in endothelial cells. importantly, the authors then went on to utilize rapamycin, a mtorc1 inhibitor in mice. as single dose given at p2 reduced ccm formation by 75%. in adult mice, chronic administration of rapamycin effectively blocked the formation of ccms in mouse models. this work suggests that there is a “triple-hit” mechanism involving the acquisition of as many as three distinct genetic mutations that culminate in ccm lof and pik3ca gof and contribute to rapidly growing, clinically symptomatic ccms. this three-hit mechanism is analogous to cancer, in which aggressive vascular malformations arise through both the loss of vascular “suppressor genes” that limit vessel growth and gain of function of a vascular “oncogene” that then stimulates excess vascular growth. importantly, these findings suggest that aggressive ccms may be treatable using clinically approved mtorc1 inhibitors. topic 3: treg cell-derived osteopontin promotes microglia-mediated white matter repair after ischemic stroke in this paper (5) the role of regulatory t cells (treg) in stroke recovery was examined. shi et al. sorted infiltrating immune cells from the ischemic brain at 5 and 14 days after transient middle cerebral artery occlusion (mcao). the numbers of t cells increased robustly from 5 to 14 days after stroke, suggesting a possible role of these cells in stroke pathophysiology and recovery. the authors subclustered and more deeply assessed the 14-day t cell population. seven subclusters were identified, including cd4+ treg cells, cd4+ memory t cells, natural killer t (nkt) cells, macrophage-like t cells, and populations of cd8+ t effector cells. the distinct treg cell subcluster expressed transcripts of canonical treg cell markers such as cd4, il2ra (cd25), foxp3, and ikzf2 (helios), which is essential for t cell regulatory function. temporal changes in the cd3+ cd4+ cd25+foxp3+ treg cell population were assessed in the ischemic brain using flow cytometry at different time points (3, 5, 7, 14, and 35 days). minimal treg cell infiltration was observed 3 and 5 days after mcao. the number of infiltrating treg cells significantly increased from 7 days onward, increasing until at least 35 days post stroke. the visualization of t-distributed stochastic neighbor embedding (visne) map of flow cytometry data revealed a prominent treg cell population among all cd4+ t cells in single-cell brain suspensions collected 14 days after stroke. these cells expressed cd25 and foxp3. consistent with recent studies, the majority of brain-infiltrating treg cells were found to be helios+, a population recently identified as phenotypically active treg cells. these cells accumulated near white matter. higher percentages of infiltrating treg cells in the ischemic brain expressed osteopontin (opn) compared with circulating treg cells from sham or stroke mice. bioinformatic analyses predicted strong interactions between treg cells and microglia through opn and integrin receptors, which were then confirmed experimentally by a series of in vivo and in vitro studies. the authors then selectively depleted tregs using a diphtheria toxin method and found that loss of these cells impaired motor recovery and reduced myelin coverage, which was confirmed by whole brain ex vivo diffusion tensor imaging and electron microscopy. the beneficial effects of treg cells were linked to oligodendrogenesis that was dependent on microglia. microglia depletion, but not t cell lymphopenia, mitigated the beneficial effects of transferred treg cells on white matter regeneration. mechanistically, treg cell-derived osteopontin acted through integrin receptors on microglia to enhance microglial reparative activity, consequently promoting oligodendrogenesis and white matter repair. these results confirm that treg cell-microglia interactions enhance a reparative microglial phenotype that promotes opc differentiation. therefore, treg cells appear to contribute to brain repair via both indirect immunomodulation of microglial responses as well as direct trophic effects. boosting treg cell numbers could be a practical approach to improve wm repair and functional recovery after stroke. topic 4: heterogeneity of meningeal b cells reveals a lymphopoietic niche at the cns borders meningeal immune cells are thought to originate from the systemic blood circulation. however, recent findings of developmentally immature b cells in the mouse central nervous system question this assumption. brioschi et al. (6) used single cell analysis from parabiotic and chimeric mouse models to demonstrate that the skull bone marrow contains a hematopoietic niche from which extravascular meningeal b cells can arise to participate in the local immune response. meningeal b cells are capable of trafficking through meningeal lymphatics to the cervical lymph nodes, enabling them to participate in antigen presentation linking brain and systemic immunity. these meningeal b cells are composed of a heterogeneous population with respect to their developmental and maturity stage, with late (b220lowcd43low), and mature (b220highcd43−) subsets representing over 80% of the total b cell population. this was confirmed by single cell rna sequencing (scrna-seq) and cytof mass spectrometry. scrna-seq analysis showed that the meningeal and skull bone marrow b cells exhibited overlapping transcriptomic phenotypes, and that these were distinct from the blood-sourced b cells. targeted gamma irradiation of the skull followed by reconstitution with donor bone marrow cells was used to generate a skull bone marrow transplantation model to investigate the origin of the meningeal b cells. the skull bone marrow chimera experiments confirmed that the meningeal b cells originate from the skull. taken together, this study showed that skull bone marrow provides an accessible source of b cells that are locally educated by cns-derived antigens. lastly, scrnaseq data showed an age-dependent accumulation of blood-derived antigen-experienced b cells (identified as b220highcd23−cd2+sca1+ cells) in the meninges. meninges from aged animals (20-25 months of age) housed a significant population of igm+ and igg+ plasma cells, indicating their antigen-experienced status. these specific “age-associated b cells” (abcs) accumulating in the meninges with aging were found to originate from the peripheral circulation, and represent a separate pool of b cells from those derived from the calvaria. given the importance of b cells in the regulation of neuroinflammation and autoimmunity, meningeal b cells may be a critical subset of the adaptive immune cells, uniquely positioned to contribute to the pool of self-reactive b cells seen with aging (see next paper). topic 5: single-cell profiling of cns border compartment leukocytes reveals that b cells and their progenitors reside in non-diseased meninges central nervous system (cns) meninges (dura, arachnoid, and pia) surround the cns tissue at its critical borders. recent studies examining the dural lymphatics have highlighted the importance of these cns interfaces and their immune landscape. schafflick et al. (7) used scrna-seq of sorted leukocytes from rat meninges, blood, and cerebrospinal fluid (csf) to identify unique cell compositions within the dura. intravenous immune labeling using fluorophore-labeled cd45 (common leukocyte marker) antibody was used to exclude intravascular immune cells. comparing scrna-seq data from different tissues revealed that the dura contained a large cluster of b cells. these dural b cells were subclustered into cxcr4high (signifying immature bone marrow b cells) versus ccl2highcxcl1/2highccl4highitgb2high (signifying chemotactically active b cells). using myelin oligodendrocyte glycoprotein (mog)35–55 peptide-induced experimental autoimmune encephalomyelitis (eae) models of neuroinflammation, the authors showed that inflammation drives phenotypic changes of dural b cells by favoring class-switched plasma cells with higher expression of iga than igg. dural b cells exhibited higher mhc class ii and other markers, indicating an increased antigen-presenting capacity under inflammatory conditions. location of meningeal b cells under homeostatic conditions was then investigated using immunofluorescence of whole mount dura preparations after intravenous injections with a fluorophore-labeled cd45 antibody. results showed that dural b cells are located both within and outside of blood and lymphatic vessels, and interestingly, the extravascular b cells were particularly abundant along the skull sutures. next, the authors confirmed the presence of meningeal b cells in human autopsy samples using cd19+cd20+cd79a+ stainings, and found that human dura also houses a relevant population of b cells. using parabiotic mouse models with different cd45 haplotypes, the precursor dural b cells were shown to be tissue resident with capacity for local proliferation, and the authors claimed their skull labeling approach challenges the presumptive skull-to-dura immune cell flux. this study provided elegant data regarding the presence of a previously-neglected, strategically-located population of meningeal b cells with high potency for antigen-presentation, chemotaxis, and regulation of cns-antigen-specific neuroinflammation. topic 6: skull and vertebral bone marrow are myeloid cell reservoirs for the meninges and cns parenchyma a third paper (8) that integrates well with the theme of the prior two, also examined skull and vertebral bone marrow, but this focused on the myeloid compartment. meninges as evidenced from this triad of papers, contain a large pool of immune cells. however, unlike the prior papers, this paper focused on innate immune cells rather than adaptive b cells. myeloid cells are some of the earlies responders to brain injury. it is well known that these cells can migrate from the periphery to invade the cns. this paper shows that these are not only “blood-derived” but can also come into the cns, rapidly, from the skull bone marrow. the perivascular spaces and the meningeal membranes that cover the cns border zones are also populated by a variety of myeloid cells. in this work, cugarra and others show that cns-associated bone marrow niches in the skull and vertebrae are also myeloid reservoirs for the meninges and cns parenchyma. under homeostasis, these bone marrow pools supply the brain and spinal dural meninges with monocytes and neutrophils via direct dural-bone marrow connections. using elegant fate mapping, dural ly6c+ monocytes and neutrophils were suspected to be from a local source rather than the blood. a calvaria bone-flap with accompanying bone marrow reservoir from gfp+ mice was transplanted into wild-type (wt) mice with an intact dura. gfp+ cells including ccr2+ monocytes, iba1+ macrophages, and cd31+ vasculature was seen in the underlying dura of the transplanted calvaria flap. thus, under homeostatic conditions, the myeloid niche distributed along the brain borders receives a substantial input from the skull bone marrow, which appears to act as a critical dispenser of myeloid cells. this provides an example of a healthy tissue hosting myeloid cells that are continuously replenished by a source that does not use blood as a major route. the authors then went one step further to determine if these cells were involved in an early response to injury using parabiotic models in three injury paradigms, experimental autoimmune encephalitis, spinal cord injury, and optic nerve crush. after injury and in neuroinflammatory diseases, the authors found that these cells can mobilize to infiltrate the cns parenchyma, and display distinct phenotypes from their blood-derived counterparts. cns-infiltrating ly6c+ monocytes (but not neutrophils, cd4 t cells, or ly6c− monocytes) were supplied not from the blood, but by adjacent skull and vertebral bone marrow. it is becoming increasingly clear that under pathological conditions, cns-infiltrating myeloid cells can originate from brain borders and display transcriptional signatures distinct from their blood-derived counterparts. these myeloid cells from bone-marrow niches in close proximity to the brain can supply innate immune cells under both homeostatic and pathological conditions. topic 7: microglia contribute to the propagation of aβ into unaffected brain tissue this very recent study revealed that microglia may act as aβ carriers in the brain of alzheimer’s disease (ad) model mice (9). d’errico and colleagues transplanted embryonic neuronal cells from wt mice into the neocortex of pre-symptomatic 5xfad transgenic mice, a mouse model recapitulating ad-related pathology. interestingly, they found that aβ plaques developed as early as 4 weeks within the grafted neuronal cells and continually increase over time. they confirmed that this was not due to anterograde transport, as very few host processes entered the graft using thy1-gfp/5xfad mice, confirming that the spread of aβ is independent of axonal transport. the greatest amyloid accumulation was along the graft border, an area with higher microglia density. therefore, the authors next investigated whether host microglia are able to invade the wt graft using cx3cr1+/−/5xfad mice. even as early as 2 weeks after transplantation, migration of host cx3cr1+ microglia from cx3cr1+/-/5xfad mice was evident within the wt graft, indicating the ability of microglia to act as an aβ carrier leading to graft invasion. using a variety of methods, the authors confirmed that the microglia were not host derived, or peripheral infiltrating monocytes. they also confirmed using both in vitro and in vivo models that a decline in microglial phagocytosis of aβ occurs in both old wt and 5xfad mice. several additional models including aged 5xfad mice (with less microglial phagocytic activity), irf8-/-/5xfad mice (with less microglial branching), and microglia-depleted 5xfad mice using csf-1r inhibition exhibited a reduction of aβ plaques in the wt grafts, further corroborating the specific role of microglia in regulating aβ propagation. this work confirms that microglia are involved in the pathogenic spread of aβ. they also went one step further in a final experiment. the authors used a model of laser-induced focal tissue injury and two-photon microscopy, and confirmed that aβ can be transported by microglia and form plaques in the lesioned tissue (figure 2). in conclusion, this study suggests a novel role of microglia in the propagation of aβ pathology in neurodegenerative and neural injury models. figure 2. embryonic neuronal cells from wild-type (wt) mice were implanted into the neocortex of pre-symptomatic 5xfad transgenic mice. aβ plaques developed as early as 4 weeks. aβ can be transported by microglia and form plaques in the lesioned tissue. microglia were involved in the propagation of aβ pathology. created with biorender.com topic 8: perivascular space dilation is associated with vascular amyloid-β accumulation in the overlying cortex this work examined the enlargement of the perivascular space (pvs) in patients with cerebral amyloid angiopathy (caa), an increasingly common form of cerebral small vessel disease. caa is a common cause of lobar hemorrhage, cortical microhemorrhages and white matter disease in the elderly (figure 3). in caa, aβ accumulates in the walls of cortical and leptomeningeal arteries, resulting in a loss of smooth muscle cells and impaired vascular function and can lead to lobar intracerebral hemorrhage in the elderly. the presence of cerebral vascular amyloid is a common neuropathological feature in patients with ad. pvs are compartments surrounding cerebral blood vessels that become visible on mri when enlarged. it has been suggested that these spaces enlarge when there are deficits in dysfunctional perivascular clearance of waste products from the brain, including amyloid. the presence of enlarged pvs (epvs) on mri is associated with aging, high blood pressure, stroke and cognitive decline in other studies. figure 3. mri and ct scan imaging of a 73-year-old woman with pathogenically confirmed cerebral amyloid angiopathy. panel a shows innumerable cortical microhemorrhages, panel b demonstrates cortical siderosis (gre), and panel c (flair) shows extensive white matter disease and cerebral atrophy. panel d is a ct scan taken 3 months after her prior mri, when she presented with left hemiparesis and mental status changes. a large lobal hemorrhage is seen in the right frontal area, along with mass effect and cerebral edema. in this paper (10) the authors used 3t and 7t ex vivo mri, semi-automatic segmentation and validated deep-learning-based models to quantify epvs and associated histopathological abnormalities in 19 caa cases and 5 controls. severity of mri-visible pvs during life was significantly associated with the severity of mri-visible pvs on ex vivo mri. this also corresponded with pvs enlargement on histopathology in the same areas. epvs were located mainly around the white matter of perforating cortical arterioles. the amount of epvs was associated with caa severity in the overlying cortex. in addition, the authors found markedly reduced smooth muscle cells and increased vascular aβ accumulation, extending into the wm, was seen in individual affected vessels with an epvs. the presence of aβ was also observed in the wm portion of the same perforating vessels, decreasing in intensity with increasing distance from the cortex. this could reflect blockage of aβ clearance towards the brain surface, consistent with the decreasing severity of caa between superficial and deep cortical layers, as previously shown histologically. these findings were confirmed in a patient with hereditary amyloid that was much younger (dutch mutation). these findings are consistent with the concept that the development of epvs reflects impaired outward flow along arterioles. this has implications for other small vessel diseases and suggests the importance of perivascular clearance mechanisms in human brain, which play an important role in the pathophysiology of both caa and ad. these results support the concept of outward flow along the walls of cortical arterioles and suggest that impaired perivascular clearance of aβ might lead to fluid stagnation and pvs enlargement around upstream portions of connected perivascular compartments. interestingly, removal of aβ plaques with anti-aβ immunotherapy can worsen cortical and leptomeningeal caa (11), suggesting that after plaque disaggregation aβ is cleared along the cerebral vessels. considering the currently unknown role of epvs and white matter hyper-intensities (wmhs) and what they signify in humans both with and without ad/caa, this paper has important clinical and pathophysiological relevance. topic 9: telomerase therapy reverses vascular senescence and extends lifespan in progeria mice aging is a major risk factor for cardiovascular disease. with aging, the vasculature is altered, and molecular and structural changes occur in endothelial cells. senescent endothelial cells show enhanced oxidative stress and dna damages, aberrant chromatin changes, alteration in the nuclear envelope, telomere erosion, and increased production and secretion of pro-inflammatory molecules that negatively affect neighboring cells. given that endothelial function regulates organ perfusion, inflammation and accommodates resident stem cells responsible for tissue repair, it is expected that restoring vascular homeostasis in aged organisms would improve organ function and extend lifespan. hutchinson-gilford progeria syndrome (hgps) is a pediatric genetic disease characterized by accelerated aging. 90% of hgps patients manifest cardiovascular disease early in life, in their teenage years. hgps is caused by a mutation in a nuclear envelop protein (lamin a or progerin), which leads to deleterious consequences in nuclear morphology and gene expression. unfortunately, treatments to mitigate this cardiovascular pathology are limited. the authors (11) used induced pluripotent stem cells (ipscs) derived from hgps patients, in addition to a hgps mouse model to determine if reversing the senescence-associated phenotype in the vasculature had beneficial effects on lifespan and behavior in mice. they found that transient delivery of modified mrna encoding for an enzyme that prevents telomere shortening (human telomerase, htert), which is a distinct feature of cell senescence, restored endothelial proliferation and function, upregulated lamin a, mitigated dna damage, and reversed the inflammatory secretome seen in cultured hgps endothelial cells (figure 4). in addition, they delivered lentiviral mouse tert (mtert) to hgps mice, and found that the aorta of treated mtert mice showed reduced levels of vcam-1 expression (an endothelial adhesion molecule that is increased with vascular inflammation), compared with control progeroid mice. these treated mice also exhibited reduced dna damages in the endothelium of different tissues (aorta, lung, and liver), and their lifespan were significantly extended, compared with hgps mice. this study demonstrates that tert treatment restores senescence-associated phenotype in human hgps endothelial cells, and expands life expectancy in a mouse model of accelerated aging. its relevance lies in the importance of specifically targeting the endothelium to prevent vascular aging and mitigates cardiovascular disease associated with aging. figure 4. telomerase (tert) therapy reverses senescence-associated phenotypes in endothelial cells differentiated from hutchinson-gilford (hgps) human ipsc lines and in a hgps mouse model. human tert (htert) mrna was delivered into hipsc-derived (control and hgps) endothelial cells by lipofectamine transfection, and mouse tert (mtert) lentivirus construct was injected in 3 and 6 months old wild-type and hgps mice via tail vein. created with biorender.com. topic 10: brain capillary pericytes exert a substantial but slow influence on blood flow the role of capillary pericytes, cells that line the capillary networks in the brain, on cerebral blood flow was investigated in this work from dr. shih’s group (12). the capillary bed has the highest flow resistance in the cerebral vasculature given their small diameter, and these vessels are lined with pericytes, unlike the larger arterioles of the brain in which neurovascular coupling is controlled largely by smooth muscle cells due to rapid blood flow demand and consist of α-sma-positive mural cells. two-photon microscopy and optogenetic stimulation was used to directly observe and manipulate brain capillary pericytes in vivo. to directly assess the contractile ability of capillary pericytes in vivo, the authors crossed pdgfrβ-cre mice with reporter mice for the light-gated ion channel, chr2-yfp. the authors confirmed that these cells, when stimulated optogenetically, can contract, decreasing their luminal diameter and reduce blood flow, but with slower kinetics than similar stimulation of mural cells on upstream pial and precapillary arterioles. importantly, this vasoconstriction was associated with a decrease in red blood cell velocity and flux in the same capillaries, showing that the induced levels of constriction were sufficient to alter blood flow, and in fact led to significant “stalling” of rbc flow. interestingly, “blebs” were seen in up to 30% of the pericytes. the authors hypothesized that these blebs reflected disruption of the actin cytoskeleton due to induced supra-physiological contraction. this slow vasoconstriction was inhibited by the clinically used vasodilator fasudil, a rho-kinase inhibitor that blocks contractile machinery and promotes vasodilation. during optogenetic stimulation of capillary pericytes, fasudil attenuated relative and absolute capillary constriction in a dose-dependent manner and also led to a dose-dependent alleviation of persistent flow stalls. this has important translational relevance as this drug is used to treat cerebral vasospasm after subarachnoid hemorrhage in japan, and could have a role in preventing the no reflow phenomenon in ischemic stroke after endovascular interventions. capillary pericytes were also slower to constrict back to baseline following hypercapnia-induced dilation, and slower to dilate towards baseline following optogenetically induced vasoconstriction compared to pre-capillary arterioles. optical ablation of single capillary pericytes led to sustained local dilation and a doubling of blood cell flux selectively in capillaries lacking pericyte contact. these data indicate that capillary pericytes contribute to basal blood flow resistance and slow modulation of blood flow throughout the brain. this could have major implications for other disorders related to cerebral hypoperfusion. figure 5. the skull bone marrow immune profile changes with aging. the composition of myeloid cell populations in the skull bone marrow changes with aging with significantly higher relative frequencies of neutrophils in aged skull (top). skull bone marrow lymphocyte compartment contains significantly higher cd8+ t lymphocytes and activated cd11b high b lymphocytes in naïve aged mice when compared with young skull (bottom). reprinted from honarpisheh et al. (13). conclusion many exciting and novel papers were published in 2021. the compendium of papers highlighted demonstrate our growing understanding of vascular malformations, neuro-immune communication, and vascular function in aging and disease. one important caveat to consider is that many of these diseases predominately affect older individuals, such as stroke and amyloid related diseases. as the field moves forward it will be important to consider the age of the animals used, and also confirming that these pathways are involved in human disease using clinical samples to ensure translational relevance. for example, recent work in our own laboratory has shown that the skull immune compartment changes dramatically with age (figure 5). age-related changes in both peripheral and cns immunity will be an important avenue of exploration in the future. acknowledgements i would like to thank felix moruno manchon, phd, juneyoung lee, phd, pedram honarpisheh, phd, aki urayama, phd, sophie ren, phd, alex choi, md, and eunhee kim, phd, for their assistance in selection of these manuscripts. ldm is supported by the national institute of health (nih) with grants from the ninds (r37 ns096493, r01 ns108779) and nia (r01 ns103592). references nikolaev si, fish je, radovanovic i. somatic activating kras mutations in arteriovenous malformations of the brain. n engl j med. 2018 apr 19;378(16):1561-1562. https://doi.org/10.1056/nejmc1802190 al-olabi l, polubothu s, dowsett k, andrews ka, stadnik p, joseph ap, knox r, pittman a, clark g, baird w, bulstrode n, glover m, gordon k, hargrave d, huson sm, jacques ts, james g, kondolf h, kangesu h, keppler-noreuil km, khan a, lindhurst mj, lipson m, mansour a, o’hara j, mahon c, mosica a, moss a, murthy a, ong j, parker ve, rivière j-p, sapp jc, sebire nj, shah r, sivakumar b, thomas a, virasami a, waelchli r, zeng z, biesecker lg, barnacle a, topf m, semple rk, patton ee, kinsler va. mosaic ras/mapk variants cause sporadic vascular 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commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. a varied neuropathology career turning west and east feel free to add comments by clicking these icons on the sidebar free neuropathology 2:20 (2021) reflections a varied neuropathology career turning west and east ralf schober st. georg teaching hospital of the university of leipzig, leipzig corresponding author: ralf schober · simsonstr. 7 · 04107 leipzig · germany ralf_schober@t-online.de submitted: 10 july 2021 accepted: 19 july 2021 copyedited by: bert m. verheijen published: 29 july 2021 https://doi.org/10.17879/freeneuropathology-2021-3448 keywords: neuropathology, reflections, autobiography i feel very honored to be included in the list of authors for the “reflections” series in free neuropathology, being that i do not really consider myself as a protagonist of our profession. my curriculum is rather variegated and adjusted to the circumstances, resilient yet as it has been tradition in my ancestry. origin and childhood my mother, while just being in labor in an upper floor of a house in bad oeynhausen/ lower saxony in 1944, refused the usual demand to take refuge in the basement when bombers were approaching. thus, the world received me with sunshine, and i believe that i had this perception ever since. my mother’s father, author of the “atlas metallographicus”, saved his position at the university of berlin during the nazi-era by falsifying his passport with the aid of a distant relative who was major in a small rural town and had the suitable seals. my father, having received a degree in chemistry, in world war ii suffered a bullet wound with temporal lobe injury and severe meningitis resulting in epilepsy and partial hearing loss, yet he resumed his beloved piano playing and later on even gave regular house concerts. survival in the post-war famine was not easy, but we were lucky to be assigned as refugees to the farm of some distant relatives (fig. 1). my father, before being able to take up a position in chemistry again, gave private school instructions to the children whilst all public schools were still closed. fig. 1. rs, showing his love for nature early on, with no fears of cjd. education being thus born in a family of the “bildungsbürgertum”, it was only natural for me to attend a “humanistisches gymnasium”, a high school with latin and greek as major subjects. but i have also good memories of the elementary school: in periods of very warm weather the teacher gave his lessons in the nearby park – i am regarding this a good example for the current covid-19 period. the high school was situated in hannover, at that time british occupation zone. the school principal, von drygalski, son of a famous polar explorer, was very proud that we were the first school class permitted to start with latin instead of english, and that the high school was named after the last german emperor. to our amusement, the name placed at the entrance repeatedly changed from “kaiser wilhelms gymnasium” to “kaiser-wilhelm-gymnasium”, due to different opinions of the city officials about the nature of this association. retrospectively, cancel-culture does not appear to be an entirely new phenomenon. we received a broad-based education and did not really suffer from gender segregation, being that the girls’ school was located in the vicinity. sports was also promoted. i spent hours in a rowing boat on the city’s “maschsee” almost daily, and our team received a number of medals. even in much later years we still could be seen as a rowing crew, exploring the landscape of the danube and other rivers on holiday occasions (fig. 2). similarly, for my school class the “abitur” was not the final event, we are still trying to have a get-together each year. fig. 2. rowing trip on the danube river with a crew of old school comrades (rs 4th from left). studies to study medicine was a family decision, especially supported by an aunt of mine who had spent her training in internal medicine in the united states. the emphasis on basic medical sciences in the german curriculum was appealing to me, and so was the relative freedom to arrange the order of courses. i was thus able to change the university three times, from marburg to berlin to göttingen. i was also able to attend voluntary general courses, for example an introduction to informatics given by the computer pioneer konrad zuse. besides i became a member of “marchia”, one of the oldest german fraternities, founded 1810 in berlin concomitant with the operative initiation of the humboldt university. in göttingen, i had the good luck to get a position as “studentischer hilfsassistent” at the university institute for pathology. this was very advantageous for several reasons. first, i could partially unburden my parents from their monthly financial support. second, i became familiar with pathological anatomy, since one of my tasks was to prepare and to display organs with characteristic changes suited to the lectures of prof. johannes linzbach, head of the institute. apparently satisfied with my work, he soon included me in his invitations of assistants and associates for the occasional barbecues in his private garden. i kept this habit in memory as a good example during my own future career. third, i had no problems to secure tissue material for the investigations of my doctoral thesis, the histochemical detection of trace elements in the organs of newborns. this work was done at the max-planck-institute for experimental medicine under the supervision of prof. friedrich timm, in neuropathology well known in connection with the histochemical timm stain for zinc in the hippocampus. graduation in medicine was in 1969, passing one written examination and then oral examinations for each medical discipline at intervals of one week. candidates were grouped into four, and one of them i was the one had to ask each of the chosen professors for the examination date. our whole group passed summa cum laude. at the end we happily invited all professors for dinner in a renowned restaurant, and to our pleasant surprise quite a number of them took part in this rather special farewell. such a ceremony would certainly not have been possible in times where virtually everything is restricted to video contacts. following graduation, a year of practical medicine was required to obtain the medical license. i completed this, the final portion at the department of internal medicine of the university of göttingen under werner creutzfeldt, son of the neuropathologist hans georg creutzfeldt. he backed my plans for a year of pathology in the united states. prerequisites were in existence, by previous medical clerkships in london and in st. louis as well as by the ecfmg certification. i was fortunate in that my successful applications included stanford university, and i also obtained a travel grant of the fulbright commission. the travel was very comfortable, and on the “ms bremen” i assisted the ship’s doctor since i was one of the few passengers that did not become seasick. professional training the planned one year stay at stanford, as it turned out, finally extended to four years, mostly due to the support and encouragement of klaus bensch. originally coming from eastern germany with rather bad experiences over there, he was one of the professors of anatomic pathology and later on followed david korn as head of the department. i was trained in electron microscopy for a project of membrane and cell fusion. we did not finally succeed in the aim of introduction of foreign material, but this research was an excellent basis for membrane research later in my career. my living came from my employment as intern and resident, the last two years in the department of neuropathology headed by lucien rubinstein, and i was working at the university hospital and in turn at the veterans administration hospital. here, lysia forno taught me both the meticulous brain cutting and the very meticulous look at h&e-stained slides, making most special stains confirmatory only. careful observation was then also the basis for a paper with mary herman, the wife of lucien rubinstein, describing brain changes following heart transplantation. it was pleasant to note that with this project we had the unrestricted support of the cardiac surgeon, norman shumway. while working in surgical pathology, i made a fine structural analysis of amyloid in a pituitary adenoma, pointing out a possible pathogenetic correlation with beta-amyloid in senile plaques. upon acceptance of the paper, i was told by the faculty that it had found the interest of bob terry. this signified to me that there were no further problems with the extension of my contract. the academic atmosphere was generally very liberal, with staff from many different countries. coming from germany i took it as a great privilege to take part. my contemporary fellows were, amongst others, samuel ludwin, william langston, bernd scheithauer, and scott vandenberg. of course, we all also enjoyed california, despite lucien rubinstein’s saying that he would not accept people that evidently came because of the good weather. with my käfer-volkswagen, imported when i became chief resident, i repeatedly made tours to big sur, to the sierra nevada and also to the christmas tree farm of jon kosek, professor of pathology at the va hospital. jon used to make this tour by bike, just as he did every day for his way to work. environmental concerns were already well developed these days, and this was also exemplified by louis fajardo, the other va-professor of pathology. he used to supply the whole department with the giant zucchinis raised in his garden. i also enjoyed a sailing course starting in sausalito and finally finished with a high-seas sailing certificate. more important, however, were other obtained certificates: the american board of pathology and neuropathology and the california medical license. furthermore, membership in the association of military surgeons of the united states, later on helping me to follow my professional career without interruption. at the end it was a difficult decision to leave for a position that was offered to me in germany, yet i kept all these certificates valid until now by implementing the continuing medical education requirements. early professional position the decision to take up a position at the max planck institute for brain research in frankfurt in 1974 was a fortunate one. not least so since in the city of frankfurt i met emilia ferrero, daughter of a piemontese family that established a new business extension there. the meeting was arranged by virgilio rolleri, a member of my fraternity and lawyer of reputation, on occasion of a grand ball. being married, we continued to enjoy dancing ever since. at the max planck institute i was in good company of international scholarship holders, coming from turkey, poland, japan, mexico, and other countries. founder of the institute in 1962 and head of neuropathology was wilhelm krücke, in conjunction with his directory of the ludwig edinger institute of neurology of the university. i was in charge of the electron microscopy group, succeeding j. michael schröder. the instrument was the zeiss em9, having good optics but requiring all adjustments to be made by hand and to photographically develop the glass plates. thus, it was good to collaborate with a japanese scholar, yasuhiro yamamura, on the experimental project, nerve regeneration of the muscle spindle after denervation. we were not successful yet to finish this project in time, and only several years later i reported the differences between motor and sensory nerves in two joint papers. meanwhile, trying to improve my skills in more up-to-date techniques, i took an embo-course on subcellular fractionation at the max planck institute for biophysical chemistry in göttingen. the ensuing collaboration resulted in several papers on membrane fusion. a paper in “science” resulted from additional collaboration with the other department of the max planck institute in frankfurt, headed by rolf hassler. diagnostic neuropathology was falling in the competence of the edinger-institute and was conducted collectively by helge gräfin vitzthum, ekkehard thomas and me. prof. krücke, apart from signing out the cases, also rejoiced in some brain cutting personally. this was always a special event for the clinicians, associates and assistants sitting around his table. while dissecting the formalin-fixed organs and laying out the brain slices, he used to tell anecdotes and life stories, often acknowledging the merits of persons that were instrumental in his career. for example, the teacher of latin at his school in dillenburg, being very popular but suddenly disappearing due to his jewish origin and having been saved only by hiding away with some neighbors. or hugo spatz and julius hallervorden, both coming from the team of walther spielmeyer but having very different character traits, one rather expansive and dominant, the other scrupulous at work, rather humble and a person of integrity. or webb haymayker, while with the nasa at moffett field, kindly providing scientific literature that at that time could not be obtained in germany. the autopsy material to be dissected and sampled for histology did not only derive from the university clinics but also from neurologic and psychiatric institutions in the vicinity of frankfurt. i spent many days performing these external autopsies and hold good contacts with the respective clinicians. a favorite destination was the landesheilanstalt eichberg, offering a very pleasant atmosphere irrespective of the dark side of its history, and also the opportunity for me and the autopsy diener to buy some bottles of rhein-wine nearby. in the context of these activities, on the other hand, i also had a very startling experience. one of the dieners that usually accompanied me was skillful at work, reliable and well disposed toward me. it was somewhat conspicuous that he was always dressed extremely well. one day he did not appear without notice, and we did not see him again. we were then informed by the police that he had died in an exchange of fire, following the discovery of his thefts in a store of elegant menswear. all dissected brains and spinal cords of interest were permanently saved at the max planck institute, adding to the ones that were already stored in the basement and up to many decades old. someday i had a request from heiko braak for slices that he would embed and cut with his new technique for large sections in the institute of anatomy. following the selection of cases according to the old documents that to a large part were also still existent as well, i willingly went with him through the dark and dusty rooms to find the right buckets. this action, i believe, may have been helpful not only for heiko braak’s further career but also for the further scientific elucidation of neurodegenerative diseases. when in later years the max planck society decided to bury all this material in a ceremony burdened with guilt, many colleagues were rather bewildered. it was also regarded as fortunate that wilhelm krücke, continuing to work for some time in the edinger institute as an emeritus together with his successor wolfgang schlote, did not live to see this anymore. i thought, in contrast, that he would not have been surprised. he used to recount a special event from his previous time in bavaria. at the end of the war, when the allied forces were approaching, one could see some formalin-fixed brains floating down the isar river. they had been discarded by his colleagues in fear of accusations, despite no guiltiness at all. my time at the max planck institute for brain research then came to an end since the max planck society closed the department of neuropathology in favor of two neuroscience departments. i was very grateful to obtain a grant for a half-year stay abroad in order to facilitate the search for a new job. advanced professional training i was successful with an application to the national institutes of health to study the pathogenesis of idiopathic polyneuritis on patient material, using the myelin protein antibodies against p0, p1, p2 and mag that were newly developed there. in 1979 i thus had the privilege to work with henry de forest webster in the laboratory of neuropathology and neuroanatomical sciences at ninds. with the permission of prof. krücke i collected appropriate paraffin blocks prior to my trip. among these was a huge one containing a furled entire sciatic nerve. i personally made sections from this block with an old tetrander in the max planck institute, anticipating that such technical facilities would not be present in bethesda. harry webster supported the study by asking for additional material from harvard university and the university of pennsylvania. in the end we did not find a pathogenetic association, but the nevertheless ensuing paper was thus co-authored by edward p. richardson and arthur k. asbury. another co-author was yasuto itoyama, a young japanese scientist staying there for a longer period. both of us were well fit for the demanded very careful work, too much so in some european eyes, for example to clean all objectives before taking photos at the zeiss axiomat microscope. harry webster also invited both of us in turn to accompany him on his frequent if not regular weekend sailings. the boat was specially designed with retractable keels on each side to suit the rather shallow banks of chesapeake bay, a challenge even for an experienced yachtsman. back in germany i found a new job in frankfurt at the department of pathology of the st. markus city hospital. though not directly subserving my career, the two years that i spent there were required to obtain the pathology license in germany since my american certificates were not fully acknowledged. due to my continuing activity in external autopsies, i could nevertheless publish a paper in virchow’s archives, a case report of a lymphoma involving the nervous system. preparing the manuscript, i gratefully acknowledged the hand-written support of ronald f. dorfman, my former teacher of surgical pathology at stanford university and an internationally recognized lymphoma expert. professional maturity i returned to neuropathology in 1982 at the university of düsseldorf, serving as assistant physician in the institute of wolfgang wechsler. in tradition with wechsler’s training by klaus joachim zülch, the research focus in the institute was neuro-oncology. this topic was strongly picked up by my companion assistant guido reifenberger, transiently scholarship holder at the sahlgrenska-hospitals in göteborg and in the later succession becoming head of the institute in düsseldorf. my own contributions, resulting from my continuing clinical-diagnostic work, were mostly case reports in collaboration with my very cooperative young colleagues. to name just a few: eva neuen-jacob, specializing in myopathology and later on being in charge of all related diagnostics at the institute; martina deckert, later on head of neuropathology in cologne; thomas bilzer, veterinary neuropathologist and specialist in borna disease; reinhard prior, later on working in italy and being coordinator of european science projects; jürgen kiwit, later on chief physician of neurosurgery in berlin-buch; hiroshi himuro, fellow in neurosurgery and later on returning to fukuoka; stan krajewski, of polish origin and later on together with his wife working at the sanford-burnham medical research institute in la jolla. a most welcome collaborator belonging to a different lifespan was henry urich, serving as locum tenens for prof. wechsler as he had done before in many other places. we signed out all cases together and published several of them, and i profited from his great experience especially in the pathology of peripheral nervous system tumors. i furthermore had a very good collaboration with the institute of pathology just some stairs below. consultations were frequently made with franz borchard, assistant medical director and later on chief physician of pathology in aschaffenburg. one of the assistants was karl-friedrich bürrig, later on chief physician of pathology in hildesheim and president of the bundesverband deutscher pathologen e.v. the director of the institute of pathology was waldemar hort, already known to me from my time as a student in göttingen. hort and wechsler were quite different characters, easily to be seen when on the way to work. the former one bicycling along the rhine river, the big front basket and the side bags fully loaden with medical papers and documents. the latter one in a porsche sports-car for the rather long trip from his home in bergisch-gladbach, and whenever the sun was shining, with open roof and a white cap on his head. in some nice and hot summer days we sometimes also saw him similarly capped, climbing up the ladder to the roof of the institute. my own primary research went in a different direction, the investigation of laser-induced tissue changes in the central and peripheral nervous system. this was a truly collaborative venture, initiated by hans-joachim schwarzmaier at the department of laser medicine. utilized was a new type of laser, the 1,32 μm nd:yag laser, in service by stefan hessel at mbb-medizintechnik gmbh münchen. the operative procedures on experimental animals were done by frank ulrich and other colleagues at the department of neurosurgery. in charge of neuroradiology was thomas kahn, continuously collaborating with me in later years in leipzig as well. the primary goal of the project was to treat brain tumors by laser-induced interstitial thermotherapy in a mini mally invasive way. after completion of the experimental phase frank ulrich indeed was successful with clinical applications, performed under stereotactic or “open” magnetic resonance imaging guidance. my histologic investigations were acknowledged as an important contribution, allowing me to participate in the gordon-research-conference on lasers in biology and medicine, meriden/usa. yet with presentations at some other conferences, i heard rather different commentaries the mission of neuropathology should be the analysis and not the destruction of tissue. i did not quite agree but in fact i saw the major utility of this laser in tissue welding, especially for peripheral nerve anastomoses. a paper in “science”, elucidating the fine structural basis for this procedure, was a major module for my habilitation and postdoctoral lecture qualification at the university of düsseldorf in 1987. not entirely with pleasure since i lost my job, due to university regulations on fixed-term contracts. consequently, i pondered an offer of my aunt to take over her practice of general medicine in her nice house in bad essen, an idyllic small town at the foot of the teutoburger wald. i had already gained some insight there from occasional holiday locums in order to supplement the budget of my family. some of the good memories were that i never again have eaten such fresh and tasty bread, butter, eggs, and bacon gifted by the grateful farmer-patients. nevertheless, i declined the offer of my aunt. this turned out to be the right decision since half a year later i could continue at the university department in a superior position, as deputy medical head. eventually, applications for an independent position were also successful. i got a very cheerful farewell ceremony when i left to the university of leipzig in 1994 (fig. 3). fig. 3. my favorite poster, compiled by my friends and colleagues in düsseldorf. the format of my neuropathology reports is remaining unchanged up to now. [please click on the figure to download a high-resolution version] independent professional position in leipzig, much of what i encountered was unusual at first, but i nevertheless liked it from the beginning. my presentation as a candidate took place in the old bologna-style auditorium of the institute of pathology, originally designed for autopsy demonstrations. speaking while standing behind the desk at the floor, i was rather confident when i saw hans hilmar göbel and jorge cervos-navarro as members of the committee, sitting at short distance in the first row. my wife, however, was sitting in the top row and became dizzy when looking down. but stepping down after my presentation, she was welcomed and accompanied by the local professors in a very warm and polite way, not always her experience elsewhere. similarly, when i started with my work, it was customary in the morning meeting to first shake hands with all colleagues. i was also very fortunate with the staff that was either assigned to me or chosen by me: my secretary annelies krob, prudent and internationally experienced from her previous work at the trade mission of the german democratic republic in japan. my laboratory technicians, starting work in their usual habit at 6:30 a.m., thus having finished some urgent tasks already when i arrived considerably later. my assistant physicians steffen kellermann and dietmar thal, greatly contributing to establish neuropathology as an independent department, the first one in the area of eastern germany. steffen kellermann had already worked in the institute of pathology and continued in my department to organize the “rätselecken”, diagnostic slide sessions that now were addressing neuropathology. to our surprise and pleasure the auditorium was filled with numerous participants, including well established colleagues coming from distant institutions, such as werner jänisch together with marlis günther from brandenburg, or rolf warzok together with silke vogelgesang from greifswald. regarding other participants coming from nearby, we were not always sure about their motives since we heard frequent praises of the dishes and cakes prepared by my wife, served in the break and afterwards. dietmar thal soon made contact with thomas arendt at the paul-flechsig-institute for brain research and with other local researchers, establishing a fruitful collaboration and the basis for his later achievements in the classification of alzheimer’s disease. quite helpful for the development of my department was furthermore a half-year stay of henry def. webster, financed by the senior scientist award of the alexander von humboldt foundation. i could only offer him a very small room as workplace, a transit room to the secretary’s office. when i tried repeatedly and in vain to get a larger room in the institute, i received the ground plan of the institute together with a small figure from a well-meaning pathology colleague, thomas friedrich, later on pathologist in zurich. my wife, sawing this, integrated it in an amusing cartoon (fig. 4). fig. 4. a unique cartoon of a problem known to many neuropathologists (“prof. schober strides through the pathology institute, searching for new rooms”). but harry webster was content with a chair and desk for his own laptop since he primarily intended to finish some publications. his stay was greatly acknowledged by the team (fig. 5).the weekends were usually reserved for excursions together with my family, in return of the hospitality that i had received in bethesda. harry and his wife marion, coming back from stays in her home country hungary, sat in the in the back row of my van, in front of them our three small children celestina, giulio and giovanni. when exploring old castles and other historical sites we soon noticed that harry was the only person having a real interest in these, and thus we spent more time in country festivals, markets, and restaurants. it was reassuring to find no pejorative words about this period in harry’s book of memoirs that he sent me 10 years later. fig. 5. the neuropathology team in leipzig confers an award to henry def. webster (upper left and lower right). professional leadership a major event in my professional life was the “international symposium and 45th annual meeting of the dgnn” in the spring of 2000, organized by my team and me when i was the elected president of the german society for neuropathology and neuro anatomy. with the generous support of the dfg, bmbf, smwk, and avhf, conveyed by otmar wiestler and other colleagues, we could invite numerous internationally leading neuropathologists and scientists as speakers and chairmen for the symposium. with some of them i hold long-lasting friendships, e.g., with davide schiffer, janus szymas, and alfonso escobar. the congress took place in the mendelssohn-hall of the gewandhaus and in representative lecture halls of the university, facilitated by the generous support of industrial and other companies that was gained by our pertinacious personal canvassing. addresses of welcome were made by volker bigl, rector of the university and previously head of the paul-flechsig-institute for brain research, and by gottfried geiler, member of the leopoldina and previously head of the institute for pathology. in the following ceremonial act, the alfons-maria-jacob-medaille was awarded to jürgen peiffer by dimitrios stavrou. refreshments were offered generously throughout, and at the social evening a free dinner was served in the “ratskeller”. the congress finished with a satellite symposium on east-west-connections in neuropathology, organized in collaboration with lothar pickenhain and with ortrun riha, director of the karl-sudhoff-institute for the history of medicine and natural sciences at the medical faculty of the university of leipzig. referees were jürgen peiffer, loránt leel-össy, irina n. bogolepova, hans-dieter mennel, ingrid kästner, igor klatzo, arcadiu petrescu, and a.o. sapetskii. in retrospect we could hear from many participants that they would not have expected such an event to take place in eastern germany 10 years after the fall of the wall. the neuropathology team in leipzig was always complemented by younger people, either research students working for their doctorate or student assistants paid with a small stipend. their selection and supervision were mostly made by vera ogunlade, a phd always good humored, quite necessary for this task. she originated from the caucasus area, was married in africa, became a member of the arnold-sommerfeld-society in leipzig, and following her job in my department she later worked with hans kretzschmar in munich. a very good graduate student was christian schulze. originating from zwickau and studying in leipzig, he successfully engaged in my continuing laser project, resulting in several publications. he later worked at harvard medical school, then in new york at columbia university medical center, and is now head of cardiology at the university of jena. other publications of my department resulted from cooperations with various clinical colleagues, especially with the neurosurgeon juergen meixensberger. with his support, my associate manfred bauer initiated a long-standing collaboration with the neuro-oncology group headed by heidrun holland, using up-to-date cytogenetic and molecular genetic methods. i personally, in addition to neuro-oncological and neuromuscular diagnostics as well as brain cutting for legal and other expert reports (fig. 6), made some investigations of historical sites and about physicians in saxony, resulting in contributions to the periodical of the dggn (german society for the history of neurology). an article about richard arwed pfeifer was made in cooperation with wolfgang schlote, another article about hugo kufs in cooperation with hans hilmar goebel. fig. 6. persistent activity of brain cutting, as usual in a standing position. additional professional obligations included frequent meetings as a member of the faculty council and of the graduation committee as well as yearly meetings in brussels as an eu expert evaluator. this activity has continued without interruption, now however in an online format. although i see the necessity for video communications, i have to admit that i am missing the personal contacts with the other experts and the concluding dinners together with the staff. attending the restaurants in brussels i was usually accompanied by my wife. we usually also went together to the social evenings of various society meetings when held in european countries and not requiring transcontinental flights. i may just name a few such social events, of which we or i have nice and lasting memories. episodes 1. at the 2nd joint meeting of hungarian-german neuropathologists, organized by samuel komoly and katalin majtényi in budapest 1999, we enjoyed a barbecue and open dance in the nice setting of a park. at the end there was a dancing competition, with emphasis on viennese waltz. and who was the winner? not surprisingly, somebody from vienna. herbert budka stood out by his extraordinary elegance and vigor with switching clockwise and counterclockwise turnings. 2. at the sixth ibro world congress of neuroscience in prague 2003, the social dinner took place in the zofin-palace, a highly representative historical building on a moldova river island. the evening was opened by eva syková, appearing on the stage in a magnificent long dress with a fur stole. the curtain swept back, and we heard some czech music very nicely played on a grand piano. the dinner came in exquisite dishes. starting with the soup, the plate at each place was covered with a high porcelain cap to facilitate a collective beginning, and the following courses of the delicious meal were all individually served just like at court. 3. the 2005 meeting of the peripheral nerve society and the italian peripheral nerve study group took place at il ciocco, a very scenic resort in tuscany. we came by car together with our youngest son and with my parents in law from torino as additional guests. having transcended the apennine mountains on a very narrow and curvy road, we were hungry and rather happy to see a great variety of delicious dishes lying out as finger food at numerous tables in the big courtyard. all participants of the meeting ate with pleasure, and the dishes as well as wines and soft drinks were replenished repeatedly. then, to our surprise, the opening ceremony ended with an invitation to walk into the building and to have dinner. this was sumptuous and was served in the traditional sequence of pasta, main dish and fruits and liquors. 4. in contrast to professional trips in europe, those to overseas destinations i usually made alone. once a year i applied for a travel grant of the daad for a presentation overseas, thus regularly meeting my american friends and colleagues again at the annual meetings of aanp and/or asip in various cities. i usually booked the flight at a considerably reduced price for a whole week, giving me the opportunity to make new friends at local rotary clubs and to explore further localities. 5. special mention deserve some very nice experiences that i made in japan. at the 39th annual meeting of the japanese society of neuropathology in fukuoka 1998, president masashi fukui invited some speakers and special guests for dinner, amongst others henry def. webster, bernd scheithauer, peter j. dyck, dimitrios stavrou, akira hori, and me. the main dish was shark fins, a highly valued delicacy. we were all seated at a round table and thus could well observe how different the skills were to eat with chopsticks and to use the small bowls with soy sauce, rather a challenge with this dish. several guests gave up and changed over to forks, but nobody was in a hurry. at the end of this cheerful company with a lot of sake, we had the choice to either take a taxi to the hotel or to continue the party in the rear of the building. looking at each other we exactly knew in advance what the choices were. the taxi was a big cab, allowing further conversations. harry webster soon spoke out and elucidated the values of the family. those of us that were neuropathologists recollected that he even in his publications had used this term allegorically, for the description of the groups of outgrowing axons. 6. another most memorable journey was to the 50th annual meeting of the japanese society of neuropathology, organized by kiyomitsu oyanagi in takamatsu 2009. this time i made a round trip, and i was accompanied by two persons. first, by my aunt, retired from general practice and at that time 87 years old but still very adventurous. second, by my older son giulio, student in theoretical physics and at that time in tokyo as an awardee of the japanese ministry of education, culture, sports, science and technology. he had asked his professor, naoto nagaosa, for an interview in his institute at the hongo campus, and we were kindly received in his office. he gave us an introduction to the new class of topological insulators, drawing a möbius strip at the blackboard and also acknowledging the origin of möbius from leipzig. at the end of this interesting but intellectually demanding recourse, my aunt drew a large old photograph out of her handbag. it was dated 1926 and showed a ceremony in japan, where her uncle walter lwowski received a golden sake-bowl engraved with the emblem of the tenno, acknowledging of the construction of the first sheet mills at kawasaki and at yawata over a period of five years. consecutively we made some further talk, and we expressed our thanks to prof. nagaosa for the long time that he had spent with this interview. we then made a sightseeing tour on the hongo campus and were fortunate to be guided very competently by masaya oda, still fluent in german from his stay at the max planck institute for brain research in frankfurt long years ago. after further sightseeing in tokyo, we took the shinkansen to takamatsu, a fast and comfortable ride. 7. the closer we came to takamatsu, the brighter blue was the sky. on our arrival at the most scenic landscape, the splendid seaside location, and the very comfortable hotel at walking distance, it almost seemed to be a pity to spend the time in conference rooms. but the meeting then proved to be extremely well organized and interesting. following the presidential lecture by kiyomitsu oyanagi and the plenary lectures by john trojanowski and dennis dickson, presentations were scheduled in several different rooms, but all participants could enjoy a bento (japanese lunch) together. a major event was the memorial celebration party. at the end, after several addresses by dignitaries and notabilities, i dared to ask kiyomitsu if i could say a few sentences in japanese that i had prepared together with my son. my japanese sentences were apparently understood, to judge by the applause. finally, to our surprise and without having been prepared, my aunt was cited to the stage. she climbed up without hesitation and reported, translated by my son, some episodes of her life as well as the connections of her family with japan, and she got even more applause. upon returning back to tokyo, accompanied by akira hori in the shinkansen and following his explanations regarding the history of the landscapes and monuments passing by, we had another very nice farewell. masaya oda invited us for dinner in a top-class restaurant, together with his family. the cook prepared the meal on hot plates just in front of each guest, separately yet perfectly synchronized (fig. 7). fig. 7. invitation for dinner in a fine restaurant in tokyo (from left: my son giulio, my aunt gisela rohlmann, rs, masaya oda and his daughter). retirement and beyond in 2010 at the age of 65, i did not feel like retiring, as it is usually required for civil servants in germany. therefore, i personally went to my employer, the smwk (saxonian ministry for science and arts) in dresden, and i asked for special permit to continue as head of the neuropathology department in leipzig. the answer of the officer in charge was affirmative. not so, however, the answer of the dean of the university in leipzig. fortunately, i soon got an offer to work as a neuropathology consultant elsewhere in leipzig, in the institute of pathology and tumor diagnostics at the municipal hospital st. georg. the director there, volker wiechmann, as well as the chief of the associated pathology practice, andreas plötner, were known to me as previous participants of my slide seminars. i was sympathetically accepted as a member of the team, and i have always been invited for social gatherings such as the annual christmas dinner party. for more than 10 years up to now i thus have continued to make diagnostic work for the neurosurgery and other departments as well as all routine brain cutting. in a rather comfortable way for several hours daily, usually in the afternoon except once a week at 7 a.m., a time that is scheduled for the tumor board. i enjoy instructing the younger colleagues at a multi-viewer microscope, and there is also the opportunity to give some lectures to students since st. georg is a teaching hospital of the university. furthermore, there are some ongoing clinical research projects in collaboration with max holzer from the paul-flechsig-institute for brain research and with other scientists. on occasion i am pleased to get visits by external colleagues and old friends, giving testimony that leipzig has become an attractive location again (fig. 8). the institute of pathology of the st. georg hospital, founded in 1913, is one example of the many historical and well restored buildings (fig. 9). fig. 8. kiyomitsu oyanagi and rs in front of dr. faustus and mephisto at the auerbach’s keller restaurant in leipzig. at the university clinics i am still participating in academic events and i am still a member in a graduation committee. asked about my hobbies and proclivities, i can say that there is no boredom. trying to stay up to date, i am reading two daily newspapers and i am browsing through the serial issues of about 10 journals including science, jnen, biospektrum, and rotary magazin. one particular field of interest is astronomy, and my collection of the pertinent literature has resulted in several presentations on galaxy evolution to a lay public. another one is history, especially in relation to the origin of religions or ideologies. i am also fond of fine arts, though not personally active anymore in painting and in piano playing. last not least i am enjoying fine cuisine, being spoiled already at home by my italian wife. my mobility is somewhat limited by a peripheral neuropathy that is associated with a protein well known to me. however, there is no change both in my general view of pathology as well as in relation to myself: a molecular detail may be important and have far-reaching implications, but it does not carry the weight of an integration of all macroscopic and microscopic aspects in correlation with the individual history. altogether it is thus a pleasure to continue with my professional activities, comprising 50 years in neuropathology. cv (short): to be found at my homepage http://www.schober-info.com/ fig. 9. the pathology staff at the st. georg hospital in leipzig (upper row from right: director volker wiechmann, rs, chief pathologist andreas plötner). copyright: © 2021 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. neuropathology studies of dementia in us persons other than non-hispanic whites feel free to add comments by clicking these icons on the sidebar free neuropathology 3:6 (2022) review neuropathology studies of dementia in us persons other than non-hispanic whites my-le nguyen1, emily z. huie1, rachel a. whitmer2, kristen m. george2, brittany n. dugger1 1 department of pathology and laboratory medicine, university of california, davis, usa 2 department of public health sciences, university of california, davis, usa corresponding author: brittany n. dugger · department of pathology and laboratory medicine · university of california, davis · 4645 2nd ave. · 3400a research building iii · sacramento, ca 95817 · usa bndugger@ucdavis.edu submitted: 26 january 2022 accepted: 04 march 2022 copyedited by: biswarathan ramani published: 10 march 2022 https://doi.org/10.17879/freeneuropathology-2022-3795 additional resources and electronic supplementary material: supplementary material keywords: disparities, dementia, neuropathology, hispanic, latino, asian, african american, brain abstract alzheimer’s disease (ad) and vascular dementia are two of the most prevalent dementias that afflict the aging population in the united states (us). studies have made great strides in understanding the neuropathology of these diseases; however, many studies are conducted in the context of non-hispanic whites (nhws), and few include the rapidly growing underrepresented populations that reside in the us. we sought to characterize current knowledge of the neuropathologic landscape of ad and vascular dementia of the largest growing us minority groups, namely latinos/hispanics, black americans, and asian americans, compared with nhws being the majority group. it is vital to note these historic categories are social constructs and cultural and social associations may underlie differences. we conducted a literature search utilizing specific criteria to yield neuropathology papers that addressed the demographics and neuropathologies of relevance, then collated the findings into this review. we reveal that while there has been much progress in neuropathological research involving latinos/hispanics and black americans in the past decade, no cohesive conclusions could be extrapolated from the existing data due to the dearth of minority participants and even smaller amount of information related to the heterogeneity within each minority group, especially latinos/hispanics. furthermore, we reveal an even greater scarcity in neuropathological studies involving asian americans, also a very heterogeneous group. we hope the presented findings will illuminate the paucity of minority representation in not just neuropathological research but the field of clinical research overall and serve to inspire clinicians and researchers to help reduce the health disparities underrepresented groups in the us face. introduction clinically, alzheimer’s disease (ad) is defined as a type of dementia distinguished by neurodegeneration that results in memory loss and deterioration of cognitive functions24, 38. neuropathologically, ad is defined by aggregations of the amyloid-β (aβ) protein, in the form of aβ plaques, and tau protein, in the form of neurofibrillary tangles (nfts)13, 25, 97. in addition to aβ plaques and nfts, neuropathologies associated with vascular dementia can also be concomitant57, 78, 101. vascular dementia is the second most common cause of dementia following ad and neuropathologically can manifest as infarcts and hemorrhages as well as other vascular pathologies such as cerebral amyloid angiopathy (caa) and arteriolosclerosis9, 17, 23, 63, 90, 101. while there has been progress with understanding disease phenotype (for review see 89) and progress with therapies having evidence of targeting and reducing aβ plaques in the brain, there have yet to be treatments that fully cure or stop the progression of the disease20, 22. although great strides have been made to combat this disease, most studies have focused on select populations or cohorts, specifically composed of persons identifying as non-hispanic whites (nhws)28, 43, 73, 97. the population of persons age 65 and over in the united states (us) has significantly increased in the past decade—as much as a 36% increase2. us ethnoracial minorities made up 20% of this age demographic at the beginning of the decade, and increased to 24% by the end of it2. the nhw population of age 65 and over is projected to increase 29% by 2040 in comparison to the 115% increase of the ethnoracial minority population2. the largest us minority population is latinos/hispanics, followed by black americans, then asian americans, which are the fastest growing demographic18, 53. other notable underrepresented groups are american indians (native americans), native hawaiians, and pacific islanders4. it is important to note in this paper we will utilize historic terms (such as those within the us census) and these categories are social constructs and cultural and social associations may underlie differences. diversity in studies maximizes variability in risk and protective factors. this can aid in studying clinically relevant transitions across the spectrum of a disease with the goal of identifying modifiable pathways to support maintenance of normal functions. as racial/ethnic groups in the us continue to grow in addition to longer life expectancies, more diverse elderly individuals will be seen for diagnosis and treatment of neurodegenerative diseases; thus, more research reflecting the population diversity is needed so that prevention, treatment, and prognosis strategies encompass all who are affected by the devastating impact of dementia. heterogeneity of race and ethnic categories race and ethnicity are two terms fundamentally distinct from one another. race historically has been used to describe the physical traits of an individual such as their eyes, hair, and skin, whereas ethnicity has been used to describe an individual’s cultural identity74. it is essential to remember these terms are socially constructed and hold no bearing on an individual’s biology. the categorizations for race and ethnicity in the us have evolved over time as self-identification shifts, immigration, and mixed racial heritage became more prevalent1.the term “race” has regretfully implied a sole focus on an individual’s morphology, and historically has not accounted for other background variables such as geographic origin, environmental factors, and sociocultural characteristics that influence these differences43, 98. while the historic terms employed in this review are not optimal given how restrictive and tentative they are, the u.s. census uses them, which many neuropathology studies have utilized as well, and thus are presented as such in this review. thankfully, there is a growing awareness that these historic terms alone do not aptly stratify these individuals in a scientific context and new approaches are being encouraged1, 6, 43, 49. furthermore, it is important to recognize there is heterogeneity within racial/ethnic categories. the terms latinos and hispanics are sometimes used interchangeably65. the term hispanics historically has been used to describe those who are from spain or other spanish-speaking country, while latino signifies those who originate from latin america, regardless of their spoken language62. moreover, it is noted the term caribbean hispanics is used to describe the latino population that resides in puerto rico, dominican republic, and cuba96. to further expand on distinctions within the latino/hispanic population of the us, some studies have partitioned this ethnicity based on area of decedents’ self-reported origin: mexican, south american, central american, and caribbean99, 100. these categories are standardized by the us census; hence studies have followed the same format64. the mexican descendent population is the largest latino/hispanic group in the us and is spread out through the country, with more density in the southwest27. individuals from south and central america also follow this distribution pattern, whereas caribbean latinos/hispanics are more concentrated in eastern states3. these geographical distributions when examining ad cohorts can be immensely important as there have been reported socioeconomic and cultural differences within these groups that are associated with risk factors for ad77. there is also heterogeneity within the black american population. the largest subgroup within the us black population is single-race non-hispanic, comprising of 87% of the total us black population95. the following largest is the multiracial non-hispanic population, constituting 8% of the overall us black population, with black hispanics making up the remainder 5%95. the majority of black americans are of west/central african and european heritage, and some also have native american roots34. an estimated 90% of the total black population was born in the us in which most are descendants of enslaved people84, 94. despite the great migration where the black population had dispersed to areas in the us away from the south, the distribution in the south has begun to grow in the past few decades92. another observed migration pattern in the last few decades has been a substantial increase among the foreign-born us black population, making up 10% of the current us black population in which the majority of foreign-born us black persons, 88%, were born in african or caribbean nations94. similarly, there is immense diversity within the asian american population. there are 21 distinct asian subgroups living in the us according to gathered data from the census american community survey, with the largest being chinese (including taiwanese), followed by asian indian, filipino, vietnamese, korean, and japanese15, 16. almost half (45%) of asian americans live in the western us, with nearly one third of the nation’s population living in california alone (30%), while 24% reside in southern states, 19% in the northeast, and 12% in the midwest15. data have shown a substantial proportion of chinese, filipino, vietnamese, korean, and japanese inhabit the western us, amounting to at least 43% distribution across all groups46. on the other hand, asian indians occupied the northeastern and southern regions of the us more frequently, accounting for over 29% distribution for each region46. by addressing the geographic, ethnic, and cultural variations that exist among race and ethnic subgroups, ad research can be refined to yield more precise methodology and analyses. differences in clinical ad based on race and ethnicity differences in the prevalence and incidence of clinically defined ad and related dementias (adrd) have been observed when comparing across race/ethnicity. ad prevalence is highest in black americans and latinos/hispanics, followed by nhws, and then asian americans21, 33, 40, 41, 61, 66-68, 70, 72, 79, 86, 96. notably, one study showed us-born nhws, hispanics, and other uncategorized races had lower frequency of dementia compared to their immigrant counterparts, except for non-hispanic blacks (nhbs), where it was an inverse correlation72. studies have also shown differences in dementia prevalence between latino subgroups. one study revealed a 4.8% prevalence of dementia among mexican americans ≥65 years residing in sacramento county, california, with up to 31% prevalence in those aged 85 years or older42. in contrast, a study of caribbean hispanics residing in north manhattan estimated a dementia prevalence for individuals aged 65-74 of 7.5%, 27.9% for those 75-84, and 62.9% for those 85 and older41. latinos/hispanics have been shown to have an earlier age of onset of ad when compared to nhws and black americans, though the difference was marginal for the latter in some cohorts21, 33, 42, 61, 72. both latinos/hispanics and black americans have a high prevalence of cardiovascular risk factors associated with adrd, while prevalence of these risk factors is lower among nhws followed by asian americans 40, 42, 61, 67, 96, 102. in a california study that examined dementia incidence in asian american subgroups, filipino americans had the highest incidence rate at 17.3 per 1000 person-years, and south asian americans (i.e., asian indian, pakistani, bangladeshi, sri lankan, or nepalese) had the lowest rate at 12.1 per 1000 person-years67. while many observable trends have been correlated with ad from a clinical perspective across different race/ethnicities, they do not confirm the presence of the hallmark protein aggregates that are currently are the gold standard for diagnoses of adrd, in other words, the neuropathology. do the neuropathological profiles of individuals with adrd of the three largest minority groups in the us differ, as there have been noted cultural and geographic differences in these groups? neuropathology studies have been conducted on predominantly nhw cohorts, so little is known about adrd in these other racial/ethnic groups6, 28, 43, 73. for this review, we will discuss the current landscape of neuropathological findings in ad and vascular dementia in latinos/hispanics, black americans, and asian americans. we seek a more comprehensive understanding of the disease profile in these groups of individuals to provide improved diagnoses and develop effective countermeasure therapies or methods to allay the risk of ad and vascular dementia. methods inclusion and exclusion criteria all literature in this review focused on signature pathologies associated with ad and vascular dementia in latino/hispanic, black american, and asian american cohorts. studies meeting the inclusion criteria were published 1995 and onwards, peer-reviewed, specifically presented ad or vascular dementia neuropathological findings, and were conducted in the us. neuropathologic evaluations of interest included but were not limited to braak neurofibrillary tangle (nft) stage, consortium to establish a registry for alzheimer’s disease (cerad) neuritic plaque score; thal amyloid phase; and national institute of aging and alzheimer’s association (nia-aa) criteria of overall ad diagnosis based on neuropathologic changes13, 48, 71, 97. for vascular dementia, pathologies of interest included, but were not limited to, infarcts, hemorrhages, arteriolosclerosis, atherosclerosis, and caa. as for staging schemes and creating a consensus for diagnosing vascular dementia, there have been multiple attempts23, 55, 83, 90, 92; however, there is no universally used system in place. exclusion criteria included papers that contained only living cohorts (i.e., cohorts not having neuropathologic evaluation of persons after death) presented data with no mention of ad neuropathologies specifically (e.g., genotyping, neuropsychological tests, etc.), studies that were not conducted on human subjects, and articles that were not sourced from peer-reviewed journals. search strategy the literature review was conducted by searching specific mesh terms in pubmed, scopus, and web of science to yield the peer-reviewed articles investigating ad and vascular neuropathology among latinos/hispanics, african americans/black americans, and asian americans on november 19, 2021. the mesh search term refinement process for these databases was guided by a uc davis health medical librarian. the full list of search terms can be found in the supplemental methods at the end of this review. results neuropathologic findings on us minority groups were often compared to nhws. for the purposes of this review, we will use latinos/hispanics as the collective term to describe this demographic unless a mentioned study further partitions out subgroups, in which the terms that are consistent with the study will be used. this same principle applies to the term black americans being used for mentions of this group unless the study denotes otherwise. details of selected main papers are within table 1 relating to neuropathological data of latinos/hispanics and/or black americans as many of these studies compared these groups. additional studies on asian americans are in table 2. click here to view a large version of this table. ad and vascular dementia neuropathology in latino/hispanics one observed neuropathological trend in latinos/hispanics was cerebrovascular pathologies, such as infarcts, caa, arteriolosclerosis, and atherosclerosis, that were typically associated with dementia and/or ad diagnoses (see table 1). a 2010 study using the national alzheimer’s coordinating center (nacc) database showed latinos/hispanics were more likely to have neurovascular pathology compared to nhws70. in persons with dementia during life, studies conducted at the alzheimer’s disease research centers (adrcs) at both university of california, davis (ucd) and the university of california, san diego (ucsd) reported a higher frequency of concomitant neurovascular pathologies compared to nhws29, 102. one comparison of interest between these studies with respect to cerebrovascular disease (cvd) pathologies—specifically microinfarcts and macroinfarcts—is filshtein et al. found occurrence was higher in latinos/hispanics compared to nhws in the ucd cohort29, while weissburger et al. discovered there were no significant differences between the two groups in their ucsd cohort102. this contradiction may be because persons with evidence of in-vivo hemorrhages, strokes, and other major agonal infarcts were excluded from the ucsd study102. a study also using the national alzheimer’s coordinating center database illustrated this theme in the specific context of caa, in which latino individuals with neuropathologically confirmed ad were more probable to have severe caa than nhws81. the study done by weissburger et al. also supported this trend102. another study by ucsd further validated this pattern by finding higher caa burden in the ad group compared to the no pathology group, which was defined as not having significant brain pathologies, and the non-ad pathology group was defined as only have tauopathies, frontotemporal dementia (ftd), progressive supranuclear palsy (psp), dementia with lewy bodies (dlb), or parkinson’s disease (pd) with neocortical lewy bodies, in an all-latino cohort91. as for hallmark ad pathologies, aβ plaques and nfts, there were more inconsistent patterns between latinos/hispanics and nhws. the study done by filshtein et al. utilizing demented cases from the alzheimer’s disease center at ucd, a california based cohort, revealed latinos/hispanics had the lowest occurrence of ad clinicopathological diagnosis without the involvement of cvd, including lower frequencies of persons at higher braak nft stage compared to nhws and black americans29. this is consistent with literature where latino cohorts tended to exhibit concomitant neurovascular pathologies with their ad diagnoses29, 102. conversely, santos et al., in a florida based cohort, demonstrated latinos/hispanics were twice as likely to have a higher braak nft stage than nhws86, while a study conducted by mehta et al., including cases with a clinical possible/probable diagnoses of ad, revealed braak nft stage did not differ significantly between latinos/hispanics and nhws70. the study by weissburger et al. also showed both groups (nhws and latinos/hispanics) had similar braak nft stage102. regarding plaques, which can include neuritic plaques (amyloid plaques containing dystrophic neurites) in some literature, mehta et al. found neuritic plaques were more frequent in latinos/hispanics compared to nhws70. however, the results by filshtein et al. revealed latinos/hispanics had the lowest proportion of cerad frequent neuritic plaque score, implying that neuritic plaques may not be as much of a major contributing pathology to their dementia29. santos et al., excluding persons that did not have autopsy confirmed ad and cases with known mutations, opted to use thal amyloid phase to categorize plaque presence, in which the phases did not differ between latinos/hispanics and nhws86. notably, latino/hispanic participants in these studies may represent diverse ethnic groups in terms of geography and nation of origin leading to seemingly contradictory findings. for instance, santos et al. had utilized the florida autopsies multi-ethnic (flame) cohort located at the mayo clinic of florida for their study, which consisted of individuals primarily from the caribbean origin for their latino group, whereas weissburger et al. and soria et al. had utilized cohorts from the ucsd adrc, which comprised of individuals primarily of mexican descent for their latino/hispanic group86, 91, 102. furthermore, these studies also had slightly different inclusion and exclusion criteria, as outlined in table 1, that may also contribute to discrepancies. ad and vascular dementia neuropathology in black americans like latinos/hispanics, cerebrovascular pathologies are also commonly observed in black americans, but the pattern is not completely consistent which may be due to cohort inclusion/exclusion criteria, demographic locations, and recruitment strategies. a study at the rush alzheimer’s disease clinical core, based in the chicago illinois area, revealed black decedents had significantly greater severity in both atherosclerosis and arteriolosclerosis when compared to nhws7. the study by filshtein et al. corroborates this finding, in which their results of persons with dementia demonstrated black participants had a higher proportion of cvd compared to nhws29. another study utilizing patient data from over 30 alzheimer’s disease centers across the country also found black americans were more likely to have had a contributing diagnosis of vascular dementia than nhws, although this study was based on small group numbers and did not account for center biases40. interestingly, mehta et al. revealed black americans had similar neurovascular pathology rates as nhws on autopsy, in contrast to the consensus of the other studies70. likewise, the results from a study on a cohort based in washington university’s alzheimer’s disease research center (adrc) also showed no differences in cerebrovascular infarcts between the nhw and black american participants107. multiple studies found caa burden did not differ significantly between black americans and nhws56, 81, 82. in contrast, graff-radford et al. observed black americans had significantly greater frequencies of caa in addition to the other vascular neuropathologies (i.e., infarcts, hemorrhages, arteriolosclerosis, atherosclerosis) in comparison to nhws40; however this study utilized data from multiple cohorts and did not control for center biases. for ad pathologies, there were also contradictions in the literature. findings from barnes et al. revealed black decedents were less likely to have ad-only pathology, defined by neuritic plaques and nfts as the single contributing pathology to their dementia diagnosis compared to nhw decedents7. along similar conclusions, filshtein et al. demonstrated mixed pathologies were more common in black decedents than in nhw decedents29. with respect to hallmark ad proteinopathy comparisons, such as neuritic and diffuse plaque counts, thal amyloid phase, and likelihood of higher braak nft stage, multiple studies showed no significant neuropathological differences in both categories for either patient demographic82, 85, 86, 107. in contrast, more than one study demonstrated black american decedents were more likely to exhibit higher braak nft stage29, 40, 70. graff-radford et al. also indicated black american participants had greater cerad-frequent scores for neuritic and diffuse plaques40, whereas mehta et al. revealed black americans had similar neuritic and diffuse plaque counts as nhws70. both studies had utilized data from the nacc database, but this conflict in findings may be due to the fact the mehta et al. study had a larger sample size from the longitudinal window from 1984 through 200570, compared to the graff-radford et al. study which recruited data from a smaller sample size from 2005 to 201540. as with the previous section, discrepancies may lie within cohort selection criteria as highlighted in table 1. ad and vascular dementia neuropathology in asian americans ad and vascular dementia pathological trends for asian americans compared to nhws are largely unexplored for all pathological categories as literature for this minority group in this specific context is still sparse. of the studies conducted, most have focused on japanese americans, specifically men, through the honolulu asian aging study (haas) which includes very few, if any other subgroups. the haas was established in 1991 and comprised surviving participants of the honolulu heart program, a prospective, community-based cohort study of heart disease and stroke established in 196536, 54, 93, 105. for neurovascular pathologies, a study revealed microinfarcts were significantly more common in japanese american men in haas compared to caucasian women in the nun study (ns)106. another finding from the haas showed that the frequency of microvascular lesions as the contributing dementia pathology was nearly the same as ad pathologies104; however, a later paper relative to this one showed that microvascular infarcts as the dominant or exclusive contributing lesion to dementia were the most frequent among decedents, then followed by ad lesions103. there were additional findings on the haas revealing dementia frequency increased with neuritic plaques in decedents with nfts and even further with cvd lesions76. interestingly, one analysis showed microinfarcts were strongly associated with poor cognitive function score in non-demented individuals, whereas nfts were strongly associated with poor cognitive function score in demented individuals59. for neuropathologic change involving ad proteinopathies, a more recent study showed that the haas was more resistant to aβ accumulation, but the ns was more resistant to neurofibrillary degeneration for individuals without aβ accumulation58. click here to view a large version of this table. native american, alaska native, native hawaiian, other groups, and points of further research the scope of this paper had focused on the neuropathology of the three largest minority groups of the us, as those were the demographics that offered adequate findings to collate into a cohesive and purposeful review. the current presented literature offers a foundation for ad and vascular dementia research in underrepresented us groups and seems to only expand each year (see figure 1), with more research being conducted on a greater variety of cohorts and sites. the existing findings are concentrated and substantial enough to serve as preliminary data for comparison of future findings, depending on the objective demographic. nonetheless, despite the upward trends of more ad and vascular dementia research being conducted in diverse cohorts, there are still many gaps that need to be filled and other demographics that need to be considered. for example, alaskan natives and american indians (native americans) constitute the fourth largest distinct (i.e. one race) population of the us4, yet there is a paucity of medical studies on persons of these backgrounds. this may be due to cultural aspects, where those who valued tradition (including religious beliefs) strongly advocated for the body to buried whole52. this dearth of information also applies to native hawaiians and other pacific islanders, despite being the fifth largest single race population and second fastest growing race in the country behind asian americans4, 45. the lack of neuropathology literature that captures the diversity of asian americans also highlights imbalances in research, as it is most probable the neuropathological trends of japanese americans would not accurately encompass the depth and breadth of diversity of persons across the asian continent. an overall paucity of literature presently exists in comprehensive studies centering on these mentioned groups and is not limited to specifically neuropathology studies. figure 1. number of papers found on pubmed as of november 19, 2021 by year using the search terms “alzheimer’s brain pathology” along with the demographic term (categories in legend, see supplemental section for further information). while there has been advances in neuropathology literature focused on latinos/hispanics and black americans in recent years compared to other minority groups, there are still limited participants in these cohorts. the existing literature is limited in sample size, which varies widely between studies and typically with the minority groups representing a small fraction of the cohort (see tables 1 and 2). furthermore, studies can have certain inclusion and exclusion criteria that may hinder participation in select groups; for example, exclusion of cvd for ad studies may decrease frequencies of certain minority groups with higher frequencies of cvd50. these constraints from low minority group recruitment may be due to numerous factors, including lack of access to healthcare, historical abuses of minority groups for medical research, mistrust of the healthcare system making participants less likely to agree to participate in clinical trials or autopsy programs, and language barriers5, 8, 10, 11, 30, 31, 37, 49, 75, 100. with respect to retention, a systematic review highlights a lack of literature that examines retention exclusively from recruitment39. socioeconomic circumstance was shown to be the most powerful contributor to the absence of participants for longitudinal studies involving ethnoracial minorities26. low socioeconomic status largely impacts access to health care resources such as regular visits to a health professional as a result of being uninsured10, 87, 88, in which patients may not only lack the direct care they need but also the general awareness of clinical study enrollment opportunities. flexible scheduling played a substantial role in participation as many individuals were restrained by work or childcare obligations for their appointments as well as transportation26, 32, 69. financial compensation was a major influencer in recruitment amongst ethnoracial minorities51, 69; it has been reported that members of the latino/hispanic community were motivated by monetary compensation for their time because they experienced economic hardships69. patients may also feel discrimination in the process of seeking care, especially among non-whites for their race, color, and/or ethnicity35. half of black americans report they have faced healthcare discrimination, and one third of asian americans and latino americans similarly report having experienced healthcare discrimination as well35. a vast majority of non-white americans believe that in the importance of having ad and dementia care providers to understand their ethnic/racial backgrounds, such as native americans, black americans, latinos/hispanics, and asian americans35. however, less than half of black and native americans are confident there are culturally competent providers, and only roughly 3 in 5 asian americans and latinos/hispanics are confident35. a few studies have attempted to understand barriers and willingness for brain donation across major us racial ethnic groups: nhws, latinos/hispanics, black americans, and asian americans11, 12. while conducting focus groups, the first study revealed concerns, attitudes and beliefs around brain donation that fell into three categories: 1) religious beliefs 2) concerns and misconceptions about brain research and 3) the role of the family7. a follow up study surveying nhws as well as 169 african americans, 50 asians, and 61 hispanics revealed older age, latino ethnicity and understanding of brain use by researchers and what participants need to do to ensure brain is donated were positive predictive factors, while the belief that the body should remain whole at burial, african/african american race, and concern researchers might not be respectful of the body during autopsy were negative predictive factors11, 12. the belief that the body should remain whole was shared amongst latinos/hispanics, african americans, and asian americans, which was a similar sentiment of alaskan natives and american indians mentioned earlier52. this further illustrates the substantial role cultural barriers may play in cohort participation from us minority groups. knowledge, stigma, and apprehension of adrd also differ across ethnic/racial groups. for example, one study revealed that nhws tended to have greater knowledge about ad compared to black americans, and black americans had same or greater levels of concern about getting ad as nhws depending on their geographic location47. another study discovered concern about developing adrd in native americans, black americans, and latinos/hispanics is noticeably lower compared to nhws35, which contradicts the finding about black americans in the aforementioned study, possibly due to region differences where the data was taken. it has also been shown that asian americans do not exhibit a strong concern of adrd as many believed it was a natural occurrence for aging people14. multiple papers have denoted that asian americans had beliefs of stigma of persons with ad, which played a significant role in seeking care from primary care providers for ad14, 19, 60. limited knowledge on not only adrd but also the brain removal process poses some hesitance on minority subject participation8, 11. as stated above, some themes that subjects or family members of subjects shared skepticism on were understanding the purpose of studying a decedent’s brain, misconceptions on how the brain is used or collected for research, and overall knowledge about the brain donation procedure11. it is important to recognize the existing inadequacies and confines of the study recruitment process for us minority groups to further advance the representation of these populations in biomedical research. fortunately, there has been progress to minimize these barriers. the uc davis adc utilized many avenues to increase diversity in enrollment in research cohorts, such as satellite clinic sites, increasing face to face screening at community events, options of in-home visits, compensation for transportation to clinic visits, dedicated drivers to transport participants to visits, and employing bicultural and bilingual individuals with proficiency of the involved populations44. these methods facilitated a substantial increase in the number of ethnic minority participants, as much as a four-fold increase44; this approach also led to more diversity in other variables as well, such as educational background44. a later study showed that mailing recruitment letters was the most successful method in a multi-modal recruitment approach in enrolling more ethnoracial minorities for adrd cohorts80. as these issues get addressed on a more widespread scale, significant advancements can be made not only in the field of neuropathology, but all fields of clinical research. acknowledgements this work was supported by the national institute on aging of the national institutes of health under award numbers ag062517, ag052132, ag050782 and ag056519, and supported by the california department of public health alzheimer’s disease program (grant # 19-10611) with partial funding from the 2019 california budget act. the views and opinions expressed in this manuscript are those of the author and do not necessarily reflect the official policy or position of any public health agency of california or of the united states 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l. brain lesions at autopsy in older japanese-american men as related to cognitive impairment and dementia in the final years of life: a summary report from the honolulu-asia aging study. j alzheimers dis 2009;18:713-725. white l, petrovitch h, hardman j, et al. cerebrovascular pathology and dementia in autopsied honolulu-asia aging study participants. ann n y acad sci 2002;977:9-23. white l, petrovitch h, ross gw, et al. prevalence of dementia in older japanese-american men in hawaii: the honolulu-asia aging study. jama 1996;276:955-960. white lr, edland sd, hemmy ls, et al. neuropathologic comorbidity and cognitive impairment in the nun and honolulu-asia aging studies. neurology 2016;86:1000-1008. wilkins ch, grant ea, schmitt se, mckeel dw, morris jc. the neuropathology of alzheimer disease in african american and white individuals. arch neurol 2006;63:87-90. copyright: © 2022 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. a hobby neuropathologist from far east resides in western europe feel free to add comments by clicking these icons on the sidebar free neuropathology 5:9 (2024) reflections a hobby neuropathologist from far east resides in western europe akira hori institute of pathology, division of neuropathology, medizinische hochschule hannover, germany corresponding author: akira hori · division of neuropathology · institute of pathology · hannover medical school · 30623 hannover · germany twothreetenah@gmx.de the author's cv has been attached as electronic supplementary material: cv supplementary material submitted: 09 january 2024 accepted: 28 march 2024 copyedited by: jared ahrendsen published: 16 april 2024 https://doi.org/10.17879/freeneuropathology-2024-5275 keywords: neuropathology, reflections, japan, autobiography preface i am not old enough and would wait till my 80 years of age to accept your invitation to "reflections" – this was my answer to prof. werner paulus, several years ago. meanwhile i lost my wife after her severe disease of several years and i needed time to regain my will and power to do something. i do not think it worthwhile to expose my autobiography, however, someone may be curious about a banal neuropathologist from far east and a permanent resident in germany. i must say that neuropathology (np) was (and is) my hobby throughout my life; sorry to the serious academics/scientists. curiosity about overseas, programmed affinity to germany, and programmed medicine man before i begin to describe my life in childhood, i will open with my family history. an ancestor of my father, in the 13th century, had been a high officer of the 96th kaiser ("tennoh" in japanese) godaigo (1288-1339), from whom he received the family name "hori"; so that his full name became "fujiwara-hori genzaemon-naofusa". later he left the big family hori and went to the country side minoshima in today’s wakayama prefecture, far south of osaka, where he began to work as a chief farmer and simultaneously as a physician. his descendants all succeeded him in this occupation. my grandfather, who died before i was born, was also a suburban medical practitioner and moved to another village, nate, near the kinokawa river. nate later became famous because of the medical doctor "hanaoka seishu" (1760-1835) who successfully practiced breast cancer surgery under general aneasthesia using the plant "mandragora" extracts (stramonium, as known today) for the first time in the world, 40 years prior to the generally known ether narcosis by william t. g. morton (1846) or chloroform anesthesia by james young simpson (1847) in the western countries. the small graves of the hanaoka family and their successors still remain where my elder brothers used to play during their school ages. one of them became a school teacher of physics and another a neurosurgeon, while my younger brother became a gynecologist in later life. my mother’s father was also a general practitioner in a country side, who was the offspring of an ancient samurai family, so far as i know. several years before i was born, my father was sent to germany (1938) by the ministry of education of the japanese government and stayed with prof. karl bonhoeffer in the psychiatric clinic of the charité hospital at berlin and with prof. paul schröder in leipzig. he devoted himself to the research theme "personality development in childhood" and wanted to be a child psychiatrist; however, there was no specialized child psychiatry in japan at that time. during his stay in germany, he visited many child-psychiatric clinics of the universities, not only in germany but also in belgium, switzerland, france, etc. however, he had to interrupt his stay because of the crisis due to the war in germany. nevertheless he brought plenty of souvenirs from his german life, including a leichert light microscope, a 8 (16) mm silent movie camera with many recorded films (his life with colleagues at the campus of charité, driving on the autobahn highway, etc.), a contax camera with many color slides (showing scenes of berlin including hitler’s flags, etc.), a portable telefunken phono-record player with many records (including beethoven’s symphony series, etc.), comic books ("father and son"), several "kinderbooks" and so on. two months after my birth in nagoya, japan, the "pacific ocean war" had begun by the surprise attack of imperial japan on pearl harbor in 1941. during the 2nd world war and afterwards till 1948, i was alone with my maternal grandparents in wakayama, while my elder brothers remained with my mother in nate because of school, although my father came back home from the war in the winter of 1945/46. our (my) normal family life began again in april 1948 when i entered primary school in nagoya at age 6. i admired and enjoyed my father’s souvenirs, namely, silent movies, color slides and books. i also listened to his music records and learned to microscope insects or leaves from our small garden. at that time, i had already a vague image of germany, together with a kind of longing, perhaps. during my school years (11 years of age) i got a pen-friend in brazil (a boy of one of the emigrated japanese families), and later (from 13 years of age) several pen-friends in philippines, thailand, as well as in (west) germany because of my curiosity about overseas. to my german pen-friend i wrote in my first letter "ich lerne deutsch" (i am learning german); she really tried in our correspondence to correct my terribly broken german sentences. fig. 1 concert somewhere in nagoya at the child music festival during my school age i began to learn painting and playing the violin (at 8 years?). every weekend i visited a sketch group for children at the nagoya univ. school of medicine (hobby painting clubs of medical staff and students) but i did not like, or even i hated, to sketch or paint – it was my joy only to play in the parks with friends and hobby-teachers. on the contrary, i liked music and learned intensively to play the violin by the suzuki method (fig. 1). every week i had a lesson by a music teacher, a member of the suzuki violin school. my father supported mr. shin’ichi suzuki personally from the stand point of child psychiatry and i was given a lesson by mr. suzuki once per year at the suzuki summer violin school and even an occasional remote personal lesson by sending/sending-back recoded tapes to mr. suzuki. i gave a "solo" concert at age 13/14 as an absolvent of the primary course: i played mozart’s violin concert no.3 and others by vivaldi, haydn, beethoven, etc., but not by j. s. bach whose compositions i did not like. a few years later in the progressed course of the violin school, i lost my interests to play the violin but liked rather to listen to the music. i told my father that i would give up my violin lessons. he answered me "your teacher gave you the violin (technique) but could not give you music" and allowed me to give up my violin lessons. i liked small children and played with them when our family friends visited us (fig. 2) though without music. nevertheless, i had several occasions to play the violin in my later life: we constructed a flute quartet group with my medical classmates (1962), or i was occasionally a helping member of a small student orchestra (because of lacking musicians for concerts). however, i was not a regular member of the orchestra because i preferred playing football as a regular student member at that time. in my later life in germany, i had a chance to play duet with a colleague whom i made acquaintance at a private home piano concert by prof. orthner in goettingen (1990). fig. 2 playing with our family friends; a girl left only with a half figure became later my wife i began my medical study at the nagoya city university in 1960. i had gotten old german textbooks: rauber-kopsch’s "lehrbuch und atlas der anatomie des menschen (1930)" which had been owned by one of my uncles who never came back home from the war. i liked these textbooks and anatomy was my favorite subject of study; i dreamed to become an anatomist. during my preclinical study i, together with several classmates, was accepted as a student assistant in the department of biochemistry where i practiced tyrosine hydroxylase extraction from potatoes and purified/quantified the activity, which was offered to our teacher for his research. after one year i changed to the department of pathology and remained till the end of my medical study. together with my best classmates, we assisted in experiments of young researchers and learned much, such as dissection techniques and interpretation of autopsy findings, care and treatment of laboratory animals, histological staining applications, microscopic and photographic techniques, and so on. medical students at that time were, after successful six-years-long medical study (under the ministry of education), obliged to practice for one year as an "internship" under ministry of welfare, prior to the physician’s state examination; that is, medical graduates were neither doctors nor students. the fact was that the graduate should work as an unpaid helper for the physicians but there was no professional-educational curriculum/program so that the internship "students(?)" or medical graduates were practically nothing other than a physicians’ and care sisters’ assistant "slave" workers. for several years the medical students and graduates had been protesting and asking for the improvement of the internship-system, namely, establishing the medical-practical training program and stopping the "slave work", in particular in the "national" hospitals under the ministry of welfare. nevertheless, the ministry ignored the graduates’ and students’ demands and this consequently led to boycotting of the internship by the graduates one year before us, refusing the "slave work" in the national, so-called "educational hospitals". they remained in univ.-hospitals and trained practically "by themselves" with support from teachers and experienced physicians of the universities. since the ministry of welfare still did nothing to reform and ignored the students’ protests, as the following generation of the graduates, we had decided to boycott the physician’s state examination, refusing the physician’s license after one year of our "autonomous" internship training. we demonstrated (i was one of the leaders of the small protesting groups) in front of the national hospitals everywhere in japan where the state examination should have been held, and we refused to take the exams – more than 95 % of all the candidates in japan had boycotted the state examination. this meant that almost 6,000 physicians became lacking in japan that year (1967). finally, the ministry of welfare gave up and decided to discontinue the "current" internship system and promised to reform the postgraduate medical training – we had definitively won but we were without physician’s license (!), although we all began our postgraduate and specialized training without license under the supporting teachers or experienced physicians. although i boycotted the state examination, and hence without physician’s license, i was successfully accepted to the postgraduate school of medicine of nagoya city university where i intended to perform scientific research during my professional training in psychiatry and neurology. among several specialized sections in psychiatry which should all be mastered by beginners, such as psychotherapy, psychopathology, psychopharmacology, psychology, neurology, etc., etc., i learned intensively neurophysiological electroencephalography (eeg) and there i found spontaneously some research themes by myself. six months after the boycott of the physician’s state examination, we had another chance to take the state examination, which the majority of boycotted candidates passed and finally became "physicians". at my oral examination, an internist professor from another university was an examiner and asked me several questions showing an eeg-record of a uremic patient. after the examination, i convinced myself i could better interpret the eeg-record than the examiner. my teacher(s) in neuropathology (np) during my specialized training in an eeg lab, i intended nearby to learn np alone as a side job, as a hobby. there was neither a lab for np-histology nor a microscope in our psychiatry department, but only one apparatus remained unused: jung’s macrotome which was used by the former (deceased) chairman of the department. all my colleagues and senior doctors in the department of psychiatry were very collegial and friendly – they supported me for my np trial and shared our limited annual budget for me alone so that i could prepare everything necessary such as reagents, glass slides, microtome knives, even photomicroscope and so on, but definitively no technical assistant. my first patient of hepatic encephalopathy died. my friend nakamura in the pathology department helped me to perform the general autopsy. after the autopsy i cleaned up the autopsy room alone and all the neuropathological technical procedures were done by myself: fixation and cutting the brain, dehydration of slices, embedding them in paraffin, slicing (including large hemispheric slides) with a macrotome/microtome, and staining. better stained were my fingers than the slides, of course. on microscoping i took microphotos (no color pictures but monochrome), developed the film, and printed the histological pictures all by myself alone in a dark chamber, followed by a case report at a local meeting. in the neighbor psychiatry department of (state) nagoya university, my father, the chairman at that time, had a np lab with a leading pathologist dr. s. iwase and several young colleagues. every tuesday evening after my daily job i visited the neighbor university lab and learned np, preferably cns aging processes including dementia and other degenerative diseases, or psychiatric disorders. the majority of the colleagues there, including k. kosaka, h. kobayashi, and h. shibayama, were also beginners and we worked diligently and discussed till late at night. we established a study club, the "nagoya np academy", inviting prof. h. shiraki from tokyo regularly and occasionally prof. r. iizuka from hokkaido, amongst the leading neuropathologists in japan at that time. "nagoya np academy" still exists today, even in a greater form and another club name, as a neuropathologists’ assembly in mid-japan. i successfully finished my postgraduate school of medicine with certified phd work on eeg, published in 1971, and now was a time for me to fly out into the wide world and in order to develop my "np". i asked prof. iizuka, staying at that time with prof. hans jacob (fig. 3) in marburg, west germany, who might be the suitable and best german teacher for me, a beginner of np. he answered me: no one other than prof. h. jacob if i intended to learn clinical np. i hesitated to come to marburg: why would i learn np in germany with the help of a "japanese" teacher? prof. iizuka wrote me: "prof. jacob should be your teacher and i have nothing to do with you." on my request, prof. jacob in the "univ.-nervenklinik (neurology and psychiatry) marburg" accepted me with his words to iizuka: "i am curiously waiting for a young japanese who writes german in 19th (or 18th?) century style." i succeeded to get the scholarship of daad (deutscher akademischer austauschdienst = german academic exchanging service) of 1971. in marburg, i devoted myself in clinical psychiatry and neurology in the morning attending clinical conferences of psychiatry, neuroradiology and other weekly programs. since there was no neurosurgical department in marburg at that time, neurosurgical patients were occasionally be examined primarily there and then sent to neurosurgery in university giessen (100 km distance), if necessary. every day at 4 pm laboratory assistants brought newly stained slides to prof. jacob when he began to microscope exclusively with me alone for 3 hours. if he had gotten brains for macroscopic examination and cutting, several clinicians always attended and discussed. i used to microscope the cases before the laboratory assistants brought them to the boss. it was really a very hard training for me to microscope, one to one, face to face, an expert and a beginner, for three hours every day. however, it was the best time in my life. he not only took me to the several national and international congresses but also he included me every time in communication with his colleagues and so i could make acquaintance with a lot of international leading scientists in eastern and western europe, such as prof. f. seitelberger, and many others. meanwhile i submitted successfully my papers to international journals, with support and guidance of prof. jacob. i should add here to describe my private life with prof. jacob. he used to take a walk through the forest/country yard with his pet dog one hour before he began his laboratory work. on request, he took me for a walk every day and this was a wonderful time for me to learn about the human hans jacob: he explained to me not only psychopathology (e.g. to my question about traditional german and french psychiatry schools) but also philosophy, his loving impressionism paintings, japanese classic and modern literature (he had deep insight), private personal communication, and so on. in the np lab in marburg there was a collection of fetal brains of prof. h. solcher who allowed me to study these slides. it was new and fascinating for me since there was no textbook on fetal neuropathology. i really recognized that standard neuroanatomy in fetal periods was always different from each other, dependent on developmental phases. prof. jacob had deep insight also in developmental neuropathology and stimulated me to study fetal "transitory" neuro-patho-anatomy, for example, transient surface granular cell layer of the brain stem (and later that of the cerebrum). through prof. jacob i could experience a large number of different kinds of np cases but i decided to devote myself in fetal np in my future since i thought i would have no chance to survive as a neuropathologist if i would do np in the same fields as others do, such as brain tumors, cns inflammatory diseases, cerebrovascular disorders, and so on. independent of my decision, prof. jacob told me at farewell after my two years stay in marburg that developmental np and electron microscopic method should be included in my future study. i can summarize what i learned from him: 1) chronological pathology ("verlaufspathologie"), and 2) multimorbidity of (aged) patients. but i dare say, that the human hans jacob was what i learned most. he was/is the teacher of my life. fig. 3 prof. dr. hans jacob, 1970 being a hobby neuropathologist (with many more teachers) if you meet three persons, you will learn something from at least one of them (confucius). returning to japan, not to my home nagoya, but to tokyo by the invitation of prof. iizuka, i got a research fellow position in the neuropathology section of the newly established tokyo institute of psychiatry and a guest lecturer position in the psychiatry dept. (chaired by prof. iizuka) at the juntendo university medical school, tokyo. while i happily enjoyed the peaceful "freedom of research" together with very friendly researchers such as (prof./dr.) m. matsushita, t. ishii, s. oyanagi and others from different departments, as well as prof. k. hirayama in the neurology of the juntendo university and sufficient funds including those from the governmental ministries, i got an enormous shock when the book "developmental neuropathology" by r. l. friede (1976) appeared because i believed at that time, i was one of the pioneers/beginners of this field of np. however, here was a man who already established this special field of np. since several years i, of course, already marked with respect r.l. friede and k. jellinger as the pioneers of this field, especially based on their publications on the concurring subject "cerebellar development" (1973/1974)* and many other papers. cooling my head and reading friede’s book precisely, i believed i should come to him to learn. i wrote him asking if i could stay with him for several months/years in zürich. his negative answer: "i know you from your publications (he cited my papers in his book), but i have no (physical) place for a guest." here i remember what prof. gisela stoltenburg-didinger, berlin, one of the best colleagues of me, once told me: prof. gullotta in bonn answered her when she would come to him to learn that she should bring her own chair since he had no place and desk for her in his lab. *friede r l: dating the development of human cerebellum. acta neuropathol 23: 1973 jellinger k: persistent matrix cell nests in human cerebellar nuclei. neuropaediatrie 5: 1974 during my practice in np at tokyo, i noticed my lacking experiences in congenital metabolic diseases. i knew that prof. j. peiffer in tübingen/germany published several works with neuro-biochemical and histochemical data, so that i applied for the scholarship of the alexander von humboldt foundation for the study of this theme staying with peiffer. the humboldt foundation and prof. peiffer accepted me in 1977 and it was nice timing since the annual german np congress would be held in tübingen under presidency of prof. peiffer with main themes including "cns malformation" which was one of my favorite subjects. at that time prof. peiffer was the rector of the university and busy with administrative responsibilities. above all i should drive very often to the partner psychiatric hospitals for autopsy (since the competent pathologist in the institute fell ill), sometimes twice a day. i had less time to study my objects and we had no fresh cases of metabolic diseases for research. instead of my intended study on congenital metabolic diseases, i found different cases of chromosomal anomalies, being untouched for research, so i tried to compose scientific papers based on these cases. during this unfavorable situation, i attended the german np congress where prof. h. orthner from göttingen, whom i knew by his works on hypothalamus, spoke to me (he already knew me by my publications) and intended to scout me to göttingen. but i expressed my anxiety about his offer, because i, primarily a psychiatrist, was against controversial psychosurgery on which orthner worked neuropathologically, especially on morphological bases of the hypothalamic function of sexual criminals after psychosurgery. he promised me that he did not intend for me to be included in this work and that i could pursue whatever i would like in research. this occasion was just a moment of the decision of my destiny. i decided to live in germany with my family as foreign residents in germany and to follow the german life style: "do in rome as romans do." prof. orthner told someone: "hori had broken all bridges between him and japan." really, i avoided every communication with my friends and colleagues in japan since then for a long time till i became used to living in german style. prof. peiffer invited me later to contribute one chapter in his editing np-textbook (1995). the department of np in göttingen, formerly one of the divisions in the psychiatric clinic of the university, belonged to the neurological center together with the neurology, neurosurgery, and functional neurosurgery departments, and not to the pathology center. i could communicate intensively with neurological and neurosurgical colleagues in every occasion. prof. orthner possessed six medical staff positions, occupied by all experienced neuropathologists including h. h. goebel (later chairman in mainz) and r. meyermann (later chairman in tübingen) as well as a younger japanese colleague kenji ikeda. the six members shared their duty for biopsy (neurosurgical pathology) and autopsy diagnostics in two months a year, that was, one should work hard alone (independently) for a month but in the following 5 months he/she was free from the duty of diagnostics. my proposal that i would take over every fetal and newborn autopsy all year long was friendly accepted by all my colleagues, so that the average numbers of autopsies i performed/diagnosed were 100 adult cases and additional 100 fetuses/newborns per year, in addition to abundant neurosurgical diagnostic cases, so that i could experience quite different kinds of cns malformations including extremely rare cases. it was no problem for me that i drove to the related psychiatric hospitals to perform full autopsies for prof. orthner (officially appointed "prosektor") several times a year, so that i could experience psychiatric or degenerative cns diseases which were generally rare in the university-pathology institutes. at autopsy i used to perform by myself preparations of cranial base, inner-ear, paranasal sinuses, full course of internal carotid arteries, occasionally also oculi, and later pharyngeal roof for the investigation of pituitary development in fetal period (fig. 4) without the help of "muscular and powerful" autopsy assistants ("preparators" or autopsy helpers), while they helped me very much by detailed and plenty of photographic documentation on my request. i had enough time to sit in the library and even to observe the hundreds of "serial thin-sections" for electron microscopy, and to learn techniques of computer-assisted morphometry, flow cytometry, chromosomal analysis with cell culture (in the department of genetics) and other methods during my "duty-free" months. colleagues, especially h.h. goebel, helped me very kindly not only with diagnoses of neurosurgical specimens but also in reviewing my scientific manuscripts. during this time, prof. orthner was going to emeritus status and prof. friede (zürich) was appointed to the new chairman of np göttingen. one year before he came to göttingen, he was invited to the annual japanese neuropathology congress as a guest lecturer by the congress president dr. ishii in tokyo (once my boss in the psychiatric institute of tokyo), who earlier stayed with friede in cleveland. dr. ishii kindly invited me, too, as a personal secretary of friede, designated my boss, so that i could personally speak with him about my research interests and many things about göttingen. on his first day in göttingen, friede said to us all that we were all experienced neuropathologists and should do further what we would like in diagnostics and research. he told me personally, i should continue my favorite, developmental np, since he would not do it as he selected his new research theme "peripheral nerves". he, as a gentleman, never influenced my activity in developmental np. once i had a big problem on fetal brain development and wanted to ask him personally and expected a clear explanation with a definitive solution. his answer, however, was: "i do not know." i was deeply disappointed because i could get no explanation from him; but on the other hand, i was glad, in secret, to have recognized: "what i do not know, he also does not know." i learned from him more so about brain tumor diagnostics than developmental neuropathology. prof. friede was, nevertheless, my later "big" teacher of np. fig. 4 i used to preparate the fetal pituitary gland together with cranial base/clivus and pharyngeal roof. this, before the ct era, led to the discovery of a unique "pharyngosellar pituitary" (acta neuropathol 89, 1995) elder colleagues had left göttingen and younger colleagues came. my monopoly of developmental np was to be given up for the training of younger people, so i decided to move to hannover (1998). after moving to hannover, we built our own small house for my family, including my wife and daughter, in the suburbs of hannover. in the cellar i constructed a room in japanese style by myself without any professional help (fig. 5a, b). this room was/is very comfortable for me and i used to sit alone and meditate whenever i had a stressful day or had some troubles in my job; this room served/serves my mental hygiene. fig. 5a, b self-made japanese style room in germany for my own mental hygiene in the institute for neuropathology of the hannover medical school, i experienced about 700 autopsy cases per year, the majority of which i examined by myself alone, including forensic cases of cns and gift fetus-cases by colleagues of external pathology institutes, and consulting cases, too. however, in the course of time, the number of autopsies reduced and reduced; this tendency, as well known, was generally in every country. thus, i could find time to visit to learn more by experts in each field, including dr. gosztony in berlin (immuno-gold-em-technique), prof. mehraein in munich (immunohistochemical application in degenerative diseases), and others. on the other hand, several guests not only from europe and the united states but also from japan visited me: prof. j. martinez (pittsburgh, us), prof. j. e. h. pittella (brazil), prof. h. ten donkelaar (netherlands), prof. yukio fukuyama, prof. haruo matsuyama, s. fushiki, h. miyata, kazuhiko ikeda, t. yamashima, later t. uchihara (japan), and many others. some oversea colleagues stayed with me at hannover as guest researchers for years or months, such as dr. sergio u. dani from belo horizonte, brazil or prof. irina bogolepova from moscow. with each of them consequently i could publish monographs: "principles of neural aging" (elsevier, 1997) with s. u. dani and g. f. walter, and several contributions as independent chapters in russian monographs (i myself cannot read/write russian medical articles) with i. bogolepova (1996). (later i visited her in the brain research institute moscow of the russian medical akademy; fig. 6). here i would like to mention one of the best seller books "clinical neuroembryology" (springer, 2006, 2014, and 2023) with hans ten donkelaar and martin lammens; this was a crystal of hard work with one of my best colleagues hans, whom i met, invited, visited, and discussed intensively in hannover, in tottori/japan, and in doorwerth/the netherlands. fig. 6 prof. bogolepov, director of the brain research institute in moscow, russian academy of medicine and his sister prof. irina bogolepova in front of the sample case in which the slides of lenin’s brain are stored along with those of some other historical personalities. my bridge to japan was, without my awareness, reconstructed and became so fine as a rainbow. i could often visit japan and see not only old and new friends or colleagues there, but also i met european fellows, sometimes as my co-visitors or as a host in japan (fig. 7, 8): prof. k. jellinger, prof. p. kleihues, m. lammens, g. stoltenburg-didinger, s. weis and many others (kyoto), later manfred oehmichen (at osaka), werner paulus (at tokyo), ralf schober (at takamatsu), markus tolnay (at nagoya/toyohashi), herbert budka (at tokyo), torsten pietsch (at tokyo), alexander stan (at tottori/tokyo), almuth brandis (at toyohashi), ingmar bluemcke (at toyohashi) as well as already mentioned hans ten donkelaar and many others. it was my great honor not only to have met and spoken but also communicated with prof. asao hirano (montefiore) (fig. 9) as well as prof. kinuko suzuki (north carolina, lastly tokyo). i was visited by several high officers from the ministry of welfare and labor of the japanese government (our enemy during my postgraduate time!). fig. 7 from the 2nd left: prof. jellinger, hori, a kyoto beauty, congress president prof. yonezawa, prof. friede, and prof. kleihues (icn 1980 at kyoto) fig. 8 japanese np congress 2007; from left: h. akiyama, m. graeber, k. ikeda, w. paulus, a. hori, k. tsuchiya, and t. arai i was requested to contribute to a np-textbook a chapter on developmental neuropathology: paulus and schröder (eds.): pathologie–neuropathologie, springer, 2012, former peiffer (ed): neuropathologie, springer, 1995. i described fetuses of atomic bomb victims. it was only one line in my chapter but i am proud of this crystal one line which was the extract of my study results after a 7-days and 6,000 double miles trip to hiroshima in 2001. i visited rerf (radiation effects research foundation), formerly abcc (atomic bomb causality commission), founded initially in 1946 by the national academy of science, us and managed by the general headquarters of the us army. this was one of my research projects on intrauterine radiation effects on fetal development, supported by dfg (deutsche forschungsgemeinschaft) and my friend in the ministry of welfare of the japanese government. fig. 9 prof. asao hirano, japanese np congress 2010 two years before my planned retirement at 65 years of age, the independent institute of neuropathology of hannover medical school was integrated under the institute of pathology. on this occasion i accepted the call from japan to one of the national hospitals in western japan, later renamed "tottori medical center" in which a clinical research institute was to be established and opened. i should contribute to its construction. i temporarily left hannover and moved alone to tottori, leaving my family in germany. one and a half years later, the research institute was opened with a young leading staff, so i gave up this "office job without medical work" although my contract was effective for 3 years. i accepted the invitation to the "research institute for longevity medicine" from a private organization with a special hospital for aged and handicapped (mostly post-apoplectic, mentally disabled and/or physically handicapped, and dementia patients), with about 500 beds, and additionally several care homes for the handicapped and aged with additional 500 beds, where i would work as a clinician and simultaneously as a neuropathologist (2005-2016). the institute and hospital were in toyohashi, not far from nagoya, my earlier home town. i was happy and satisfied with my dream-job: clinical struggles and np autopsy (deceased patients treated by myself could be examined pathologically and critically by myself!). to tell the truth, i became ill with a stress ulcer because of highly demanding clinical care for critical patients, mostly at end stages. in the latter five years, i worked the first 3 months in this hospital/institute and the following 3 months again in the institute of pathology, hannover medical school, where, at that time, only one neuropathologist, my colleague dr. almut brandis, worked. pivoting more than 9000 km distance between japan and germany every 3 months was hard but i enjoyed the intercontinental flight and, on one occasion, a surface route round trip by the trans-siberian railway and the connecting ferry between vladivostok and sakai/tottori. i made several stops in siberia visiting several cemeteries in the suburbs for war-captured, not guilty, and fallen japanese "citizen" soldiers of my former generation. fortunately, i could read russian kyril letters and could speak broken russian sufficiently for tourism so i had no difficulty finding the cemeteries in irkutsk, khabarovsk, and other siberian towns. submission of papers to medical journals back to my younger, np-beginner time. i tried to submit papers to different international journals independently. once i got an answer from a psychiatric journal with the reviewer’s arguments: acceptable after revision. the reviewer not only proposed some corrections of scientific descriptions but also explained to me in detail, how the paper should be constructed, how the legends to the figures should appear and so on. for a beginner like me, it was very helpful and i was very thankful. although anonymous, it was easy to identify the reviewer. later i told these facts to my colleague stoltenburg-didinger who explained me that the identified referee was known as an ideal boss (of another institute) who was not only an excellent np teacher but everything could be learned from him, such as skills and techniques of a successful application for research funds or manners of candidate for a new position in an institute, and so on. when i submitted my paper to an american journal, the editor sent me back the manuscript with comments of a referee and proposal of revision. the referee pointed out not only some scientific problems but also proposed very detailed grammatical and stylistic corrections of my sentences, pointing out the unsuitable expressions or errors, which many "japanese" tended to make. in this case i was also thankful for the very kind and friendly review with "encouragement" for a beginner by the referee, whom i could, of course, identify: a famous us-japanese neuropathologist, whom i made personal and very friendly acquaintance and communication many years later. i remember my experiences as a requested referee in my later np life. i should review a case report of a less known type of cns malformation. the discussion of the authors about formal pathogenesis was, however, not compatible with my interpretation. but i could not reject the paper only because of the different interpretation by the authors and the referee. i criticized the paper with the contradictive argument of the authors and explained another aspect (my opinion), suggesting that the authors should consider another aspect in their discussion. my decision was to accept this work for publication after revision. today i still believe my interpretation was right and that of the authors was incorrect. this reference was based on a bitter experience: an early submission of mine was simply rejected by one of the referees without rational reasons, only saying "this is a well-known fact and nothing new" which was absolutely incorrect. i did not respond and i myself rejected to (re)-submit my papers, for a long time, to this qualified journal with one disqualified referee. once i tried to examine the intoxicated neuronal migration disturbance experimentally in my younger np life. after i killed the first two of several animals alone in my lab, i asked myself if it was ok to do so, even if i could perhaps expect some fine results in my experimental research i decided i would never again kill lovely animals. i enjoyed my np job, as well as related publications, as one of my hobbies and always with joy and humor. for example, i eagerly looked for a plan for publishing in new journals and i tried to submit my works several times in "volume 1" of the journal and, if possible, its "issue 1". one of my best colleagues, prof. a. matsumura, a neurosurgeon and now in emeritus, found it amusing and helped me later. trials in success were: brain dev vol. 1 no. 2 (1979); clin neuropathol vol. 1, no. 1 (1982); child’s nerv syst vol. 1, no. 6 (1985); j med imag vol. 1, no. 1 (1987). today there are so many new "online journals", some of which seem to be, unfortunately, dangerous pirate journals. with humor and joy in np, i am proud of my own photographic documentation, some examples of which include: one, three, and four mamillary bodies (?) (fig. 10); anatomic variability of anterior commissure fiber course (fig. 11). fig. 10 from my hobby collections: different features of mamillary bodies in human brain. a: unimamillaria? fusion of mamillary bodies (the first report by dr. pittella in 1985). b: trimamillaria or quintimamillaria? no! numbers indicate three pieces of ganglionic hamartoma. ordinary mamillary bodies are seen in the shadow, posterior to hamartomas. c: again trimamillaria? no! 3 is "eminentia grisea" (tuber cinereum) of the hypothalamus, a normal anatomical structure. the pituitary stalk is seen near chiasma opticum. d: quadrimamillaria? morphometric studies showed insignificant difference in neuron cell count both in the lateral and medial subnuclei of the corpora mamillaria between this case and control cases; the size of neurons in this case was larger than that of control cases, but statistically insignificant, so that this case remained unreported. e: again quadrimamillaria? 1 and 2 are regular mamillary bodies, 3 and 4 are ganglionic hamartomas. (acta neuropathol 56, 1982). f: diastematomamillaria? mamillary bodies separated from each other and not side-by-side (1, 2) in a case of median cleft face syndrome with double pituitary. (acta neuropathol 59, 1983) arrows in upper and lower pictures indicate double pituitary stalk and double pituitary glands, respectively. fig. 11 another example from my hobby collection: anatomic variants of fiber crossing of the anterior commissure in relation to the fornix. of 100 examined adult brains, 66 showed type i, 20 type ii, 13 type iiia+b, and the last one showed a unique loop formation. according to a personal mri, examined in 2013, my own anterior commissure showed a type i course. prof. c. hartmann, chief of the division of neuropathology at hannover medical school, allows me to visit his lab regularly. he kindly accepts my hobby np, which remains so in my life further in germany, while still keeping my japanese nationality. my old colleague prof. uros roessman from cleveland, emigrated from former yugoslavia to austria and finally to the us, once answered my question, what home is: "home is where you are now and feel at home." copyright: © 2024 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. viral infection and dementia: a brief synthesis feel free to add comments by clicking these icons on the sidebar free neuropathology 2:15 (2021) opinion piece viral infection and dementia: a brief synthesis clayton a. wiley division of neuropathology, department of pathology, university of pittsburgh, pittsburgh, pa, usa corresponding author: clayton a. wiley · division of neuropathology · department of pathology · university of pittsburgh · 200 lothrop street · scaife hall s-701 · pittsburgh, pa 15213 · usa wileyca@upmc.edu submitted: 17 may 2021 accepted: 4 june 2021 copyedited by: shino magaki published: 8 june 2021 https://doi.org/10.17879/freeneuropathology-2021-3347 keywords: dementia, emergent infections, immunosenescence, neurodegeneration, syphilis, viral encephalitis abstract for the past 400 years, the most common cause of dementia was tertiary syphilis [1]. its prevalence declined dramatically with the advent of potent antibiotics in the 20th century, but these same antibiotics also helped increase our average lifespan, leading to dramatic increases in the prevalence of age-related dementias. abundant progress has been made connecting early onset dementias with mutations in neural genes. late onset dementias have been linked to a more enigmatic set of genes, some of which have been connected to neuroinflammation, begging the question: are age-related dementias linked to infection? numerous studies have reported an association between dementia and infections in general and viral infections in particular. while these associations have been subject to extensive reviews, the purpose of this synthesis is to examine the hypothesized link of viral infections and dementia from the opposite perspective: what do we know about acute and chronic encephalitides that could forge a link with dementias? there appears to be little support for the concept that viral infections are a major contributor to today’s common dementias. however, the emergence of new central nervous system (cns) viral infections, coupled with senescent immune and nervous systems in our aged population, create new opportunities for infections to contribute to dementia. introduction there may be as many clinical definitions for dementia as there are pathological definitions for neurodegeneration. a simple clinical definition of dementia might be “a chronic irreversible encephalopathy affecting multiple cognitive domains” (memory being the most prominently involved domain in the most common form of dementia, alzheimer’s disease [ad]). clinical dementias can be measured with a variety of clinical scales but are most commonly appreciated from the perspective of impaired capacity to perform activities of daily living. a simple pathological definition of neurodegeneration could encompass loss of neurons, their axonal connections or synapses, but given difficulties in quantifying these degenerative changes, current pathological diagnoses rely on distinctive histopathology (figure 1) [2, 3]. with recent refinements in our understanding of dementia and neurodegeneration, is there support for an infectious etiology in age-related dementias? figure 1. bielschowsky-stained section of neocortex from a patient with alzheimer’s disease. individual pyramidal neurons show intense staining of neurofibrillary tangles in cell bodies and proximal processes. neuropil plaques show staining of dystrophic neuritic processes. there are no infiltrating adaptive immune cells. syphilis: the most common form of dementia it has been hypothesized that syphilis was brought to europe with the return of various european conquistadors [1]. in the old-world, syphilis spread mercilessly infecting up to 20% of the population, 5% of whom eventually developed dementia [4]. so prevalent was tertiary syphilis that sir william osler held it as the paradigmatic neurologic disease saying, “know paretic neurosyphilis in all aspects and you know all of psychiatry.” [5] by the beginning of the 20th century tertiary syphilis was the most common form of dementia [4]. fortunately, the advent of antibiotics in the 20th century coupled with the exquisite sensitivity of treponema pallidum, led to rapid decline in prevalence and incidence of infection and commensurate decrease in syphilitic dementia. which is not to say syphilis is gone. today it infects 0.5% of the world’s population with 6 million new infections per year (~100,000 in north america). the agent treponema pallidum (figure 2) causes a protean complex of diseases of which its tertiary form, mediates dementia. without antibiotic therapy, syphilis can establish a lifelong infection that evades and exhausts the immune system. tertiary syphilis is the result of years of organism replication to the point that unfettered growth throughout the brain along with macrophage activation causes a global chronic encephalitis with neurodegeneration and dementia [5]. it is an unusual form of encephalitis, distinct from the acute viral infections described below, with little evidence of cell mediated immunity and instead a remarkable abundance of activated microglia in gray matter regions of abundant t. pallidum. as with most infections, perivascular infiltrates are prominent and with the spirochetal tropism for vascular walls can progress to an end arteritis infarctive syndrome. histopathologic changes are widespread with minimal evidence of a system specific distribution except for a frontal/temporal cortical predominance. there is extensive neuronal loss and reactive astrocytosis but no intraneuronal protein aggregates. figure 2a. warthin starry stain of rabbit testes infected with treponema pallidum. short thin corkscrew like organisms (black) are seen adjacent to a small blood vessel. figure 2b. immunostain (red) for t. pallidum shows a dense collection of microorganisms. age-related dementia perversely, the cure of the most common cause of infectious dementia was followed years later by an increasing prevalence of age-related dementias that plague us today. the same antibiotic development that fueled elimination of syphilis also protected people from common pathogens and thus (along with improvements in sanitation) indirectly increased our average life span. prior to the introduction of agriculture, the average life span of homo sapiens has been estimated to be less than 35 years [6]. with agriculture and the industrial revolution, by 1900 the average lifespan increased in the us to 47 years. add in modern sanitation and medicine and by 1950 the average lifespan was 66 for males and 71 for females. a mere 70 years later the average bumped another decade to 76 and 81 for males and females, respectively. unfortunately, one thing we know about dementia today with absolute certainty is, it is age dependent. the alzheimer’s association estimates that in the us, one in 10 people over the age of 65 has ad (one of the most common forms of dementia) (https://www.alz.org/alzheimers-dementia/facts-figures, accessed 12.15.20). despite many of those dying 3-5 years after diagnosis, by 85 years of age 30% of the population is demented. said another way, syphilitic dementia disappeared and was more than replaced by age-related dementia. is there evidence that this age-related dementia is related to an infectious agent? infection-related dementia there have been scattered but intense investigations to discover an infectious etiology for today’s common dementias. these studies have spanned simple attempts to morphologically identify an infectious agent within the brains of affected individuals, to sophisticated and sensitive metagenomics sequencing studies. numerous reports, some of which were replicated, have identified a gamut of bacterial or viral agents in the brains of deceased previously demented individuals [7]. however, given the prevalence of dementia and the abundance of opportunities to find an associated infectious agent, it is difficult to imagine that a prevalent direct etiologic agent has been missed. that conclusion should be stated with some diffidence given the famous example of helicobacter pylori linked to gastric and duodenal ulcers [8], but it is a fair summary to say, no infectious agent has been consistently associated with common forms of age-related dementia known today. but is presence of the infectious agent within the brain tissue the sine qua non linking an agent to dementia? it is theoretically possible that an infectious agent could initiate the neurodegenerative process or mediate it remotely without directly invading the brain. for example, direct infection of neurons perturbs cellular metabolism that could potentially disrupt normal protein synthesis or proteasome/autophagy degradation leading to accumulation of aggregated proteins and neuronal dysfunction. indirect effects of systemic infection on neurophysiology could include decreased perfusion secondary to general cytokine induced blood coagulation or altered neuronal gene expression induced by specific cytokines like interferons. this brief review will be limited to assessing potential direct viral etiologies of human dementias from the perspective of what we know about acute and chronic viral encephalitis that could link them to dementias. the reader is referred to a selection of reviews assessing potential of bacterial etiologies [7]. prions (proteinaceous infectious particles) which are infectious agents known to cause dementia will also not be discussed because they are exquisitely rare, part of the host genome rather than a viral agent and have been the subject of many comprehensive reviews [9]. why do we care about viruses? our biosphere has evolved from competition amongst replicating agents. uniand multicellular hosts have evolved numerous defenses to ward off invasion by infectious agents. this dynamic state persists with continuous evolution in host defense and agent offense. viruses are one class of infectious agents. perhaps most succinctly defined by peter medawar as a piece of bad news wrapped in a protein [10], viruses have evolved to co-opt the host cell energy and machinery to replicate the viral genome. the strategies for accomplishing this are legion and in rare cases involve infecting the host brain. how do viruses infect the brain? viral infection can occur through a variety of pathways [11]. to move from infected to naïve host, viruses must traverse the environment and access the surface of living cells which, in the case of droplet and aerosol transmission, implies contact with a mucous membrane, but in the case of arthropod born viruses, is the result of direct subcutaneous injection. at the cell surface, specific host membrane ligands bind viral proteins vastly facilitating viral entry. intracellular replication increases the number of infectious particles (virion) that can disseminate to sites for a secondary amplification (frequently in lymphoid structures) before greater dissemination throughout the host (e.g., the brain). viral entry into a host cell immediately triggers the host cell’s innate immunity, launching a race between the virus and host immune system that ends with either cessation of viral replication and clearance of the virus, or a variety of debilitating outcomes for the host. upon cell entry cytoplasmic and membrane bound host proteins of the innate immune system immediately detect viral proteins and nucleic acids initiating a cascade of antiviral machinery (e.g., interferon) to block viral replication in the infected and adjacent cells. unfortunately, evolution has selected for an array of viral mechanisms to evade the host innate immune response. the partial thwarting of explosive viral replication gives the host’s second line of defense, adaptive immunity, a chance to mount a complex humoral and cellular response. adaptive immunity takes precious days to develop, so if the virus gains entry to the cns during that window, it may gain a temporal advantage over the growing adaptive immune response resulting in a costly destructive battle inside the cns. the cns is protected from viral invasion by an elaborate system of physical, chemical and physiological barriers [12]. encased within the cranial vault, direct introduction of a viral agent is essentially impossible, so viruses take two other routes to enter the brain: hematogenous or transaxonal. both of these other routes require initially establishing an infection in the periphery. hematogenous dissemination requires that the virus reach a concentration (titer) in the blood that can broach the blood brain barrier (bbb) by passage through, or infection of, endothelial cells. an intriguing variation on this route is viral entry into white blood cells and transit through the bbb hidden within a monocyte trojan horse. a second route, transaxonal, has been perfected by many viruses and requires infection of peripheral nerves that naturally extend cellular processes into the brain, such that the virus can utilize intracellular transport mechanisms to rapidly enter the cns sanctuary. viral encephalitis viruses that successfully circumvent peripheral innate immunity and access the cns through pathways described above, have the opportunity to encounter cns cells [11]. if viral surface proteins bind to host neuroglial membrane receptors, the virus is taken into the cell where it can be detected by innate immune machinery similar to that in the periphery. neuroglial elements may not be able to unleash the same potent anti-viral innate immunity as peripheral cells. within a closed chamber the brain can tolerate only limited cytotoxic edema. additionally, given exquisite balance of rna processing within the cns, it may not tolerate potent interferon related arrest of rna metabolism. like other organs, the brain has histiocytes (microglia) that are specialized in detecting pathogens and attracting peripheral immune surveillance [13]. as adaptive immunity is brought to bear on infected cns cells, cellular destruction leads to neurological dysfunction which is accentuated by cytotoxic edema. simple brain edema associated with immune attack is enough to cause neurological dysfunction and through swelling, brain herniation and death. add to that the viral and cytotoxic effector arms of the immune system and it is not surprising that encephalopathy is the clinical outcome. viral-host duals display a spectrum of histopathologies. rapidly growing viruses with the capacity to lyse cells combined with robust immune responses can lead to frank necrosis, while less dramatic infectious battles result in non-necrotic inflammatory reactions [14]. most viral encephalitides are acute infections ending with either host death or viral clearance achieved in a matter of days. chronic viral encephalitis is a more nuanced outcome, characterized by persistent adaptive immune response in the context of continued viral replication. do acute viral encephalitides lead to dementia? viruses mediating acute encephalitis cover a wide range of families but are mostly rna viruses. because of their temporal time course and systemic symptoms, acute viral encephalitides are not likely to be confused clinically with a progressive dementia. as the host immune response brings viral replication under control, cerebral edema can mediate global neural dysfunction, clinically manifest as delirium. with clearance of virus and abatement of the immune response, a brain lesion resulting from the inflammatory process can manifest as a static neurologic deficit, but the clinical picture is not one of a progressive neurodegenerative disease. firstly, the time course of acute encephalitis is in days not months to years. secondly, distribution of infection within the cns, with rare exception, is global rather than system specific as seen with most dementias. thirdly, with the exception of immune compromised hosts, acute encephalitis pathology demonstrates a severe inflammatory response rather than subtle neurodegenerative changes with pathognomonic aggregated proteins (figure 3). figure 3a. immunostain for cd3 of neocortical tissue from the brain of a child who died of coxsackie viral encephalomyocarditis shows dense infiltration of lymphocytes (red). figure 3b. immunostain for coxsackie viral antigens shows staining of multiple neuroglial cells in both the nucleus and cytoplasmic processes. do chronic viral encephalitides lead to dementia? the spectrum of viruses mediating chronic encephalitis is more limited and has proportionally more representation from dna viruses. some chronic encephalitides (e.g., herpes simplex virus [hsv]) are the end product of millennia of co-evolution between host and virus. while hsv can cause an acute encephalitis in the immunologically compromised, in the immune intact host hsv has achieved a unique latent infection in the peripheral nervous system (pns) where viral synthesis is shut down and re-awakened in times of stress permitting active disease and transmission. regardless of organ, chronic infection results from a dynamic balance where the virus does not kill the host and the host does not develop an adequate immune response that can eradicate infection. in addition to outflanking the innate immune response, many viruses have developed the means to mediate some level of general or specific immunosuppression. if this down regulation occurs prior to viral eradication, it could result in viral persistence with or without some low level of immune reaction. alternatively, rather than causing immunosuppression themselves, viruses can take advantage of genetic or iatrogenic compromises of the host immune system. finally like all other organs (including the brain), the immune system undergoes a natural senescence [15]. with age, stem cell elements dissipate and immunological memory fades. loss of immunological surveillance can result in selective holes in adaptive immunity permitting chronic viral infections (e.g., progressive multifocal leukoencephalopathy, figure 4). figure 4. in situ hybridization (red) for jc viral nucleic acids in the brain of a patient succumbing to progressive multifocal leukoencephalopathy. individual oligodendroglial nuclei are filled with viral nucleic acid while surrounding parenchyma shows no t-cell infiltration. potential chronic viral infections associated with dementia an example of a rare chronic viral encephalitis seen in children and young adults can be caused by two different viruses: rubeola and rubella. childhood infection with these agents is usually followed by a self-limiting systemic disease marked by a distinctive skin rash. in rare children, for unknown reasons, but hypothesized to be related to an aberrant immune response, chronic viral infection persists in the cns [16]. unlike acute viral encephalitis, the immune system only dampens exponential viral growth, without destroying infected host cells to eradicate the virus. for years viral replication and a low-grade immune response compromise neuronal function and lead to atrophy and a global encephalopathy. pathologically these diseases show muted inflammatory changes. hsv: of perhaps all chronic viral infections potentially associated with neurodegeneration, herpesviruses may be the most studied. herpesviridae are ancient viruses that have co-evolved with many mammalian species. of the 8 known human herpesviruses, hsv is the most notorious for mediating neurological disease. in the fetus, newborn and immunocompromised, hsv mediates an acute and frequently necrotic pan-encephalitis. for unknown reasons, rare apparently immunologically intact individuals also develop an acute necrotizing encephalitis predominantly focused in the frontal and temporal lobes. while readily treatable, there are case reports where this acute infection transitions into a smoldering chronic infection, raising the prospects that it could be associated with a chronic neurodegenerative disease. the literature on this topic is immense and controversial with proponents on both sides of the debate for and against hsv involvement in neurodegeneration (reviews [17, 18]). while there is no consensus, the cumulative evidence would suggest that while low levels of hsv are detectable in cns and pns (latent infection), there is minimal to no evidence that active hsv replication is temporally or spatially associated with common neurodegenerative diseases. whether hsv infection could initiate a cascade of inflammatory events that subsequently drives a degenerative process awaits construction of a pathogenically logical and testable hypothesis. sars-cov-2: in 2021 it is almost impossible to write anything without mentioning covid-19. much heat and little light has been published on the effect of sars-cov-2 infection on the nervous system. as with many severe infections, a variety of neurological signs and symptoms have been observed in covid patients [19-21]. observations of olfactory neuroepithelial infection [22] have led to the conjecture that sars-cov-2 enters the cns by ascending axonal connections to the olfactory bulb. such a dissemination has been demonstrated in transgenic murine models expressing human angiotensin converting enzyme 2 (ace2; the human viral receptor) under a keratin promoter [23]. but this convenient animal system does not seem to actually model the human disease [24]. intensive attempts have been made to identify sars-cov-2 infection in human autopsy tissue to little avail. the preponderance of evidence would suggest a non-direct infection effect on the nervous system such as overwhelming systemic immune activation (cytokine storm) leading to dissolution of the bbb and compromised cns perfusion. in addition to these effects of acute infection, more chronic cns symptoms have been documented in a subset of patients recovering from covid (long-haulers). while early in our studies of covid pathology, clearly this disease is open to the same theories that have developed with hsv associated neurodegeneration. the challenge will be to convert these theories into testable hypotheses. is hiv the infectious dementia of our generation? before aids was even proven to be caused by a viral infection, it was clearly associated with severe neurologic disease. beyond the devastating opportunistic infections related to severe immunosuppression, late-stage aids patients also exhibited a unique dementia. in retrospect, the histopathology of aids dementia was exactly what should have been expected for a macrophage tropic virus in a patient with severe immunosuppression, minimal to no lymphocyte infiltration in the context of abundant virus [25] (figure 5). indeed, the concentration of virus in the cns exceeded that in lymphoid organs [26]. the big mystery that has yet to be solved is, how did infection of microglia in the absence of neuroglial infection lead to a clinical dementia [27]? it has been hypothesized that infected microglia either produced a neurotoxin (e.g., quinolinic acid [28]) or were unable to carry out critical physiological functions that non-infected microglia normally perform (e.g., synaptic stripping). hypotheses range from remote or local responses to immunologic stimulation to hypothesized abortive infection of astrocytes. before intensive investigation could elucidate the pathogenesis of aids dementia, highly effective anti-retroviral therapy eradicated hiv encephalitis. while hiv infected individuals still experience neurologic symptoms of unknown etiology, aids dementia, like syphilis dementia, disappeared. figure 5a. low power h&e-stained section of cortical tissue from a patient with hiv encephalitis. figure 5b. higher power h&e-stained section showing a multinucleated giant cell. what would make a dementia virus? with what we have learned from infections of the nervous system, what would be the characteristics of a host-viral interaction that would result in a neurodegenerative disorder of dementia? the abundance of rna viruses that infect the brain would suggest that something about their biology makes them particularly effective at replication in the cns. to effectively infect cns cells, the hypothetical dementia virus would need a viral coat protein capable of binding host cns cell membranes. through its intricate specialization and regionalization there are an abundance of potential cns region and system specific target proteins (e.g., neurotransmitter receptors). infection of cns cells would not necessarily lead to lysis of neuroglial elements but would incapacitate normal cell physiology leading to dysfunction or degeneration. competition between normal host cns rna metabolism, viral rna metabolism and innate immune responses could mimic many of the neurodegenerative mechanisms being investigated in neurodegenerative disorders like amyotrophic lateral sclerosis [29]. alternatively, as seen with syphilis and hiv, infection of microglia might indirectly mediate neuronal dysfunction [27, 30]. how would the hypothetical virus reach the cns and propagate between hosts? while many pathways could be used, aerosol transmission is the most effective means of spreading between high density host populations (figure 6). additionally, for entirely mysterious reasons, aerosol dissemination is highly effective at transferring virus to the brain. viruses that normally are limited to peripheral infections, when aerosolized, lead to severe pan-cns infection (e.g., rift valley fever virus, figure 7). figure 6a. in situ hybridization autoradiography (black grains) for influenza a in the brain of a ferret 8 days after aerosol exposure to h5n1. numerous foci throughout the brain demonstrate abundant flu infected cells. figure 6b. higher power image showing regions of intense neuroglial infection. but the most important feature of the hypothetical dementia virus infection is that it would occur in the context of immunosuppression. many viruses elicit general or specific immunosuppression, with hiv notable for a particularly severe cd4 t-cell suppression. however, it is also possible that rather than causing immunosuppression on its own, the hypothetical virus could take advantage of senescence of the human immune system accompanying the aging of our population. with greater than 20% of our population over the age of 65 by 2050, there is no shortage of potential hosts. the covid-19 pandemic has taught us the cost of not being prepared to combat emergent infections. the highly effective mrna vaccines are the result of decades of intensive investigation. we need to broaden and deepen our understanding of how vaccination can be employed to confer protection of the brain particularly from aerosol infection in the immunosenescent host. figure 7. when rift valley fever virus (rvfv) is transmitted by mosquito bite, infection does not cause an encephalitis. however, when delivered as an aerosol, rvfv causes a global encephalitis. in situ hybridization autoradiography (black grains) for rvfv in the brain of a mouse 7 days after aerosol exposure to rvfv. essentially all of the neurons are infected. conclusion historically infectious agents have been the primary cause of dementia. introduction of antibiotics eradicated syphilitic dementia while at the same time helping humans live longer allowing them to develop even more prevalent age-related dementias. the recent emergence of hiv has once again proved the potential for an infectious agent to mediate a dementing illness. fortunately, we discovered pharmaceuticals to arrest hiv infection and block development of immunosuppression and aids dementia. in the context of an increasingly aged population, living with senescent immune and nervous systems offers up new targets for emergent viral infections to wreak havoc on our cognitive capacities. however, for the most common age-related neurodegenerative diseases (e.g., ad, lewy body disease, frontotemporal dementia) there is little in the histopathology defining these disorders to connect them to a viral etiology. references [1] tampa m, sarbu i, matei c, benea v, georgescu sr. brief history of syphilis. j med life. 2014;7:4-10. 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[30] mcmanus rm, heneka mt. role of neuroinflammation in neurodegeneration: new insights. alzheimers res ther. 2017;9:14. copyright: © 2021 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. cellular activation patterns of cd10+ fibro-adipogenic progenitors across acquired disease states in human skeletal muscle biopsies feel free to add comments by clicking these icons on the sidebar free neuropathology 5:3 (2024) original paper cellular activation patterns of cd10+ fibro-adipogenic progenitors across acquired disease states in human skeletal muscle biopsies peter w. schutz1, simon cheung2, lin yi3, fabio m. v. rossi3 department of pathology, university of british columbia, vancouver, canada department of pathology, vancouver general hospital, vancouver, canada school of biomedical engineering, university of british columbia, vancouver, canada corresponding author: peter w. schutz · vancouver general hospital · department of pathology · 910 west 10th avenue · vancouver, bc, v5z 1m9 · canada peter.schutz@vch.ca submitted: 01 november 2023 accepted: 27 january 2023 copyedited by: deborah mcintyre-hofmann published: 09 february 2024 https://doi.org/10.17879/freeneuropathology-2024-5162 keywords: fibro-adipogenic progenitor, inflammatory myopathy, denervation, cd10, periarteriolar, type 2 atrophy abstract background: fibro-adipogenic progenitors (fap) are muscle resident mesenchymal stem cells pivotal for regulation of myofiber repair. experimental results show in addition involvement in a range of other pathological conditions and potential for pharmacological intervention. fap histopathology in human muscle biopsies is largely unknown, but has potential to inform translational research. methods: cd10+ faps in 32 archival muscle biopsies from 8 groups (normal, dermatomyositis, inclusion body myositis (ibm), anti-synthetase syndrome, immune-mediated necrotizing myopathy (imnm), denervation, type 2 atrophy, rhabdomyolysis) were visualized by cd10 immunohistochemistry and their histology compared. groups are compared by semi-quantitative scoring. results: histological activation of endomysial cd10+ faps includes prominent expansion of a network of cell processes surrounding muscle fibers, as well as endomysial cell clusters evidencing proliferation. prominence of periarteriolar processes is a notable feature in some pathologies. fap activation is often associated with fiber degeneration/regeneration, foci of inflammation, and denervation in keeping with experimental results. type 2 atrophy shows no evidence of fap activation. dermatomyositis and anti-synthetase syndrome associated myositis demonstrate diffuse activation. conclusion: assessment of cd10+ fap activation is routinely possible using cd10 immunohistochemistry and demonstrates several patterns in keeping with preclinical results. prominent expansion of fap processes surrounding myofibers suggests enhanced interaction between myofiber/basement membranes and faps during activation. the presence of diffuse fap activation in dermatomyositis biopsies unrelated to fiber repair raises the possibility of fap activation as part of the autoimmune process. future diagnostic applications, clinical significance and therapeutic potential remain to be elucidated. introduction fibro-adipogenic progenitor cells (faps) are muscle resident mesenchymal stem cells with potential to differentiate along a variety of lineages (fibrogenic, adipogenic, osteogenic) as well as several defined functions in muscle regeneration and tissue homeostasis. elegant mouse studies have conclusively outlined their central role in determining muscle fiber repair or fibrosis after acute injury via cellular interactions with myogenic muscle stem cells (satellite cells) and macrophages [18]. subsequent studies have demonstrated their involvement in mediating neurogenic atrophy, highlighting involvement in two very different types of tissue response [9, 20]. more specific pathomechanistic roles have been demonstrated experimentally in the context of muscular dystrophies (dystrophinopathies; dysferlinopathy, facioscapulohumeral muscular dystrophy, fibrodysplasia ossificans progressiva, posttraumatic fibroadipogenic muscle degeneration (rotator cuff tears), and fibro-fatty muscle degeneration in the context of systemic disease (chronic renal insufficiency, type 2 diabetes), and senescent cell states in inflammatory myopathies [5, 6, 14, 16-19, 21, 22, 25]. quiescent faps are present in endomysial, perimysial, epimysial and perivascular locations [3]. within the endomysium, faps are situated in the interstitial space outside basement membranes. endomysial faps form an interconnected network of processes on the surface of muscle fibers and extending along capillaries. cd10 is a robust and widely available marker for faps in human muscle [12]. in response to activating signals, faps can initiate intercellular communications, clonal expansion, differentiation, or subsequent apoptosis [3, 30]. morphological changes in response to activation in human skeletal muscle are not well delineated. while the involvement of faps in various pathogenic contexts is coming to light, single-cell rna sequencing has uncovered transcriptomic heterogeneity of molecular fap subpopulations in animal models and in humans [7, 13, 15, 21]. recent evidence indicates, for instance, that a cd90+ fap subpopulation increases selectively in skeletal muscle of patients with type 2 diabetes [6]. in particular, cd10 has been recently reported to identify a subset of these cells with high adipogenic potential [7]. these data suggest that quiescent faps can evolve into a variety of transcriptomic subtypes in the context of a given pathological condition, whose exact biological significance and potential for therapeutic intervention often remain to be elucidated. experimental proof of concept studies have shown that targeting faps pharmacologically has therapeutic potential. direct interruption of specific fap signaling pathways with tyrosine kinase inhibitors can reduce fibrosis in a dystrophinopathy mouse model [18], indirect modulation of the muscular cytokine environment can result in fap mediated augmentation of muscle regeneration [4], and the beneficial effects of exercise on senescent muscle are partly mediated by faps [25]. in view of these results, understanding pathogenetic contributions of faps in a wider range of human myopathies is of translational interest. little is known about the histological appearance of activated faps in human skeletal muscle biopsies across various myopathies. the majority of fap studies have been performed in well controlled mouse models, mostly with changes in fap cell numbers as a measure of activation [17, 18, 20, 21, 25]. better understanding of histopathological reaction patterns of faps on muscle biopsy may help to screen a wide range of conditions for potentially pathological fap activation. the aim of this study was to delineate histological activation patterns of faps in human skeletal muscle biopsies to allow for immunohistochemical screening and evaluation of fap activation in various conditions. we compared histopathological changes of cd10+ faps in six frequent disease conditions seen in clinical muscle biopsies. methods the study was approved by the clinical research ethics board of the university of british columbia. chart review and biopsy selection muscle biopsies taken between 2010 and 2018 were selected from the archive of the muscle pathology laboratory at vancouver general hospital for nine groups: normal (n), anti-synthetase syndrome associated myositis (asa-m), dermatomyositis (dm), inclusion body myositis (ibm), immune mediated necrotizing myopathy (imnm), type 2 fiber atrophy (t2a), neurogenic atrophy (na), and rhabdomyolysis (rm). pathological categories were selected to represent histopathologically well defined acquired myopathic conditions. electronically available medical records were reviewed. normal biopsies were chosen based on the absence of abnormalities on routine clinical biopsy evaluation and chart review negative for muscular conditions. histology and chromogen immunohistochemistry paraffin sections (4 µm) were used for evaluation in order to take advantage of established cd10 immunohistochemical protocols. he stained sections were prepared in the usual manner. cd10 was used as marker for faps based on poor performance of pdgfr-α immunohistochemistry in human muscle samples and previous work by hejbol et al lending support to the use of cd10 as fap marker in human muscle tissue [12]. all immunohistochemical stains were performed on a dako omnis instrument with heat epitope retrieval for 30 min at 97c and ph 9.0. the cd10 antibody used was dako ready to use monoclonal, clone 56c6 (agilent, santa clara, usa), with dab chromogen. fluorescence immunohistochemistry normal human muscle frozen section (8 µm) was fixed in cold acetone/ethanol (1:1) for 10 min, followed by washing in pbs for 30 min. after blocking in 20% normal goat serum for 30 min, sections were concomitantly incubated overnight at 4°c with cd10 antibody, clone 56c6 (bio-rad, mca1806t, isotype mouse igg1, dilution 1:40), and laminin-2-alpha antibody, clone 4h8-2 (abcam, ab11576, isotype rat igg1, dilution: 1:200). after washing, section was stained for 2 hrs. at room temperature with secondary antibodies goat anti-mouse igg1 alexa fluor 647 (thermo fisher, 21240, dilution 1:1000) and goat anti-rat igg alexa fluor 488 (thermo fisher, a-11006, dilution 1:1000). hoechst 33342 was used for nuclear staining. histopathological review stained sections were reviewed by an experienced myopathologist (pws). qualitative changes were captured descriptively on initial review. the type of qualitative changes found was suggestive of a semi-quantitative scale to compare changes between biopsies (table 2. figure 4.). this semi-quantitative scale could be consistently applied to all biopsies. the scoring system is entirely descriptive at this stage and any potential pathological significance could be addressed in subsequent studies. results patient characteristics we reviewed muscle biopsies from 32 patients summarized in table 1. the age range was 39 – 82 years, biopsied muscles included quadriceps, biceps, gastrocnemius, and, in a single case, deltoid. gastrocnemius biopsies are over-represented in the neurogenic atrophy group since these biopsies were often taken concomitantly with sural nerve biopsies. there is a slight male predominance in the ibm group in keeping with disease epidemiology. the selection of normal controls was based on normal myopathological examination and absence of neuromuscular disease upon retrospective chart review. clinical indications for biopsies with normal muscle histology are listed. serological information for patients with inflammatory myopathies was available in 6/9 cases and is presented in table 1. diagnosis of inflammatory myopathies in cases without serological information is based on clinico-pathological correlation. case diagnosis age /years sex muscle clinical msa 1 n 58 f q chronic fatigue   2 n 56 m q sensory-motor polyneuropathy   3 n 71 f b sle, sclerosing cholangitis, and liver tx   4 n 51 f q chronic fatigue   5 n 67 f b suspect mitochondrial disease   6 asa-m 42 f q anti-synthetase syndrome jo1 7 asa-m 52 m b polymyositis jo1 8 asa-m 52 m q polymyositis with ild jo1 9 dm 81 f b dermatomyositis n/a 10 dm 61 f b dermatomyositis mi2 11 dm 50 m b dermatomyositis n/a 12 ibm 67 m b ibm   13 ibm 82 m b ibm   14 ibm 73 m b ibm   15 ibm 74 m b ibm   16 ibm 61 f b ibm   17 ibm 82 f q ibm   18 imnm 53 m q statin associated myositis hmgcr 19 imnm 57 m b statin associated myositis n/a 20 imnm 66 f b statin associated myositis hmgcr 21 t2a 58 f n/a multiple morbidities   22 t2a 74 f b suspect parkinson plus syndrome   23 t2a 82 m q gait disturbance   24 t2a 44 f q leg pain   25 t2a 75 m d recurrent lung carcinoma   26 na 39 f g peripheral neuropathy   27 na 57 m g als   28 na 67 m q motorneuron-syndrome   29 na 58 f g polyneuropathy   30 rm 72 m q rhabdomyolysis uncertain cause   31 rm 68 f n/a rhabdomyolysis and renal failure   32 rm 72 f n/a rhabdomyolysis and sepsis table 1. clinical and serological characteristics of study cases. n: normal; asa-m: anti-synthetase autoantibody associated myositis; dm: dermatomyositis; ibm: inclusion body myositis; imnm: immune mediated necrotizing myopathy; t2a: type 2 fiber atrophy; na: neurogenic atrophy; rm: rhabdomyolysis; msa: myositis-specific-antibody; q: quadriceps femoris; b: biceps brachii; d: deltoid; g: gastrocnemius. cd10+ faps in normal skeletal muscle biopsies the histological appearance of faps revealed by cd10 immunohistochemistry was consistent with expectations based on previous studies [12]. cross sections of skeletal muscle showed scattered concave triangular or quadrangular cell bodies in endomysial corners between muscle fibers (fig. 1a). linear endomysial spaces between muscle fibers show occasional wispy lines of cd10 positivity, corresponding fap cell processes running along the surface of muscle fibers in a transverse plane. these processes are clearly visible on longitudinal sections tangential to the surface of muscle fibers, which show delicate branching processes embracing muscle fibers (fig. 1b). longitudinal sections through the body of muscle fibers demonstrate dot-like staining in the endomysial space, representing delicate processes in cross section. there is band-like or coarsely granular staining along capillaries, which likely reflects the known proclivity of faps to arrange along capillaries. small vessels with thicker walls, likely arteriolar, show delicate sparse perivascular staining (fig. 1g). localization of cd10+ faps outside basement membrane was confirmed on immunofluorescent triple labelling (fig. 2). figure 1. resting and activated histology of cd10+ fibro-adipogenic progenitors in human skeletal muscle. a-b: normal muscle demonstrates occasional triangular staining cell bodies in endomysial corners (black arrow) and delicate linear staining processes along endomysial lines (white arrow) on cross section (a). longitudinal sections (b) show plump cell bodies (black arrow) with processes extending around muscle fibers (white arrow). these appear as dot-like staining on deeper planes (white arrowhead). c-d: activated faps can show segmental ropey expansion of endomysial processes (black arrow) along one side of a muscle fiber only (c), or circumferentially (d, black arrows). e-f: endomysial corners are significantly expanded in e (black arrow), indicating enlarged or aggregated cell bodies. multiple nuclei in endomysial corners in f (black circle) are consistent with cell proliferation. g-h: periarteriolar staining is sparse in normal biopsies (g, black arrow), but can increase significantly in activated states (h, black arrow). figure 2. normal muscle biopsy with immunofluorescent labelling for laminin, cd10, and hoechst demonstrates location of plump shaped cd10+ cells bodies and wispy processes outside the basal lamina in the endomysial space, in keeping with the expected location of cd10+ faps. cellular changes of cd10+ faps in pathological muscle biopsies qualitative review showed changes to the appearance of cell processes as well as to cell bodies in pathological biopsies. processes frequently appear as ropey and coarse linear staining along endomysial lines, either segmental or circumferential around muscle fibers, indicative of expanded, hypertrophic fap process networks (fig. 1c, d). longitudinal sections demonstrate broad sheets of cd10 positivity instead of delicate thread-like processes. endomysial corners can show enlarged, prominent, bulky staining, indicative of expanded or conglomerated cell bodies. several biopsies show clusters of nuclei in a cd10 positive background, indicating cell clusters as opposed to single, slender angulated cell bodies in normal biopsies (fig. 1e, f). a subset of biopsies demonstrated strong circumferential staining around small vessels with a thin muscularis, consistent with enhanced periarteriolar cell processes. this contrasts with sparse delicate periarteriolar staining in normal biopsies (fig. 1g, h). overall, the observed changes can be placed into three categories: (i) expansion of fap processes represented by ropey thickening and circumferential staining along endomysial lines; (ii) proliferation and hypertrophy of cell bodies in endomysial corners, represented by the presence of cell clusters; (iii) expansion of periarteriolar processes, indicated by circumferential coarse staining around thicker-walled small vessels. these changes did not occur in unison and each component could be seen in isolation or in conjunction with others. for semi-quantitative evaluation of the cellular activation of faps we developed a scoring system based on these observations, capturing process expansion, cell clusters, and periarteriolar staining. the scoring system is summarized in table 2. exemplary images for semi-quantitative evaluation are presented in fig. 3. figure 3. exemplary photographs representing semiquantitative scores 1 to 4 of endomysial fap process activation as defined in table 2. with increasing severity, ropey processes are seen along increasing numbers of myofibers, eventually showing circumferential staining around a few and many fibers for scores 3 and 4, respectively. endomysial fap score fap processes 0 wispy processes along rare fibers 1 ropey processes along occasional fibers, not circumferential 2 ropey processes along numerous fibers, not circumferential 3 ropey processes along numerous and circumferential processes around occasional fibers 4 circumferential ropey processes around numerous fibers fap cell clusters 0 none 1 single isolated 2 occasional (1-2/mm2) 3 few (3-6/mm2) 4 frequent ( >6/mm2) periarteriolar fap score 0 none 1 sparse 2 occasional 3 moderate 4 frequent definitions and comments scoring highest score typically associated with areas of pathology on a given section in an area of 1 mm2. ropey thickening of cell processes along periphery of muscle fiber, often with expanded staining of endomysial corners; exclude staining related to vessels or to schwann cells of endomysial nerve twigs. cell cluster two or more adjacent nuclei with cd10+ cell bodies, often in endomysial corners. periarteriolar circumferential arteriolar cd10+ processes. table 2. semi-quantitative scoring system for endomysial and periarteriolar fap activation on cd10 immunohistochemically stained cross sections of skeletal muscle. endomysial activation involves cell process expansion and/or evidence of proliferation in the form of cell clusters. periarteriolar activation is represented by arterioles entirely surrounded by cd10+ processes. for illustration of histological changes see figure 1. for exemplary photos of endomysial fap process activation scores see figure 4. immunoreactivity for cd10 of non-fap structures in muscle biopsies cd10 labels faps in human muscle biopsies, but it is not a specific marker. its utility derives from a combination of the characteristic morphology of faps with cd10 positivity. other histologically easily identifiable structures can label for cd10. sarcoplasm of regenerating fibers can show positivity for cd10 [2], as was apparent in biopsies for imnm and rhabdomyolysis (fig. 4e, h). myelin sheaths and perineurium label with cd10 [12], seen in a small nerve branch in the normal biopsy represented in fig. 4a. although cd10 is expressed in early lymphoid progenitors and germinal center cells, these are not normally part of inflammatory infiltrates in muscle, unless histologically distinct lymph-follicle-like-structures are present as part of an inflammatory process, or there is involvement by cd10+ lymphoma. figure 4. exemplary patterns of cd10+ fap activation across pathological groups. a: normal muscle biopsy. occasional triangular cell bodies in endomysial corners and granular staining along capillaries running transversely. there is in addition a cross section of peripheral nerve with cd10 immunoreactivity of perineurium and myelin sheaths (black arrowhead). b: anti-synthetase syndrome. strong immunoreactivity of endomysial processes and expansion of endomysial corners with circumferential staining around several fibers. c: dermatomyositis. diffuse presence of ropey endomysial staining, some circumferential. several expanded endomysial corners. d: inclusion body myositis. multifocal ropey positivity associated with endomysial cellular infiltrates and fiber size variability. e: immune mediated necrotizing myopathy. multifocal ropey positivity associated with scattered degenerating and regenerating fibers with sarcoplasmic staining in regenerating fibers. f: type 2 atrophy. no significantly abnormal cd10 staining. g: neurogenic atrophy. increased ropey staining in particular in regions of atrophic fibers. h: rhabdomyolysis. multifocal ropey positivity or expansion of endomysial corners. note strong sarcoplasmic labelling in several regenerating fibers. endomysial and periarteriolar cd10+ fap activation across pathological groups patterns of cd10+ fap activation differed across disease groups. typical photographs are presented in fig. 4, scoring results are summarized in fig. 5. detailed correlation of scoring results, he pathology, and distribution of cd10+ fap activation is presented in table 3. biopsies from the anti-synthetase syndrome group showed strong diffuse activation of faps with often circumferential processes and prominent fap cell clusters. on he stained sections, scattered or perifascicular deand regenerating fibers were apparent, which demonstrated sarcoplasmic labelling on cd 10 immunohistochemistry. fap activation was diffuse and as such included activation around normal appearing fibers as well as activation adjacent to degenerating and regenerating fibers (fig. 4b). dermatomyositis biopsies showed impressive diffuse circumferential ropey staining in regions of fiber atrophy but notably also in areas of fibers without evidence of deor regeneration. in addition, there were clusters of cell proliferation, though less pronounced than in asa-m biopsies. the cause of diffuse and pronounced fap activation was not obvious on this study in the absence of evidence of fiber deor regeneration, inflammatory infiltrates, or neurogenic change (fig. 4c). ibm biopsies showed staining associated with inflammatory foci or regions of chronic myopathic change and fibrosis only, often with cell clusters. this could be interpreted as in keeping with a role of faps in fiber repair and fibrosis (fig. 4d). interestingly, ibm biopsies tended to show prominent periarteriolar staining, the significance of which is uncertain (fig. 5b). immune mediated necrotizing myopathies showed multifocal increased staining restricted to degenerating/regenerating fibers, with a mix of linear coarseness and expanded endomysial corners, consistent with fiber repair (fig. 4e). type 2 fiber atrophy did not show significant abnormal staining and has the same profile on scores as normal biopsies (figs. 4f; 5a). in contrast, biopsies with neurogenic atrophy clearly exhibited increased ropey staining surrounding muscle fibers without evidence of fap proliferation, as is also apparent on the scoring profile (figs. 4g; 5a). neurogenic biopsies showed in addition significant periarteriolar activation (fig. 5b). experimental evidence suggests a causal role of faps in neurogenic fiber atrophy. the absence of evidence of fap proliferation raises the possibility that this effect may primarily go along with cell process expansion in human skeletal muscle. biopsies from patients with rhabdomyolysis represent a histologically more diverse group with biopsies taken at various stages of degeneration or regeneration and with overall variable severity. these biopsies showed variable interstitial staining centered on abnormal fibers and frequent fibers with sarcoplasmic positivity in keeping with fiber regeneration (fig. 4h). figure 5. a: endomysial fap activation scores for cell processes and cell proliferation across pathological groups as defined in table 2; median with interquartile range; circles represent individual cases. b: periarteriolar fap activation scores across pathological groups as defined in table 2; median with interquartile range; circles represent individual cases. case diagnosis fap processes fap cell clusters fap periarteriolar he pathology distribution of cd10 staining 1 n 1 0 0 normal normally scattered 2 n 1 0 0 normal normally scattered 3 n 0 0 0 normal normally scattered 4 n 1 0 0 normal normally scattered 5 n 0 0 1 normal normally scattered 6 asa-m 4 3 3 pf and scattered d&r, numerous, lymphocytic clusters around d&r; focally without obvious correlation with fiber pathology 7 asa-m 2 3 2 scattered d&r, occasional around d&r; focally without obvious correlation with fiber pathology 8 asa-m 4 3 0 pf and scattered d&r, numerous, lymphocytic clusters around d&r; focally without obvious correlation with fiber pathology 9 dm 4 2 2 pf atrophy, fibrosis, pm lymphocytes diffusely without obvious correlation with fiber pathology or atrophic regions 10 dm 2 0 0 pf atrophy, focal mild diffusely without obvious correlation with fiber pathology or atrophic regions 11 dm 2 1 0 pf atrophy, focal mild diffusely without obvious correlation with fiber pathology or atrophic regions 12 ibm 2 2 2 cm, endomysial lymphocytes, mild regions of lymphocytic inflammation 13 ibm 2 1 2 cm, endomysial lymphocytes, mild regions of lymphocytic inflammation 14 ibm 3 3 3 cm, endomysial lymphocytes, moderate regions of lymphocytic inflammation 15 ibm 2 1 3 cm, endomysial lymphocytes, moderate regions of lymphocytic inflammation 16 ibm 1 1 3 multifocal endomysial lymphocytes regions of lymphocytic inflammation 17 ibm 2 2 4 cm, endomysial lymphocytes, moderate to severe regions of lymphocytic inflammation 18 imnm 2 1 0 scattered d&r, mild around d&r 19 imnm 1 1 0 scattered r, mild around d&r 20 imnm 2 2 0 scattered d&r, prominent capillaries, moderate around d&r 21 t2a 0 0 0 biphasic fiber size distribution, moderate normally scattered 22 t2a 0 0 0 biphasic fiber size distribution, severe normally scattered 23 t2a 1 0 0 biphasic fiber size distribution, moderate normally scattered 24 t2a 0 0 0 biphasic fiber size distribution, moderate normally scattered 25 t2a 1 0 0 biphasic fiber size distribution, mild normally scattered 26 na 2 0 3 regional angulated atrophic fibers, severe around atrophic fibers 27 na 3 0 4 severe neurogenic atrophy, mild compensatory hypertrophy around atrophic fibers; distinct staining around hypertrophic fibers 28 na 2 0 1 moderate neurogenic atrophy around atrophic fibers; some clusters without cd10 reaction 29 na 2 0 2 rare small clusters of angulated atrophic fibers normally scattered 30 rm 2 2 0 early necrosis, numerous, calcification, scattered regen, severe around injured fibers 31 rm 2 0 0 scattered fibers in late myophagocytosis/early regeneration around injured fibers 32 rm 2 1 1 d&r, focal inflammation around vessels around injured fibers table 3. endomysial and periarteriolar activation scores for each case in correlation with he pathology and observed distribution of cd10 staining. n: normal; asa-m: anti-synthetase autoantibody associated myositis; dm: dermatomyositis; ibm: inclusion body myositis; imnm: immune mediated necrotizing myopathy; t2a: type 2 fiber atrophy; na: neurogenic atrophy; rm: rhabdomyolysis; cm: chronic myopathic change, in particular fiber size variability and endomysial fibrosis; d: degenerating/necrotic fibers; r: regenerating fibers; pf: perifascicular. discussion morphological changes of cd10+ faps upon activation across a range of pathological conditions in human skeletal muscle biopsies in our study combine expansion of endomysial cell processes, clustering of cell bodies between muscle fibers, and prominent circumferential periarteriolar cell processes. fap proliferation in response to myofiber injury is well described in experimental studies [18, 25]. expansion of fap processes has received less attention, but is a prominent histological feature in our study of endomysial cd10+ fap activation in human muscle biopsies. morphologically, the expanded network of processes surrounding myofibers suggests enhanced fap-myofiber/basement membrane interactions, including surveillance and signaling. this is in keeping with a general role for multipotent stromal cells as damage sensors and responders, modulating the local cellular environment [27]. several observations of cellular fap reactions are in keeping with previously reported results. normal muscle biopsies showed delicate cd10+ faps without features of histological activation or at most segmental ropey processes along occasional fibers [3, 12]. disease groups with muscle fiber degeneration and regeneration as part of their histopathology (imnm, asa-m, rm) revealed fap activation of processes and some evidence of cell proliferation mostly in proximity of degenerating or regenerating fibers, in keeping with their role in muscle fiber repair [18]. neurogenic atrophy biopsies demonstrate activation of endomysial fap processes without evidence of fap proliferation in regions of atrophy and, interestingly, around hypertrophic fibers in one biopsy. mouse work has previously suggested a distinct and pivotal role of fap activation for denervation atrophy involving il6-stat3 signaling, possibly reflected in an expanded network of processes [20]. it is thought that fap activation may be triggered by disintegration of neuromuscular junctions as a result of nerve degeneration. taken together, our findings confirm the applicability of major preclinical insights to clinical muscle biopsies, and support the validity of our approach. they also suggest that the type of cellular reaction (proliferation and expansion of processes) could be modulated by the pathogenetic context. it is tempting to speculate that some of this variability could be related to transcriptomic plasticity emerging in several studies [7, 13, 15, 21]. dermatomyositis biopsies reveal extensive, diffusely distributed cd10 staining of endomysial circumferential processes and cell clusters. the overall degree of cd10 staining was to some extent related to the general severity of underlying pathological change in terms of fiber atrophy, size variability and fibrosis on he stained sections. however, normal appearing fibers and fibers without evidence of necrosis, regeneration, or inflammatory infiltrates showed pronounced adjacent and circumferential fap activation. this pattern cannot be easily explained by experimentally and clinically delineated roles for known fap activation in fiber degeneration, neurogenic atrophy, or post-traumatic or systemic fibrofatty degeneration. rather it suggests extensive pathological fap activation in dermatomyositis related to an unknown cause. abnormal ifn1-signaling activation is an important component of the partially understood pathogenesis of dermatomyositis [8, 10, 23], and diffuse distribution of fap activation could indicate its involvement in the underlying autoimmune process. this would be in keeping with a posited role of faps as damage sensors interacting with the immune system [18, 27]. biopsies of type 2 fiber atrophy showed the same scoring profile as normal biopsies, indicating the absence of fap activation in this pathology group. this result forms a contrast to obvious fap activation as part of neurogenic atrophy. type 2 fiber atrophy is a common pathological reaction pattern in muscle disuse or in various, often metabolic or toxic, myopathic conditions. the absence of fap activation in type 2 atrophy as opposed to neurogenic atrophy indicates a different level of involvement of interstitial cells in both types of atrophy. antisynthetase-antibody associated (asa) myositis shows the highest scores for endomysial fap activation. this can partly be explained by the presence of degenerating and regenerating fibers. in addition, several regions without obvious fiber necrosis or regeneration demonstrated signs of fap activation, which is more difficult to understand. asa-myositis is known to show interferon activation on the analysis of cell homogenates in a different pattern from dermatomyositis, with more prominent interferon 2 and less prominent but active interferon 1 activation [1, 23]. this may be part of an explanation for similarly diffuse fap activation in antisynthetase syndrome and dermatomyositis. overall, results point to fap activation as part of an autoimmune process, perhaps in addition to activation caused by fiber necrosis. periarteriolar fap scores revealed variable activation across groups with prominent activation in neurogenic atrophy and ibm biopsies, some activation in dermatomyositis and anti-synthetase syndrome, and no activation in type 2 atrophy and immune mediated necrotizing myopathy. periarteriolar prominence of fap processes suggests cellular interaction with perivascular basement membrane, vascular mural cells, or endothelial cells. experimental evidence supports a specific compartmentalized interaction between faps and capillaries mediated by vegf in a model of ischemic skeletal muscle injury. this interaction mediates capillary remodeling and vascularization [11, 26]. periarteriolar prominence of fap processes would be in keeping with the spatial organization of endothelial cell interaction. in addition, the role of vascular mural cells for skeletal muscle tissue homeostasis has been suggested [24]. in this context, periarteriolar fap processes raise the possibility of mural cell – fap interaction. while experimental data support the existence of spatially compartmentalized interaction between faps and vascular structures, it remains unclear why there is differential periarteriolar fap activation across groups of more chronic myopathies, for instance prominent periarteriolar activation in ibm and none in imnm. it is tempting to speculate that a specific patho-mechanistic context plays a role. the use of cd10 as immunohistochemical marker to visualize faps in muscle biopsies has the advantage of easy applicability to a wide variety of archival biopsies. the fact that the method works well on formalin-fixed, paraffin embedded tissue, expands the spectrum of immunohistochemical stains for paraffin embedded muscle biopsies [28, 29], with the advantage that these can be used in laboratories outside dedicated muscle pathology centers. although cd10 is not specific for faps, their characteristic histological appearance allows reliable identification of reaction patterns. other cd10-positive, non-fap structures in muscle biopsies were easily identified in our study. recently, single cell sequencing technologies have yielded insights into transcriptomic heterogeneity of fap subpopulations in response to various pathological stimuli [7, 13, 15, 21]. these studies suggest that fap activation results in a dynamic profile of fap subtypes, which varies with pathological context. more specifically, a recent publication demonstrated in this regard that cd10+ faps in humans represent a large fap subpopulation with adipogenic potential [7]. it is therefore possible that our approach does not capture the entire population of cells, which could be identified as faps on a transcriptomic level. bearing these limitations in mind, cellular reaction patterns delineated by cd10 immunohistochemistry remain a valid insight into cd10+ fap activation in various disease groups, even if it may not capture the entire fap spectrum. conclusion in conclusion, cd10 immunohistochemistry is a robust technique to analyze histological reaction patterns of cd10+ faps in routine clinical muscle biopsies. histopathological features of activation of endomysial cd10+ faps in human muscle biopsies include prominent expansion of the network of cell processes surrounding muscle fibers as well as endomysial cell clusters as evidence of proliferation. in addition, prominence of periarteriolar processes is a notable feature in some pathologies. a scoring system for endomysial and periarteriolar activation is proposed. analysis of biopsies 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to myotrauma. semin cell dev biol 119: 23-31. https://doi.org/10.1016/j.semcdb.2021.07.013 copyright: © 2024 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. cerebrovascular disease lesions are additive and tied to vascular risk factors and cognitive impairment feel free to add comments by clicking these icons on the sidebar free neuropathology 3:7 (2022) original paper cerebrovascular disease lesions are additive and tied to vascular risk factors and cognitive impairment john l robinson1, hayley richardson3, sharon x xie1,3, brian alfaro1, nicholas loh1, virginia m-y lee1, edward b lee2, john q trojanowski✝ 1 center for neurodegenerative disease research, department of pathology and laboratory medicine, institute on aging, university of pennsylvania perelman school of medicine, philadelphia, pa, usa. 2 translational neuropathology research laboratory, department of pathology and laboratory medicine, university of pennsylvania perelman school of medicine, philadelphia, pa, usa. 3 department of biostatistics, epidemiology and informatics, university of pennsylvania perelman school of medicine, philadelphia, pa, usa. ✝ passed away on 08 february 2022. corresponding author: edward b. lee · 613a stellar chance laboratories · 422 curie blvd · philadelphia, pa 19104 · usa edward.lee@pennmedicine.upenn.edu submitted: 24 january 2022 accepted: 04 march 2022 copyedited by: lauren walker published: 14 march 2022 https://doi.org/10.17879/freeneuropathology-2022-3792 keywords: cerebrovascular disease, alzheimer’s disease, infarcts, amyloid angiopathy, arteriolosclerosis, vascular risk factors, ageing abstract cerebrovascular lesions are prevalent in late life and frequently co-occur but the relationship to cognitive impairment is complicated by the lack of consensus around which lesions represent hallmark pathologies for vascular impairment, particularly in the presence of alzheimer’s disease (ad). we developed an easily applicable model of cerebrovascular disease (cvd), defined as the presence of two or more lesions: moderate to severe cerebral amyloid angiopathy, moderate to severe arteriolosclerosis, infarcts (large, lacunar, or micro), and/or hemorrhages. ad was defined as intermediate or high ad neuropathologic change. the contribution of vascular risk factors such as atherosclerosis and/or a health history of heart disease, hyperlipidemia, stroke events, diabetes, or hypertension was also assessed. logistic regression analysis reported the association of cvd with increasing age, vascular risk factors, ad, and cognitive impairment in this study of 1,485 autopsied individuals. cerebrovascular lesions were present in 48% and 16% had cvd. increasing age associated with all lesions (p<0.001), except hemorrhages (p=0.41). cvd was more likely in individuals with vascular risk factors or ad (p<0.01). cvd, but not individual cerebrovascular lesions, associated with impairment in cases without ad (p<0.01), but not in cases with ad (p>0.61). from this, we conclude that a simple, additive model of cvd is 1) age and ad-associated, 2) is associated with vascular risk factors, and 3) clinically correlates with cognitive decline independent of ad. introduction alzheimer’s disease (ad) and cerebrovascular disease (cvd) are the two most common causes of late-life dementia (1). ad is best described as the widespread distribution of β-amyloid deposits, tau-positive neurofibrillary tangles, and cortical neuritic plaques as primary pathologies along with the cerebral amyloid angiopathy (caa), lewy bodies, and tdp-43 inclusions also prevalent as secondary pathologies (2,3). cvd is also considered the accumulation of multiple pathologies including infarcts (large, lacunar, and micro), arteriolosclerosis, perivascular space dilation, perivascular hemosiderin leakage, myelin loss, caa (leptomeningeal, parenchymal and capillary), hemorrhages (micro and larger), fibrinoid necrosis, and microaneurysms (4). unfortunately, while consensus criteria exists to determine the level of ad neuropathologic change by the examination of three hallmark pathologies, it is not clear which cerebrovascular lesions relate to impairment and which accumulate due to conditions such as cardiac atherosclerosis, monogenic stroke disorders, embolic disease, vasculopathies, haematological disorders, or metabolic disorders or simply ‘brain ageing’ (4–7). to address this, the vcing study reported seven pathologies that reproducibly correlate with cognitive impairment to varying degrees in a study of 113 individuals (55 to 100 years) including leptomeningeal caa, large infarcts, lacunar infarcts, microinfarcts, arteriolosclerosis, perivascular space dilation and myelin loss (8). a simplified model was proposed highlighting just three cerebrovascular lesions: large infarcts, moderate/severe occipital leptomeningeal caa, and moderate/severe arteriolosclerosis in the occipital white matter. inspired by this attempt at consensus, we screened for three lesions in the occipital cortex – moderate to severe leptomeningeal caa, moderate to severe white matter arteriolosclerosis, and microinfarcts – in a cohort of 1,485 individuals (31 to 103 years old). we then compiled available autopsy data for the presence of moderate to severe caa in other cortical areas, global infarcts (large, lacunar and micro), and large hemorrhages. we describe an additive model of cvd, defined as a presence of multiple cerebrovascular lesions. the goal of this study is to test the hypothesis that our easily replicable, histological definition of cvd associates with increasing age, with cognitive impairment and/or dementia, and with vascular risk factors such as atherosclerosis and/or a health history of heart disease, hyperlipidemia, stroke events, diabetes, or hypertension. materials and methods cohort the cohort consisted of individuals with known ages at death of ≥30 years, and available cerebrovascular and ad pathology measures who participated in the autopsy program at the center for neurodegenerative disease research (cndr) at the university of pennsylvania. of 1,785 individuals age ≥30 at time of death, ad pathology measures were known for 1,659. as of december 2020, cerebrovascular measures were assessed in 1,512 of these individuals. 7 were excluded for having rare clinical diagnoses and 20 were excluded for rare neuropathological diagnoses, resulting in a cohort of 1,485 individuals. clinical diagnoses included no impairment (ni) for individuals without a neurodegenerative disease, ad (probable and possible), amyotrophic lateral sclerosis (suspected, possible, probable, and definite), corticobasal syndrome, cognitive impairment (including amnestic and non-amnestic mild cognitive impairment), frontotemporal degeneration (including behavioral variant, primary progressive aphasias, and not otherwise specified), multiple system atrophy, parkinson’s disease (with and without cognitive impairment), parkinson’s disease dementia or dementia with lewy bodies, progressive supranuclear palsy, schizophrenia, and vascular dementia. the frequency of each is listed in table 1. neuropathologically, the cohort includes neuropathologic ad (intermediate or high level of ad neuropathologic change), low ad (low level of ad neuropathologic change), not ad (including primary age related tauopathy and unremarkable cases), amyotrophic lateral sclerosis, corticobasal degeneration, frontotemporal lobar degeneration, lewy body disease, pick disease, and progressive supranuclear palsy, with the frequency of each is listed in table 2. since our brain bank consists of such a diverse population of neurodegenerative diseases, two subgroups were defined to assess the clinical impact of ad and cerebrovascular pathology (table 3). the ad spectrum subgroup (n=743) is comprised of cases with underlying ad neuropathology (from not ad to high ad) as the primary neuropathological diagnosis regardless of clinical symptoms. the ni to ad subgroup (n=566) is comprised of cases in the ad spectrum subgroup but excludes clinical frontotemporal syndromes and other atypical ad presentations. informed consent for autopsy was obtained in accordance with state laws and protocols approved by the university of pennsylvania. vascular risk factors vascular risk factors included atherosclerosis and/or a history of heart disease, hyperlipidemia, stroke events, diabetes, or hypertension, extracted from health records spanning three decades. heart disease (n=867) included diagnoses of atrial fibrillation, aortic stenosis, cardiac arrhythmia, coronary artery disease, and/or cardiac murmur. stroke events (n=845) included diagnoses of transient ischemic attacks and/or cerebrovascular accidents. hyperlipidemia (n=577) included diagnoses of hyperlipidemia or hypercholesterolemia. diabetes (n=867) and hypertension (n=846) status were also obtained. atherosclerosis was defined as moderate to severe atherosclerosis observable in the circle of willis at autopsy and was available for the majority of cases (n=1,480). 846 cases had enough health history data to define them as having any single risk factor or multiple risk factors. pathology sixteen brain regions are routinely examined in the cndr neuropathology evaluations (9). each region was assigned a semi-quantitative score, i.e. none, rare, mild, moderate, or severe for neurofibrillary tangles based on immunohistochemistry against tau (mouse antibody phf1, a gift from dr. peter davies), or for plaques and caa based on immunohistochemistry against amyloid-β (mouse antibody nab228, generated in cndr). furthermore, arteriolosclerosis scores were determined based on hematoxylin and eosin histology. moderate to severe caa and arteriolosclerosis was assessed as per deramecourt et al (10). all cases were reviewed by a board-certified neuropathologist (jqt and/or ebl) for quality assurance and accurate grading. the level of ad neuropathology was assessed by the amount of tau-positive neurofibrillary tangles, tau-positive neuritic plaques, and amyloid-β positive plaques according to consensus criteria (2). neuropathologic ad was defined as an intermediate or high level of ad neuropathologic change. cerebrovascular lesions were assessed by examination of histological and immunohistochemically stained slides and compiled from post-autopsy gross and microscopic reports and as described below. data on large infarcts were collected from reports detailing infarcts ≥1 cm in diameter visible at autopsy. lacunar infarcts were also determined from records that reported grossly visible infarcts <1 cm in diameter or that were described as lacunar infarcts at time of autopsy. microinfarcts were defined as infarcts <0.5cm in diameter that were not observed grossly and were collected from microscopic reports. in addition, the occipital hematoxylin and eosin slides were examined for all cases for the presence of microinfarcts. hemorrhages refers to large hemorrhages described grossly. arteriosclerosis in the occipital white matter was assessed for all cases. caa severity was primarily assessed in the occipital cortex and was reported as a maximum of scores from three additional neocortical regions if available: middle frontal cortex, superior temporal cortex, and angular gyrus. four neocortical caa scores were available for the majority of cases (n=1,258). statistical analysis r 3.6.2 was used for all regression analyses. univariate analysis was performed to quantify the association between variables. logistic regression analysis was used to build a model of dementia to assess each measure’s contribution. odds ratios and effect sizes for age were assessed for each 5-year increase with confidence intervals reported at 95%. likelihood ratio tests for nested models were used to assess the effect of variables with multiple categories, such as clinical diagnosis. we investigated the effect of missing vascular risk factor data through inverse probability weighting (11). this procedure aims to correct potential bias incurred by considering only subjects with available risk factor data. inverse probability weighting results were very similar to those using available data, illustrating the robustness of results. as such, complete case analyses are presented. unadjusted analysis of cases with cognitive impairment or dementia, and with no cvd, with a single cerebrovascular lesion and with cvd, between participants with and without dementia was performed using pearson’s χ2 tests using two degrees of freedom. all statistical tests were two-sided with statistical significance set at <0.05 level. results full cohort demographics demographic and neuropathological data was available for 1,485 cases (table 3). ni individuals, comprising 9% of the cohort, were without a history of clinical neurodegenerative disease. of the remaining individuals, 51% were clinically late-onset cases with an average age of onset of 64.9 years and an average age of death of 74.5 years. 66% had cognitive impairment or dementia including ad, frontotemporal syndrome, and parkinson’s disease dementia or dementia with lewy bodies (see table 1 for the breakdown by primary clinical diagnosis). pathologically, over ten primary neuropathological diagnoses were represented, with ad and lewy body disease representing the most frequent underlying neurodegenerative diseases (see table 2 for the breakdown by primary neurodegenerative disease). vascular risk factors – such as the presence of moderate to severe atherosclerosis or a health history that included diabetes, heart problems, hypertension, high levels of lipids, or stroke events – were present in 64% of individuals. atherosclerosis, hypertension, and high lipids were the most common risk factors. these risk factors frequently co-occurred. individually, diabetes, heart disease, and stroke had a >90% co-occurrence with other vascular risk factors while atherosclerosis, hypertension, and high lipids had a >60% co-occurrence with other vascular factors. overall, multiple risk factors were present in 35% of individuals. neuropathologic ad, defined as the presence of intermediate or high ad neuropathologic change, occurred in 48% of all cases, representing 28% with ad dementia, 8% with atypical ad clinical phenotypes such as frontotemporal degeneration, 10% of cases as a co-pathology, and 1% of ni cases. fig. 1 representative cerebrovascular lesions moderate to severe (a) leptomeningeal caa was frequently observed in the occipital cortex. (b) severe white matter arteriolosclerosis was not uncommon particularly in the occipital white matter adjacent to the ventricle. (c) infarcts were observed in all subcortical and cortical regions, including this large infarct affecting occipital cortex. scale bar is 100um in (a) and (b) and 1mm in (c). cerebrovascular lesions the presence and severity of cerebrovascular lesions were histologically assessed (figure 1, table 4). the most common lesions affected blood vessels including the presence of moderate to severe caa or arteriolosclerosis which affected 29% and 18% of the overall cohort, respectively. infarcts and hemorrhages were less common, but when present, the majority occurred when concurrent cerebrovascular lesions were described. large infarcts, for instance, were noted in only 5% of all cases, but 86% of cases with large infarcts had other cerebrovascular lesions. similarly, the majority of cerebrovascular lesions co-occurred 52-86% of the time, with only caa occurring with other lesions in a minority of cases 38% of the time. given the preponderance of concurrent cerebrovascular lesions, cvd was defined when more than one lesion was present. overall, cerebrovascular lesions were observed in 48% (n=720) of cases, including 32% (n=482) with a single lesion and 16% (n=238) with multiple lesions. we next assessed, which, if any, neuropathological or clinical groups had elevated frequencies of cvd. as age at death significantly associated with the presence of either single or multiple cerebrovascular lesions (or 1.27, 1.21-1.34, p<0.001), we adjusted for age in our analysis. neuropathologically, the cases with a primary diagnosis of amyotrophic lateral sclerosis, corticobasal degeneration, frontotemporal lobar degeneration, lewy body disease, pick disease, and progressive supranuclear palsy, low ad , and neuropathologic ad were compared to the cases with ‘not ad’ as their primary diagnosis. only neuropathologic ad had an increased prevalence of cvd (or 2.59, 1.44-4.67, p<0.01). other neurodegenerative diseases were not more likely to have cvd (table 2). similarly, clinical ad dementia cases were twice as likely as ni cases to have either single or multiple cerebrovascular lesions (or 2.24, 1.50 3.36, p<0.001). other clinical groups were not more likely to have cvd (table 1) except for corticobasal syndrome cases (or 1.90, 1.05-3.41, p=0.03). since many of the corticobasal syndrome cases were neuropathologically diagnosed as ad, to further assess the impact of clinical diagnoses, we performed a likelihood ratio test comparing the fit of a model including both age and neuropathologic ad to a model including age, neuropathologic ad and clinical diagnoses. in this analysis, the model including clinical diagnoses did not improve the model fit (p=0.33). thus, it appears that only cases with neuropathologic ad had an increased likelihood for cvd. age and ad associations with cvd since an increased incidence of cvd only occurred with neuropathologic ad and the level of ad neuropathologic change was assessed in all cases, we next fit logistic regression models using neuropathologic ad and age as predictors for each cerebrovascular lesion (table 5). in this analysis, increasing age associated with an increasing likelihood of each lesion except hemorrhage (p<0.001) and neuropathologic ad associated with an increasing likelihood of the presence of caa (p<0.001) but not the other cerebrovascular lesions (p>0.07). overall, single cerebrovascular lesions were not age associated (p=0.09), but were neuropathologic ad-associated (p<0.001), while cvd associated with both increasing age and neuropathologic ad (p<0.001). we conclude that infarcts and arteriolosclerosis are more likely with increasing age, while caa is more likely when ad pathology is present, and cvd is associated with both increasing age and neuropathologic ad. vascular risk factors and cvd vascular risk factor information was available for a majority of the cohort (57%; n=846). unsurprisingly, the presence of any or multiple vascular factors increased with age (any risk factor = or 1.48, 1.37-1.59, p<0.01; multiple risk factors = or 1.32, 1.23-1.42, p<0.01). to better understand the role of vascular risk factors in the development of cerebrovascular pathology, we next performed logistic regression analysis incorporating vascular risk factors, age and ad in a model of cvd (table 6). when any vascular risk factors are present, there is an association with cvd (p<0.001) but not single cerebrovascular lesions (p=0.37). vascular risk factors had the strongest association with the presence of infarcts (p<0.001), and also associated with arteriolosclerosis (p<0.01), but did not associate with caa (p=0.55). in these analyses, age consistently associated with all cerebrovascular lesions (p<0.01), while neuropathologic ad strongly associated with caa (p<0.001), arteriolosclerosis (p=0.001), but not infarcts (p=0.23). in all cases, multiple risk factors were not significantly more likely to associate with cerebrovascular lesions in the presence of any risk factor. ad spectrum and ni to ad demographics because cognitive impairment or dementia due to causes other than neuropathologic ad was common in our cohort, we hypothesized that the clinical impact of cvd would only be apparent in cases with ad or cerebrovascular pathology as their primary pathology. these cases are the ad spectrum and ni to ad subgroups (table 3). the ad spectrum subgroup includes approximately half of the full cohort (n=743) including 56% with ad dementia, 17% with atypical dementias and 3% with neuropathologic ad in the absence of cognitive impairment. the ni to ad subgroup (n=566) is the ad spectrum subgroup excluding the atypical ad cases and includes 74% with ad dementia. the ad spectrum and ni to ad groups shared similar demographics. both were older at onset (68.1 and 69.1 respectively) and at death (77.0 and 77.3 respectively) compared to the overall cohort. more women were present in the ad spectrum and ni to ad groups (53% and 54% compared to 44%). cvd was elevated in these groups. cvd was present at 22% and 23% respectively compared to 16% for the overall cohort. the presence of one or more risk factors was also higher in the ad spectrum and ni to ad groups. any vascular risk factor was present at 73% and 77% respectively compared to 64% for the full cohort, while multiple risk factors were present in 46% and 50% respectively compared to 35% for the overall cohort. clinical relevance of cvd we next asked if the presence of cvd associated with cognitive impairment in the ad spectrum and ni to ad subgroups (table 7). in the full cohort, cvd did not correlate with a higher incidence of impairment in cases with (p=0.13) or without (p=0.77) neuropathologic ad. in the ad spectrum subgroup, neuropathologic ad cases with cvd did not differ by their incidence of impairment (p=0.90) compared to neuropathologic ad cases without cvd. however, cases without neuropathologic ad but with cvd were substantially more likely to have impairment than cases without neuropathologic ad or cvd (p<0.001). in the absence of neuropathologic ad, the incidence of impairment was 56% if definite cvd was present, but only 19% if limited cvd was present, and 12% if cvd was absent. a similar result was observed in the ni to ad cohort. neuropathologic ad cases, with and without cvd, did not differ by their incidence of impairment (p=0.61), but cases without neuropathologic ad, but with cvd, were substantially more likely to have impairment than cases without cvd (p<0.01). in the ni to ad cohort, in the absence of neuropathologic ad, the incidence of impairment was 30% if cvd was present, but only 6% if a single cerebrovascular lesion was present, and 5% if no cerebrovascular lesions were observed. discussion that cerebrovascular disease is the second most common cause of dementia after alzheimer’s disease is well known (12), but the term “cerebrovascular disease” has various meanings depending on the clinical, neuroimaging or neuropathological context. even within the field of neuropathology, dozens of lesions have been reported, many of which are dependent on laboratory-specific sampling and histological techniques (4,6). furthermore, while understanding of individual lesions has improved (13–17), the co-occurrence of lesions confounds efforts to correlate the lesion-specific effects with cognitive impairment (8,18). thus, it is important to define a model of cvd that is straightforward and easily reproduced (figure 2). fig. 2 cvd associates with cognitive impairmentthe possibility of either ad (or 1.11, 95% ci 1.02-1.20, p<0.01) or vascular co-morbidities (or 1.46, 95% ci 1.30-1.64, p<0.01) is more likely with increasing age. individual cvd lesions frequently occur when ad or vascular risk factors are present (see tables 3 and 4). vascular risk factors also increase the likelihood of multiple cvd lesions (or 1.94, 95% ci 1.07-3.52, p=0.03) as does ageing itself (or 1.15, 95% ci 1.02-1.30, p = 0.03). multiple cvd lesions (or 2.36, 95% ci 1.23-4.52, p<0.01), but not single lesions (or 0.93, 95% ci 0.54-1.60, p=0.80), associate with cognitive impairment or dementia. logistic regression modelling the effect of age, ad pathology, vascular risk factors and cvd on cognitive impairment for ad spectrum cases with available vascular risk factors (n=421). similar results were obtained when modelling for dementia (not shown). our approach examined six cvd pathologies including moderate/severe cortical caa, moderate/severe white matter arteriolosclerosis, large infarcts, lacunar infarcts, microinfarcts and hemorrhages, and defined a moderate to high level of cvd as the presence of two or more of these pathologies. the vcing study’s model of three pathologies weighted large infarcts twice as strongly as moderate/severe occipital leptomeningeal caa or moderate/severe occipital white matter arteriolosclerosis and defined a moderate to high level of cvd as the presence of either large infarcts, both caa and arteriolosclerosis, or large infarcts with one or both other lesions. since large infarcts were associated with other cvd lesions 86% of the time, our additive model of cvd is functionally similar to the vcing model that defines all cases with large infarcts as having a moderate level of cvd. indeed, the correspondence between the vcing model and our study was high (pearson’s correlation coefficient = 0.803). cases with a moderate or high level of cvd by vcing were also defined as a moderate to high level of cvd by cndr with 91-100% agreement (table 8). however, with the inclusion of additional lesions in our model, many more cases have cerebrovascular pathology than are defined by the vcing model, so 36-44% of our moderate to high level cvd cases would not be captured by the vcing model (table 8). defining cvd as the presence of multiple cerebrovascular lesions correlated well with cognitive impairment in the absence of definite ad, while single cerebrovascular lesions did not (table 7). other studies have also reported that a moderate or high burden of cerebrovascular lesions is a better correlate of cognitive decline than low levels (17,19,20). by age 90, for instance, close to 50% of individuals without dementia have microinfarcts, while just over 50% of individuals with dementia have infarcts (19). however, three or more microinfarcts were present in 26% of those with dementia compared to 7% without, indicating that it is the presence of multiple infarcts that associated with dementia, while one or two were tolerated without concomitant dementia. similar results have been reported for younger cohorts (17,20), suggesting that a more severe cvd burden is a better correlate of impairment. it should also be pointed out that an additive model of cvd is functionally similar to the consensus criteria for ad (2). in ad, an individual with only β-amyloid plaques has a low level of ad neuropathologic change, but an individual with additional tau-positive neurofibrillary tangles who also develop tau-positive cortical neuritic plaques is more likely to have an intermediate or high level of ad neuropathologic change. most older individuals without cognitive impairment have a low level of ad neuropathologic change but most individuals with dementia have a high level of ad neuropathologic change (3). our study attempted to disentangle the contribution of ageing itself or ad pathology to the development of cerebrovascular lesions (table 5). individual lesions such as infarcts, moderate/severe arteriolosclerosis, and moderate/severe caa associated with increasing age, but only moderate/severe caa associated with the presence of ad pathology. however, it should be noted that ad and cvd rarely occur in isolation. not only is the common co-morbid lesion frequency estimated at >90% for caa, ~50% for arteriolosclerosis, >30% for microinfarcts, >10% for larger infarcts and >10% for hemorrhages (18), but both ad and cerebrovascular pathologies may accumulate a decade or even decades before clinical symptoms (21–24). recent imaging studies have even suggested that hypoperfusion (25) and blood brain barrier dysfunction (26) are early events in the development of ad, much as they are speculated to be in the development of cvd. nonetheless, our data is consistent with ageing as a risk factor for infarcts and arteriolosclerosis, and ad as a risk factor for caa. cvd also associated with vascular risk factors in our study (table 6). both cvd and ad are ageing-related diseases, but cerebrovascular lesions such as infarcts or moderate/severe arteriolosclerosis were more likely when co-morbidities such as hypertension, diabetes or atherosclerosis were present. while these associations between vascular risk factors, cerebrovascular pathology, and cognitive impairment are widely assumed to exist, they are rarely reported [7, 11, 18]. there are multiple reasons for this, but the largest confound may be the overlap between risk factors associated with both cvd and ad. indeed, several ‘vascular’ risk factors are also ad risk factors and previous studies have suggested that up to 35% of late-life dementia may be preventable by targeting multiple risk factors such as diabetes, hypertension, obesity, smoking, depression, cognitive inactivity, and physical inactivity (1,27,28). hypertension and diabetes are potentially modifiable risk factors for clinical ad [17], atherosclerosis is increasingly seen as contributing to dementia in a mode similar to ad [28], and vascular risk factors have correlated with increasing ad biomarkers in preclinical ad [3]. in our study, multiple vascular risk factors were often present in the same individual, but, surprisingly, the presence of multiple risk factors was not more likely than the presence of a single risk factor to associate with cvd pathology. the present work is best understood in the context of the strengths and limitations of our study design. for this study, as recommended in the preferred model of the vcing study (8), we screened the occipital cortex for caa, arteriolosclerosis and micro-infarcts. it should be noted that our model is minimalistic, and other models of cerebrovascular disease also deserve closer attention for their relevance to the study of dementia in older adults. strozyk et al examined a 6-point vascular score (involving large infarcts, lacunar infarcts and leukoencephalopathy) that associated with dementia (29); deramecourt et al reported a 20-point vascular score (involving arteriolosclerosis; amyloid angiopathy, perivascular hemosiderin leakage; perivascular space dilation, myelin loss and cortical micro-infarcts and large infarcts in multiple regions) that correlated well with vascular and mixed dementia (10); the newcastle categorization describes cerebrovascular disease as heterogeneous and proposes six subtypes to best describe the presence and distribution of cerebrovascular lesions (30). one obvious limitation is that we graded leptomeningeal, parenchymal and capillary caa as a single cortical caa score when each type of caa may have differential effects in a cvd model of cognitive impairment (32,33). relatedly, reporting infarcts by subtype – i.e. strategic infarcts versus multiple small lacunes versus cortical infarcts – may have revealed which type of infarct most correlated with cognitive impairment (29, 30). the primary benefit of screening the occipital cortex for arteriolosclerosis, micro-infarcts and caa is that it appears to be susceptible to both caa (5) and micro-infarcts (19). nonetheless, the inclusion of additional regions or stains may have also affected the overall picture of cvd in our cohort, as the differences in postmortem sampling and staining methods is a well-known problem in the field (4). in our analysis of vascular risk factors, we describe atherosclerosis in the circle of willis as a risk factor, while other studies report atherosclerosis as a neuropathologic change (23, 31). in fact, both views on atherosclerosis are valid. atherosclerosis as an intracranial pathologic change may be a consequence of other risk factors such as hyperlipidemia, and atherosclerosis as a risk factor may contribute to the intracerebral parenchymal lesions in our study. finally, the majority of cases in our brain bank are composed of individuals with a neurodegenerative disease who were enrolled for brain tissue donation through tertiary referral clinics, and therefore our cohort has a younger age at death than many communityor population-based cohorts, with an overall lower incidence of cerebrovascular lesions than is observed in older cohorts (34). as a consequence, the number of ad-associated cvd lesions is perhaps overrepresented compared to the number of age-associated cvd lesions. in conclusion, we report an additive model of cvd that accounts for the presence of global infarcts, moderate/severe caa, and moderate/severe arteriolosclerosis. when multiple cerebrovascular lesions are present, cvd associates with cognitive impairment or dementia in the absence of other neurodegenerative diseases and, importantly, cvd also associates with vascular risk factors. as a straightforward to apply definition of cvd, our model could be easily reproduced in community‐based cohorts for a more complete understanding of the frequency of cvd in the population. future studies may be better able to disentangle the overlap between risk factors, ageing, and ad pathology with cerebrovascular pathology and thus improve the detection and treatment of vascular cognitive impairment and dementia. author contributions jqt conceived and designed the study. jlr and ebl also contributed to the study design. jlr, ba and nl performed immunohistochemistry and semi-quantitative grading which were verified by jqt and ebl. hr and sxx performed the statistical analysis. jqt, hr, sxx, vmyl, ebl and jlr contributed to data analysis and interpretation. jlr wrote the initial draft of the manuscript, with all authors contributing to the final version. all authors read and approved the final manuscript. potential conflicts of interest ebl is an editorial board member but was not involved in the editorial handling of this manuscript. jlr (no conflict); hr (no conflict); sxx (no conflict); ba ((no conflict), nl (no conflict); vmyl (no conflict); jqt (no conflict) funding us national institute on aging (national institutes of health u19 ag062418, p30 ag010124, pi: jqt; p01 ag017586, pi: vmyl; p01 ag066597, p30 ag072979, pi: ebl). acknowledgements we are indebted to patients and families who donated tissue to the cndr. we also thank all past and current referring neurologists, pathology assistants and pathologists for making this research possible. in addition, we would like to thank theresa schuck for helping us procure and stain all samples. references livingston g, sommerlad a, orgeta v, costafreda sg, huntley j, ames d, et al. dementia prevention, intervention, and care. lancet. 2017 dec;390(10113):2673–734. montine tj, 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these icons on the sidebar free neuropathology 2:27 (2021) reflections lessons learned from a career in neuropathology roy o. weller faculty of medicine, southampton university, united kingdom address for correspondence: roy o. weller, bsc md phd frcpath, emeritus professor of neuropathology · clinical neurosciences · ld66, mail point 806, south academic block, level d · southampton university hospital southampton so16 6yd · united kingdom rcn@soton.ac.uk submitted: 09 october 2021 accepted: accepted: 12 october 2021 published: 25 october 2021 https://doi.org/10.17879/freeneuropathology-2021-3634 additional resources and electronic supplementary material: supplementary material keywords: clinical neuropathology, research, students, lymphatic drainage of the brain, alzheimer’s disease, cerebral amyloid angiopathy, personal reflections, family introduction writing an autobiography was probably the last thing on my mind when i was asked to add mine to a growing list of personal accounts published in free neuropathology. it was no easy task to summarise my life in what i hope will be an interesting account for my readers. but, in the end, it has been rewarding for me at least to state what i think have been the important events, ideas and attitudes that have shaped my life and career in neuropathology and to recognise some of the many people who have taught, influenced, helped me and offered their friendship over many years. as for the lessons learned, i hope they will become apparent as the account proceeds. early years i was born in a south-eastern suburb of london a year before the outbreak of the second world war. our house was two miles from the river thames and woolwich arsenal but, despite this vulnerable position, our home suffered no damage from bombing. it did mean, however, that there were many periods when we spent nights in an air-raid shelter in the garden. i was too young to appreciate the deprivations of wartime england and the terrible time that my parents endured. the first school that i attended was partially destroyed by a v2 rocket and my classmates and i transferred to another school that had already been half destroyed by incendiary bombs. despite this, all i can remember is a very happy childhood and education. at the age of 11 years, i transferred to a grammar school in the centre of london, close to tower bridge. founded in 1588, st olave’s and st saviour’s school that i attended had moved to a beautiful early victorian building in 1840 to make room for the construction of london bridge station. the school is now in kent and the original 1840 building is a 5-star hotel. i received a classical education with some concentration on languages; english, latin, french, greek and german have all been of great value to me, not only in medicine but also when i travelled widely in europe as an undergraduate and during my postgraduate career. science was also strong in my school and this enabled me to enter guy’s hospital medical school in london as a medical student in 1956. my motivation for studying medicine was mainly the attraction of the basic sciences, particularly biochemistry, but i soon became enthralled with patient contact and clinical medicine. during my undergraduate studies i spent a year, gaining a bachelor of science degree in anatomy under excellent tuition from roger warwick and peter williams who were both editors of gray’s anatomy. i visited many of the medical schools in london for lectures and demonstrations during this year which gave me a clear insight into academic medicine and was probably a major factor in determining my postgraduate career in neuropathology. early postgraduate career having completed my medical education, i took a teaching post in anatomy at guy’s and a research post with professor john cavanagh (figure 1) to work on peripheral neuropathies, most especially experimental diphtheritic neuropathy in chickens (much to the amusement of my friends). my objective was to determine the ultrastructural sequences involved in the initial stages of segmental demyelination. john was an ideal supervisor at the beginning of my research career as he would present me with a problem and leave me to solve it, only giving advice when asked; john also taught me the basis of the scientific method and how to view earlier findings in my field with scepticism. i was introduced to the technical aspects and analysis of transmission electron microscopy (tem) that was still a relatively new technique. the skills i learnt at that time have served me throughout my career. in my first paper in 1965 1, i showed that, prior to breakdown of the myelin sheath in segmental demyelination, acid phosphatase-containing lysosomes gathered in cytoplasmic pockets on the inner aspect of the myelin sheath. this gave some indication of how the myelin was removed without damaging the axon that it encompassed. figure 1. john b cavanagh 1997. my second post at guy’s was with professor colin adams, who was a lipid histochemist of world renown. he had a gift for adapting techniques to identify individual lipids, particularly in atherosclerosis and in multiple sclerosis. the major task for my phd was to define the ultrastructure of intracellular lipid accumulations in atherosclerosis. this was no easy matter as the lipids are soluble in the organic solvents used in preparation for tem. by a combination of polarised light microscopy, negative staining and routine tem (figure 2), i identified the multi-lamellated structure of the liquid crystalline lipid droplets that appeared to be the template for esterification of the cholesterol that they contain 2,3. it was during this period, that i started my education in clinical neuropathology with henry urich at the london hospital. henry’s teaching was direct and very suitable for me in the early stages of my career. figure 2. intracellular lipid droplets in atherosclerosis. top left and then clockwise: tem showing removal of lipid from cells during preparation. birefringent liquid crystalline droplets of cholesterol and phospholipids isolated from an atherosclerotic lesion. similar droplets examined as negative stained tem showing lamellated structure 2,3. us-nih-fellowship at the albert einstein college of medicine in new york having gained my ph.d. in 1967, it was time for a change, so my wife, francine, and our two children, adrienne, aged three and timothy aged two years embarked on a boat for the usa. i had gained an us-national institutes of health (us-nih) postdoctoral fellowship in neuropathology with dr robert terry at the albert einstein college of medicine in new york. it was a very different but highly stimulating environment. bob terry was very generous with his time and ideas and this led to numerous interesting discussions about pathology of the nervous system. i learned much about clinical neuropathology from bob and his colleagues, especially kinuko suzuki. for my research, i investigated the effects of acute hydrocephalus upon the brains of adult rabbits, infant dogs and in biopsies from very young children. my main teacher and collaborator in this project was henryk wisniewski who was an excellent animal surgeon and experimenter. henryk devised a technique for producing hydrocephalus by injecting highly viscous, but non-inflammatory, silicon oil through a lumbar intrathecal catheter into the posterior fossa of adult rabbits and young dogs to compress the fourth ventricle and impede the drainage of csf. i examined the brains in large-area 1 μm resin sections and by tem. by sequential examination of the olfactory bulbs in adult rabbits and the walls of the lateral ventricles in infant dogs, we showed that as the ventricles dilate in the early stages of hydrocephalus, the ependyma ruptures and the flow of csf into the periventricular white matter results in extensive csf oedema with a mild degree of axonal degeneration. as hydrocephalus progresses, the csf oedema subsides and the periventricular white matter displays extensive reactive astrocytosis, and axonal degeneration 4,5. a similar sequence of changes was revealed in biopsies from very young children with acute hydrocephalus 6. these observations suggested that treating children with shunts in the early stages of hydrocephalus would preserve axons and improve prognoses. our findings were confirmed several years later when ct scans clearly revealed the presence of periventricular oedema in the acute stages of hydrocephalus. the sequence of changes of ependymal rupture, increasing csf oedema and destruction of periventricular white matter is well demonstrated in a congenital model of hydrocephalus in mice that i bred later when i returned to guy’s (figure 3). figure 3. progressive hydrocephalus in the mouse showing (a) normal mouse brain. (b) early stages of hydrocephalus with csf oedema of the white matter. (c) severe oedema with destruction of the white matter and preservation of grey matter. (d) sem showing almost total destruction of the cerebral white matter and rupture of the ependyma 7. my family and i spent a most rewarding year in new york. we lived in an apartment block in the bronx very near the einstein. as the months progressed we adapted to the very low temperatures in the winter in new york and the high temperatures and humidity in the summer. our children became bilingual through contact with their friends at nursery school; they would speak with a new york accent and vocabulary between themselves and their friends but spoke english when communicating with francine and me. we explored manhattan widely and travelled to a variety of cities in the us to appreciate the diversity of the country. as we finally left new york in september 1968, we travelled by greyhound bus to a conference in new orleans before flying home – quite an adventure! i have very pleasant memories of the team of people with whom i worked at the einstein (figure 4). through meetings of the american association neuropathologists (aanp), i remained in contact with bob terry for many years. similarly, i retained contacts with henryk wisniewski, who had a very successful research career in demyelinating diseases and in alzheimer’s disease. figure 4. neuropathology group at the albert einstein college of medicine 1968. first row, left to right: drs kinuko suzuki, mariene lenger, krystyna wisniewska, kytja voeller and anne johnson. second row, left to right: drs roy weller, henryk wisniewski, robert d. terry, john andrews, jan leestma and ivan herzog. third row, left to right: drs steven shayvitz, michael shelanski, richard snyder, john prineas and carlos araoz. i have kinuko-suzuki to thank for my much of my training in diagnostic neuropathology and for her friendship that lasts to the present day. of the younger members of the team, michael shelanski became professor of pathology at columbia university in new york, john prineas returned to australia as a neurologist in sydney to continue his research on multiple sclerosis. jan leestma developed a very successful career in forensic neuropathology in chicago; we are still in contact. after the photograph in figure 4 was taken, cedric raine and peter spencer joined bob terry’s department. cedric developed a very successful career in multiple sclerosis research. peter spencer moved into toxicology and together with herb shaumburg published a large multi-author book on clinical and experimental neurotoxicology to which my colleague in southampton, john mitchell, and i contributed a chapter on a fascinating mexican segmental demyelinating neuropathy due to ingestion of fruit and seeds of the buckthorn plant (karwinskia humboltiana) 8. it was rumoured that mexican women would administer small amounts of the fruit or seeds to their husbands so that that they would feel weak and stay at home rather than spending the evenings drinking with their friends. return to england in the fall of 1968, my family and i returned to england and i was appointed lecturer in pathology at guy’s hospital medical school. i continued my close connections with neuropathology by working in peter daniel’s department at the institute of psychiatry in london. my joint appointment allowed me to learn much from peter and from his colleagues. i also broadened my knowledge of clinical diagnostic pathology in general by engaging in the analysis of renal biopsies in collaboration with david turner, a fellow pathologist, and renal physicians, stewart cameron and chisholm ogg. this was a very fruitful time as correlation of the ultrastructural changes in renal biopsies with clinical syndromes was still in its early stages. together with barry nester, i published mid-20th-century diagnoses for three post-mortem kidneys that had been preserved by richard bright in the 1820-30s 9. for me, this was a valuable time for establishing contacts in london and elsewhere in the uk and for considering my ultimate career. during the 1970s i started to regularly attend conferences abroad, mainly in europe. this was essential for my continuing education and for maintaining contact with colleagues. i also benefitted by visiting many interesting countries and cities and meeting a great variety of different people. in 1971, i was fortunate to spend a short period in berlin with jorge cervós navarro that not only greatly increased my experience in neuropathology, but also allowed me to appreciate life in east berlin, then in the soviet bloc, and to visit the famous pergamon museum and its marvellous exhibits from ancient greece. it was through jorge that i was introduced to the german neuropathology society. at this time, trips to interesting nearby attractions were a feature of the german conferences. following a meeting in heidelberg, for example, we visited bad dürkheim and the dürkheimer wurstmarkt (sausage market but in reality the world’s largest wine festival) (figure 5). i retained contact with jorge for many years and together we wrote a book on the “pathology of peripheral nerves” that was published in 1978. figure 5. bad dürkheim (germany), 1971, fotoschiessen. as our colleague in the centre hit the target, the photo was taken with jorge cervós navarro (left) and roy weller (right). the text book – “pathology of peripheral nerves” (lower right) – was one product of my collaboration with jorge. university of southampton school of medicine in early 1972, the opportunity arose to join the new medical school that had been established in 1971 in the university of southampton on the southern coast of the uk. it was a combined national health service (nhs) and university post for which the job description was brief and to the point: “provide a diagnostic service for neuropathology for the wessex regional neurological centre, teach neuropathology and perform research”. following a thorough assessment of the post and the environment in southampton, i applied and was appointed to start in january 1973. i have never regretted the move to southampton. my family and i moved to live in winchester, just north of southampton; an ancient roman city and mediaeval capital of england, one hour by train from london and 40 min by car from heathrow. clinical neuropathology service the 30 years that i worked in southampton were delightful and i greatly appreciated the friendship of my colleagues. my relationships with clinical and university colleagues worked through collaboration and mutual respect for each others’ areas of expertise. the two neurosurgeons, jason brice and john garfield, who were working in the wessex neurological centre (there are now 14 neurosurgeons) when i arrived and the three neurologists, stanley graveson, peter robinson and lee illis (now 21 neurologists) had very professional attitudes. their aim was clearly to provide an excellent clinical service that required a high quality and rapid diagnostic neuropathology service from me, together with weekly multidisciplinary team meetings to discuss individual patients. i had soon realised that my clinical colleagues had many challenges and that my role was to provide them with all the assistance that i could. as we built up an atmosphere of mutual collaboration my life in southampton remained stable and pleasurable. neuropathology in southampton had a defined laboratory with its own staff but it was part of a larger department of histopathology/cell pathology with prof dennis wright at its head. this arrangement had many advantages, not least the wide range of expertise and facilities. regular clinical pathology and research meetings allowed the sharing of ideas and information about new developments. social activities were enthusiastically pursued which helped with cohesion of the department; there was also a constant circulation of pathology trainees from the uk and other countries through neuropathology providing a very interesting international environment. there were some disadvantages, such as competition for space and staff but the advantages heavily outweighed the disadvantages. from the time that i arrived in southampton, i was responsible for neuropathological services for the wessex region of some 3 million people. richard goodbody, who had long been involved in brain tumour diagnoses and in analysing post-mortem brains, very helpfully continued as part-time in this field. i covered brain, nerve and muscle biopsies, ophthalmic biopsies together with post-mortem brains and teaching sessions for trainee pathology staff, clinicians, medical students and nurses. my workload was very high and i was careful to prioritise so that i could cover all fields for which i was employed. i soon acquired a very extensive experience of diagnostic neuropathology and felt able to compile textbooks on neuropathological subjects (see later section) during the 1970s, there were rapid advances in clinical neurology, neurosurgery and neuroradiology with the introduction of ct scanning and mri that had a huge influence on diagnostic neuropathology. tumours and other lesions in the brain and spinal cord were well visualised, but instead of reducing the workload in neuropathology, the number of biopsies steadily increased. there were significant advances in the technical aspects of neuropathology. immunocytochemistry, for both frozen and paraffin sections, and later genetic techniques were incorporated into the diagnostic pathology and neuropathology services. ray hunt and barbara davis led the technical team for neuropathology and together with phillip steart, jean buontempo and judy mepham were always very anxious and able to advance the scope and quality of the service. trainees, fellows and staffing in clinical neuropathology in addition to trainee pathologists and neurosurgeons from southampton rotating through neuropathology, a number of fellows from other institutions joined me over the years. ricardo campora came in the 1970s from seville and worked with me for a year before returning to spain to practice neuropathology. ricardo is extremely able and became professor of pathology and head of department in seville. i am still frequently in touch with ricardo and his wife conchita. for a few years i taught a neuropathology course in seville and received tremendous hospitality there; we also meet on occasion in london. laura chavez from mexico city worked with me as did markus tolnay from basel and jim lowe from nottingham; all have had successful careers in neuropathology and we remain in contact. in the 1980s john grant came to work with me as a trainee in neuropathology. this changed my life as it gave me much more flexibility in how i spent my time. john was always very reliable and a great inspiration; when he left southampton john gained experience in zürich before embarking on a very successful career as a consultant pathologist in cambridge, where he has been head of department for a number of years. patrick gallagher was a consultant cardiac pathologist who also joined the neuropathology diagnostic team which again was of great benefit to the service. david ellison arrived as a trainee in neuropathology in the early 1990s and soon made significant progress in research into the genetics of brain tumours. david became a consultant neuropathologist in southampton before gaining a professorship in newcastle and then moving to memphis, tennessee, to pursue his world-renowned career in the pathology and genetics of childhood brain tumours. soon after david ellison left southampton, james nicoll came from glasgow; he brought tremendous expertise in dementia research, particularly in the roles of microglia. james has made a considerable contribution to the study of post-mortem brains in patients following immunotherapy for alzheimer’s disease. i retired in 2003 and left clinical neuropathology in southampton in the extremely capable hands of james nicoll. after my retirement the clinical neuropathology service was augmented by two very competent consultants, mark walker and mark fabian. research in this account, i take a personal view of how my research progressed in southampton and i include the names of some, but by no means all, of the many people involved. serendipity and the views of my colleagues worldwide played a major role in how my research progressed. i am indebted to the students and postdocs who contributed so much to our research programme and stimulated its progress. a number of themes initially emerged from my research including structural changes in peripheral neuropathies and myopathies, regeneration in nerves and muscle and detailed analyses of brains with growth hormone-related creutzfeldt-jakob disease. postdoctoral fellows, lawrie haynes and gary caine used tumour biopsy material to investigate the effects of pharmacological agents on tissue culture preparations of gliomas. with the introduction of immunocytochemistry for paraffin sections developed by dennis wright and his colleagues in our department, we were able to investigate the uptake of proteins by normal and neoplastic astrocytes. major research themes four major interconnected themes dominated most of my research in southampton and led to advances and revision of scientific concepts. (i) the structure of the leptomeninges and their relationships to the brain. (ii) the detailed pathways for lymphatic drainage of the brain. (iii) the relationship between age-related failure of lymphatic drainage and the accumulation of amyloid-β (aβ) in artery walls in cerebral amyloid angiopathy (caa) and in the brain in alzheimer’s disease. the latter advances were mainly through the work of roxana carare, who following completion of her phd formed her own research group that included cheryl hawkes and many others (figure 6). roxana’s work progressed to identify potential therapies for alzheimer’s disease and caa and to early clinical trials. roxana is now professor of clinical anatomy in southampton. (iv) through a very fruitful and lasting collaboration with britta engelhardt in bern, switzerland, we also investigated the relationship between lymphatic drainage of the brain and neuroimmunological diseases with relevance to disorders such as multiple sclerosis. a list of over 200 peer-reviewed papers that my colleagues and i published can be obtained from pubmed. i will only cite a few of those papers in the following text. figure 6. roxana carare and her group. students and technical staff with cheryl hawkes (right front) next to roy weller and roxana carare. why do research? for me, the main reasons for engaging in research are to satisfy my curiosity and to take the opportunity to present unique data to colleagues at scientific conferences and in scientific journals. i had been exposed to research during my bsc course at medical school and in my early postgraduate years working with john cavanagh and colin adams. bob terry encouraged research in all his fellows and led by example. by the time i arrived in southampton, research was well ingrained within me. southampton has been, for me, an ideal environment for research through excellent facilities and multiple disciplines within the university available for consultation and collaboration. with a heavy diagnostic workload in biopsies and post-mortem brains, i was exposed to many unsolved problems in human neurobiology and neuropathology that provided a stimulus for hypothesis-driven experimental research. another important stimulus for research was the requirement that each medical student in southampton perform a research project from october to may in the fourth year of the course. this meant that a constant stream of two or three highly intelligent, hard-working enthusiastic 22 year old students worked in neuropathology each year with their individual projects but collaborating closely with bsc and phd students, post-docs and research fellows. my strategy was to begin a research theme with straight-forward observational projects for the medical students that generated hypotheses to drive experimental research for the phd students and postdoctoral fellows. many students presented papers at british neuropathological society (bns) meetings and published papers in peer-reviewed scientific journals. all groups benefitted their future careers by acquiring skills in spoken and written communication. how did i benefit from students and others involved in research projects? the students educated me by their observations, the papers that they read and from the knowledge that they had gained in their recent medical education. retaining an open mind was very important: each student was given a problem to solve but advised not to read the previous literature until they had results and could form their own opinions. there were numerous occasions upon which students burst into my office crying “the literature is wrong!” in this way they built up a healthy scepticism for previous findings. i advised students not to believe anything they read and not to believe anything that i told them until they had verified it for themselves. structure of the human leptomeninges and their relationship to the brain and blood vessels although my interest in fluid balance in the brain started with my work on hydrocephalus in new york, it was reawakened in southampton by student projects using scanning electron microscopy (sem) to examine human leptomeninges. margaret upton 11 identified the intricate pattern of channels by which csf passes through arachnoid granulations to the blood in venous sinuses. margaret hutchings made a startling discovery in 1986 using sem on the surface of human brain by showing that the pia mater is reflected from the surface of the brain on to arteries and veins in the subarachnoid space thus separating csf in the subarachnoid space from the brain 10 (figure 7). this exploded the longstanding concept that there was a direct communication between csf and brain. margaret also showed that erythrocytes in subarachnoid haemorrhage do not penetrate the pia mater to enter the brain. we later confirmed that solutes and macrophages do pass through the pia mater that forms a continuous sheet on the surface of well-fixed normal brain as shown by ruth alcalado 12. no pores in the pia have been identified by tem and the holes seen in sem images are probably post mortem or fixation artefact. figure 7. sem of the meningeal surface of the human cerebral cortex showing the arachnoid mater (ar) and branches of an artery (a) spreading over the surface of the pia mater (p). the circle depicts the point at which a branch of the artery enters the cortex and how the pia mater is reflected on to the artery thus separating the subarachnoid space from the brain. (b) is an enlarged view of the reflected pia mater (p). artefactual holes are seen in the pia in this post-mortem specimen. arrow (lower left) identifies a filiform trabecula crossing the subarachnoid space 10. in the 1980s, en-tan zhang came from beijing to work with me in southampton. en-tan is a medically qualified anatomist and he made a major contribution to establishing the relationships between the leptomeninges and arteries as they enter the brain. using well fixed human biopsy brain material, en-tan showed that a single layer of pia mater coats arteries as they enter the surface of the cerebral cortex and that there is no “periarterial space” between the glia limitans and the artery wall 13. astrocytes in the glia limitans, pia mater, smooth muscle cells and endothelium together with the intervening layers of basement membrane form a compact structure, with no perivascular space (figure 8). the long-described virchow-robin spaces do not exist in the cortex and if present they are mainly due to swelling of astrocyte processes from poor fixation as emphasised by milton brightman in the 1960s. en-tan’s observations completely changed our perception of the relationship between csf and the brain. subsequently, roxana carare’s group showed that tracers from the csf enter the brain, not along perivascular spaces (as there are none) but along basement membranes between the glia limitans and the single layer of pia mater that surrounds each artery as it enters the brain 14. basement membranes between smooth muscle cells in the tunica media were later shown by roxana carare to be the pathways for flow of fluid and solutes out of the brain. due to the absence of periarterial spaces, there are no periarterial pathways for the entry of inflammatory cells from the csf into the brain. periarterial spaces do form in other the parts of the brain, especially in the basal ganglia and around arteries in the white matter. at both of these sites, arteries are surrounded by two layers of pia mater and the periarterial space develops between them 15,16. figure 8. tem of a well-fixed human cortical artery showing part of the wall and surrounding brain. the layers of glia limitans, pia mater, smooth muscle cells and endothelium adjacent to the lumen are all compacted with no preriaterial space (see also 13). lymphatic drainage of the brain there are two extracellular fluids associated with the brain and spinal cord: (i) csf in the ventricles and subarachnoid spaces and (ii) interstitial fluid (isf) in the extracellular spaces of the brain and spinal cord. pathways for lymphatic drainage of csf are almost completely separate from lymphatic drainage of isf from the brain. lymphatic drainage of csf shinya kida, a neurosurgeon from japan, joined us in southampton in the early 1990s and embarked upon a search for the drainage pathways for csf in the rat with a medical student andreas pantasis. lymphatic drainage of csf to cervical lymph nodes had been demonstrated by schwalbe in 1869, and bradbury and cserr showed in the 1980s that lymphatic drainage of csf occurred through the cribriform plate of the ethmoid bone but the details were unclear. shinya and andreas 17 demonstrated direct drainage of indian ink tracer from the subarachnoid space along distinct vessels that pass through the cribriform plate alongside olfactory nerves into the nasal submucosa en route to cervical lymph nodes. such channels were also shown to exist in post mortem humans brains by the injection of myodil tracer into the csf 18 and by reconstruction from serial sections of the human cribriform plate and nasal mucosa by effie djuanda in her fourth year medical student project 19. other channels for drainage of csf identified in these studies included dural lymphatics and the sheaths of cranial nerves 17,20. drainage of csf by the nasal route has been demonstrated in humans using pet scanning 20. the balance between lymphatic drainage of csf to cervical or lumbar lymph nodes and drainage through arachnoid granulations has yet to be determined. the small size and paucity of arachnoid villi in the rat suggests that they are quantitatively less important for the drainage of csf in rodents than lymphatic drainage. in humans, arachnoid granulations do not develop until the age of two years which suggests that lymphatic drainage may also be an important route for the drainage of csf in humans. arachnoid granulations may compensate for variations in csf pressure that occur with change of body position; this speculation still requires direct verification. lymphatic drainage of interstitial fluid (isf) and solutes from the brain there are no conventional lymphatic vessels in the brain or spinal cord. in the 1980s helen cserr and her colleagues 21 injected minute amounts of radioactive human serum albumin as a tracer into the basal ganglia of the rat brain, and showed that the tracer drained to cervical lymph nodes along the walls of cerebral arteries. only 10-15% of tracer leaked into the csf emphasising that the pathways for lymphatic drainage for csf from the subarachnoid spaces are separate from drainage of isf from the brain itself. en-tan zhang attempted to define the drainage pathways for isf in more detail in the late 1980s by injecting indian ink particles directly into the basal ganglia of the rat brain. the ink particles tracked along the outside of arteries, were taken up by perivascular macrophages and remained in situ 22. this technique did not identify the drainage pathways for isf but did identify a pathway that was later used for convection-enhanced delivery (ced) of drugs directly into the brain 23. the results from en-tan’s work led to discussions with alex roher from phoenix, arizona, who suggested that cerebral amyloid angiopathy (caa) may be related to periarterial lymphatic drainage of the brain. we subsequently published a joint paper in american journal of pathology in 1998 with the title “cerebral amyloid angiopathy: amyloid β accumulates in putative interstitial fluid drainage pathways in alzheimer’s disease“ 24. breakthrough a major breakthrough in our research on lymphatic drainage of the brain came from the work of roxana carare in her phd project that i supervised jointly with hugh perry. roxana injected formalin-fixable fluorescent dextrans or soluble amyloid-β (aβ) into mouse brains and examined the drainage pathways by confocal microscopy. in her paper, “solutes, but not cells, drain from the brain parenchyma along basement membranes of capillaries and arteries: significance for cerebral amyloid angiopathy and neuroimmunology” 25, roxana defined the pathways for the lymphatic drainage of fluid and solutes from brain tissue that we later termed the “intramural peri-arterial drainage (ipad) pathways” 14. following the injection of minute volumes (0.5 μl) of tracer into the caudate nucleus of the mouse brain, tracer spread through the extracellular spaces of the brain and within 5 mins had entered the lymphatic drainage pathway (ipad) in the basement membranes of capillaries and basement membranes between smooth muscle cells in the tunica media of cerebral arteries. the drainage pathway (ipad) extended into leptomeningeal arteries. no tracer was seen around veins in normal animals following intracerebral injections although animals with age-related impairment of ipad did show some tracer around veins 26. confusion considerable confusion in the scientific literature has resulted from the injection of excessive volumes of tracer into the mouse brain that results in leakage of tracer into the csf giving the false impression that lymphatic drainage of the brain occurs via the csf. furthermore, injecting excessive amounts of tracer into the brains of mice and not examining the animals until 1 hour later, after ipad is complete, result in pooling of tracer around veins and has given the erroneous impression that lymphatic drainage of the brain is along the walls of veins. supporting evidence for ipad in the human brain caa in mice and humans provides convincing supporting evidence that ipad is the route for lymphatic drainage of fluid and solutes from the brain parenchyma. (i) the pattern of distribution of aβ in capillary and artery basement membranes in caa exactly mirrors the distribution of aβ and other tracers in ipad pathways in the animal experiments 27. (ii) caa occurs in transgenic mice in which there is excessive production of aβ in neurons; in these mice the origin of the aβ is definitely from cells in the brain 28. age-related failure of elimination of aβ by ipad is associated with caa and alzheimer’s disease by combining experimental studies in the mouse with observations of human caa and alzheimer’s disease, the following main conclusions have been reached as summarised in 29. ipad is impaired by age-related changes in the walls of arteries and by caa 26. both theoretical models and experimental studies suggest that the motive force for ipad is derived from waves of smooth muscle cell contraction (vasomotion) that pass along the walls of cerebral arteries in the opposite direction to blood flow, but in the same direction as ipad 29,30. age and the presence of apolipoprotein e ε4 (apoe4) is associated with the impaired ipad 31 and this correlates with age and apoe4 as major risk factors for alzheimer’s disease and caa in humans. caa also affects capillaries; painstaking cutting of hundreds of serial transverse sections of arteries in regions of capillary caa by hong yeen yow (bsc student) suggest that capillary caa is associated with thrombotic occlusion of penetrating cortical arteries 19. age-related impairment of ipad appears to be associated with loss of homeostasis in the brain as reflected by accumulation of fluid in white matter hyperintensities on mri and the failure of elimination of aβ with age and in alzheimer’s disease. dilatation of periarterial spaces in the white matter is associated with caa and probably reflects failure of ipad. translational neuroscience: therapeutic strategies for caa and alzheimer’s disease: facilitating ipad for the past 20 years there have been many trials of immunotherapy for alzheimer’s disease but with little success in reducing cognitive decline in the treated patients. studies of post mortem brains following aβ immunotherapy have shown that although aβ plaques are removed from the cerebral cortex both arterial and capillary caa are increased in severity 32,33. it does appear that age-related failure of ipad is a rate-limiting step in aβ immunotherapy and this emphasises the importance of facilitating ipad in the treatment of caa and alzheimer’s disease. potential new therapeutic strategies could act via ipad by modulating vasomotion as the postulated motive force for ipad. this may be accomplished by stimulating contraction of vascular smooth muscle cells directly or through their nerve supplies. another therapeutic possibility is the use of chaperone molecules to enhance the clearance of aβ along the basement membranes that form the ipad pathways 29. lymphatic drainage of the brain and neuroimmunology the brain is an immunologically privileged site 34 as shown by experiments in which skin allografts implanted in the brain survive for longer periods than allografts in other organs. rapid rejection of brain allographs does occur, however, when similar allografts are implanted into the animal’s skin. the leptomeninges and csf spaces do not show the same immunological privilege as brain tissue 34. one of the major differences is that csf drains directly into lymphatic vessels that allow fluid, antigens and antigen presenting cells to drain directly from the csf to lymph nodes. ipad pathways are too narrow to allow the traffic of apcs from the brain to lymph nodes 25. this may be a major factor in immunological privilege of the brain. cervical lymph nodes have a major role in immunological reactions in the brain as shown by marian phillips, dong sun (ph.d. students), jonathan lake and michelle needham (bsc medical students) and by jon laman and his group 35-37. our first task was to enhance experimental autoimmune encephalomyelitis (eae), in the cerebral hemispheres of rats so that accurate measurements of inflammation could be made. in most models of eae following injection of antigen into the footpads, inflammation is concentrated in the spinal cord. however, shinya kida had previously shown that a wound to the surface of the rat cerebral cortex in the form of a cryolesion resulted in activation of perivascular macrophages throughout the cerebral hemispheres. thus we showed that a cryolesion on the surface of the cortex in rats, 7 days post injection (dpi) of eae antigens into the foot pad of the rat resulted in enhanced inflammation in the cerebral hemispheres at day 15 dpi. removal of cervical lymph nodes on day 7 dpi resulted in a 50% reduction in eae-related inflammation at day 15 dpi. these results suggest that cervical lymph nodes play a key role in cerebral eae 35. furthermore, adoptive transfer of lymphocytes from animals with cryolesion eae resulted in a predominance of eae lesions in the cerebral hemispheres in naive recipients rather than spinal cord eae 36. our involvement in neuroimmunology brought us into contact with britta engelhardt in bern and her excellent studies on the receptor-mediated entry of lymphocytes into the brain through post-capillary venules. we are still in collaboration 34. figure 9. summary of pathways for the flow of fluid and solutes into and out of the brain. (a) csf flows into the brain along basement membranes shared by pia mater and glia limitans on outer surfaces of cortical arteries as they penetrate the brain. (b) interstitial fluid (isf) and solutes such as aβ flow out of the brain to lymph nodes along ipad pathways. (c) details of the pathway by which csf flows into the cerebral cortex along pial-glial basement membranes and then enters the brain to flow out along ipad. reproduced from 29. original design by roy weller and graphic by artfact graphics. award of the gertrud-reemtsma prize for international translational neuroscience 2020 figure 10. the prize winners of the international prize for translational neuroscience 2020: left to right: roy weller, maiken nedergaard, and mathias jucker. i was greatly honoured by the award of the international prize for translational neuroscience by the council of the gertrud reemstma foundation, administered by the max planck society. the prize had been the k. j. zülch-prize until 2019 with many famous and worthy winners from 1990. i was awarded the prize together with maiken nedergaard and mathias jucker (figure 10). my award was for the southampton group’s “ground-breaking scientific discoveries in the field of lymphatic drainage of the brain and its relationship to the aetiology of alzheimer’s disease”. of course the prize was awarded as much of the work performed by the many medical students, ph.d. students and postdocs who worked with me in southampton. roxana carare, cheryl hawkes, en-tan zhang, shinya kida and dong sun deserve special mention. nevertheless, i felt it was a great honour for our group. due to covid-19 i could not attend the award ceremony on september 10th 2020 in cologne, germany, in person so i presented my lecture on zoom. management roles throughout most of my career in southampton i was involved in management in the nhs, southampton university, and in regional, national and international organisations. it was part of my role to assist with the organisation of the institutions to which i belonged. furthermore i was rewarded by meeting many interesting people while performing management tasks. within two years of my arrival in southampton, i became chairman of the whole of pathology; this was in a very active period when all pathology departments were moving into a newly-built pathology block. at that time, in the mid-1970s, there were virtually no management courses and i learnt the necessary skills by observing others, how they chaired meetings, coordinated events and managed people so that the whole system ran smoothly and within budget. soon after my promotion to professor of neuropathology in 1978, i became deputy dean of medicine, which included, among other very interesting tasks, chairing the committee for selection of medical students; this gave me insight into the great variety of skills exhibited by the applicants to medical school. following my four-year term as deputy dean, i was appointed chairman of the university library committee. not only did this coincide with the delivery to the library of the papers of the duke of wellington, but it was also the time that the library acquired the papers of lord louis mountbatten. the papers were markedly different in their condition. the wellington papers were written with indian ink on parchment such that they could be cleaned with large india rubber balls in sinks of cold water. the mountbatten papers, on the other hand, were the fourth or fifth typed copy and required urgent transcription. for some 20 years i chaired various regional and national committees that ensured high quality training of pathologists locally in wessex and nationally through the royal college of pathologists. this was the time during which neuropathology became an independent discipline within the royal college of pathologists and training and examinations were formalised. the establishment of the european confederation of neuropathological society (eurocns) also allowed a european program of education and qualifications in neuropathology to be established. british neuropathological society the british neuropathological society (bns) plays valuable roles in neuropathology in the uk, both in monitoring the quality of clinical neuropathology and in encouraging research. the annual bns scientific meeting is always very interesting and informative. in 2000, the bns organised an international neuropathology society congress in birmingham, uk, with some 700 delegates. i chaired the committee that organised the scientific programme and david graham was president. due to the hard work and diligence of my many colleagues, the congress was a great success. i was elected president of the bns for 2001-2002. major figures such as hume adams and david graham in glasgow, james ironside and jeanne bell in edinburg, raj kalaria in newcastle, james lowe in nottingham, seth love in bristol, peter lantos in london and many others played very significant roles in organising the bns such that the bns conferences attracted significant numbers of neuropathologist from europe, usa and other countries. editor of neuropathology and applied neurobiology in 1975, john cavanagh founded the journal neuropathology and applied neurobiology (nan), that was adopted by the bns as the society journal. i followed john as editor in chief of nan between 1988 and 1998. this was a most rewarding time for me, organising the publication of research articles and review articles from an international body of authors. i learned much about the organisation of scientific journals and gradually nan started to make a profit that has benefited the bns financially. john had organised nan with the original publishers, blackwell, so that the profits were shared between the publisher and the bns. elizabeth whelan was my main contact with the publisher and our relationship was excellent throughout my time as editor, largely due to elizabeth’s skill and dedication. james lowe followed me as editor in 1999 and there has been a succession of very successful editors since. the impact factor of nan has increased steadily from 3.9 in 2014 to 7.09 in 2019. tom jacques is the present editor in chief and nan continues to play a significant role in international neuropathology. editorial boards i have been a member of editorial boards of a number of journals but the ones i remember in particular are the boards of brain pathology, acta neuropatholgica and more recently, free neuropathology. werner paulus made a particular impression on me as editor of acta neuropatholgica; his very inspired decisions on which papers to accept for the journal ensured that the impact factor for acta neuropatholgica rose to 18. werner is the founding editor in chief of free neuropathology. books published 1978-2013 i decided to harness the clinical material associated with the diagnostic service to publish a number of text books; this was partly as a self-education exercise. some books i wrote alone or with another single author or i acted as editor as well as a contributor. the rewards for my efforts were that writing and editing consolidated my knowledge of neuropathology and built lasting friendships with multiple authors who showed great tolerance for my editorial changes. my first was “pathology of peripheral nerves” by r o weller and j cervós-navarro, published by butterworths, london in 1978 and my second resulted from a course that we ran in southampton: “clinical neuropathology.” a multi-author book edited by r o weller, m swash, dl mclellan and c l scholtz, published by springer verlag, berlin in 1983. the japanese edition of “clinical neuropathology” was translated by t mientani and published by nishimura co ltd in 1983. “a colour atlas of neuropathology” by r o weller, published by harvey miller and oxford university press in 1984 and i contributed to “mcalpine’s multiple sclerosis”, by w b matthews, e d acheson, j r bachelor and r o weller. edited by w. b. matthews, published by churchill livingstone, edinburgh in 1985. as part of “systemic pathology”, third edition: general editor, w st c symmers. i edited a multi-author “volume 4: nervous system, muscle and eyes”, published by churchill livingstone, edinburgh in 1990. in 2002 i contributed to “diagnostic pathology of nervous system tumours”, by j w ironside, t h moss, d n louis, j s lowe and r o weller, published by churchill livingstone, edinburgh. i was appointed director of the book series sponsored by the international society of neuropathology (isn), publishing “neurodegeneration. the molecular pathology of dementia and movement disorders”, second edition. edited by d w dickson and r o weller. series director, r. o. weller. published by wiley-blackwell, chichester, uk in 2011. as “neurodegeneración”, the book was published in spanish by editorial medica panamericana, madrid and buenos aires. the second book in the isn series was “muscle disease: pathology and genetics”, second edition. edited by h h goebel, c a sewry and r o weller, series director, r. o. weller. published by wiley-blackwell, chichester, in 2013. i also contributed chapters to a number of books including: gray’s anatomy, greenfield’s neuropathology, clinical and experimental neurotoxicology and histology for pathologists. international conferences and visits to other neuropathology units attending conferences was essential for my continuing education and for maintaining contact with colleagues. i also benefitted from visiting many interesting countries and cities throughout the world and meeting a great variety of different people. i regularly participated in the annual meetings of the british, french and german neuropathology societies and occasionally, the scandinavian and italian neuropathology society meetings. i developed strong friendships with many members of those societies; francoise gray, jacqeline mikol and homa adle-biassette to name but a few. a particular favourite conference of mine was the meeting of the swiss society of neuropathology in st. moritz every two years. not only was the meeting held amidst beautiful scenery, the train journey was spectacular and there was ample opportunity for skiing and social interaction for discussing scientific data. i made many friends at these meetings. in addition to european meetings, i attended many conferences in the usa, including annual meetings of the american association of neuropathology. other conferences to which i contributed include those in india, australia and brazil. photographs of many of the friends that i made at these international meetings appear in an excellent autobiography by sam ludwin 38. in 1988 paul kleihues kindly invited me to work for four months in zürich. there was a most interesting group of young neuropathologists working with paul at the time, otmar wiestler, andreas von deimling and adriano aguzzi all of whom subsequently developed very successful careers and with whom i developed lasting friendships. while working for six weeks in harare in zimbabwe in 1991, i was involved mainly in teaching with occasional and most stimulating visits by car to other parts of the country. martin lewis was there at the time; he was an english pathologist working in florida and we made a long tour around zimbabwe in a 1968 mercedes car that he had borrowed. martin had extensive experience of africa that is outlined in his book “the call of africa”. particularly rewarding were my visits to beijing after my retirement at the invitation of wei wei zhang. quite apart from her tremendous hospitality, wei wei and her husband accompanied me on very interesting trips to other chinese cities to meet many researchers and clinicians and to see the sights. i shall ever be indebted to professor zhang for introducing me to so much chinese culture and cuisine. post-retirement when i was about to retire, i realised that leaving what seemed like a lifetime in neuropathology would be very difficult. through the kindness of my colleagues, i took gradual retirement over 4-5 years moving to part-time involvement in clinical diagnostic neuropathology. i also remain involved on a consultancy basis with professor roxana carare’s group who, following her ph.d., developed a very successful research programme in southampton with substantial grant income. i watched with great interest and some advice to the development of the ipad work and i was involved in the discussion during the preparation of papers for publication. this was very helpful in my disengagement from neuropathology. in addition, i was asked to be chairman of the biomedical grants advisory board for the alzheimer’s society from 2013 to 2018 which kept me in touch with developments in research into alzheimer’s disease. living in winchester gave me the opportunity to become a guide in the cathedral. this 900-year-old building and its contents are a mine of information regarding british history in the last 2000 years. i edited the winchester cathedral record that published research articles concerning the cathedral for four years and i have been actively involved in researching the cathedral for my own benefit and the benefits of my fellow guides and visitors. family if it had not been for the support that i have received from my family, my career in medicine and neuropathology would not have been possible. my loving parents greatly encouraged me in my education and my wife, francine, has offered her full support to me and to our moves around the uk and abroad. throughout our 60 years of happy marriage, i have admired francine’s independence and i am indebted to her for expanding my knowledge and involvement in the arts, especially music and literature. we have both sung in choirs for almost the whole of our married life and continue to do so with great enjoyment. our two children, adrienne and timothy have been a huge joy to us as have their spouses and our four grandchildren. acknowledgments i would like to thank professor roxana carare for all her help in the final editing and submission of the paper to free neuropathology. references weller ro. diphtheritic neuropathy in the chicken: an electron-microscope study. j pathol bacteriol 1965; 89: 591-8. weller ro. cytochemistry of lipids in atherosclerosis. j pathol bacteriol 1967; 94(1): 171-82. weller ro, clark ra, oswald wb. stages in the formation and metabolism in intracellular lipid droplets in atherosclerosis. an electron microscopical and biochemical study. j atheroscler res 1968; 8(2): 249-63. weller ro, wisniewski h. histological and ultrastructural changes with experimental hydrocephalus in adult rabbits. brain 1969; 92(4): 819-28. weller ro, wisniewski h, shulman k, terry rd. experimental hydrocephalus in young dogs: histological and ultrastructural study of the brain tissue damage. j neuropathol exp neurol 1971; 30(4): 613-26. weller ro, shulman k. infantile hydrocephalus: clinical, histological, and ultrastructural study of brain damage. j neurosurg 1972; 36(3): 255-65. weller ro. pathology of cerebrospinal fluid and interstitial fluid of the cns: significance for alzheimer disease, prion disorders and multiple sclerosis. j neuropathol exp neurol 1998; 57(10): 885-94. mitchell j, weller ro, evans h, arai i, daves gd, jr. buckthorn neuropathy: effects of intraneural injection of karwinskia humboldtiana toxins. neuropathol appl neurobiol 1978; 4(2): 85-97. weller ro, nester b. histological reassessment of three kidneys originally described by richard bright in 1827-36. br med j 1972; 2(816): 761-3. hutchings m, weller ro. anatomical relationships of the pia mater to cerebral blood vessels in man. j neurosurg 1986; 65(3): 316-25. upton ml, weller ro. the morphology of cerebrospinal fluid drainage pathways in human arachnoid granulations. j neurosurg 1985; 63(6): 867-75. alcolado r, weller ro, parrish ep, garrod d. the cranial arachnoid and pia mater in man: anatomical and ultrastructural observations. neuropathol appl neurobiol 1988; 14(1): 1-17. zhang et, inman cb, weller ro. interrelationships of the pia mater and the perivascular (virchow-robin) spaces in the human cerebrum. j anat 1990; 170: 111-23. albargothy nj, johnston da, macgregor-sharp m, et al. convective influx/glymphatic system: tracers injected into the csf enter and leave the brain along separate periarterial basement membrane pathways. acta neuropathol 2018; 136(1): 139-52. doi: 10.1007/s00401-018-1862-7. macgregor sharp m, bulters d, brandner s, et al. the fine anatomy of the perivascular compartment in the human brain: relevance to dilated perivascular spaces in cerebral amyloid angiopathy. neuropathol appl neurobiol 2019; 45: 305-8. pollock h, hutchings m, weller ro, zhang et. perivascular spaces in the basal ganglia of the human brain: their relationship to lacunes. j anat 1997; 191(pt 3): 337-46. kida s, pantazis a, weller ro. csf drains directly from the subarachnoid space into nasal lymphatics in the rat. anatomy, histology and immunological significance. neuropathol appl neurobiol 1993; 19(6): 480-8. johnston m, zakharov a, papaiconomou c, salmasi g, armstrong d. evidence of connections between cerebrospinal fluid and nasal lymphatic vessels in humans, non-human primates and other mammalian species. cerebrospinal fluid research 2004; 1: 2-15. weller ro, djuanda e, yow hy, carare ro. lymphatic drainage of the brain and the pathophysiology of neurological disease. acta neuropathol 2009; 117(1): 1-14. de leon mj, li y, okamura n, et al. cerebrospinal fluid clearance in alzheimer disease measured with dynamic pet. j nucl med 2017; 58(9): 1471-6. szentistvanyi i, patlak cs, ellis ra, cserr hf. drainage of interstitial fluid from different regions of rat brain. american journal of physiology 1984; 246: f835-44. zhang et, richards hk, kida s, weller ro. directional and compartmentalised drainage of interstitial fluid and cerebrospinal fluid from the rat brain. acta neuropathol 1992; 83(3): 233-9. barua nu, bienemann as, hesketh s, et al. intrastriatal convection-enhanced delivery results in widespread perivascular distribution in a pre-clinical model. fluids barriers cns 2012; 9: 2. weller ro, massey a, newman ta, hutchings m, kuo ym, roher ae. cerebral amyloid angiopathy: amyloid beta accumulates in putative interstitial fluid drainage pathways in alzheimer’s disease. am j pathol 1998; 153(3): 725-33. carare ro, bernardes-silva m, page am, nicoll ja, perry vh, weller ro. solutes, but not cells, drain from the brain parenchyma along basement membranes of capillaries and arteries: significance for cerebral amyloid angiopathy and neuroimmunology. neuropathol appl neurobiol 2008; 34(2): 131-44. hawkes ca, hartig w, kacza j, et al. perivascular drainage of solutes is impaired in the ageing mouse brain and in the presence of cerebral amyloid angiopathy. acta neuropathol 2011; 121(4): 431-43. carare ro, hawkes ca, jeffrey m, kalaria rn, weller ro. review: cerebral amyloid angiopathy, prion angiopathy, cadasil and the spectrum of protein elimination failure angiopathies (pefa) in neurodegenerative disease with a focus on therapy. neuropathol appl neurobiol 2013; 39(6): 593-611. herzig mc, van nostrand we, jucker m. mechanism of cerebral beta-amyloid angiopathy: murine and cellular models. brain pathol 2006; 16: 40-54. carare r, aldea r, agarwal n, et al. clearance of interstitial fluid (isf) and csf (clic) group—part of vascular professional interest area (pia). cerebrovascular disease and the failure of elimination of amyloid-β from the brain and retina with age and alzheimer’s disease-opportunities for therapy. alzheimer’s dement 2020; 12: e12053. aldea r, weller ro, wilcock dm, carare ro, richardson g. cerebrovascular smooth muscle cells as the drivers of intramural periarterial drainage of the brain. front aging neurosci 2019; 11: 1. hawkes ca, sullivan pm, hands s, weller ro, nicoll ja, carare ro. disruption of arterial perivascular drainage of amyloid-beta from the brains of mice expressing the human apoe ε 4 allele. plos one 2012; 7(7): e41636. nicoll ja, wilkinson d, holmes c, steart p, markham h, weller ro. neuropathology of human alzheimer disease after immunization with amyloid-beta peptide: a case report. nat med 2003; 9(4): 448-52. boche d, nicoll j, weller r. immunotherapy for alzheimer’s disease and other dementias. curr opin neurol 2005; 18(6): 720-5. engelhardt b, vajkoczy p, weller ro. the movers and shapers in immune privilege of the cns. nat immunol 2017; 18(2): 123-31. phillips mj, needham m, weller ro. role of cervical lymph nodes in autoimmune encephalomyelitis in the lewis rat. j pathol 1997; 182(4): 457-64. lake j, weller ro, phillips mj, needham m. lymphocyte targeting of the brain in adoptive transfer cryolesion-eae. j pathol 1999; 187(2): 259-65. van zwam m, huizinga r, heijmans n, et al. surgical excision of cns-draining lymph nodes reduces relapse severity in chronic-relapsing experimental autoimmune encephalomyelitis. j pathol 2009; 217(4): 543-51. ludwin s. autobiography series: in search of knowledge and joy: my life as a neuropathologist. j neuropathol exp neurol 2018; 77(2): 162–75. 🎉 🎂 celebrating professor roy weller: 🎊 🥂 83 years old, over 5 decades of contributions to neuropathology prof johannes attems consultant neuropathologist, newcastle university and editor, acta neuropathologica the first time i met professor roy weller was at the british neuropathological society winter meeting in 2001; in the break after the session where i had presented, he the famous professor approached me the unknown junior doctor from vienna to discuss my talk! it was a nearly 20 minute discussion about the pathogenesis of cerebral amyloid angiopathy and professor weller listened to what i had to say and even produced some drawings illustrating the perivascular clearance. this first meeting with professor weller was hugely inspiring and motivating and certainly had a big impact on my decision to continue with research into neuropathology and i still think often back to this day in 2001! naturally many more stimulating discussions between us followed (on all topics, professional and private) and i am looking forward to many more discussions in the future. very many thanks for all your support roy! prof roxana carare clinical neurosciences, university of southampton roy weller has catalyzed and energized the research into lymphatic drainage of the brain, inspiring and allowing us to pursue this with translational value for cerebral amyloid angiopathy and alzheimer’s disease. he has taught me and many others the scientific method and testing hypotheses based on a solid overview of past and present observations on the human brain. the research group that i lead is a result of his continuous efforts to help with the direction of research, interpreting results, analytical, critical and forward-thinking discussions. we are grateful for his attention to detail and educating us to be researchers of high integrity and a deep quest for advancing science. prof mony de leon weill cornell medicine director, brain health imaging institute i am delighted to celebrate professor roy weller’s 80th birthday by reflecting on his contributions to pathology and to radiology. however, using just a few words to do this, owing to his extensive and influential scholarly history, is a remarkably difficult but a highly refreshing task. taking advantage of a personal and arbitrary starting point, my first encounter with roy’s work was on his work in dementia and hydrocephalus. weller, a longtime developer of animal models of hydrocephalus and an advocate for innovative dementia treatment options, would write in the j neurol neurosurg psychiatry 1989, “when confronted with a patient with dementia, gait dyspraxia, and incontinence, we suggest that it is more realistic to look for a remediable hydrocephalic component to the illness than to consider that the patient must have either alzheimer’s disease, multi-infarct dementia, or normal pressure hydrocephalus alone.” in the late 1970s i was a gerontology student studying alzheimer disease (ad) and in 1980 i received an nyu-brookhaven national lab (bnl) neuroimaging post-doctoral fellowship. with the emergence of human imaging studies during the late 1970s, my mentor and colleague neuroradiologist ajax george and i were awarded our first nih-nia funded studies of ct atrophy in dementia and in 1980, collaboration with alfred wolf and joanna fowler at bnl, nih-ninds funded fdg-pet studies of ad began. at the time, it was commonly believed that imaging had little to contribute to the ad diagnosis due to background aging effects. one real-world test of our ad diagnostic imaging work was to evaluate if our measurements differentiated between ad and hydrocephalus. we quantified ct scans for regional ventriculomegaly, evaluated compressed sulci and csf pooling, we published and post mortem validated a ct scale for periventricular white matter pathology, and tested whether the hippocampus, a site of early ad pathology, was preserved in hydrocephalus. we ran fdg-pet scans before and after shunting looking for metabolic recovery, and collected biopsy samples during shunting for evidence for ad. this work led to another point of connection between my work and roy weller’s, interactions with two pathologists henry wisniewski and robert terry (both deceased) who both worked with roy and who would become my valued friends and advisors. today, the radiological diagnosis is an essential part of the differential dementia examination, and multiple avenues of imaging research, guided by roy’s insights, continue. one such insight, and clearly for me, roy’s most important contribution was for his anatomico-physiological studies examining the normal and pathological clearance of csf and isf from the brain. these studies, which continue today, are now led at the university of london in southampton by professor roxana carare, a former weller mentee. historically, these studies revealed the anatomical pathways for isf drainage along basement membranes surrounding smooth muscle cells of cerebral arteries and along capillaries, and provided evidence that impaired interstitial flow contributed to parenchymal and vascular amyloid accumulations as well as being negatively impacted by amyloid accumulations. this mechanistic message was heard by many others, including the radiological community where currently pet and mri imaging studies are underway today translating the weller and carare observations of a direct link between impaired vascular biology and ad and most recently other neurological diseases defined by misfolded proteinopathies. thus, we wish roy a happy birthday and applaud him and his colleagues for their continued efforts to treat dementia by providing pathology grounded data and testable hypotheses. prof margaret esiri consultant neuropathologist, john radcliffe hospital, oxford i first encountered roy at my first bns meeting in (probably) 1971 or 1972. he came over as a calm, confident deliverer of a presentation – i’ve forgotten what it was on. i think he may have been at guy’s at the time. i was very impressed! i have gone on being impressed with roy’s very significant contributions to neuropathology ever since. long may they continue! happy birthday, and many more to come, roy! prof masafumi ihara head of the cardiovascular centre, osaka, japan it is my great pleasure to contribute to an article celebrating prof. weller's 80th birthday. prof. weller is an internationally recognized neuropathologist and well-known to have established the important concept of perivascular drainage pathway (now called ipad) as an abeta clearance system. i had been inspired by the concept and had a great opportunity to meet him in the xviith international congress of neuropathology in salzburg (icn 2010). i remember i had a good discussion with him in front of a poster of his team. i visited his and prof. carare’s laboratory in southampton with three of my phd students in 2012 and have been collaborating with the southampton team. our collaboration led to my investigator-led clinical trial of a vasoactive drug for mci patients to determine whether ipad can be facilitated clinically. dr. satoshi saito, my former phd student, joined the southampton team in 2018, came back to japan in 2020 after learning a lot not only from prof. carare but also from prof. weller, and is now working as a talented physician scientist in my department. thus, prof. weller academically guided us as an established neuropathologist but not only that, he very kindly acted as a tour guide of winchester for me a few years ago! prof. weller has continued impacting my research team for over 10 years with his plentiful neuropathological knowledge and we would like to send our heartfelt congratulations to prof. weller. prof raj kalaria neurovascular pathologist, newcastle university and chairman, international society for vascular behavioral and cognitive disorders (vascog) i had first heard about roy from helen cserr, many moons ago! besides the ‘art’ of neuropathology, roy’s profound writings on drainage pathways of the brain intrigued me. in the late 1980s when i was still at case western reserve university training in vascular neuropathology, i had communicated with roy about a paper in neuropathology and applied neurobiology. at the time, roy was editor-in-chief of the journal. few years later, i met roy face-to-face when i was interviewed for a job in southampton with the hope that the family and i could quickly return to england. i did not get the post but i thought it was such a privilege and an honour to meet the pleasant and humble professor weller! i had also thought somehow i should still keep in touch, especially that i developed a strong interest in cerebrovascular mechanisms and vascular pathology. i was absorbed in mechanisms of cerebral amyloid angiopathy (caa) although at the time i searched for explanations on cystatin c caa, which causes profound intracerebral haemorrhages. cystatin c is enriched in the brain and csf but it was also found in the cervical lymph glands of icelandic patients. using the cystatin c reasoning, i was determined to disprove the giants selkoe and wisniewski, who were both proposing at the time that cerebral aβ amyloidosis was initiated by a protein(s) from the circulation (even papers in nature) akin to the classical pathways discussed by glenner and others how amyloidosis is caused in end organs. large nih funds were even spent on studies how aβ could be transported from the blood-to-brain rather than searching for routes how it leaves the brain. all above was proven to be inaccurate and i was glad that roy’s seminal work glued it altogether suggesting that aβ is not only produced in the brain but ‘pouring’ out of it along perivascular pathways as one of the main routes. roy and later roxana beautifully demonstrated the mechanisms involved in caa and how proteins including aβ, prions, cystatin c drain out formulating the ipad hypothesis. this is also of importance because we have used the tonsils (lymphatic nodes) to test individuals who might have prions and infected with new variant cjd. to show, i understood the ‘drains’ of the brain, i was proud to give a talk on weller’s hypothesis of caa at the isn conference in turin in 2004. even though, i did not end up in southampton, i am very fond of roy and he has been an excellent mentor, advisor, collaborator and a good friend with whom one could relax, have a drink or two in the pub and discuss the ‘nuts and bolts’ of vascular neuropathology rather than daily mundane matters. over the years, it has been one of the most exciting journeys with roy. our regular interactions at the bns meetings, vascog and isn conferences including the one roy organized in 2000 in birmingham were refreshing, delightful and enjoyable. having dinners at such gatherings and other times is something i always looked forward to and will cherish. thank you roy and congratulations on reaching 80, true to what the scriptures say ‘the span of our life is 70 years or 80 if one is especially strong…’ (psalms 90:10). prof dietmar thal consultant neuropathologist, leuven, belgium roy weller: a pioneering neuropathologist. the first time i heard a talk given by roy weller, he was explaining the contribution of perivascular clearance on several brain disorders. this was 1994. perivascular clearance mechanisms became of clear importance for amyloid pathology in alzheimer’s disease. here, prof. weller had a strong impact on my thinking about how cerebral amyloid angiopathy develops in ad, namely as a result of perivascular clearance. for me, this could also explain interactions between cerebral small vessel disease and hypertension with ad by impairing the perivascular clearance. prof. weller’s concept of perivascular clearance is not only the basis of our understanding of cerebral amyloid angiopathy pathogenesis but had even a bigger impact on the understanding of fluid and protein drainage of the brain in general. prof hugh perry emeritus professor, university of southampton it was my pleasure to meet and get to know roy weller when i moved to southampton in the late 1990s. roy was a generous and welcoming colleague and as a basic scientist, mostly involved in pre-clinical models, he was there to remind us of the need to be aware of the human perspective. he had a great breadth of knowledge in neuropathology and also an understanding of the history behind many key observations in neuropathology. his research into how interstitial fluid carrying proteins from the brain drained from the brain was one of his passions. this is, of course, a very important component of understanding interactions between the immune system and the central nervous system. as the rapidly growing field of neuroimmunology advances so the research that roy championed will remain relevant and new discoveries will amplify the importance of his favoured research topic. prof stephen wharton consultant neuropathologist, sheffield institute of translational neuroscience, and ex-editor, neuropathology and applied neurobiology i first came across roy in the 1980s. i was presenting some (rather incomplete) results from my intercalated bsc project at a meeting of the anatomical society in southampton. i received a kind and supportive question from an audience member and was told, “that’s roy weller”. later, when i began training in neuropathology i used his textbook on nervous system, muscle and eyes, part of the comprehensive symmers series and enjoyed its clarity and approachability. roy of course was editor of neuropathology and applied neurobiology for ten years or so, and i had the opportunity to work with him more closely when i subsequently became editor after jim lowe. roy agreed to remain on the editorial board where he was always an enthusiastic contributor with good ideas to move the journal forward. he was also a great source of advice, both scientific and for those tricky situations that sometimes arise with papers. since then i have continued to enjoy conversations about neuropathology at various meetings. i have always enjoyed these stimulating conversations and i, like many others, have appreciated his kindness and advice over many years. copyright: © 2021 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. heterogeneity of dna methylation profiles and copy number alterations in 10782 adult-type glioblastomas, idh-wildtype feel free to add comments by clicking these icons on the sidebar free neuropathology 5:7 (2024) original paper heterogeneity of dna methylation profiles and copy number alterations in 10782 adult-type glioblastomas, idh-wildtype david e. reuss1,2, daniel schrimpf1,2, asan cherkezov1,2, abigail k. suwala1,2, tereza lausová1,2, matija snuderl3, david capper4, martin sill5,6, david t. w. jones6,7,8, stefan m. pfister6,8, felix sahm1,2,6, andreas von deimling1,2,6 department of neuropathology, institute of pathology, university of heidelberg, heidelberg, germany clinical cooperation unit neuropathology, german cancer consortium (dktk), german cancer research center (dkfz), heidelberg, germany department of pathology, new york university langone health and school of medicine, new york, new york, usa department of neuropathology, charité universitätsmedizin berlin, berlin, germany; german cancer consortium, berlin, germany; german cancer research center (dkfz), heidelberg, germany division of pediatric neurooncology, german cancer consortium (dktk) and german cancer research center (dkfz), heidelberg, germany hopp children's cancer center heidelberg (kitz), heidelberg, germany pediatric glioma research group, german cancer research center (dkfz), heidelberg, germany department of pediatric oncology, hematology and immunology, heidelberg university hospital, heidelberg, germany corresponding author: andreas von deimling · department of neuropathology · university of heidelberg and clinical cooperation unit neuropathology · german cancer consortium (dktk) · german cancer research center (dkfz) · heidelberg · inf224 · 69120 heidelberg · germany andreas.vondeimling@med.uni-heidelberg.de additional resources and electronic supplementary material: supplementary material submitted: 02 february 2024 accepted: 13 march 2024 copyedited by: vanessa s. goodwill published: 22 march 2024 https://doi.org/10.17879/freeneuropathology-2024-5345 keywords: glioblastoma, copy number variations, cnv, methylation, classification, 7/10 signature, egfr, cdkn2a/b, amplification, homozygous deletion abstract the morphological patterns leading to the diagnosis of glioblastoma may also commonly be observed in several other distinct tumor entities, which can result in a mixed bag of tumors subsumed under this diagnosis. the 2021 who classification of cns tumors has separated several of these entities from the diagnosis of glioblastoma, idh-wildtype. this study determines the dna methylation classes most likely receiving the diagnosis glioblastoma, idh wildtype according to the definition by the who 2021 classification and provides comparative copy number analyses. we identified 10782 methylome datasets uploaded to the web page www.molecularneuropathology.org with a calibrated score of ≥0.9 by the heidelberg brain tumor classifier version v12.8. these methylation classes were characterized by the diagnosis glioblastoma being the most frequent classification encountered in each of the classes according to the who 2021 definition. further, methylation classes selected for this study predominantly contained adult patients. unsupervised clustering confirmed the presence of nine methylation classes containing tumors most likely receiving the diagnosis glioblastoma, idh-wildtype according to the who 2021 definition. copy number analysis and a focus on genes with typical numerical alterations in glioblastoma revealed clear differences between the nine methylation classes. although great progress in diagnostic precision has been achieved over the last decade, our data clearly demonstrate that glioblastoma, idh-wildtype still is a heterogeneous group in need of further stratification. introduction the term glioblastoma multiforme was introduced by percival bailey and harvey cushing in 1926. however, the definition of glioblastoma has experienced major change in the last decades accompanying the evolution of the current who classification for cns tumors. initially based purely on histology (1) with gradual inclusion of molecular findings (2), who grading of brain tumors has now transformed (3,4) to a classification system heavily relying on molecular parameters (5). in this process the relevance of the idh mutation status for glioblastomas has been recognized (6) and consequently, idh-mutant glioblastomas were separated from glioblastoma, idh-wildtype. further, h3f3a k27m mutations were recognized within malignant brain stem gliomas (7) and had been categorized as the first pediatric highly aggressive brain tumor group designated diffuse midline glioma, h3 k27-altered. major overhaul was introduced in the 2021 (5th) edition (5) which radically separated pediatric from adult high-grade gliomas. the distinct tumor types of infant-type hemispheric glioma, diffuse pediatric-type high-grade glioma, h3-wildtype and idh-wildtype (8), and diffuse hemispheric glioma, h3 g34-altered were added. the latter tumor type, however, also is frequently seen in adult patients and the exclusive sorting with pediatric tumors may be subject to change in the future. also, high-grade astrocytoma with piloid features was elevated to a separate tumor entity stripping many cerebellar high grade gliomas from the glioblastoma, idh-wildtype pool. several approaches to the molecular subclassification of glioblastomas have been taken ranging from initial detection of chromosomal alterations (9), gene expression (10) and cpg methylation (11). here we focus entirely on cpg methylation by analyzing an unprecedented series of tumors representing who adult glioblastoma idh-wildtype employing data generated by the illuminahumanmethylation450 (450k) or methylationepic (850k) array platforms. materials and methods selection of cases only datasets derived from analysis with illumina 450k or illumina epic chips were included in this study. the vast majority of the data were collected from uploads to the www.molecularneuropathology.org webpage for prediction by the heidelberg brain tumor classifier. in a first step we selected 5719 datasets submitted to the webpage with the diagnosis of glioblastoma and a calibrated score ≥0.9 for any methylation class (mc) included in the v12.8 version of the brain tumor classifier. the v12.8 version has been updated for data from the infinium methylationepic v2.0 platform, however, also allows analyses of data from older chip generations up to 450k without compromising. this approach yielded a list containing 83 different mc (supplementary table 1). from this list, mc were selected in which the most prevalent diagnosis was glioblastoma. because who in its 2021 edition separated pediatric from adult gliomas, only methylation groups with a majority of patients in the adult age range were included. further, mc corresponding to distinct who tumor types other than glioblastoma idh-wildtype were excluded; examples of excluded mc are astrocytoma, idh-mutant and diffuse hemispheric glioma, h3 g34-mutant. this approach resulted in nine mc: glioblastoma, idh-wildtype, rtk2 subtype (mc gbm_rtk2); glioblastoma, idh-wildtype, typical mesenchymal type (mc gbm_mes_typ); glioblastoma, idh-wildtype, rtk1 subtype (mc gbm_rtk1); glioblastoma, idh-wildtype, atypical mesenchymal type (mc gbm_mes_atyp); adult-type diffuse high grade glioma, idh-wildtype, subtype e (mc hgg_e), adult-type diffuse high grade glioma, idh-wildtype, subtype b (mc hgg_b); adult-type diffuse high grade glioma, idh-wildtype, subtype f (mc hgg_f); glioblastoma, idh-wildtype, with primitive neuronal component (mc gbm_pnc); and high-grade diffuse glioma of the midline/posterior fossa, h3/idh-wildtype (mc gbm_cbm). in a second step we screened the heidelberg brain tumor data base for tumors with a prediction for these 9 mc and a calibrated score ≥0.9. inclusion was based on prediction only and independent from accompanying diagnosis. this procedure yielded the study group including a total of 10782 tumors from independent patients. generation of tsne, cnv and summary cnv plots tsne analyses were performed using the r-package rtsne (https://github.com/jkrijthe/rtsne) employing the 20,000 most variable cpg sites according to standard deviation; 3000 iterations and a perplexity value of 10. copy number variation (cnv) data were calculated from the output of the illumina 450k or 850k/epic platforms. cnv plots are based on the raw data subjected to analysis by the 'conumee' r package (12) (https://github.com/mwsill/conumee-2). assessment of copy-number alterations was automated using the results from conumee after additional baseline correction. amplifications were called if the respective probes exhibited a value higher than 0.52 on a log2 scale. homozygous deletions were called if the respective probes exhibited a value lower than −0.4 on a log2 scale. statistics given the large number of parameters, tests for deviations from normal distributions were not performed. the supplementary files provide all information for executing targeted statistical analyses. results methylation classes with frequent glioblastoma diagnoses applying the inclusion criteria 8 mc were identified with glioblastoma being the predominant accompanying histological diagnosis. one mc, hgg_f, actually fails the inclusion criteria because the most frequent diagnosis was glioma including lower grade tumors. however, the preliminary nomenclature implies close relation to mc hgg_b and mc hgg_e. therefore, mc hgg_f was not excluded from these analyses and the present series contains 10782 tumors falling into 9 mc. figure 1 depicts a tsne analysis of the study set. figure 1. tsne showing the distribution of 9 mc subsumed within the who 2021 diagnosis of glioblastoma, idh-wildtype diagnoses in 9 methylation classes pathological diagnoses submitted at time of upload to www.molecularneuropathology.org were categorized. table 1 depicts 27 categorized diagnoses and the prevalence for each in the 9 mc. no diagnosis was provided at upload to the webpage for 3690 of 10782 tumors. the frequency for tumors submitted without a diagnosis was rather evenly distributed among the 9 mc ranging from 22 % to 37 %. this provided a basis for searching for frequencies of other diagnoses showing major deviations among the 9 mc. the bulk of the tumors (9857 of 10782 cases) belonged to the three mc gbm_mes_typ, mc gbm_rtk1 and gbm_rtk2 corresponding to the mc gbm_mes mc gbm_rtk1 and gbm_rtk2 defined in the first public version v11b4 of the brain tumor classifier (13). the other 923 tumors split up in 6 mc which were first introduced in version 12.5 of the brain tumor classifier. diagnosis simplified hgg_b hgg_e hgg_f gbm_mes_atyp gbm_mes_typ gbm_rtk1 gbm_rtk2 gbm_pnc gbm_cbm n 117 153 150 329 3668 2267 3924 124 50 angiocentric glioma 0 0 1 0 0 0 0 0 0 astroblastoma 0 1 0 0 2 0 0 0 0 desmoplastic melanoma 0 0 0 1 0 0 0 0 0 ependymoma 1 6 0 1 6 4 2 0 0 epithelioid glioblastoma 0 0 0 8 12 0 2 0 0 evn 0 0 0 0 1 0 1 0 0 giant cell glioblastoma 1 0 0 5 11 7 7 0 0 glioblastoma 22 42 19 69 1578 1086 1760 37 17 glioma 22 18 54 15 275 150 241 7 5 glioneuronal tumor 4 5 3 1 18 3 7 1 0 gliosarcoma 0 0 1 46 61 15 5 2 0 hgap 0 4 0 0 1 0 1 0 2 high grade glioma 15 27 18 33 283 162 342 19 3 low grade glioma 1 1 6 0 10 0 9 0 0 lymphoma 0 0 0 0 1 2 0 0 0 medulloblastoma 1 0 0 0 0 2 1 1 2 meningioma 0 0 0 5 4 6 3 0 0 metastasis 0 0 0 0 6 3 1 2 0 no diagnosis 39 45 33 98 1239 742 1434 42 18 no tumor diagnosis 1 0 5 1 10 0 1 0 0 oligodendroglioma 2 1 2 0 11 9 16 0 0 pilocytic astrocytoma 4 0 0 0 3 0 2 0 0 pxa 0 0 1 6 40 1 4 0 0 pnet 0 0 0 0 1 1 1 3 2 sarcoma 0 0 0 15 2 0 1 0 0 tumor 4 3 7 25 93 74 83 10 1 table 1. diagnoses provided with submission of the methylation datasets to the www.molecularneuropathology.org webpage. (abbreviations: evn, extraventricular neurocytoma; hgap, high grade astrocytoma with piloid features; pxa, pleomorphic xanthoastrocytoma; pnet, primitive neuroectodermal tumor). the diagnosis of glioblastoma was most frequently given for tumors belonging to the three, mc gbm_mes_typ, mc gbm_rtk1 and gbm_rtk2 with incidences of 43 %, 48 % and 45 %. in contrast, only 21 % of tumors in mc gbm_mes_atyp received the diagnosis glioblastoma. alternatively, mc gbm_mes_atyp collected the highest proportions of epithelioid glioblastoma, gliosarcoma, pleomorphic xanthoastrocytoma (pxa), and sarcoma diagnoses. this indicates that the morphology of tumors in this mc is more heterogeneous than that of the three "canonical" mc gbm_mes_typ, mc gbm_rtk1 and gbm_rtk2. the novel mc gbm_pnc (glioblastoma with primitive neuronal component) (14) and gbm_cbm collected the majority of the medulloblastoma and pnet diagnoses. overview on copy number variation in 9 methylation classes summary cnv plots were calculated for all 9 mc providing an overview on frequent gross numerical aberrations (figure 2). visual inspection reveals substantial differences. as expected, the 7/10 signature appears evident in the three canonical mc gbm_mes_typ, mc gbm_rtk1 and gbm_rtk2, as well as in gbm_mes_atyp and in gbm_pnc. as previously reported, gbm_pnc profiles frequently show a chromosome 1 gain and a less frequent gain of 7, whereas losses of chromosomes 10 and 13q are highly prevalent (14). in addition, loss of chromosome 16 is more common in mc gbm_pnc. a clear increase of likely combined chromosome 19 and chromosome 20 gains is seen in mc gbm_rtk2 and loss of chromosome 15 is most common in mc gbm_rtk1. in the mc gbm_mes_atyp loss of chromosomes 18 is more common than in other mc. figure 2. summary cnv plots cnv in the 9 methylation classes and prevalence of the 7/10 signature combined gain of chromosome 7 and loss of chromosome 10, dubbed 7/10 signature, is a very frequent molecular feature of glioblastoma. summary copy number plots cannot provide precise numbers on the occurrence of the 7/10 signature, because this approach does not allow differentiation between the number of tumors with either only one or the combination of both lesions. therefore, analyses of the individual prevalence of chromosomal alterations and their combinations have been performed. an overview of regions most differentially affected is given in table 2. a compilation of alterations in individual cases is provided in supplementary table 2. mc (n) 1p 1q 7p 7q 10p 10q 13q 19p 19q 20p 20q 7/10 sig.   gain gain gain gain loss loss loss gain gain gain gain n ( %) hgg_b (117) 3 % 9 % 9 % 4 % 10 % 15 % 20 % 3 % 3 % 15 % 11 % 2 (2 %) hgg_e (153) 5 % 15 % 5 % 10 % 6 % 12 % 34 % 3 % 2 % 2 % 8 % 5 (3 %) hgg_f (150) 2 % 13 % 21 % 18 % 22 % 21 % 15 % 0 % 0 % 2 % 1 % 23 (15 %) gbm_mes_atyp (329) 16 % 11 % 62 % 57 % 53 % 47 % 46 % 2 % 1 % 18 % 16 % 125 (38 %) gbm_mes_typ (3668) 5 % 5 % 76 % 70 % 87 % 85 % 33 % 9 % 12 % 28 % 22 % 2667 (73 %) gbm_rtk1 (2267) 6 % 10 % 83 % 81 % 82 % 85 % 41 % 11 % 8 % 13 % 13 % 1739 (77 %) gbm_rtk2 (3924) 6 % 8 % 88 % 87 % 93 % 92 % 24 % 43 % 42 % 48 % 46 % 3495 (89 %) gbm_pnc (124) 48 % 46 % 58 % 57 % 90 % 90 % 47 % 10 % 10 % 40 % 41 % 71 (57 %) gbm_cbm (59) 6 % 22 % 24 % 26 % 38 % 26 % 34 % 6 % 4 % 10 % 12 % 6 (12 %) table 2. gains and losses on selected chromosomal arms as suggested by visual inspection, gains of chromosome 1 are most prominent in mc gbm_pnc observed in nearly half of the cases. gains on chromosomes 19 and 20 are most frequent in mc gbm_rtk2 also approaching 50 % in these tumors. the best parameters for defining the 7/10 signature have not yet been established. in this study, a previously published algorithm has been employed (15). presence of the 7/10 signature was scored if gain of at least 50 % of the short or the long arm of chromosome 7 coincided with loss of at least 50 % of the short or the long arm of chromosome 10. employing this algorithm revealed clear differences in the frequency of the 7/10 signature in the different mc. the overall prevalence for the 7/10 signature in the present series was 8133 of 10782 cases corresponding to 75 %. however, this high frequency is mainly due to the very frequent occurrence in the three canonical mc. highest prevalence was observed in mc gbm_rtk2 with 89 % followed by the two other frequent mc gbm_rtk1 (77 %) and gbm_mes_typ (73 %). clearly lower frequencies were observed in gbm_pnc (57 %) and gbm_mes_atyp (38 %) (table 1). in the mc hgg_b, hgg_e and hgg_f and gbm_cbm the 7/10 signature was not in the foreground, although in mc hgg_f chromosome 7 gains and chromosome 10 losses were seen in approximately 20 % of cases. however, the combination of chromosome 7 gain and chromosome 10 loss in individual cases, which cannot be recognized in sumcnv plots, was only 15 % in the latter. prevalence of gene amplifications and cdkn2a/b homozygous deletions in 9 mc various gene amplifications and homozygous deletions on chromosome 9q containing the cdkn2a/b loci are hallmarks of glioblastoma. a detailed analysis of selected candidate genes and regions was performed on all 10782 datasets. canonical glioblastoma subtypes are known to harbor specific gene amplifications in different frequencies. for example, egfr amplifications are most prevalent in transcriptomically "classic" glioblastoma roughly equivalent to methylation class gbm_rtk2, whereas pdgfra amplifications are enriched in transcriptomically "proneural" glioblastoma roughly equivalent to mc gbm_rtk1 tumors (10,16,17). however, reported frequencies in the literature vary and are likely influenced by the composition of study cohorts and applied methods. here we analyzed the so far largest cohort enabling the most definitive determination of reference values. amplification of egfr occurred in 77 % of gbm_rtk2 but only in 20 % of gbm_rtk1 and 27 % of gbm_mes_typ. the overall frequency in these three mc approximates 40 %. in contrast egfr amplification was present at low frequencies in the other 6 mc. cdk4 and mdm2 were amplified most frequently in mc gbm_rtk1. amplification of pdgfra was pronounced in mc hgg_b, mc gbm_rtk1 and especially in mc gbm_cbm. the mc with the lowest overall frequencies of gene amplifications were mc hgg_f and mc gbm_mes_atyp. table 3 summarizes the most frequent gene amplifications. the distribution in individual tumors is provided in supplementary table 3. mc (n) cdk4 egfr mdm2 mdm4 mycn pdgfra hgg_b (117) 5 % 3 % 1 % 0 % 3 % 16 % hgg_e (153) 1 % 0 % 0 % 0 % 3 % 9 % hgg_f (150) 1 % 1 % 0 % 2 % 2 % 2 % gbm_mes_atyp (329) 4 % 2 % 2 % 1 % 0 % 1 % gbm_mes_typ (3668) 5 % 27 % 6 % 5 % 0 % 3 % gbm_rtk1 (2267) 26 % 20 % 16 % 8 % 1 % 20 % gbm_rtk2 (3924) 11 % 77 % 7 % 8 % 1 % 4 % gbm_pnc (124) 5 % 3 % 9 % 9 % 10 % 1 % gbm_cbm (50) 10 % 6 % 12 % 2 % 0 % 34 % table 3. gene amplifications in methylation classes contained in glioblastoma idh-wildtype substantial underrepresentation of chromosomal portions was most frequently observed on the short arm of chromosome 9 in the region containing the cdkn2a/b loci. we called homozygous deletions upon reading values for regions lower than −0.4 on a log2 scale. this cutoff value results in inclusion of borderline cases, however, more stringent criteria can be applied using the data provided in supplementary table 3. homozygous deletion in the cdkn2a/b region is most frequent in mc gbm_rtk2 (82 %) followed by gbm_rtk1 (61 %). mc gbm_cbm and mc gbm_mes_atyp exhibit this alteration in approximately half of the cases. in contrast, homozygous deletion of cdkn2a/b was detected in only up to one in four cases belonging to mc hgg_b and mc gbm_pnc, and only up to one in ten cases in mc hgg_e and mc hgg_f. other regions are less commonly affected and calling homozygous deletions in these areas should be done conservatively. table 4 provides according data. mc (n) cdkn2a/b nf1 pten rb1 tp53 hgg_b (117) 25 % 13 % 1 % 13 % 7 % hgg_e (153) 11 % 3 % 1 % 15 % 8 % hgg_f (150) 7 % 3 % 1 % 2 % 2 % gbm_mes_atyp (329) 45 % 5 % 10 % 7 % 3 % gbm_mes_typ (3668) 49 % 4 % 6 % 3 % 1 % gbm_rtk1 (2267) 61 % 2 % 30 % 19 % 8 % gbm_rtk2 (3924) 82 % 4 % 34 % 14 % 4 % gbm_pnc (124) 23 % 2 % 55 % 47 % 15 % gbm_cbm (50) 52 % 6 % 4 % 4 % 4 % table 4. chromosomal deletions in mc contained in glioblastoma idh-wildtype age distribution information on age was available for 5153 of 10782 datasets. an age of 0 years was encountered in all mc with entries for subsequent years missing in several mc (figure 2). this was assumed a result of submitters intention to communicate unknown age of the patient. therefore, all data referring to age 0 were ignored for age related analyses. the frequent idh-wildtype glioblastoma mc gbm_mes_typ, gbm_rtk1 and gbm_rtk2 showed an age distribution matching that of tumors typically occurring in elderly adults. this was also seen for mc gbm_pnc. upon closer inspection, the median and average ages did show differences, notably the higher median and average ages of patients with mc gbm_rtk1 tumors. patients with gbm_mes_atyp and gbm_cbm tumors were markedly younger. further, gbm_mes_atyp patients showed also more frequent occurrence in younger patients with an additional, albeit lower, peak in the age group 16 to 20 years old. age distribution in three mc hgg_b, hgg_e and hgg_f was quite different with hgg_f exhibiting a distribution similar to the frequent gbm mc. tumors belonging to mc hgg_b were most frequent in middle aged patients while mc hgg_e tumors were observed in all age groups ranging from infants to the elderly. data for mc gbm_cbm were sparse not allowing to determine ages for peak incidence. however, data on age for 29 patients suggests that patients in all age groups are affected. figure 3 and table 5 depicts age distributions in 9 mc. figure 3 v12.8 prediction (n) median age average age hgg_b (60) 31,5 35 hgg_e (72) 42,5 41,7 hgg_f (93) 65 62,2 gbm_mes_atyp (170) 57 54,2 gbm_mes_typ (1805) 61 60,2 gbm_rtk1 (1094) 65 63,7 gbm_rtk2 (1827) 60 60,3 gbm_pnc (47) 61 61,2 gbm_cbm (28) 54 50,2 table 5. median age and average age of patients in the respective mc given in years. cases (n) in each group are without patients aged 0. mgmt methylation in 9 mc methylation of the mgmt promoter is a highly relevant predictor for alkylating chemotherapy (18). thus, determination of the mgmt methylation status is a routine procedure in neuropathological diagnosis. mgmt methylation status was comparable between the mc gbm_mes_typ, gbm_rtk1, gbm_rtk2, gbm_pnc and gbm_cbm. fewer cases with mgmt methylation were encountered in mc gbm_mes_atyp and mc hgg_e. patients belonging to mc hgg_f exhibited even lower methylated cases, whereas mgmt methylation in mc hgg_b was barely detected. table 6 compiles data on the distribution of mgmt methylation in 9 mc. mc (n) methylated unmethylated undecided missing hgg_b (117) 3 % 96 % 1 % 0 % hgg_e (153) 35 % 61 % 2 % 2 % hgg_f (150) 19 % 79 % 1 % 1 % gbm_mes_atyp (329) 33 % 64 % 2 % 1 % gbm_mes_typ (3668) 44 % 53 % 2 % 1 % gbm_rtk1 (2267) 46 % 51 % 1 % 1 % gbm_rtk2 (3924) 51 % 47 % 2 % 1 % gbm_pnc (124) 50 % 48 % 2 % 1 % gbm_cbm (50) 44 % 52 % 4 % 0 % table 6. mgmt methylation status comparison of predictions for cases of 9 methylation classes from v12.8 with classifier version 11b4 the brain tumor classifier v12.8 has experienced addition of many new entities. six of the 9 mc here under investigation are novel and have not been present in classifier version 11b4. we compared v12.8 predictions with v11b4 predictions. predictions from v11b4 were available for 10778 cases. for the vast majority of cases belonging to one of the six novel mc, no predictions with calibrated scores higher than 0.9 have been generated by version v11b4. for the six novel mc only 97 of 923 predictions reached a score >0.9 and 81/97 were for the methylation family glioblastoma. the predictions for the three canonical mc showed a high overlap between v11b4 and v12.8 output. of 9859 tumors, 8647 have been predicted with a score ≥0.9 by version 11b4. in these three groups median score for tumors with <0.9 predictions were 0.72 for gbm_mes_typ, 0.78 for gbm_rtk1 and 0.85 for gbm_rtk2. data are compiled in supplementary table 4. discussion the aim of this study was assessing the homogeneity of tumors currently subsumed under the diagnosis glioblastoma, idh wildtype according to the who classification scheme 2021 (5). basis for analysis was a set of 10782 methylation datasets matching mc given by the heidelberg brain tumor classifier version 12.8 which by histology predominantly are classified as glioblastoma within who 2021. no clinical data were available apart from a diagnosis for 7092 and age for 5153 of the 10782 patients. this lack of clinical data poses a grave limitation. however, those parameters which could be assessed without clinical data allowed for sound observations based on the large number of samples. diagnosis for many of the lesions were tentative at time of submission of the methylation data to www.molecularneuropathology.org. the present data clearly show that the who 2021 diagnosis of glioblastoma, idh wildtype encompasses a mixture of tumors belonging to different tumor types. further, the data provide evidence of high variation of distinct genetic alterations in tumor groups believed to be very closely related. the eight mc with tumors predominantly diagnosed as glioblastoma and one additional mc were identified with very different numbers. while there is a bias for pediatric tumors submitted to methylation analysis, the selected mc mostly occurred in adult patients. due to similarities between the mc gbm_mes_typ, mc gbm_rtk1 and mc gbm_rtk2 the brain tumor classifiers assign a family score for glioblastoma. idh-wildtype. this ensures, that tumors, for example with scores of 0.5, 0.2 and 0.15 for these three mc still would receive a glioblastoma prediction although each individual score would fail the mark of 0.9. while selecting the present series the family score of glioblastomas was not taken in account. this means, that glioblastomas belonging to the mc gbm_mes_typ, mc gbm_rtk1 and mc gbm_rtk2 are underrepresented in this study. however, it was felt that no additional information would be gained by enlarging the numbers for these three mc. this selection bias, however, implies that the actual occurrence of the other 6 mc may be lower than can be assumed from the relation to the mc gbm_mes_typ, mc gbm_rtk1 and mc gbm_rtk2 tumor numbers in this study. nevertheless, tumors belonging to the mc gbm_cbm, mc hgg_b, mc hgg_e, mc hgg_f and mc gbm_pnc are very infrequently encountered in routine practice, thus making their recognition dependent on data collections from large number of institutions. six of the nine mc under investigation in this study are not represented by a distinct tumor type in who 2021 and are in need of future clinical and histopathological characterization. currently best studied within this group is mc gbm_pnc typically exhibiting gains on chromosome 1 and fewer gene amplifications, lower incidence of the 7/10 signature and fewer homozygous deletions of cdkn2a/b than the majority of glioblastoma idh wildtype tumors (14). tumors from the mc hgg_f have recently been characterized as frequently growing with a gliomatosis-like pattern, lacking microvascular proliferation or necrosis, frequent promoter tert and pik3r1 mutations and a longer overall survival (19). tumors from the mc hgg_b and hgg_e shared several features. the patients were distributed across a wider age range and the frequency of chromosomal gains and losses was comparable. this differed in hgg_f; patients in this group were older with very few pediatric and juvenile individuals affected. interestingly, the dominant diagnoses provided for these patients was glioma and not high grade glioma or glioblastoma, indicating that the majority of submitters had the impression that high malignancy was not obvious in many of the cases, compatible with the reported lack of microvascular proliferation and necrosis in this mc. copy number alterations varied strongly among the 9 groups. the 7/10 signature predominated in the canonical gbm_mes_typ, gbm_rtk1 and gbm_rtk2, however, was also present with a lower incidence in gbm_mes_atyp and gbm_pnc. it was very rare in hgg_b and hgg_e and seen in 12 % and 15 % in gbm_cbm and hgg_f. several mc exhibited virtually unique features in sumcnv plots. gbm_rtk2 had striking gains of chromosomes 19 and 20, a feature which previously has been associated with long time survival in glioblastoma (20). gbm-pnc showed chromosome 1 gains in nearly half of the cases, which was not a prominent alteration in all other mc (14). the variance in cnv profiles, gene amplifications and deletions also among the three canonical glioblastoma mc remain poorly understood. these may become of future relevance with more personalized targeted therapies emerging, possibly affecting prognoses or prediction for the different mc. in conclusion, tumors from adult patients subsumed under the diagnosis glioblastoma idh-wildtype in the most recent who brain tumor classification can be separated in at least nine groups differing in both methylation and cnv profiles. clinical characterization of novel subgroups is a pressing issue. it can be expected that glioblastoma idh-wildtype will be further stratified into several different more narrowly defined brain tumor entities. acknowledgement supported by deutsche forschungsgemeinschaft, sfb 1389, a05/c06. disclosure m. snuderl, d. capper, d. schrimpf, m. sill, d.t.w. jones, s. pfister f. sahm and a. von deimling are co-founders and shareholders of heidelberg epignostix gmbh. supplementary tables supplementary table 1. predictions in tumors accompanied by the diagnosis of glioblastoma or gbm at submission to www.molecularneuropathology.org. supplementary table 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neuropathol. 2015;130(3):419-34. https://doi.org/10.1007/s00401-015-1427-y copyright: © 2024 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. neuropathology of the alzheimer’s continuum: an update feel free to add comments by clicking these icons on the sidebar free neuropathology 1:32 (2020) review neuropathology of the alzheimer’s continuum: an update kurt a. jellinger institute of clinical neurobiology, vienna, austria address for correspondence: kurt a. jellinger · institute of clinical neurobiology · alberichgasse 5/13 · a-1150 vienna · austria kurt.jellinger@univie.ac.at submitted: 06 october 2020 accepted: 07 november 2020 copyedited by: nicole schwab published: 11 november 2020 https://doi.org/10.17879/freeneuropathology-2020-3050 keywords: alzheimer’s disease, β-amyloid, tau pathology, oligomers, amyloid angiopathy, alzheimer subtypes, regional vulnerabiliy, co-pathologies abstract alzheimer’s disease (ad), the most common form of dementia worldwide, is a mixed proteinopathy (amyloid and tau). originally defined as a clinicopathological entity, it is a heterogenous, multifactorial disorder, currently referred to as the alzheimer’s continuum. its cardinal pathological features are extracellular β-amyloid (amyloid plaques) and intraneuronal tau aggregates forming neurofibrillary tangles, which are accompanied by vascular amyloid deposits (cerebral amyloid angiopathy), synapse and neuronal loss, as well as neuroinflammation and reactive astrogliosis. in addition to “typical” ad, various subtypes with characteristic regional patterns of tau pathology have been described that show distinct clinical features, biomarker levels, and patterns of key network destructions responsible for cognitive decline. ad is frequently associated with other age-related changes including lewy and tdp-43 pathologies, hippocampal sclerosis, argyrophilic grain disease, cerebrovascular lesions, and others. these additional pathologies influence the clinical picture of ad, may accelerate disease progression, and can cause a number of challenges in our understanding of the disease including the threshold of each individual pathology to cause dementia and the possibility of underlying common etiologies. this article provides an up-to-date overview of ad neuropathology, its heterogeneity, and additional pathologies in order to explain the difficulties in the diagnosis and the failure of clinical trials in ad patients. abbreviations ad – alzheimer’s disease, adnc alzheimer’s disease neuropathological changes, ap amyloid plaque, app amyloid precursor protein, aβ β-amyloid peptide, aβo aβ oligomer, caa cerebral amyloid angiopathy, cbs corticobasal syndrome, cerad consortium to establish a registry for alzheimer disease, csf cerebrospinal fluid, cvd cerebrovascular disease, dlb dementia with lewy bodies, eoad early-onset ad, ftld frontotemporal lobar degeneration, ftld-tdp frontotemporal lobar degeneration with tdp-43, gvd granulovacuolar degeneration, hcsp-ad hippocampal sparing ad, hp-tau hyperphosphorylated tau protein, late limbic-predominant age-related tdp-43 encephalopathy, late-nc limbic-predominant age-related tdp-43 encephalopathy neuropathological change, lc locus ceruleus, load late-onset ad, lp-ad limbic-predominant ad, lppa logopenic primary progressive aphasia, ma-ad minimal-atrophy ad, mci mild cognitive impairment, mtl medial temporal lobe, nft neurofibrillary tangle, nia-aa national institute of aging/alzheimer's association, np neuritic plaque, nt neuropil thread, part primary age-related taupathy, pca posterior cortical atrophy, phf paired helical filament, sf straight filament, tdp-43 43-kda tar dna binding protein 43. 1. introduction alzheimer’s disease (ad) is the most common form of dementia, currently affecting around 50 million people worldwide. it accounts for 60-70% of dementia cases in clinical and autopsy series, but it is often associated with other confounding pathologies in the elderly. its incidence increases from 2/1.000 at age 65-74 years to 37/1.000 at age 85+ [1], and doubles every five years after age 65, with peaks in the tenth decade and slight decrease afterwards [2, 3]. the point prevalence of ad among individuals aged 60+ is 40.2/1,000 persons, the pooled annual period prevalence is 30.4/1,000, and the incidence rate is 15.8/1,000 person-years [4]. with the disproportional increase of the elderly population, the prevalence of ad will approach around 132 million worldwide and up to 16 million cases in the usa by 2050 [5, 6], ad has become a tremendous public health and socio-economic challenge of the 21st century [5]. as available treatments only target symptoms and neither slow nor reverse the progression of the disease, the development of disease-modifying therapeutic procedures is urgent [7]. ad was originally defined as a clinicopathological entity, characterized by progressive memory deficit, involvement of multiple cognitive domains, and a defining pathological substrate with deposition of amyloid-β peptide (aβ) in extracellular plaques and cerebral vasculature (cerebral amyloid angiopathy/caa), neuritic plaques defined by the presence of microtubule-associated hyperphosphorylated tau protein (hp-tau), intraneuronal aggregations of hp-tau manifesting as neurofibrillary tangles (nfts) in the cell soma, and neuropil threads (nts), which occur mainly in dendritic compartments and, to a lesser degree, in the axonal domain. these changes are accompanied by early synaptic loss [8], activated microglia [9], mitochondrial dysfunction causing energy loss [10], neuroinflammation [11], neurovascular dysfunction [12], disruption of the blood-brain barrier [13], neuronal loss and reactive astrogliosis [14]. ad, a mixed proteinopathy (amyloid, tau, tdp-43, and others), is a heterogenous disorder currently referred to as the alzheimer’s continuum [15] with several pathobiological subtypes and various co-pathologies [16]. the final definite diagnosis of ad rests with post-mortem neuropathology despite the advent of more sensitive neuroimaging and the use of reliable biomarkers [17]. even though the classical morphological features of ad have been known for many years, the recently used more sensitive immunohistochemistry techniques for aβ and hp-tau have replaced silver-staining techniques and have not only forwarded the diagnosis of ad but allowed a more scientific evaluation of the disease's pathology. for the neuropathological diagnosis of ad, the updated national institute on aging/alzheimer's association (nia/aa) 'abc' criteria are used [17]. the morphological changes involving brain regions and neuronal cell types following a stereotypical pattern [18] result from selective cellular and regional vulnerability to pathogenic factors and their progression through functionally integrated regions of the brain [19-23] as well as functional networks that result in progression of ad [24, 25]. however, ad is a heterogenous continuum with a variety of clinically and morphologically defined subtypes, currently referred to as alzheimer’s clinical syndrome [15], which presents major challenges for both diagnosis of ad, monitoring and targeting of disease progression [26]. the new definition of ad as a biologically defined spectrum, using the nia/aa framework [15], enables recognition and diagnosis of the various subtypes of ad [16]. research consensus guidelines have been proposed for the intra vitam biologically-based categorization termed 'atn', which uses combinations of in vivo biomarkers for aβ deposition (a), tau pathology (t), and neurodegeneration (n). they use cerebrospinal fluid (csf) or plasma biomarkers, pet, and functional and structural mri. the biomarker profiles and categories of the alzheimer’s spectrum referring to ad neuropathological changes (adnc) have been summarized recently [27]. 2. pathology of alzheimer’s disease 2.1. macroscopic features the ad brain often has decreased weight and at least moderate cortical atrophy most marked in the medial temporal lobes (mtls) with relative sparing the primary motor, somatosensory and visual cortices and enlargement of the lateral ventricles (ex vacuo hydrocephalus). brain atrophy often involves posterior cortical areas, most notable in precuneus and posterior cingulate gyrus in the preclinical stage of ad [28]. however, none of the macroscopic features are specific to ad, and healthy elderly people often show moderate cortical atrophy especially affecting the frontal lobes, with volume loss of the white matter [29]. medial temporal atrophy affecting amygdala and hippocampus with enlarged temporal horn is typical of ad (fig. 1). however, this is also seen in other age-related disorders such as hippocampal sclerosis [30]. figure 1. comparison between formalin-fixed brain slices of the left hemispheres (level of posterior hippocampus) of an aged nondemented individual (a) and an ad patient (b). note the marked atrophy (thinning of the gyri and deepening of the sulci) in b, in particular hippocampal atrophy (arrow in b) with widening of the inferior horn of the second ventricle (asterisk in b). photographs by courtesy of simon fraser and arthur oakley. 2.2. microscopic features the definite diagnosis of ad requires microscopic examination of multiple brain regions with semiquantitative assessment of the density of lesions and their topographical distribution. extracellular amyloid plaques (aps) and intracellular nfts that are essential for the neuropathological diagnosis, are associated with tau-positive nts, dystrophic neurites and neuritic plaques (nps), caa, reactive astrocytes and activated microglia, and neuroinflammation are present. these lesions result in loss of synapses and neurons in vulnerable regions leading to brain atrophy and the characteristic clinical picture of the disease. hirano bodies, granulovacuolar degeneration (gvd), tdp-43 deposits, and other lesions may also be present [31, 32]. 2.3. amyloid deposits aps are formed by the abnormal extracellular nonvascular accumulation and deposition of aβ peptides of varying length including those with 40 or 42 amino acids (aβ-40 and aβ-42), resulting from the sequential cleavage of the amyloid precursor protein (app) by the enzymes βand γ-secretases [33]. app, from which aβ is cleaved by endoproteolytic processing, is a large single transmembrane protein, encoded by the app gene on chromosome 21 [34, 35]. proteolytic cleavage of app develops mainly via two exclusive pathways, the amyloidogenic and the non-amyloidogenic pathway, but other alternative pathways (η-secretases, δ-secretase, etc.) have been described for the physiological processing of app [36]. the initial cut at the β-site of app is due to the β-secretase activity enzyme bace1, a transmembrane enzyme with aspartyl protease activity. clearance by β-secretase yields a slightly shorter soluble fragment (sappβ) and a correspondingly longer c-terminal fragment (ctfβ) or c99 [37]. app undergoes constitutive shedding by a protease activity called α-secretase, which appears to be a metalloprotease of the adam family. tace (adam17) is one of the α-secretase, but adam10 is more important for α-secretase activity and sappα production. adam10 is the physiologically relevant constitutive of α-secretase in primary neurons [38], as has been demonstrated in vitro and in vivo [39]. cleavage of app by α-secretase releases the soluble ectodomain of app, called sappα, and a membrane-tethered intracellular c-terminal fragment, termed ctfα of c83. the amyloidogenic (or β) cleavage of app is in direct competition with an alternative non-amyloidogenic pathway (cleavage by the α-secretase within the aβ sequence) which precludes the formation of amyloidogenic peptides and leads to soluble sappα, and has neuroprotective properties preventing aβ production [40]. however, aberrant sappα production may tilt the cells toward unregulated growth, but the underlying mechanisms are still unknown [41]. lastly, γ-secretase, a high molecular weight complex that consists of presenilin (ps1, ps2), an aspartyl membrane protease, aph-1, nicastrin and presenilin enhancer (pen2), cleaves app terminal fragments (ctfs) such as c83 and c99, releasing 3 or 4 amino acid peptides from the transmembrane fragment of app. notably, γ-secretase is active on app only following the antecedent αor β-secretase. the products of γ-secretase cleavage of c83 are a 3-kda peptide, termed p3 and an app intracellular domain (aicd), while γ-secretase cleavage of c99 yields the infamous aβ peptide and an identical aicd fragment. besides cleavage by α-, β-, and γ-secretase, other n-terminal fragments (ntfs) of app have been identified that are generated by unknown proteases [42, 43]. mounting evidence suggests that astrocytes that have increased levels of app, β-secretase (bace1), and γ-secretase play an additional role in ad by secreting significant amounts of aβ and contributing to overall aβ burden in the brain [44]. bace1 inhibition more effectively suppresses the initial process of plaque formation, rather than the subsequent phase of plaque growth, which has implications for therapeutic efficiency for the treatment of ad [45]. ad is driven by intraneuronally retained aβ produced by the ad-specific βapp-independent pathway [46]. neuronal aβ-42 is enriched in small vesicles at the presynaptic side of synapses [47]. aβ deposits contain a mixture of various isoforms. the most common are aβ-40 (under physiologic conditions around 90%), aβ-38 and aβ-42 (less than 10%). aβ-40 is produced within the trans-golgi network (tgn) whilst aβ-42 can be made in either the tgn or the endoplasmic reticulum [48]. the specific production of aβ-42 in the endoplasmic reticulum of neurons links this compartment with the generation of aβ and explains why primarily endoplasmic reticulum localized proteins such as presenilin could induce ad [49]. increased production of aβ-42 at the expense of aβ-40 is a common feature in both familial and sporadic ad [50]. the latter is believed to be more toxic than aβ-40 because of its tendency to aggregate and to form fibrils [51]. the phosphorylation of app by extracellular-regulated kinase (erk) and protein kinase c (pkc), in the proteolytic processing of app has been demonstrated to be critically modulating the generation of aβ [52]. the c-terminal app fragments (app intracellular domain) are generated by γ-secretase cleavage [53]. γ-secretase was shown to cleave near the cytoplasmic membrane boundary of app, called ε-site cleavage, as well as in the middle of the membrane domain, called γ-site cleavage, indicating that γand ε-site cleavage are regulated independently [54]. ubiquilin-1 has been shown to modulate γ-secretase-mediated ε-site cleavage and thus may play a role in regulating γ-secretase cleavage of app and other proteins [55]. further cleavage of app intracellular domain (aicd) fragments by caspase or caspase-like proteases results in additional fragments which, however, does not seem to require antecedent proteolysis of app [41]. figure 2. amyloid and neuritic plaques. a; a1. multiple diffuse amyloid plaques in the neocortex (antibody 4g8). b, b1. neuritic plaques that contain aβ and tau in distended processes (i.e. dystrophic neurites). gallyas silver stain visualizes both aggregated aβ and tau and is therefore ideal to detect neuritic plaques (ring in b, neuritic plaque; arrow in b1, dystrophic neurite; arrowhead in b1, neurofibrillary tangle). scale bars: 200 μm. from [71]. truncated aβ fragments are deposited in aps due to axonal linkage and release of app [56]. chemical imaging of evolving ap pathology in a transgenic mouse model for ad suggested initial plaque formation to be seeded by aβ-42, followed by plaque maturation upon deposition of aβ-40 as well as deposition of others [57]. due to its higher rate of fibrillization and insolubility, aβ-42 is its major component in addition to other aβ peptides [58]. a recent report demonstrated the role of hif-1alpha/lncrna bace1-as axis in the transactivator of transcription (tat)-mediated induction of astrocytic amyloidosis [59]. advanced biophysical examination of aβ derived from ad brain tissue showed polymorphic structures [60]. the terminology of aβ plaques is confusing, since a myriad of non-vascular aβ deposits have been described, but five major types can be distinguished: (a) primitive or immature plaques are spherical deposits of predominantly aβ-42 in the neuropil without a dense core and neurites; (b) diffuse plaques, usually large (50µm to several hundred µm), slightly immunoreactive and ill-limited, contain loose amyloid bundles in the neuropil without degenerating neurites and accompanying microglia (fig. 2a); (c) stellate deposits probably related to astrocytes [61]; (d) focal deposits with dense and spherical accumulations of aβ-42, surrounded by a neuritic corona containing dystrophic tau-positive neurites and astrocytic components, constituting the “cored”, “classical” or “neuritic” plaques (nps) (fig. 2b, 2b1); and finally (e) compact or burnt-out plaques with a dense core of aβ-40, absent or tau-negative, ubiquitin-positive neurites. nps have compact dense amyloid cores composed of more fibrillated forms of aβ (fig. 3). they contain tau-positive dystrophic neurites and are accompanied by synaptic loss, activated microglia and reactive astrocytes [62, 63]. there are differences in the composition of the aggregates, for example, the aβ in nps has a more varied composition with the presence of aβ 40, 42, 43, n-terminus truncated aβ and other post-transitionally modified forms [64, 65]. tau-positive nps begin early in ad, but major tau deposition follows the aβ deposition and the clustering of activated microglia [66]. recent studies unequivocally demonstrated that plaque-associated myeloid cells are derived exclusively from resident microglia [67]. in ad, microglia can eliminate aps through phagocytosis with apoe lipoprotein at an early stage of disease progression [68]. scanning transmission electron microscopy (stem) showed three types of fibrillary network structures: amorphous network, fibril bundles, and amyloid stars [69]. although diffuse non-neuritic plaques are generally present before nps, whether an individual diffuse plaque can actually transfer into an np or whether these two types develop differently, is not clear at present. a recently described type called the coarse-grained plaque, a relatively large deposit (diameter about 80 µm) characterized by multiple cores and aβ-devoid pores, is prominent in the neocortex and associated with homozygous apoeε4 status and caa. this divergent ap type is similar to caa, predominantly composed of aβ-40, and has been observed particularly in early-onset ad (eoad) [70]. figure 3. em image of amyloid core of a neuritic plaque. radiating bands of amyloid fibrils comprise the core (x). note the adjacent abnormal fibrils filled with dense bodies (arrows) and surrounding damaged myelin sheaths (x 4000). “burnt out” plaques are composed of dense cores lacking neuritic components, while the astrocytic processes penetrating the plaque core may represent a regressive stage (“remnant plaques”) [72]. “cotton wool plaques” are non-compact deposits, made of aβ-42 with sparse glial components and variable neurites but not surrounded by a neuritic corona. they can be detected with h&e staining [61]. aβ and tau each begin to aggregate in separate neuroanatomical locations and meet in the cerebral cortex in the np. this “collision” of both proteins mediated by microglia has devastating consequences in terms of neuronal loss, promoting neurodegeneration and the consequent development of cognitive decline, but this is still under investigation [73]. 2.4. distribution of amyloid deposits aps in ad brain show a typical distribution with brain areas that are connected via the “default network” typically affected early. in animal models some demonstration of “propagation” along neuronal systems has been observed [74, 75], suggesting some axonal transport of seeds that lead to extracellular deposits. most aβ deposits are located in the gray matter, while some diffuse or lake-like deposits may be seen in the subpial white matter. cortical soluble aβ protein is a neurotoxic agent [76, 77], and aβ oligomers (aβos) may trigger the early phase of the aβ seeding process, while depletion of aβos delays the aggregation process leading to a transient reduction of seed-induced aβ deposits [78]. the topography of aβ deposits depends on the stage of the disease, which leaded to several staging schemes. three stages were distinguished: stage a with amyloid deposits in the basal portions of the frontal, temporal and occipital cortex; in stage b all isocortex is involved, with primary cortices spared and the hippocampus only mildly affected; while stage c shows deposits in the whole isocortex including sensory and motor core fields [18]. others proposed five amyloid “phases” using sensitive silver staining or aβ antibodies: stage 1 or isocortical, stage 2 with additional involvement of hippocampus and entorhinal cortex, stage 3 plus striatum and diencephalic nuclei, stage 4 several brainstem nuclei and medulla oblongata, and stage 5 presenting amyloid deposits in the pons and molecular layer of the cerebellum [79, 80]. these can be reduced to three stages: 1 isocortical, 2 allocortical or limbic, and 3 subcortical. usually involved is the total isocortex, layers ii-v more than layers i and vi [18]. in advanced cases band-like diffuse aβ deposits are also seen in the subpial surface of the cortex or in the white matter close to layer vi [71]. amyloid pet-based staging of aβ pathology in vivo confirmed its progression in ad [81], and revealed higher plaque counts in entorhinal and occipital regions of typical ad, while other phenotypes showed more severe aβ deposition in frontal and parietal cortices [82]. post-mortem analysis of (18)fflutemetamol and (11)cpib pet signal showed that it is influenced by both diffuse plaques and cored plaques and, therefore, is likely a function of plaque size and density of aβ fibrils in plaques. brain regions with large volumes of diffuse plaques could yield pet retention levels comparable with lower volume/frequency of cored plaques [83]. 2.5. cerebral amyloid angiopathy aβ peptides also involve the vessel walls, as with caa, with the more soluble aβ-40 as the major constituent. 85-90% of confirmed ad cases have some degree of caa [84]. it mainly accumulates in the interstitium between the smooth cells of the tunica media. small arteries, arterioles and even capillaries in the cerebral cortex and leptomeningeal vessels are affected [85]. stage 1: vessels are affected in the isocortex, stage 2: involvement of allocortex, and stage 3: basal ganglia, thalamus, pons and medulla oblongata [86]. others distinguished four patterns [87]: type 1: aps with or without caa in the leptomeninges alone; type 2: caa in both leptomeningeal and deeper penetrating arteries (fig 1a); type 3: caa affects both precapillaries and arterioles; type 4 shows aβ deposition in and around blood vessels. genetically, type 3 (capillary subtype) is more strongly associated with the apoeε4 allele [87, 88]. two other types were distinguished: type 1 affecting capillaries, arterioles and small arteries is associated with apoeε4, whereas type 2 not involving capillaries is more likely associated with apoeε2, its most frequent form [89]. both severe caa and ad are associated with apoeε4-positive patients [88]. a more recent staging system is based on the severity of caa in a single vessel: grade 0: absence of staining, grade 1: a congophilic ring around the otherwise normal-appearing vessel, grade 2: complete replacement of the tunica media by congophilic material, grade 3: involving >50% of vessel circumference, giving a “double-barrel” appearance, and grade 4 or fibrinoid necrosis of the vessel wall with additional amyloid deposits in the surrounding neuropil (“dyshoric changes”) [88]. the parietal and occipital cortices are more vulnerable than the frontal and temporal lobe, and the leptomeningeal vessels more than the parenchymal ones [84]. aβ deposition shrinks the cerebral blood vessels by about 8% and reduces the energy supply resulting from decrease of blood flow [90]. caa can cause small infarcts in the cerebral cortex, while severe caa may lead to lobar hemorrhages in the frontal and occipital lobes and to diffuse white matter lesions (fig. 4) [91]. brain hemorrhage does not appear to be directly linked to amyloid burden in patients with caa-related intracerebral hemorrhage, because amyloid burden was similarly distributed across the brain hemispheres and no interhemispheric difference was observed for aβ burden nor for mri markers of small vessel disease [92]. caa and deep perforating arteriopathy are similar and interact with blood-brain barrier breakdown, endothelial damage, and impaired perivascular aβ drainage. both may cause ischemic lesions and intracerebral hemorrhages [93]. chronic treatment of a mouse model of ad with fungicides produced aβ fibril formation and impairment of aβ clearance through neprylisin, suggesting that fungicide residues could be a risk factor for ad via caa [94]. although several pathogenic mechanisms, including the disbalance between production and clearance of aβ creating a self-reinforcing cycle of increased vascular aβ and further caa and ad progression, have been shown, they do not explain completely the disease pathogenesis [95]. the intersection between caa and ad points to a crucial role for improving vascular function in the treatment of ad [96]. figure 4. multiple large hemorrhages in both frontal lobes (a) and occipital lobe (b). diffuse white matter destruction (c). caa in many vessels in the cerebral white matter; scale bar 70 µm (d). from [97]. 2.6. tau pathology tau protein is encoded by the mapt (microtubule-associated protein tau) gene on chromosome 17 [98], which generates a total of 6 isoforms through alternative splicing of exons 2, 3 and 10 in the cns [99]. tau protein, the main constituent of nfts, is involved in the stabilization of neurotubules that leads to the appropriate function of the neuron. its microtubule-binding regions are made of 3 or 4 repeats (3r or 4r tau), their second repeat (exon 10) being spliced in some isoforms. combined phosphorylation of ser202, thr205, and ser208 forms a unique post-translational modification configuration that promotes tau aggregation, accelerating the formation of tau filaments and eventually resulting in nft formation. tau adopts different stable conformations, consistent with the notion of 'strains' as may be seen with the concept of phenotypic diversity or with different environmental stimuli [100, 101]. truncation of tau by caspases-3 or -4 is an early event in the development of nfts [102]. the molecular mechanisms leading to the accumulation of tau are characterized by numerous translational modifications that change its conformation and structural state. recent studies indicate that the dysregulation and dislocation of splicing factor proline and glutamine rich (sfpq), the subsequent dna anomalies and aberrant dynamics of tia-1-positive stress granules in association with pathological tau may represent a critical pathway which contributes to the rapid progression of ad [103]. abberant phosphorylation and truncation make tau protein into a pathological entity; paired helical filaments (phf), the major structural constituents of nfts, exhibit a greater degree of phosphorylation than normal tau [104].tau monomers can aggregate to form oligomers and higher-order fibrils. whilst aβ can largely self assemble, tau phosphorylation is believed to be important for its aggregation [105]. phosphorylation of ser208 likely occurs at different disease stages from phosphorylation of ser202 and thr205. hp-tau accumulation causes synaptic impairment, neuronal dysfunction, and formation of nfts. tau with site-specific posttranslational modification/soluble hp-tau species impact mitochondria and facilitate neurodegeneration [106]. recent studies support the hypothesis that tau phosphorylation at ser208 strongly contributes to unique types of tau aggregates, and may be a reliable marker for the presence of mature nfts [107]. in ad, tau protein usually accumulates in the somato-dendritic and, to a lesser degree, in the axonal domains of the neuron. nfts and pretangles are due to accumulation in the soma; nts occur in dendrites, and the neuritic corona of core plaques is constituated by axonal processes filled by tau proteins (fig. 2). as major constituents of nfts and nts, they are hyperphosphorylated and aberrantly misfolded, have lost their microtubule stabilizing functions, and contribute to axonal transport deficits [105]. phfs in ad contain all 6 isoforms of tau protein including those with 3 and 4 repeats (3rand 4r-tau) in the microtubule binding domain, forming the core of phf [108]. the tau isoforms show a chronological shift: initially, early pretangles are positive only for 4r, gradually 3r is involved in mature tangles, and finally 4r is replaced by 3r in ghost tangles [109]. ultrastructurally, nfts appear as phfs, i.e., fibrils of ca. 28 nm in diameter that form pairs with a helical tridimensional conformation and a regular periodicity of 65-80 nm [110] or as helical or twisted ribbons [111]. straight filaments (sfs) show a longer crossover distance and modulations in width from 10 to 15 nm. both lesions are different from those seen in other tauopathies [112]. phfs and sfs differ in their inter-protofilament packing, and are ultrastructurally polymorph [113]. visible with cryo-em, phfs and sfs are made of two c-shaped protofilaments with a combined cross-β-β-helix structure, without variations in the filamentous structures between sporadic and inherited ad [114]. nts have an ultrastructure and immunohistochemistry similar to nfts. why, despite its axonal origin, phf tau accumulates primarily in the neuronal cell body and dendrites, is unknown. it shows in three stages: (a) pre-nfts composed of diffuse, or punctuate tau staining occur within the cytoplasm of otherwise normal-looking neurons with well-preserved neurites; or (b) mature intraneuronal nfts consist of cytoplasmic filamentous aggregates of tau displacing the nucleus toward the periphery of the soma and extending to the proximal segment of the axon. they appear as “flame-shaped tangles” in pyramidal neurons of the hippocampus (fig. 5) and layer v of association cortices and as “globose tangles” in subcortical nuclei; (c) extraneuronal “ghost” nfts in dead neurons, showing loss of their nucleus and of stainable cytoplasm [115]. total loss of functional microglia in advanced late-onset ad (load) promotes widespread intraneuronal neurofibrillary degeneration leading to brain failure [116]. neuronal tau pathology has been linked to neuronal death and cognitive decline in ad [117], while others suggested that neuronal cell loss is associated with dementia and not the presence of plaques and tangles [118]. it is generally thought that nfts impede neuronal functioning, but recent data indicate that they can be found in functionally intact neurons integrated in cortical circuits [119-121]. how hp-tau specifically mediates its toxic effects is still unknown, but oligomeric tau species, analogues to aβos, are potential toxic species besides nft tau. recent proteomic studies have identified specific proteins that interact with hp-tau, showing novel potential pathogenic mechanisms that are relevant in ad and providing insight into how hp-tau mediates its toxicity in ad [122]. figure 5. in ad, high amounts of neurofibrillary tangles and neuropil threads are seen in the hippocampus (a). ca1, ca2, and ca4 hippocampal cornu ammonis (ammon’s horn) sectors 1, 2, and 3, respectively; gr, granule cell layer of the dentate gyrus. immunohistochemistry with antibody at8. scale bar: 50 μm. from [71]. 2.7. topography and spreading pattern of tau the extent of tau pathology (nfts and nts) follows a predictable spatiotemporal progression through functionally integrated brain regions [18], which had been interpreted as a cell-to-cell spreading through prion-like propagation [123-127] or a transneuronal spread through functional networks, associated with a trigger, possibly aβ and/or neuronal network activity that could lead to progression of nft pathology [128]. since tau is expressed predominantly in neurons rather than glial cells, the detection of tau aggregates in astrocytes and oligodendroglia has given support to the concept that the release of misfolded tau from neurons (or oligodendroglia) may result in uptake into other cells [129]. microglia could potentially play a role in spreading of tau pathology [130]. transcellular progression of tau seeds has been observed in early braak stage in regions predicted to be free of hp-tau [131]. according to the original staging [18], the first nfts consistently occur in the transentorhinal (perirhinal) region (stage i) along with the entorhinal cortex, followed by the ca1 region of the hippocampua (stage ii), indicating a preclinical phase of ad which can last up to 20 years. limbic structures, such as the subiculum of the hippocampal formation are affected next (stage iii), followed by the amygdala, thalamus, and claustrum (stage iv). stages iii and iv are often correlated clinically with mild cognitive impairment (mci). in stage v, nfts spread to isocortical areas with the association areas being affected prior and more severely, followed in stage vi by the primary sensory, motor and visual areas, which is usually associated with overt dementia (fig. 6). this nft staging has been widely accepted in routine pathology and appears well correlated with the clinical status, at least in the amnestic ad. imaging in vivo tau pathology with tau-specific pet tracers identified nft pathology reflecting braak stages iv or higher. it rendered it possible to study the temporal progression of tau pathology in vivo, and, therefore, can be used as a reliable biomarker of tau pathology [132-137]. there is an inverse correlation between the accumulation of nfts and cognitive status; the spread and level of tau accumulation reflects the severity of dementia with time [61, 138-140]. the seeding activity is suggested to begin in the transentorhinal/entorhinal regions and anticipates hp-tau pathology in ad, whereas the locus ceruleus (lc) showed seeding only in later nft stages [141]. however, immunohistochemistry has detected pre-tangle material in multiple subcortical regions, especially in locus ceruleus (lc) neurons [142-144]. involvement of the subcortical nuclei, not considered in the original braak scheme, however, occurs in early stages of the disease and has important clinical consequences. the cholinergic nucleus basalis of meynert and axons of the adrenergic lc projecting neurons are affected already in braak stages 0/i, associated with severe neuronal loss, while moderate to severe deposition of tau in the lc was only seen in braak stages above iv [142]. the intralaminar nuclei of the thalamus, the pontine parabrachial region, the medullary reticular formation, the dorsal raphe nucleus, the oculomotor system, and the autonomous nuclei are also affected early and increase with disease progression [142, 145-147]. nigral pathology including hp-tau (nfts) accumulation and α-synuclein aggregates is common in elderly patients with and without ad, and may be related with extrapyramidal symptoms [148-150]. figure 6. spreading pattern of neuritic ad pathology. modified from [18]. 2.8. aβ and tau pathology chicken or egg? the causes of sporadic ad are far from being understood, while the hallmarks that distinguish ad from other neurodegenerative diseases – namely aβ plaques and nfts have been known for many years. the physiological and pathological roles of tau and aβ, and their implications for ad pathology and therapeutics have been reviewed recently [151]. many studies have linked aβ and tau and raised the possibility that protein-protein interactions are the key for both spreading and toxicity of these two abnormal proteins [152]. several models of interaction have been suggested: (1) the seeding of toxic tau is enhanced by the presence of aβ; (2) the toxicity of aβ depends of the presence of tau; (3) aβ and tau enhance each other's toxicity. modern network-based models revealed ways in which aβ and tau protein might interact with each other to enhance the propagation of ad, thus shedding light on the importance of protein clearance and protein interaction mechanisms in the development of ad pathology [153, 154] [155]. soluble oligomeric aβ is hypothesized to be a possible cause of the hyperphosphorylation of tau and the development of nfts. the presence of aps accelerates both the formation of hp-tau aggregates [156] and its interneuronal transfer [157]. the aβo hypothesis was introduced in 1998, suggesting that the brain damage leading to ad was initiated by soluble ligand-like aβos [158]. the extension of tau pathology is different from the spread of aβ deposition that is related to diffusion of soluble aβ in the extracellular space [159, 160]. quantification of adnc in formalin-fixed post-mortem human brain tissue detected high amounts of aβ in the frontal cortex and striatum, and of hp-tau in the frontal cortex and hippocampus of cases with high adnc pathology load [161]. the most recent version of the amyloid cascade hypothesis assumes ad arises from synaptic toxicity mediated by soluble aβos, leading to synaptic dysfunction and loss. age-related aggregation of aβ and its apparent downstream effects on microglia, astrocytes, and neurons, including the post-translational modification of the tau protein, seems necessary for ad symptom expression [162]. while an optimal concentration of aβ is thought to likely maintain synapses, alterations in the proteolytic processing of app may cause dyshomeostasis of aβ, increasing the levels of aβ-42, and initiating ad by setting off a chain of events that leads to the accumulation of tau and downstream neuronal cell death [163]. soluble aβos are now suggested to cause neuronal damage [76]. they are believed to insert into membranes, while others support ligand-like accumulation at particular synapses, providing a substantial molecular basis for the cause of ad [164]. recent data support the hypothesis that aβ enhances tau pathology through increased spreading of tau induced by phf in vivo [165-168], and that aβos promote tau seeding potentiating intracellular tau aggregation [169, 170]. intraneuronal aβ accumulation is suggested to precede tau pathology in the entorhinal cortex [171] and to interact with hippocampal and cortical tau pathology, while in the absence of aβ tau deposition may be insufficient for the neurodegeneration process that leads to ad [172]. many data supporting a toxic role for aβos have backed the aβo hypothesis for ad pathogenesis, but further advances in aβo structure-function studies are needed [158]. recent studies point to a role for exosomes in the spreading of toxic aβos and the associated disease progression in the ad brain [173]. however, the traditional consensus of the amyloid paradigm as a singular cause of ad has been under revision, with the accumulation of new pathobiological evidence [174]. new theories suggest that various mechanisms, including prion-like spread of aβ and tau, vasoconstrictions, growth hormone secretagogue receptor 1α (ghsr1α), and neuroinflammation, come together at a crossroad that ultimately leads to ad [11], while others suggested that extracellular aβ and tau act in parallel and upstream of app [175, 176]. however, recent findings have shown that the soluble form of app binds directly to gababr1a and modulates synaptic transmission [177], while that of aβ aggregates do not need app overexpression [178] but are performed by extracellular exosomes [173]. aβos are deposited inside synaptic terminals [179], enriched in small vesicles at the presynaptic side [47], and enhance synaptic dysfunction in ad [180]. according to others, aβ and hp-tau may develop concomitantly within synaptic terminals [181, 182] and cause abnormalities at synapses [183]. on the other hand, preclinical evidence indicates that tau pathology can progress independently of aβ accumulation and arises downstream of genetic risk factors for ad by an aberrant metabolic pathway [184]. the argument that insoluble aβ and tau deposits begin forming concomitantly in the cerebral cortex of ad brains would be consistent with the argument in favor of the pathogenic importance of tau deposition. recent quantitative studies did not find regional association between aβ-42 and insoluble tau, but a higher regional association between total aβ-42 and soluble tau phosphorylation. this provides evidence supporting the local interplay between aβ and soluble hp-tau in ad brains [185], and accumulating evidence suggests that both pathologies have synergistic effects. the complex aβ-tau interaction is important for elucidating disease pathogenesis and the design of next-generation ad therapeutical trials [153]. targeting the common epitope could be a more effective treatment strategy than targeting only aβ or tau alone [186]. mounting data suggest that the prion-like spreading of diffusible oligomers and other protein aggregates from cell to cell within the brain, probably through specific neuronal networks, may contribute to ad progression [128, 187]. app overexpression is not a prerequisite for the prion-like induction of cerebral aβ deposition that may contribute to disease progression in ad [178], and the multiple failures of previous anti-aβ drugs may suggest that in the ad brain, the accumulation of aβ could be secondary to an unknown 'initial disrupting event' [188]. processing and clearance of aβ and tau could be related to a bidirectional relationship between adnc and autophagy [189]. seeded templating and neurotoxicity are two of the most critical properties attributed to oligomers that have been documented for misfolded proteins in neurodegeneration [190]. it has been speculated that cellular prion protein (prpc) is a critical player in the interplay between aβ and tau propagation in a large group of ad cases. pre-existing hp-tau pathology interacting with prpc appears to be a prerequisite for aβ function as a hp-tau pthology acceleration via prpc [165]. toxic tau oligomers (tauos) and toxic oligomeric aβ assemblies (aos) have prionoid characteristics and are responsible for cell-to-cell spreading in the brain. both extraand intracellular aβos and tauos (not nfts and aps) may represent novel targets of ad research and therapeutic trials [191]. preventing soluble aβo formation and targeting their n-terminal residues with antibodies could be an attractive combined therapeutic approach [178]. recent studies found striking patient-to-patient heterogeneity in the hyperphosphorylated species of soluble oligomeric seed-competent tau. its seeding capacity correlates with the aggressiveness of the clinical disease, and some post-translational modification sites appeared to be associated with both seeding activity and worse clinical outcomes, suggesting that different individuals with “typical” ad have distinct biochemical features of tau that correlated with differences in the aggressiveness of clinical course [192], supporting an important causal role of tau as a driver of clinical dysfunction in ad [193]. the synergism between aβ deposition, nft neurodegeneration, and caa may be a better predictor of cognitive decline or disease progression than either pathology alone [194] (fig. 7). figure 7. staging of aβ, nft, and caa in non-demented (pre-ad) and demented ad patients. from [195]. 2.9. synaptic and neuronal loss essential neuropathological features of ad are loss of synapses and selected neuronal cells (20-40% in neocortex and 25-65% in hippocampus) as the main pathological substrate of cortical atrophy. its regional and laminar pattern parallels the distribution of nfts and has been suggested to be a better correlate of cognitive deficits than the aβ burden [139, 196]. little is known about the molecular basis of selective neuronal vulnerability in ad and the molecular pathways that lead to neurodegeneration, a key characteristic of the disease. it is the result of multiple molecular changes of interacting genes and pathways within vulnerable neurons [25]. the relationship between cellular senescence in the context of aging and ad have been reviewed recently [197]. age-related intraneuronal aggregation of aβ is colocalized with mitochondria and endosomes and less so with lysosomes and autophagosomes. understanding age-related changes in intraneuronal aβ may lead to application of countermeasures to prolong dementia-free health span [198]. the intraneuronal accumulation of aβ may involve synaptic dysfunction and the formation of aps in ad; intraneuronal aβ-42 has been reported to disrupt the normal cytoarchitecture of neurites. recent studies indicate that in ad, vulnerable-neuron-specific dysregulation of polypyrimidine tract binding protein (ptb) (ncbi gene id 5725), a regulator of alternative splicing [199], is the protein most highly correlated to tau in the principal neurons of the entorhinal cortex layer ii (ec ii). ptb could precipitate a 3r/4r tau imbalance in these neurons and explain the premature accumulation of nfts, thus explaining the vulnerability of ec ii neurons [25]. the neurotoxic effect of astrocyte-derived exosomes (ade) is evident with the overlap of ap density and c3/4 fragments (complement factors) observed in early ad [200]. although tangle-bearing neurons can be long lasting in regions where nfts occur at a presymptomatic stage, neuronal loss occurs early in the course of the symptomatic disease [201]. two mechanisms of neuronal death in ad have been discussed: one affecting tangle-bearing neurons that will lead to ghost extracellular tangles, another affecting tangle-free neurons, at least in part by apoptosis [202-204]. inflammation-induced hyperphosphorylation of tau destabilizes the microtubule-actin network and impairs axonal transport and disturbs energy metabolism in the axon, inducing further tau phosphorylation. accumulating data point to the fact that this facilitates the formation of phfs, further impairs axonal transport leading to complete blockage and axonal leakage, and induces loss of synaptic contacts promoting activation of microglia and reactive astrogliosis [56]. microglia have been shown to instigate tau pathology in diverse ways, inducing tau aggregation by proinflammatory cytokine release [205, 206], and spreading hp-tau oligomers or nfts through exosome secretion [130]. synaptic loss that is possibly driven by aβ and tau pathology has been suggested to precede neuronal loss [207]. synapses are present in aps and their total number decreases with time [61, 208]. their loss has been demonstrated ultrastructurally and immunohistochemically [209]. in late stages of ad synapse loss ranges from 10 to 60%, most severely in the frontal and mesiotemporal regions. synapse loss by activated astrocytes producing different secretomes reduce protein synthesis for synapse formation, resulting in synaptic loss found in ad [210]. there is a close relationship between aβ accumulation and synaptic loss that may provide direction for the development of potential disease-modifying treatments of ad [211]. eoad is associated with a higher burden of adnc and a higher rate of neocortical atrophy and synapse loss than the much more common and apparently sporadic load [212]. however, synaptic loss is not a unique hallmark of ad and occurs in many other brain diseases [213]. 2.10. neuroinflammation activated microglia operating as phagocytes are frequently observed around aβ plaques driving an inflammatory response, which can be activated by multiple factors in the local environment [214], in particular by the presence of aβ in the cortex, indicating a “toxic” response which corrupts neurons as collateral damage (“bystander effect”) [215]. tau-positive nps being early in ad, however, major tau deposition follows the accumulation of aβ and clustering of activated microglia. an increase in membrane attack complex formation leads to increased tau pathology and neoronal loss [216]. on the other hand, microglia may contribute to elimination of tau deposits by phagocytosis [217, 218]. different states of microglia activation, corresponding to regional activation of aβ and tau, are present simultaneously in the same brain. the clustering of activated microglia is greatest in the primary motor cortex, a region relatively spared compared to the severely affected inferior temporal cortex in ad. this suggests that microglial activation is not prominent in the early phase of ad pathophysiology [66]. recent studies in hp-tau mice demonstrated that microglia are not the agitators of tau aggregation, but different results about the involvement of microglia in tau aggregation and clearance were presented [219, 220]. thus, the functional role of microglial activation with hp-tau oligomers still remains elusive. gene-profiling technologies applied to isolated microglia have challenged the hypothesis that there is one acute-type (microglial drivers) of inflammation in the human brain causing accelerated proinflammatory damage in ad. these studies have shown that many of the microglia genes expressed in increased levels reflect a response to restore homeostasis and limit inflammatory damage [221]. on the other hand, there is an early microglia reaction to ad pathology, but a loss of healthy microglia is the prominent feature in severely affected regions of the ad brain [222]. in addition, there is a non-disease-specific response of microglia to neuronal damage, with upregulation of phagocytotic activity to remove damaged neurons and synapses by cd68 immunoreactivity of lysosomes [73]. their numbers increase on promotion to neuronal damage associated with nfts [62], which is due to enhanced production of inflammatory cytokines, such as il-21 and increase in t follicular helper cells. the strong immune response is insufficient at clearing up aβ and instead exacerbates inflammation [223]. reactive astrocytes that may react to cytokines and other agents produced by pro-inflammatory microglia, are observed around aps, though less frequently compared to microglia. reactive astroglia burden occurs later in ad and correlates mainly with tau pathology [31]. 2.11. pathology of preclinical ad amyloid and neuritic plaques and nfts occurring in non-demented elderly individuals, represent asymptomatic or preclinical ad (pre-ad), while clinical ad affects subjects with late stages of adnc. both ad and pre-ad cases often exhibit caa, which is also observed in non-ad cases, i.e., those without adnc. patients with mci do not always have adnc even though they have a risk of developing dementia in 10-12% and sometimes do not have any discernable pathology [224, 225]. the presence of nfts and caa in cases without aps, classified as non-ad, suggests that they may precede ap pathology or may present a pre-amyloid plaque stage not yet included in the current criteria for the neuropathological diagnosis of ad [226]. increased soluble/dispersible aβ in pre-ad compared to fully developed suggests that, in addition to more severe and widespread adnc, soluble aβ aggregates play a role in the conversion of pre-ad to clinical ad [31]. cognitively impaired individuals presenting with an early onset ad phenotype showed higher rates of tau pet accumulation, while among cognitively unimpaired individuals higher rates of tau accumulation were associated with faster rates of memory decline [227]. 2.12. neuropathological diagnosis of alzheimer’s disease histopathological examination of the brain has to establish that adnc are present in sufficient densities and extensions to distinguish ad from other age-related disorders [61]. because the disease affects the whole brain, it is not sufficient to make the diagnosis of ad just on one or two brain blocks; instead, multiple brain areas have to be examined and a staging protocol has to be established. the current algorithms for the pathological diagnosis of ad are based on semiquantiative assessment of aps and nfts providing reasonable interrater agreement when using standardized criteria [228]. current guidelines include (a) cut-off quantitative values for aps and tangles [17, 229]; (b) the semiquantitative assessment and age-adjustment of nps in the consortium to establish a registry for alzheimer's disease (cerad) protocol [230]; (c) topographic staging of neuritic/tau pathology [18], re-evaluated by immunohistochemistry [231]; and (d) the progress and distribution of aβ phases [79]. in order to develop a system that combines all the above pathological features, the nia/aa established a composite score comprising the extent of involvement/spread of cerebral aβ based on the progression model by the thal phases: (a), that of nfts based on the progression model of braak, (b), and the cerad score, which describes the density of neuritic amyloid plaques based on certain key locations in the neocortex, (c) (table 1). from this combination, it gave a likelihood for the degree of ad neuropathological changes in an individual case. sufficient agreement in ad diagnosis could be reached only when the lesions are considerable (braak nft stage v and vi) with 91% agreement, while for mild lesions it was poorer (for braak stage i and ii, agreement was only around 50%) [228, 232, 233]. combined braak and cerad scores in the nia-ri (national institute on aging and reagan institute) criteria that apply only to demented persons, relate dementia to adnc with high, intermediate and low likelihood [234]. they have been widely used in anglo-american neuropathology and are now replaced by the nia/aa guidelines. although the sensitivity and specificity of the nia-ri criteria has been proposed to be around 90%, only 30 to 57% of the brains of patients with the clinical diagnosis of probable ad showed “pure” adnc, thus reducing their predictive value to 38% [235]. an evaluation of the nia-ri criteria identified between 54 and 97% of ad cases with high braak or cerad stages, and eliminated between 60 and 100% of non-ad with low braak or cerad stages [232, 233]. another autopsy study reported diagnostic sensitivity ranging from 70.9 to 87.3% and specificity from 44.3 to 70.8% [236]. the recent updated nia/aa guidelines for the neuropathological assessment of ad consider adnc levels regardless of the clinical history of a given individual [17]. they include (1) the recognition that adnc may occur in the apparent absence of cognitive impairment; (2) the use of an “abc” score for adnc that incorporates histological assessment of aβ plaques (a), based on its phase assessment [79], staging of nfts, (b) based on the braak staging system [231], and scoring of nps, based on their semiquantitative assessment in at least three neocortical regions, and (c), based on cerad criteria [230]. table 2 shows how each of the three scores are transformed to state the level of adnc on a four tiered scale (non, low, intermediate, and high). the entire process of the neuropathological diagnosis of ad can be followed along the pathways shown in fig. 8. (3) more detailed approaches for assessing co-morbid conditions, such as lewy pathology, vascular brain injury, tdp-43 immunoreactive lesions, argyrophilic grain disease, and others that can complicate the pathological diagnosis and can sometimes co-exist with ad, are also considered [17]. testing of the revised nia/aa guidelines in 390 autopsy cases distinguished pure ad and non-ad dementia from non-demented cases with a sensitivity of 91% and a specificity of 99%. the sensitivity increased after exclusion of non-ad dementia cases, indicating that the revised nia/aa criteria appear practicable for distinguishing pure ad from non-ad dementia, preclinical ad, and controls [226]. the revised nia/aa guidelines for the severity score for adnc used in the ad centers program achieved an excellent agreement (κ=0.88, 95% ci 0.77-0.95), and good-to-excellent agreement for the three supporting scores [237]. figure 8. pathway of the combination of different pathological features that allows a classification of adnc according to the nia-aa guidelines. a comparative study of clinical and neuropathological diagnoses of ad in three epidemiological samples reported a sensitivity for probable ad of 93% [238]. meta-analysis of 20 (out of 1,189) studies to distinguish autopsy-verified ad from other dementias or healthy controls showed a sensitivity of 85.4% (95% ci 80.8-90%) and a specificity of 77.7% (95% ci 70.2-85.1%). values were higher for neuroimaging procedures and slightly lower for csf biomarkers, while the combination of both resulted in better results [239]. 3. pathobiological subtypes of ad recent studies showed that the neuropathology of ad is heterogenous [240-242]. the current guidelines for the neuropathological diagnosis of ad only consider the classical “plaque and tangle” phenotype but not other subtypes such as the “plaque only but without tangle formation/predominant” type with abundant amyloid, or the “little or no tau pathology” type limited to the hippocampus and abnormal hp-tau in neocortical pyramidal cells. this type, observed in 3.4-8.0% of demented subjects over age 85 years [243], frequently represents a specific type of dementia with lewy bodies (dlb)/dlb-ad [244]. the recently described “primary age-related taupathy” (part) [245], previously referred to as “nft-predominant dementia” [246], involves people over 85 years old and is associated with mild to moderate cognitive impairment [247, 248] it reveals tau pathology restricted to the mtl (braak stages 0-iv), relative absence of amyloid (thal aβ phases 0-2), total absence of nps, and rare caa [249]. the composition of nfts in part both for 3r and 4r tau isoforms is identical with those in classical ad [246], while pattern of hippocampal tau pathology differs significantly between part and ad [250, 251]. tau aggregates influence cognition and hippocampal atrophy in the absence of aβ [249]. positive correlations were reported in part between the braak nft stage and phosphorylated 43-kda tar dna-binding protein (ptdp-43) stage and density [252]. part is considered either a prodromal form or a subtype of ad [253, 254] (see table 3). mapt h1h1 genotype frequency is high in both part and limbic-predominant ad (lp-ad), and similar to typical ad, while apoeε4 is rather rare in part [255]. other genetic differences between part and ad have been described [256]. it seems that lower concentrations of aβos cause less severe tau deposition due to the fact that they can potentiate tau aggregation by promoting tau seed uptake [170]. cognitive decline in part is usually milder than in ad and correlates with tau burden. biomarkers and neuroimaging studies will be important to define part ante-mortem and to follow its natural history [257]. while the incidence of classical ad increases from the 7th to the 9th decade and later shows a mild decrease, the frequency of part increases after the age of 85 years [3]. the limbic-predominant age-related tdp-43 encephalopathy (late), a recently described disease entity mainly involving elderly people (>75 years at death), is associated with an amnestic dementia syndrome that may mimic ad [258, 259]. it shows pathogenic mechanisms of both frontotemporal lobar degeneration with tdp-43 (ftld-tdp) and ad, but there are different molecular patterns of tdp-43 pathology in various clinical phenotypes with a higher chance of ftd-like symptoms in ad + full-length tdp-43 cases [32]. recent studies indicated that in most cases, limbic-predominant age-related tdp-43 encephalopathy (late-nc) and ftld-tdp can be differentiated by applying single neuropathological criteria, e.g., the severity of cortical tdp-43 inclusions [260]. biomarkers for ante-mortem diagnosis of this syndrome are currently not available [261]. recent clinicopathological studies have enabled the identification of several pathophysiologically defined subtypes of ad. one distinguished three ad subtypes based on nft density: typical ad with balanced nft counts in the neocortex and hippocampus (75%), hippocampal sparing (hcsp), with nft counts predominantly in association cortices (11%), and limbic-predominant (lp) ad mainly involving the hippocampus (14%) [262]. these subtypes had different clinical phenotypes, with different ages at onset and rates of progression (fig. 9). patients with hippocampal sparing ad (hcsp-ad) were youngest at onset, had a higher proportion of men, and progressed more quickly than typical ad. lp-ad patients were older, more often female, and showed slower progression. age at death of the lp form was highest, while patients with hcsp-ad were youngest, indicating this type as the most aggressive. this could be related to the contribution of tdp-43 pathology, hippocampal sclerosis, and the microtubule-associated protein tau (mapt) h1h1 genotype to lp-ad, factors related to temporal lobe atrophy, older age, and slower disease progression. apoeε4 carriers more frequently had lp-ad and typical ad, whereas non-carriers more frequently presented as hcsp-ad. vascular co-pathology (ranging from 16 to 36%) was highest in the lp and lowest in the hcsp cases. typical ad had higher ap burden in occipital regions compared with lp-ad [262], while in contrast to specific tau accumulation and brain atrophy patterns among ad variants, aβ accumulation appeared rather diffuse and similarly across groups, except the ma group [263, 264]. tau pathology was closely associated with sites of neurodegeneration and brain atrophy corresponded well with nft topography and neuronal loss. [265-267]. clinical symptoms correlate with neuronal hypometabolism [262, 268, 269]. similar results were reported in a study of 933 autopsy cases of ad, all with neuritic braak stage > iv [270]. typical ad was more frequent than in the mayo series (82.5 vs 75%), while the other two subtypes were slightly less frequent. minimal-atrophy ad (ma-ad) was not included in this study. the lp-ad cases shared some morphological features with part [245], although later studies demonstrated significant pathological differences between part and lp-ad [240]. figure 9. main factors and characteristics of the four major subtypes of ad. ad: alzheimer’s disease; nft: neurofibrillary tangle; wmh: white matter hyperintensity; caa: cerebral amyloid angiopathy; eoad: early-onset alzheimer’s disease; load: late-onset alzheimer’s disease; lp-ad: limbic-predominant ad. typical ad showed greater white matter hyperintensity (wmh) burden, which may be due to wallerian degeneration induced by cortical tau pathology [271, 272], small vessel disease, or both [273-275]. tau pathology and neurodegeneration can disrupt key brain networks, which may induce memory impairment comparable to lp-ad and typical ad in the absence of overt brain atrophy of the mtl in ma-ad that shows [276-278]. distinct patterns of nft deposition in young-onset versus older-onset ad give evidence for variability in regional deposition patterns and demonstrate that different disease phenotypes have different patterns of tau pathology [279]. other atypical non-amnestic syndromes, referred to as focal ad [26, 280, 281], include logopenic primary progressive aphasia (lppa), showing higher nft density in superior temporal gyrus but thal amyloid plaques similar to amnestic ad [282-284]. the proportion of apoeε4 carriers was elevated in amnestic but not in non-amnestic forms of ad, suggesting that apoe is a selective risk factor that increases the vulnerability of memory-related medial temporal areas rather than language-related neocortices [285]. further atypical forms are posterior cortical atrophy (pca) [286], non-amnestic ad with tdp-43 pathology [287], syndromes resembling behavioral variant fronto-temporal lobe degeneration with tau pathology (bvftd-tau) [266, 288], and the corticobasal syndrome (cbs) subtype of ad that shows a higher nft density in the perirolandic cortices and greater neuronal loss in substantia nigra which may contribute to parkinsonism that uncommon in classic ad [289]. behavioral/dysexecutive ad revealed temporo-parietal-predominant atrophy. in the mayo series, pca, lppa and bvftd variants were more common in hcsp-ad than in lp and typical ad [262, 290]. accumulation of nfts and activated hypertrophic microglia associated with low neuron densities suggests that they may collectively contribute to focal neurodegeneration characteristic of primary progressive aphasia ad [291]. the pathogenic factors underlying ad subtypes are unclear and cannot be explained by aβ pathology alone, because the distribution of aβ pet retention is quite similar in all subtypes [263]. however, solid-state nuclear magnetic resonance measurements showed qualitative differences between aβ-40 and aβ-42 aggregates in the brain tissue of patients with two atypical ad clinical subtypes posterior cortical atrophy variant and a typical prolonged-duration form indicating that there are structural variations in aβ fibrils from ad clinical subtypes [60]. ma-ad, although aβ-positive, shows less tau pathology. according to recent studies, ad “subtypes” may be linked to different tau protein modifications, suggesting that ad patients may have multiple molecular drivers of an otherwise common phenotype [192]. this suggests that multiple subtypes are parts of the same ad continuum [266], which may have consequences for personalized therapeutic approaches. 4. the impact of co-pathologies ad pathology rarely occurs in isolation, while complex pathologies frequently lead to cognitive decline. the number of co-morbidities increases in the aging brain, causing mixed pathologies [3, 248, 274, 292-298]. the challenges of pathological mimics and concomitant pathologies in the neuropathologic diagnosis of ad have been critically reviewed recently [174]. the most frequent co-pathologies are cerebrovascular disease (cvd) and lewy and tdp-43 proteinopathies [31, 258, 299, 300]. in a consecutive autopsy series of 2,060 elderly demented patients and those with the clinical diagnosis of ad, adnc were present in 82.9% of all demented and in 92.8% of clinically diagnosed ad cases, but only 33.6% and 47.6%, respectively, showed pure adnc (abc 3/3/3). the others were either atypical ad forms or subtypes (including part) (7 and 6%, respectively) or exhibited additional cvd (24.3%), lewy (12.5%) or other mixed pathologies. vascular dementia in this cohort accounted for only 12.2% and 3.3%, respectively; other non-ad pathologies were present in 7.2% and 3.7% [301]. another study of demented elderly persons reported pure adnc in only 31% and multiple pathologies in 63% [302]. a review of 12 studies with 3,574 patients, irrespective of the clinical symptoms, reported adnc between 19% and 67%, lewy pathologies in 6% to 39%, vascular pathologies in 28% and 70%, tdp-43 proteinopathy in 19% to 78% [290], hippocampal sclerosis between 3% and 13%, and mixed pathologies between 8% and 70% [295]. among 447 patients with probable ad, only 3.13% showed pure adnc, 27.3% ad+cvd + other, 3% ad + cvd, 7.6% ad + other degenerative lesions, and 47% ad+cvd + other neurodegenerative lesions [293]. this list of combinations is not complete and there are other combinations with rare entities that need specific attention [174]. among 673 autopsy cases, including 320 demented, the majority showed mixed pathologies [274]. late-nc was present in 57% of ad cases and was associated with more rapid disease progression [259]. increased tdp-43 pathology in typical ad and lp-ad compared to hcsp-ad [262] was due to a strong association between hippocampal sclerosis and tdp-43, but clinical presentation seemed to be driven by morphological subtypes and not by tdp-43 pathology [290]. among 61 autopsy-proven ad cases, late-nc was present in 67.2% (ad, late-nc), however, it was not associated with an increase of the burden of early or late tau nor aβ pathology. late-nc showed a lower final mini mental state examination (mmse) score independent of tau pathology [303]. among 172 autopsy-confirmed ad cases, 19% were classified as non-amnestic, 69% of which had typical adnc, 31% were hcsp-ad, 36% tdp-43-positive, while there were no lp-ad cases [287]. in a recent study of 46 autopsy-confirmed ad cases, 63% exhibited late-nc (ad+) and a higher burden of hp-tau. this indicates a possible progression of the disease, whereas ad plus late-nc was not associated with differences in cognitive scores [304]. late-nc may also occur in isolation and has been viewed as a common brain disease in aging [258]. among 574 individuals with complete measurements of mmse and the clinical dementia rating scale sum of boxes (cdr-sb) from 39 ad centers across the usa, 63% of those given the 'gold standard' diagnosis of ad, possessed either tdp-43 proteinopathy or caa of sufficient severity to independently explain the majority of their cognitive impairment. aβ and/or tau burden, particularly in braak stages iv to vi, and small cerebral vessel disease may synergistically affect cognitive decline [305], and a significant interaction was found between braak nft stages, caa status and cognitive decline, suggesting that there is a significant interaction between tau pathology and caa on cognition within the ad clinical spectrum [306]. hence, interventions targeting caa may contribute to delay the onset of cognitive impairment, particularly in individuals with intermediate adnc [307]. this suggests that many individuals diagnosed with ad may actually suffer from a mixed dementia, and therapeutic targeting ad-related processes only may have limited efficiency in these co-morbid populations [298]. based on data from the nacc, 1,854 participants with a clinical diagnosis of ad and adnc at autopsy (confirmed ad) were studied; 204 with the clinical ad diagnosis had no adnc (ad-mimics), while 253 participants with negative clinical ad diagnosis had adnc (unidentified ad). compared to confirmed ad cases, ad-mimics (ftld-tau, hippocampal sclerosis, cerebrovascular pathology, etc.) had less severe cognitive impairment [308]. special practical considerations for the diagnosis of essential co-pathologies and their relations with ad were given recently [174]. argyrophilic grain disease (agd), a limbic-predominant 4r-tauopathy, with grain-like deposits in neuritic dendrites, oligodendroglial inclusions (“coiled bodies”), ramified astrocytes, and ballooned neurons in the amygdala, hippocampus and mtls [309], represents an age-related disorder and has been reported in up to 25% ad cases [310], and rarely occurs before the age of 75 [311]. aging-related tau astrogliopathy (artag) is defined by the presence of two types of tau-bearing astrocytes: thorn-shaped and granular/fuzzy astrocytes in the brains of old-aged individuals in different locations and anatomical regions (subependymal, subpial, perivascular, white and gray matter [312, 313]. among additional pathological changes in ad is granulovacuolar degeneration (gvd), characterized as 3-5 μm vesicles bound by a unit membrane, most frequently occurring in the pyramidal neurons of the hippocampus, usually in association with nfts. their origin and significance are unclear. despite the strong association between tau aggregation and granulovacuolar degeneration body (gvb) formation [314], intracellular aggregates of proteins other than tau can also induce gvb formation, which needs further elucidation [315]. the granule of the gvd is immunolabeled by antibodies against tubulin, ubiquitin, neurofilament, and tau [31]. they correlate with nft density, suggesting that they may be a cellular response to neuronal damage or late-stage autophagic vacuoles [316]. necrosome complex detected in gvd is associated with neuron loss in ad [317]. recent clinicopathological studies have shown that the complex cascades of the underlying pathologies in most elderly patients may lead to cognitive decline, and that the number of possible combinations due to co-morbidities increases with aging [248]. these concomitant pathologies may be harmful to individuals with low cognitive reserve such as patients with ma-ad. they can cause a number of challenges including the evaluation of the significance of each pathological entity in the manifestation of the clinical symptoms, and the threshold of each individual pathology to cause dementia [174]. total burden of comorbid pathological abnormalities, rather than any single lesion, is the most important cause of cognitive impairment, often despite clinical diagnosis of “only” ad [318]. 5. conclusions ad is a heterogeneous, multifactorial disorder, manifesting clinically and morphologically as several subtypes that have a distinct signature of network disruptions associated with their atrophy pattern and reflecting the differential spread of nft pathology and neuronal loss due to different vulnerability patterns of affected brain regions, which relates to specific molecular-functional properties of the affected neuronal systems [22, 23]. the severity of lesions corresponds to the “n” category in the new a/t/n classification for biomarkers [319]. the heterogeneity of the alzheimer’s syndrome is related to multiple pathogenic factors which induce misfolding tau, aβ, tdp-43, and other proteins, the synergetic or additive action of which results in various disease phenotypes [320]. several factors such as brain resilience may help compensate for these pathologies up to a certain level, although their relevance is still poorly understood. these problems and the increasing incidence of ad illustrate its consequences on public health and the resulting challenges for future medicine. increased sensitivity and specificity of new atn biomarker systems and more extensive clinicopathological studies in well-defined populations are needed, with post-mortem studies using the updated nia/aa criteria. the recent advent of tau pet and novel imaging and fluid-based (csf) biomarkers allows us to study the temporal progression of tau pathology in vivo [321, 322]. improving methods for disease detection and monitoring its progression may hopefully lead to the development and refinement of tau-based therapeutics. in the interest of optimizing the clinical diagnosis of ad and related disorders, neuropathological studies should use a wide range of molecular pathological methods and should evaluate multiple cns regions. an optimal and less cost-intensive strategy would be to screen specifically neurodegeneration-related proteins and to examine their 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international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. 5th asian oceanian congress of neuropathology along with the 5th annual conference of the neuropathology society of india (aocn-npsicon) meeting abstracts feel free to add comments by clicking these icons on the sidebar free neuropathology 3:14 (2022) meeting abstracts 5th asian oceanian congress of neuropathology along with the 5th annual conference of the neuropathology society of india (aocn-npsicon) hosted by: department of neuropathology, nimhans, bangalore, india meeting abstracts september 24–26, 2021 submitted: 12 may 2022 accepted: 12 may 2022 published: 19 may 2022   the 5th asian oceanian congress of neuropathology along with the 5th annual conference of the neuropathology society of india (aocn-npsicon) was held in virtual mode on september 24–26, 2021, at national institute of mental health and neurosciences (nimhans), bangalore, india, hosted by the department of neuropathology. it had 361 attendees from 20 countries from asia and oceania including india. the event brought together pathologists, clinicians and neuroscientists from all over asia and oceania with invited speakers from the usa, germany and canada. the program was very comprehensive and covered advances in the fields of neurooncology with emphasis on the upcoming who 2021 classification of cns tumors, neuromuscular disorders, epilepsy and neurodegenerative disorders through key note addresses and symposia that featured 78 distinguished international and national faculty sharing their expertise. in addition, there were case-based learning modules, opportunities for paper presentations and poster sessions for young faculty and postgraduates with several awards for young investigators, best papers and posters. a highlight of the conference was a unique debate on the hot topic of the decade: methylation-based classification of cns tumors and a panel discussion on covid-19. the participants were highly appreciative of the academic content. https://doi.org/10.17879/freeneuropathology-2022-4121 keywords: neuropathology society of india, npsi, meeting abstracts contents 1. clinicopathologic analysis of diffuse midline gliomas: a single centre study 2. low frequency of ezhip overexpression in diffuse midline gliomas, h3 wildtype 3. location specific kiaa1549::braf fusion variants in pilocytic astrocytoma 4. epithelial to mesenchymal transition (emt) in meningiomas 5. programmed death ligand 1(pd-l1) expression and tumor infiltrating immune cell subpopulations association with clinicopathological and prognostic parameters in diffuse gliomas 6. insights into molecular biology and immune micro-environment of pleomorphic xanthoastrocytoma 7. dynamics of cell-free dna in predicting response in adult diffuse glioma on chemoradiotherapy 8. regional heterogeneity in mitochondrial function in human brain ageing: implications for region-specific vulnerability to neurodegeneration 9. ryr1-related myopathies: a series of 5 patients from an indian tertiary care centre 10. utility of respiratory chain complex assays in the diagnosis of mitochondrial disorders a phenotype, histopathological and genotype correlation 11. myelination changes in white matter following severe traumatic brain injury (stbi): a neuropathological study 12. is cerebral malaria an astrocytopathy? a post mortem study of bbb dysfunction 13. does mitochondrial dysfunction play a role in pathogenesis of mesial temporal lobe epilepsy secondary to hippocampal sclerosis? 14. diffuse midline gliomas with h3k27 mutation clinicopathological correlates 15. significance of nestin and cd133 as cancer stem cell markers in diffuse gliomas and its association with idh-1 status and p53 expression 16. immunophenotypic profile of pediatric brain tumors reflecting molecular alterations 17. pdl1 expression in cns tumours 18. droplet digital pcr: a robust technique for detection of idh1 r132h mutation in formalin fixed tissue samples 19. tert promoter mutations in meningiomas: a clinicopathological correlation 20. evaluation of tert promoter mutation status in meningiomas 21. pitfalls in diagnosis of oligodendrogliomas on squash cytology 22. low prevalence of braf v600e mutations in pleomorphic xanthoastrocytoma 23. unusual primary intracranial sarcomas – ewing’s sarcoma and synovial sarcoma 24. relevance of the 4-variable risk stratification model in cns solitary fibrous tumours 25. oropharyngeal psammomatous melanotic schwannoma, non-syndromic an unusual tumour at an unusual site 26. silent corticogonadotroph adenoma (scga): a silent monster 27. molecular profiling: a key to clinical and histological enigma in an ambiguous case 28. pseudotumoral hemicerebellitis masquerading as lhermitte–duclos disease – a case report 29. anoctamin-5 muscular dystrophy: report of 2 cases with different phenotypes and genotypes from indian subcontinent 30. dysferlinopathy in a cohort of north indian patients: clinical histopathological and mutational spectrum 31. role of mitochondrial respiratory chain complexes in pathogenesis of temporal lobe epilepsy 32. gliotic and destructive brain lesions associated with drug resistant epilepsy a clinicopathological study 33. cytopathic changes of herpes zoster encephalitis in csf mimicking malignant cells 34. resolving the diagnosis of hmsn with nerve histopathology and genetics 35. understanding the er stress response to predict clinical outcome in focal cortical dysplasia patients 36. candida meningitis mimicking tuberculous meningitis in an immunocompetent patient diagnostic conundrum 37. rare case of dorsally located multiple neurenteric-cyst without spinal dysraphism – neuropathology insights and systematic review 38. efficacy of dimethyl fumarate in chronic constriction injury induced neuropathic pain in rats 39. moya moya disease: an autopsy case study 40. hematolymphoid malignancies presenting with neurological manifestations and hand-mirror cells in peripheral blood: report of two cases 41. mullerian choristoma as a cause of tethered cord syndrome: a case report in a 13-year-old worsening after the onset of menarche 42. plurihormonal pit-1-positive adenoma: a short series 43. an interesting case of extradural tumour in a pediatric patient 44. case report of rare extra axial cerebellopontine angle medulloblastoma, a meningioma mimicker: caution advised 45. potpourri of five cases of rare central nervous system tumors with review of literature 46. primary yolk sac tumor of cerebellar vermis: a case report 47. a diagnostic dilemma astroblastoma 48. meningioangiomatosis: a rare cause of refractory temporal lobe epilepsy 49. isolated cerebral rosai dorfman disease with granulomatous angiitis 50. twin tales of anaplastic ependymoma with extensive vacuolation/signet cell change and lipomatous differentiation     abstract 1 free neuropathol 3:14:5 clinicopathologic analysis of diffuse midline gliomas: a single centre study shiva soma1, megha s uppin1, arvind suman2, suchanda bhattacharjee2 1 department of pathology, nizam’s institute of medical sciences, hyderabad, india 2 department of neurosurgery, nizam’s institute of medical sciences, hyderabad, india background: diffuse midline gliomas are primary cns tumors found in midline location of brain and spinal cord. these are who grade 4 tumors with characteristic h3k27m mutation and worse prognosis. objectives: to determine clinical, pathological, radiological, surgical outcome of patients diagnosed as dmgs. materials and methods: all patients diagnosed as dmg from january 2017 to july 2020 were included following ethical approval. clinical presentation, radioimaging details were taken from medical records. histopathologic features were noted. immunohistochemistry (ihc) was performed with idh1r132h (dianova, dilution 1:200), atrx (sigma aldrich, dilution1:500), h3k27m (milipore, dilution 1:500), p53 (sigma aldrich, ready to use) using poly hrp technique on fully-automated immunostainer (x matrix, biogenex). results: study included a total 29 patients with mean age of 21±12.5years, m:f ratio of 2:1. tumor distribution was thalamus (58.6%), brain stem (27.6%), cerebellum (20.7%), dorsal spine (10.3%) and cervical spine (6.9%). in 14 patients, tumor was biopsied and in the rest stereotactic or open excision was attempted. morphology was variable in all cases. gemistocytes, low cellularity, giant cells, pxa like pattern were some of the highlights in morphology. h3k27m showed dark nuclear expression in all with loss of atrx in 21 cases. twenty-two patients were treated with radiotherapy and 21 patients died on follow up at 6 months. high survival duration was seen in patients of >18 years age (p=0.02) who received rt (p=0.05). conclusion: dmgs are aggressive tumors. ihc with surrogate molecular marker helps in diagnosis. prognosis is poor irrespective of surgery and radiotherapy.   abstract 2 free neuropathol 3:14:6 low frequency of ezhip overexpression in diffuse midline gliomas, h3 wildtype poonkodi manohar1, shilpa rao1, nandeesh bn1, yasha tc1, anita mahadevan1, vani santosh1 1 department of neuropathology, nimhans, bangalore, india background: few diffuse midline gliomas (dmgs) with h3k27me3 loss, lack h3k27m mutation. these h3 wildtype tumors have been shown to either overexpress ezhip or harbour other mutations, thus categorised as dmg, h3k27 altered. however, studies on the frequency of ezhip overexpression in dmg is sparse. objectives: the purpose of the present study was to analyse dmgs in paediatric and adult patients and study the frequency of ezhip overexpression in dmg, h3 wildtype tumors. material and methods: histological and immunohistochemical profiles of dmgs diagnosed between the years 2018 and 2020 (n=123) were reviewed and segregated into two groups; h3k27m mutant and h3 wildtype tumors. immunohistochemistry was performed on the h3k27m wildtype tumors using rabbit monoclonal antibody to ezhip(cxorf67). results: of the 123 cases, 22 were in children and 101 in adults. 20/ 22 (91%) paediatric tumors and 64/101 (63.4%) adult tumors harboured h3k27m mutation. the common location of h3k27m mutant and h3wildtype tumours was thalamus in both age groups. out of a total 39 h3wildtype tumours, only 2 showed ezhip overexpression along with loss of h3k27me3. both the tumours were located in the thalamus, with glioblastoma histology and occurred in young adults (30 and 36 years). conclusion: in our study, the majority of dmgs in paediatric age group and about two thirds in adults harboured h3k27m mutations. a very small subset of h3 wildtype tumours overexpressed ezhip. this study highlights the low frequency of ezhip overexpression in dmg h3 wildtype tumors.   abstract 3 free neuropathol 3:14:7 location specific kiaa1549::braf fusion variants in pilocytic astrocytoma sumitra sivakoti1, harsha sugur1, arivazhaghan a2, saini j3, yasha tc1, vani santosh1 1 department of neuropathology, national institute of mental health and neurosciences, nimhans, bengaluru, india 2 department of neurosurgery, national institute of mental health and neurosciences, nimhans, bengaluru, india 3 department of neuro imaging & interventional radiology, nimhans, bengaluru, india background: pilocytic astrocytomas (pa) are characterized by constitutive activation of the ras/mapk signalling pathway. the most common underlying genetic alteration is kiaa1549::braf fusion, followed by nf1, brafv600e, fgfr1 mutations, braf fusion with other proteins, ntrk family and kras mutation. objectives: to study the frequency of the most common braf genetic alterations, particularly kiaa1549::braf fusion variants and v600e mutation in pa and correlate with clinical features, and particular tumour site. material and methods: retrospective analysis of 61 pa cases was undertaken, and they were tested for kiaa1549::braf fusion and v600e mutation by qpcr and ihc respectively. whole rna from ffpe tissues was reverse transcribed to cdna. using taqman assay, kiaa1549::braf fusion testing at 16-9, 15-9 and 16-11 exons was performed by qpcr. ihc was performed on tissue microarray sections. results: braf genetic alterations were seen in majority of pa (80.3%) -kiaa1549-braf fusions accounting for 77% and v600e mutation for 3.3%. fusion at exon junction 16-9 was the most common (68%) and majority were cerebellar tumors in children less than 15years age. fusion at exon junction 16-11 (15%) was restricted to the cerebellum alone. we noted fusion at exon junction 15-9 (23%) frequently in midline location and few in cerebellum. multiple fusions (16-9, 15-9) were seen in two. brafv600e mutations (2) were restricted to supratentorial location. kiaa1549::braf fusion and brafv600e mutation were mutually exclusive. conclusion: braf alterations are common in pa, especially in children. different sites exhibit different kiaa1549::braf fusion transcripts in varying frequencies16-9 mainly in cerebellum, 15-9 mainly in midline, and 16-11 exclusively in cerebellum. infrequent supratentorial pa show brafv600e mutation. these have prognostic and possible therapeutic implications.   abstract 4 free neuropathol 3:14:8 epithelial to mesenchymal transition (emt) in meningiomas sanjay sriram1, swati mahajan1, mc sharma1, chitra sarkar1, ashish suri2, vaishali suri1 1 department of pathology, all india institute of medical sciences, new delhi, india 2 department of neurosurgery, all india institute of medical sciences, new delhi, india background: epithelial-to-mesenchymal transition (emt) is a process involved in invasion and metastasis of tumors. its activation has been well documented in various malignancies and is associated with poor prognosis; however, data is limited in meningiomas. objectives: to analyse the expression of emt related proteins and transcription factors in various grades of meningiomas and to correlate with clinical parameters. materials and methods: seventy meningiomas of various histopathological subtypes and grades (who grade i=31, ii=31, iii=8) were analysed by immunohistochemistry for emt related proteins (e-cadherin, β-catenin, n-cadherin) and transcription factors (snail-1, slug). results: downregulation (loss of expression) of e-cadherin, β-catenin and upregulation of snail-1 was seen in higher frequency (82%, 72% and 69%) in who grade ii/iii meningiomas as compared to grade i meningiomas (6%, 3% and 13%) (p<0.05), thus indicating high frequency of emt pathway activation in high grade meningiomas. n-cadherin and slug expression was seen in only minority of cases. no difference in emt phenotype existed between different histomorphological subtypes of grade i meningiomas. on survival analysis patients with emt activation across all grades had shorter progression free and overall survival. further, in four paired samples analysed, emt activation was seen in all the principal tumors. conclusion: meningiomas exhibiting emt contribute to the aggressiveness and increased recurrence risk of these tumors. hence emt markers can be used for predicting the behaviour of meningiomas.   abstract 5 free neuropathol 3:14:9 programmed death ligand 1(pd-l1) expression and tumor infiltrating immune cell subpopulations association with clinicopathological and prognostic parameters in diffuse gliomas swati mahajan1, muhammed shafeeq1, mehar c sharma1, ashish suri2, vaishali suri1 1 department of pathology, all india institute of medical sciences, new delhi, india 2 department of neurosurgery, all india institute of medical sciences, new delhi, india background: recent discoveries have revealed that the glioma microenvironment includes a wide variety of immune markers that play an important role in the process of tumorigenesis. objectives: we aimed to analyze the utility of immune markers in prognostic stratification and understand the potential candidature of diffuse infiltrating gliomas for immune checkpoint blockade. material and methods: one hundred gliomas (who grade ii–iv) were analyzed by immunohistochemistry for pd-l1 expressing tumor cells, tumor-infiltrating lymphocyte subsets (tils; cd4, cd8, foxp3, ctla4) and tumor-associated macrophages (tams; cd68, cd163). results: expression of pd-l1 was more frequent in adults, idh1 wild type gliomas (76%) and was significantly high in glioblastomas (gb, 74%) followed by grade iii (50%) and ii (27.5%) astrocytomas. median cd8+ tils, cd68+ and cd163+ tams density was higher among grade iv (5, 39 & 11/mm2) and grade iii (4, 35 & 10/mm2) as compared to grade ii tumors (1, 22 & 3/mm2). foxp3 and ctla-4 positive t cells were observed in minority of gbs only. three-paired cases analyzed showed upregulation of pd-l1, tams, ctla-4 and foxp3 in recurrent tumors, indicating a role of the immune markers in recurrence. further, positive correlation between pdl1 expression and cd8+ tils and tams was noted. on survival analysis, increase in pdl1 expression, tils and tams were associated with a shorter overall survival. conclusion: immune markers are frequently expressed in gliomas in a grade-dependent pattern. their analysis could aid in predicting the prognosis of patients and add potential value for immunotherapy treatments.   abstract 6 free neuropathol 3:14:10 insights into molecular biology and immune micro-environment of pleomorphic xanthoastrocytoma iman dandapath1, jyotsna singh1, swati mahajan1, prerana jha1, nidhi shukla1, rahul gupta4, amit katiyar8, vikas sharma8, sujata chaturvedi2, arvind ahuja3, meenakshi bhardwaj3, ravindra saran4, ajay garg5, mehar c sharma1, niveditha manjunath6, ashish suri6, ritu kulshreshtha7, chitra sarkar1, vaishali suri1 1 neuropathology laboratory, neurosciences centre, all india institute of medical sciences, new delhi, india 2 department of pathology, institute of human behaviour and allied sciences, new delhi, india 3 department of pathology, pgimer & dr. rml hospital, new delhi, india 4 department of pathology, gb pant institute of post graduate medical education and research, new delhi, india 5 department of neuro radiology, all india institute of medical sciences, new delhi, india 6 department of neurosurgery, all india institute of medical sciences, new delhi, india 7 department of biochemical engineering and biotechnology, indian institute of technology delhi, new delhi, india 8 ccrf, all india institute of medical sciences, new delhi, india background: pleomorphic xanthoastrocytomas (pxas, grade ii) are rare, accounting for less than 1% of astrocytomas and commonly occur in young patients. some tumours which occur or recur with malignant change are known as anaplastic (apxa, grade iii). there is limited data on their molecular characteristics and immune microenvironment. objectives: a comprehensive study highlighting the underlying molecular biology in pxas for future development of a robust and cost-effective panel of biomarkers for risk stratification and discovery of novel drug targets. methods: genome-wide expression profiling of 14 pxa and 6 apxas was performed by microarray. amongst differentially expressed genes (degs), cyclin dependent kinase 14 (cdk 14) and mitochondrial fission process 1 (mtfp 1) were validated by qrt-pcr. immune profile was analysed using immunohistochemistry for pdl1 and ctla4. results: unsupervised hierarchical clustering revealed two distinct molecular clusters (cluster 1: 10 pxa, 3 apxa and cluster 2: 4 pxa, 3 apxa), indicating molecular heterogeneity within same grade. 10 differentially upregulated and 418 downregulated genes were identified between the clusters. qrt-pcr validation of cdk 14 (upregulated in cluster 2) and mtfp 1 (upregulated in cluster 1) showed strong concordance with expression array. there was no significant difference in age, sex, immunohistochemical profile, frequency of braf mutation or cdkn2a deletion between two clusters. significantly worse progression-free survival was observed in cluster 2 (p=0.003). mrna profiling-based prediction of recurrence was efficient, independent of histological grade and of braf mutation or cdkn2a deletion. pdl1 and ctla4 expression was higher in pxa and apxas than primary glioblastomas. conclusion: study highlights distinct molecular subgroups of pxas. degs between two clusters may be used for histology independent classification, prognostication, and prospective therapeutic targets. higher up-regulation of pdl1 and ctla4 suggests candidature for immunotherapy.   abstract 7 free neuropathol 3:14:11 dynamics of cell-free dna in predicting response in adult diffuse glioma on chemoradiotherapy adil husain1,3, sridhar mishra1, rahat hadi2, nuzhat husain1 1 department of pathology, dr. ram manohar lohia institute of medical sciences, lucknow, india 2 department of radiation oncology, dr. ram manohar lohia institute of medical sciences, lucknow, india 3 department of biosciences, integral university, lucknow, india background: adult diffuse glioma (adg) is a heterogeneous primary brain tumor with a poor prognosis and treatment response. tissue biomarkers are available for diagnostic and prognostic purposes. however, obtaining tissue is invasive and has limitations. cell-free dna (cfdna) may help to meet these challenges in the management of adg. objectives: the study aimed to quantify cfdna in adg on radiotherapy/chemotherapy and mutational profiling. material and methods: the study group comprised histopathologically confirmed adg (n=50), including grade ii, iii and iv glioma, and controls (n=25). serum cfdna was extracted using charge switch gdna 1ml serum kit (invitrogen, usa) and quantified using sybr based quantitative polymerase chain reaction (qpcr). next-generation sequencing (ngs) was performed in 07 pre-operative and 05 post-operative cfdna and tissue dna on an ion personal genome machine (ion pgm) with an in-house designed ngs panel (including tp53, atrx, and idh1 and idh2). results: in patients with adg, the pre-radiotherapy cfdna level was significantly higher (median; 113.46ng/ml), (iqr; 50.73-238.71) than normal controls (median; 74.37ng/ml), (iqr; 49.67-120.00) (p=0.048). non-responders had significantly higher cfdna levels (median; 184.4ng/ml), (iqr;73.84-631.10) than responders (median; 68.12ng/ml), (iqr; 26.55-165.4)), (p=0.023). tp53 gene mutation was most common in both pre-operative and post-operative cfdna samples. conclusion: pre-radiotherapy cfdna levels are associated with survival outcomes independent of other prognostic factors. targeted ngs in pre-operative cfdna matches the results of ihc analysis with high concordance, and it may be helpful in inoperable cases or have recurred.   abstract 8 free neuropathol 3:14:12 regional heterogeneity in mitochondrial function in human brain ageing: implications for region-specific vulnerability to neurodegeneration anusha y kiran1, praseedha mol3, firdouz a bhat3, oishee chatterjee3, srinivas bharath m.m2, t.s. keshava prasad4, anita mahadevan1 1 department of neuropathology, nimhans, bangalore, india 2 department of clinical psychopharmacology and neurotoxicology, nimhans, bangalore, india 3 institute of bioinformatics, bangalore, india 4 centre for systems biology and molecular medicine, yenepoya research centre, yenepoya, india background: region specific vulnerability is implicated in neurodegenerative disorders. mitochondrial dysfunc-tion is implicated, while ageing is the greatest risk factor. it is unknown if age dependant variation in mitochon-drial function exists across brain regions. objectives: this study investigates regional differences in mitochondrial function across different neuroanatom-ical regions in healthy ageing. material and methods: five regions from post-mortem human brains (frontal cortex, cerebellum, striatum, hip-pocampus and medulla; age=080yrs) was evaluated by mitochondrial complex assays. enzyme histochemical staining and quantitative proteomics was performed in young (25±5 yrs) and old (≥65 yrs) ages. results: mitochondrial enzyme assays revealed lowered activity of complexes i & iv with age in all anatomical regions, complex ii showed increasing trend and complex iii remained unchanged. medulla revealed highest activity, followed by cerebellum. on enzyme histochemistry, nadh labelled neuronal cytoplasm of frontal, stria-tum and cerebellar glomeruli and reduced with age. sdh showed increasing intensity with age, in neurons of striatum, cerebellum and medulla whereas cox showed reduced neuronal labelling intensity in hippocampus, cerebellum and medulla, with age. mitochondrial protein expression profile in frontal, striatum and hippocampus was similar, compared to cerebellum and medulla. medulla showed highest expression energy metabolism pro-teins and antioxidants irrespective of age, followed by cerebellum. synaptic transmission & calcium transport pathways were enriched in frontal, striatum and hippocampus. conclusion: the study clearly demonstrates brain region specific mitochondrial metabolism, and redox homeo-stasis which could potentially contribute to region specific vulnerability to neurodegeneration.   abstract 9 free neuropathol 3:14:13 ryr1-related myopathies: a series of 5 patients from an indian tertiary care centre saumya sahu1, swati mahajan1, aishwarya dhall1, bandana jassal1, mohammed faruq3, vaishali suri1, rohit bhatia2, vishnu v2, mehar c sharma1 1 department of pathology, all india institute of medical sciences, new delhi, india 2 department of neurology, all india institute of medical sciences, new delhi, india 3 csir-institute of genomics and integrative biology (csir-igib), new delhi, india background: ryr1-related myopathies (ryr1-rm) are described as a rare, clinically and histopathologically heterogeneous, and slowly progressive neuromuscular disorders. they constitute the most common class of congenital myopathies. clinical phenotypes are diverse and include king-denborough syndrome, exercise-induced rhabdomyolysis, lethal multiple pterygium syndrome, adult-onset distal myopathy, atypical periodic paralysis, mild calf-predominant myopathy, and dusty core disease. objectives: to report clinicopathological features of a group of the patients diagnosed with ryanodinopathy in a tertiary care centre from india. material and methods: a retrospective study (2019-2020) was performed on the clinical, histopathological and genetic features of all pediatric and adult patients, in whom ryr1 mutation was detected using next generation sequencing. results: five cases of ryr1-related myopathies were identified with age at onset varying from infancy to adulthood (2-39 years). a range of overlapping clinical phenotypes was noted: predominant proximal muscle involvement, facial weakness, external ophthalmoplegia, winging of scapula and mild tetraparesis associated with joint laxity. two different histopathological patterns were recognized: centronuclear (n= 2) and central core (n = 3). each case exhibited a different ryr1 mutation variant of which two novel genetic variants were revealed. further one genetic variant was reported to be associated with malignant hyperthermia susceptibility. conclusion: there is a striking clinical and genetic heterogeneity among ryr1-related myopathies with identification of two new ryr1 variants. its recognition is essential for genetic counselling and improving patient’s safety during anaesthesia to avoid episodes of hyperthermia.   abstract 10 free neuropathol 3:14:14 utility of respiratory chain complex assays in the diagnosis of mitochondrial disorders a phenotype, histopathological and genotype correlation deepha s1,3, ponmalar jn1,3, shivani sharma1,3, nagappa m2,3, govindaraj p3,5, bindu ps2,3,6, taly ab2,3, bharath mm4, gayathri n1,3 1 department of neuropathology, nimhans, bangalore, india 2 department of neurology, nimhans, bangalore, india 3 neuromuscular laboratory, department of neuropathology nimhans, bangalore, india 4 department of clinical psychopharmacology & neurotoxicology, nimhans, bangalore, india 5 centre for dna fingerprinting and diagnostics (cdfd), hyderabad, india 6 the children’s hospital at westmead clinical school, sydney medical school, the faculty of medicine and health, the university of sydney, sydney, nsw, australia background: mitochondrial respiratory chain (mrc) enzyme complex assay play a crucial role in the diagnostic workup in a patient with suspected mitochondrial disorders. determining the enzyme activities assist in defining isolated or multicomplex deficiency disorders and guide in identifying the molecular basis of the disease. objectives: to assess mitochondrial function in patients with suspected mitochondrial disorders. material and methods: this retrospective study (2014-2021) analysed a large cohort (n=1046) of cases with clinical diagnosis of mitochondrial disorders and /or other genetic and acquired skeletal muscle diseases. fresh skeletal muscle tissue was subjected to mrc enzyme complex assay by spectrophotometry as a part of routine diagnostics. results: a total of 268/1046 cases (25.6%) revealed complex deficiencies. amongst these, 208 cases [136 children (age range:10months-18years; m:f= 81:55) and 72 adults (age range:19-62 years; m:f= 39:33)] were primary mitochondrial disorders with syndromic and non-syndromic phenotypes. the syndromic phenotypes (n=65; 31.2%) include cpeo (n =15), mitochondrial myopathy (n=10), melas (n =9), leigh syndrome (n=8), progressive myoclonic epilepsy (n=11), leukodystrophy (n=4), leukoencephalopathy (n=5), encephalomyopathy (n=1), sensory ataxia neuropathy (n=1) and narp (n=1). non-syndromic presentations constituted 143 cases (68.75%). muscle histopathology findings was diagnostic in 33 (15.8%), while normal in 175 (84.13%). the deficiency included isolated complex i (n=127, 61%), isolated complex iv (n=31,14.9%), isolated complex iii (n=5, 2.4%) and multiple complexes (n= 45, 21.6%). complex i was the most common respiratory chain deficiency followed by multiple complex deficiencies, complex iv and complex iii. genetics done in over 100 cases revealed mutations across subunits of the complexes. conclusion: mrc enzyme complex assay enhanced the diagnostic yield in a large number of patients, in particular those with non-syndromic presentations as compared to conventional enzyme histochemical methods. correlation of clinical, biochemical, pathological and genetics findings will be presented.   abstract 11 free neuropathol 3:14:15 myelination changes in white matter following severe traumatic brain injury (stbi): a neuropathological study meenakshi sharma1, arulselvi subramanian2, vaishali suri3, deepak agrawal4, rajesh malhotra5, sanjeev lalwani1 1 division of forensic pathology and molecular dna, jai prakash narayan apex trauma centre, aiims, new delhi, india 2 department of laboratory medicine, jai prakash narayan apex trauma centre, aiims, new delhi, india 3 department of pathology, aiims, new delhi, india 4 department of neurosurgery, jai prakash narayan apex trauma centre, aiims, new delhi, india 5 chief, jai prakash narayan apex trauma centre, all india institute of medical sciences, new delhi, india background: white matter injury after tbi involves both axonal injury and myelin pathology that evolves throughout the post-injury time course. objectives: to examine myelination changes in post stbi patients. materials and methods: 64 post mortem brain tissues (corpus callosum), 24 non-tbi and 12 control samples from the patients who died due to stbi were collected. patients with initial gcs score ≤ 8 and age above 18 years with positive ct findings were included. routine h&e grading (3), myelin assay (lfb-pas) and ihc for myelin basic protein (mbp) microscopic changes were graded on the basis of percentage of demyelinated area. results: among stbi group, h&e grading shows moderate demyelination in 39.06% patients and normal grade in 26.5% cases, whereas lfb-pas & ihc –mbp grading, depicts severe demyelination in 29.8% patients and shows moderate demyelination in 26.5% patients. comparative to non-tbi group where 91.7% h&e grade and 83.3% lfb-pas &ihc –mbp grade shows normal grade, control cases express 100% normal myelination grades. both grading’s found to be significant in stbi compare to non-tbi and control groups. conclusion: though h&e showed subtle demyelination changes, lfb-pas & ihc –mbp grading depicted maximum changes following stbi. marked demyelination started in first week (3-10 days) after stbi with reduced demyelination after 10 days which might suggest remyelination of demyelinated areas. clinical evaluation of tbi will need to address the challenge of accurately detecting the extent and stage of myelin damage for identifying broader range of therapeutic opportunities to improve outcome after tbi.   abstract 12 free neuropathol 3:14:16 is cerebral malaria an astrocytopathy? a post mortem study of bbb dysfunction aditi goyal1, anita mahadevan1, netravathi m2, jitender saini3, satish chandra4, sk shankar1 1 department of neuropathology, nimhans, bangalore, india 2 department of neurology, nimhans, bangalore, india 3 department of neuroimaging and interventional radiology, nimhans, bangalore, india 4 department of neurology, apollo speciality hospital, bangalore, india background: pathogenesis of edema and seizures in cerebral malaria (cm) leading to mortality is unresolved. microvascular pathology with sequestration of parasitized rbcs, endothelial activation, inflammatory mediator release and disruption of blood brain barrier (bbb) is a central pathogenetic event. however, role of astrocytes, major regulators of bbb, that protect from vasogenic edema via aqp4 channels in their end feet is unexplored. objectives: to evaluate histomorphological alterations in astrocytes and their role in pathogenesis of cerebral malaria. materials and methods: clinical, demographic and neuropathological changes of six patients who succumbed to cerebral malaria were reviewed. immunohistochemistry was performed for glial (gfap, s100b) bbb (aqp4, igg) and immune (p65 (nfκb)) markers. results: patients [age range:14-40yrs; male:female=2:1; duration of illness (doi):2-21days] were arbitrarily divided into group1 (doi <10days-hyperacute) and group 2 ( >/= 10days, acute). brain showed marked cerebral edema with slate-grey discoloration. histopathologically, all showed sequestration of parasitized rbcs in microcirculation, fresh ring and/or ball haemorrhages (6), resolving haemorrhages (4), and durck granulomas (dg) (1). astrocytic alterations were striking. in hyperacute stage (group1), astrocytes were markedly stunted with attenuated processes, prominent dystrophic beading and retraction of processes, with reduced ensheathing of vessels. progressive reduction in gfap, s100b and aqp4 expression occurred with increasing doi. loss of aqp4 expression demonstrable around haemorrhages and dg, with leakage of igg from vessels in acute stage. p65 (nfkb) expression was limited to resolving haemorrhages and dg. conclusion: astrocytopathy results in bbb dysfunction in cm. understanding the pathogenetic events at microvascular interface could aid design effective therapy to prevent mortality and morbidity in cm.   abstract 13 free neuropathol 3:14:17 does mitochondrial dysfunction play a role in pathogenesis of mesial temporal lobe epilepsy secondary to hippocampal sclerosis? shwetha sd1, anita mahadevan1, srinivas bharath mm2, keshav prasad ts3, m.ravindranadh chowdary4, raghavendra k4, ajay asranna4, arivazhagan a5, malla bhaskara rao5, jitendra saini6, rose dawn bharath6, sinha s4 1 department of neuropathology, national institute of mental health and neurosciences (nimhans), bengaluru, india 2 department of clinical psychopharmacology and neurotoxicology, nimhans, bengaluru, india 3 department of centre for systems biology and molecular medicine, yenepoya research centre, mangalore, india 4 department of neurology, nimhans, bengaluru 5 department of neurosurgery, nimhans, bengaluru department of neuroimaging and interventional radiology, nimhans, bengaluru, india background: studies in animal models of temporal lobe epilepsy suggest a pathogenetic role for mitochondrial dysfunction, although validation studies in humans are scarce. objectives: we chose to evaluate the mitochondrial status in the hippocampus resected from patients with mesial temporal lobe epilepsy (mtle) through proteomic approaches. material and methods: crude mitochondrial preparations from human hippocampus samples, resected from patients with mtle, who underwent amygdalohippocampectomy (early-onset <10years of age, n=9 and late-onset >11years of age, n=9), compared with age matched normal controls (n = 9) were subjected to quantitative proteomics using high-resolution mass spectrometry (ms). ms data was validated by mitochondrial respiratory chain complex assays (ciciv). results: the ms identified 7,961 proteins among which, 190 proteins and 60 mitochondrial proteins differentially over expressed in early and late onset respectively (p<0.05). proteins associated with biological processes such as mitochondrial electron transport chain, mitochondrial translation and branched-chain amino acid catabolic process were differentially overexpressed in cases with early onset mtle, suggesting a pathogenetic role. fatty acid beta-oxidation and glutathione metabolic processes were common to both early and late onset mtle. mitochondrial respiratory complex iactivity was higher in early onset compared to late onset mtle and controls, validating the proteomics data. the activities of mitochondrial complexes ii-iv remained unaltered. conclusion: mitochondrial dysfunction in hippocampus appears to have a role in the pathogenesis of early onset mtle, in particular, mitochondrial complex i subunits. evidence for role of mitochondrial dysfunction may aid in development of novel therapeutic strategies for treatment of mtle.   abstract 14 free neuropathol 3:14:18 diffuse midline gliomas with h3k27 mutation clinicopathological correlates john abha1, chacko ag2, moorthy r2, joseph bv2, john r3, bindra m1, gowri mahasampath4, chacko g1 1 department of general pathology, christian medical college, vellore, india 2 department of neurosurgery, christian medical college, vellore, india 3 department of pediatric oncology, christian medical college, vellore, india 4 department of biostatistics, christian medical college, vellore, india background: h3k27m mutant diffuse midline gliomas are infiltrative, midline high-grade gliomas with a k27m mutation in either h3f3a or histih3b/hist1h3c and have been reported to have a poor prognosis. objectives: to assess the morphology, the h3k27m status and clinical outcomes in h3k27m positive and negative diffuse midline gliomas. material and methods: seventy-three cases of diffuse midline gliomas of who grades ii-iv from 2012 to 2020, underwent immunohistochemical evaluation using h3k27m mutation specific antibody and h3k27me3 trimethylation antibody. morphological parameters, clinical details and outcome were correlated with mutational status. results: forty four of the 73 tumours were positive for the h3k27m mutation.h3k27m mutant diffuse midline gliomas occurred more commonly in adults and in the thalamus. they corresponded most often to a who grade iii. the mib-1% was significantly higher in the mutant group of tumours. when considering the entire cohort, the h3k27m mutant group showed better overall survival and recurrence free survival compared to the wild group. however, rfs in the mutant group was worse in the supratentorial tumours compared to the infratentorial tumours and spinal cord h3k27m mutant tumours showed shorter os and rfs compared to wild type tumours. in the cohort of h3k27m mutant tumours, children showed worse os when compared to adults. conclusion: contrary to reported literature h3k27m mutant diffuse midline gliomas occurred more commonly in adults and in the thalamus. h3k27m mutant tumours had a better rfs and os than the wild-type cases. however, amongst h3k27m mutant tumours, children had worse os than adults. spinal cord h3k27m mutant tumours had a shorter rfs and os when compared to brainstem and thalamus.   abstract 15 free neuropathol 3:14:19 significance of nestin and cd133 as cancer stem cell markers in diffuse gliomas and its association with idh-1 status and p53 expression sivaranjani s1, srinivas bh1, surendra kumar verma1, gopalakrishnan ms2 1 department of pathology, jipmer, puducherry, india 2 department of neurosurgery, jipmer, puducherry, india background: based on the cancer stem cell (csc) theory, they have self-renewal, uncontrolled proliferation, multi-directional differentiation properties. we have studied cd133 and nestin, which are the most commonly used two csc markers with consistent expression in diffuse gliomas. objectives: to assess of the level of expression of csc markers; nestin and cd133 and identify the correlation among various grades of diffuse glioma, idh status and p53. material and methods: a cross-sectional retrospective study conducted in department of pathology and neurosurgery with 102 subjects on expression of cscs and correlation with that of p53 and idh1 status in adult diffuse gliomas by immunohistochemistry on ffpe sections. the scoring of expression of cd 133 and nestin was adopted from zhang et al and p53 from aruna et al. the data was further analysed. results: the diffuse gliomas were graded based on who into grade ii, iii and iv. the expression of cd133 and nestin was compared with the increasing grades of diffuse gliomas and plotted on roc curves with auc of 0.6806 and 0.6119 respectively. this expression also showed a positive correlation with the idh status of tumor. conclusion: csc markers are expressed in diffuse gliomas and have higher expression with increasing in who grade and have significant association with idh-1 mutant status. hence, it can be inferred that diffuse gliomas with the higher expression of csc markers have poorer prognosis. further, they have the potential to be used as therapeutic targets in the future.   abstract 16 free neuropathol 3:14:20 immunophenotypic profile of pediatric brain tumors reflecting molecular alterations karuna balakrishnan1, srinivas bh1, surendra kumar verma1, gopalakrishnan ms2 1 department of pathology, jipmer, puducherry, india 2 department of neurosurgery, jipmer, puducherry, india background: pediatric brain tumors are the most common solid pediatric tumors. currently, there has been a drive towards “personalised medicine” or “precision medicine,” where chemotherapy is targeted against specific driver mutations. the revised 4th edition and the yet-to-be-released 5th edition of who has considered this and included genetic information into the classification for a combined phenotypic-genotypic approach. as molecular analyses are not available in many centres in india, surrogate immunohistochemistry (ihc) markers corresponding to genetic alterations have been developed. objectives: to study the immunophenotypic profile of pediatric brain tumors corresponding to molecular alterations material and methods: this is a cross-sectional descriptive study of immunophenotyping of 51 patients with pediatric brain tumors reflecting molecular alterations on ffpe sections. immunohistochemistry for beta-catenin, gab-1, yap-1, ini-1, p53 and lin28a was done for 22 embryonal tumors. atrx, brafv600e and h3k27m was done on 27 glial tumors and idh-1 wherever necessary. brafv600e was done on 2 gangliogliomas. results: using beta-catenin, gab-1 and yap-1, 19 medulloblastomas were classified into non-wnt/shh pathway activated (16/19), shh pathway activated (3/19) and) wnt pathway activated (0/19). out of the 2 etmrs diagnosed morphologically, one showed lin28a expression, the other etmr showed ini-1 loss and hence reclassified as atrt. out of 9 pilocytic astrocytomas, 2/9 showed atrx loss, 4/9 showed brafv600e expression. h3k27m was positive in 2/2 diffuse midline gliomas. of the 3 anaplastic astrocytomas and 6 glioblastomas, 1/3 anaplastic astrocytoma was idh positive with atrx loss. hence it was reclassified as idh mutant grade 3 astrocytoma. rest all were idh negative. 1/3 anaplastic astrocytoma and 4/6 glioblastomas showed loss of atrx; hence they were assumed to be h3g34 altered diffuse hemispheric gliomas. the remaining 1/3 anaplastic astrocytoma and 2/6 glioblastomas showed retained atrx expression and hence considered diffuse pediatric high-grade glioma, h3-wildtype and idh-wildtype. conclusion: for embryonal tumors, currently, many clinical trials are underway based on this molecular classification. for example, for wnt-activated medulloblastomas, the dose of radiation has been reduced. for shh medulloblastomas, smo inhibitors have been introduced. brafv600e and h3k27m mutated tumors can also be given targeted therapies. hence, in a resource-limited setup, these surrogate ihc markers can help fine-tune the diagnosis so that appropriate treatment can be given.   abstract 17 free neuropathol 3:14:21 pdl1 expression in cns tumours divyangi paralkar1, ashwani tandon1, rekha singh2, adesh shrivastava3, neelkamal kapoor1 1 department of pathology and lab medicine, all india institute of medical sciences bhopal, india 2 department of endocrinology and metabolism, all india institute of medical sciences bhopal, india 3 department of neurosurgery, all india institute of medical sciences bhopal, india background: immune checkpoints like pdl-1 regulates tumour microenvironment. pd-1 is expressed on activated t cells while pdl-1 is expressed on antigen presenting tumor cells. pdl-1 inhibits t cell activation; decreases proliferation and cause t cell apoptosis. tumour survival is a balance between immune surveillance and cancer cell proliferation. it may be immunomodulated by pd-l1. anti pd-l1 have be emerged in the treatment for multiple cancers in advanced stage objectives: detection of pdl-1 expression in cns tumour. material and methods: cross sectional study was conducted on 99 cases of cns tumour (50 diffuse astrocytoma; 6 pilocytic astrocytoma; 7 ependymoma; 2 oligodendroglioma; 27 meningioma; 07 embryonal tumour) between january 2014 to august 2020 in department of pathology and lab medicine, all india institute of medical sciences, bhopal. results: diffuse astrocytoma total 50 cases of which 1/9 of grade ii, 1/6 of grade iii (1/1 til), 7/19 of grade iv (2/3 til) and 10/16 of recurrent glioma (4/4 til). pilocytic astrocytoma (3/6 cases with 1/1til); ependymoma 7 cases (0/ 1 supratentorial, 3/3 posterior fossa and 2/3 spinal cord (0/1 til); oligodendroglioma (0/2); meningioma 27 cases (6/18 grade i (2/3 til); 8/8 grade ii (2/2 til), 1/1 grade iii (1/1 til)); medulloblastoma 0/6 cases; atypical teratoid/rhabdoid tumour 1/1 were pdl1 positive. (43/99 with til 13/16) conclusion: 43.3% cns tumour showed pdl-1 positivity with higher percentage in high grade. 16.16 % tumour showed til with 81.25% pdl-1 expression.   abstract 18 free neuropathol 3:14:22 droplet digital pcr: a robust technique for detection of idh1 r132h mutation in formalin fixed tissue samples rituparna chakraborty1, swati mahajan1, jyotsna singh1, mehar c sharma1, chitra sarkar1, vaishali suri1 1 neuropathology laboratory, neurosciences centre, all india institute of medical sciences, new delhi, india background: mutations involving isocitrate dehydrogenase 1 (idh-1) occur in a high proportion of diffuse gliomas (≈90%), with implications on clinicopathologic diagnosis and prognosis. ihc is an easy and quick method of detecting idh1-r132h mutations, but sometimes there may be some discrepancies. traditional approaches, such as sanger sequencing is laborious and lack sensitivity due to tumor heterogeneity and low tumor purity of glioma samples. the recently developed droplet digital pcr (ddpcr) technique generates a large amount of nanoliter-sized droplets, each of which carries out a pcr reaction on one template. therefore, ddpcr provides high precision and absolute quantification of the nucleic acid target. objectives: the present study compares results of ihc with ddpcr in diffuse gliomas. material and methods: 50 diffuse infiltrating idh1 immunopositive gliomas and 25 control samples (meningiomas) were included in the study. all cases were assessed for idh1 mutations by ddpcr. a cut off criteria which includes mutant allele fraction was standardized. the detection limit was calculated using serially diluted positive mutant dna in a background of wild-type dna. results: there was 100% concordance of results between ihc and ddpcr. all control samples detected negative for idh1 mutation by ddpcr. further compared with sanger sequencing, ddpcr was less time consuming (≈3 hours) and less laborious. conclusion: ddpcr is a reliable, rapid, robust,100 % sensitive and specific method for screening the idh1 mutation. it can be used to detect even low-frequency mutation burden.   abstract 19 free neuropathol 3:14:23 tert promoter mutations in meningiomas: a clinicopathological correlation ganga kundeti1, nupur karnik1, mamta gurav1, sneha janjal1, omshree shetty1, ayushi sahay1, vijay patil2, prakash shetty3, aliasgar moiyadi3, tejpal gupta4, sridhar epari1 1 department of pathology, actrec and tata memorial hospital, tata memorial centre, homi bhabha national institute, mumbai, india 2 department of medical oncology, actrec and tata memorial hospital, tata memorial centre, homi bhabha national institute, mumbai, india 3 department of neurosurgery, actrec and tata 4emorial hospital, tata memorial centre, homi bhabha national institute, mumbai, india 4 department of radiation oncology, actrec and tata memorial hospital, tata memorial centre, homi bhabha national institute, mumbai, india background: mutations in the non-coding promoter region of tert gene has now been clearly established as one of the most dominant non-histological biomarker for aggressiveness in meningiomas. objectives: to study the pattern of tert promoter (ptert) mutations across different histological grades of meningiomas. materials and methods: diagnosed cases of meningiomas, which had been evaluated and interpretable for ptert mutations by direct target (c228 and c250) sequencing were analysed for their correlation for clinicopathological features. results: 155 cases (with age-range: 18-75 years & male to female ratio: 0.98 [males=58, females= 59]) formed the study cohort. commonest location was cerebral convexity. histologically, 63 were grade 1 (angiomatous:1; transitional:38; meningothelial: 14, fibroblastic:2 and nos:8) , of these 28 had subtle atypical features ( ≥1 and ≤ 3 atypical histological findings i.e. high cellularity, small cell change, prominent nucleoli, necrosis, mitotic activity <4/10 high power fields (hpf) and necrosis). 79 were grade 2 (clear cell:1; chordoid: 1, 77: atypical) and 13 (papillary:2; rhabdoid:1) were grade 3. 8/155 (5.2%) showed tert promoter mutation (c228t: 5, and c250t: 3). 2 (of 13; 15.4%) were grade 3, 3 (of 79; 3.8%) were grade 2 and 3 (of 63; 4.8%) were grade 1. all three ptert mutant histologically grade 1 meningiomas, showed subtle atypical features, none (n=35) of the typically grade 1 showed ptert mutations. conclusion: ptert mutations are uncommon in meningiomas and are seen only in cases with presence of histologically aggressive features.   abstract 20 free neuropathol 3:14:24 evaluation of tert promoter mutation status in meningiomas jyotsna singh1, swati mahajan1, afreen khan1, swati singh1, ashish suri2, niveditha manjunath2, mehar c sharma1, vaishali suri1 1 neuropathology laboratory, neurosciences centre, all india institute of medical sciences, new delhi, india 2 department of neurosurgery, all india institute of medical sciences, new delhi, india background: meningiomas are the most common benign intracranial tumours. approximately 20% of the patients show aggressive phenotype with significant patient morbidity and mortality. tert promoter (ptert) mutations have been associated with upregulation of telomerase activity and tert mrna expression in diverse cancer types including glioblastomas and oligodendrogliomas. few studies on meningiomas have documented that presence of ptert mutations is associated with higher tumour grade, enhanced risk for recurrence and progression. objectives: to analyse the frequency of ptert mutation and assessed its prognostic significance in meningiomas. material and methods: a total of 125 cases (57 males and 68 females) of grade 1 (n=60), 2 (n=42), 3 (n=13) and control (n=10) were included in the study (2012 to 2020). they were assessed for c228t and c250t hotspot mutation in the ptert region by using sanger sequencing. samples were stratified into two groups tert mutated vs tert wild type. results: there were 111 adult and 4 paediatric cases. the mean age of the patients was 38.8 years (range 10–70 years). ptert c228t mutation was found in only 1.7% (2 of 115) meningiomas. both were grade 2 and adults. on follow up, both the patients died of tumour recurrence. conclusion: ptert mutations are infrequent in meningiomas and associated with aggressive biology. further, the predictive power of ptert status essentially allow the clinician to identify aggressive meningiomas, patients at risk for early recurrence and provides biomarker for new therapeutic interventions.   abstract 21 free neuropathol 3:14:25 pitfalls in diagnosis of oligodendrogliomas on squash cytology tista basu1, mou das1, uttara chatterjee1 1 department of pathology, institute of post graduate medical education and research, kolkata, india background: oligodendrogliomas (odg) are one of the less common gliomas. odg presents with a broad morphological spectrum and may mimic a variety of glial and non-glial neoplasms. the diagnostic peri-nuclear halo on histopathology is not seen in squash smears at all, thereby posing diagnostic difficulties in squash smears. objectives: to evaluate the diagnostic accuracy, limitations and pitfalls of intraoperative cytology in oligodendrogliomas. materials and methods: intraoperative squash smears of cns tumours stained with haematoxylin and eosin stain were evaluated followed by corresponding histopathology sections. appropriate immunostains were utilised where they were relevant. the findings were reviewed to determine the diagnostic pitfalls in twelve cases of odg from our institution in the last two years. results: amongst the 12 cases of histomorphologically proven odg, 5 were correctly diagnosed as odg grade 2 on squash smears. amongst four cases diagnosed as astrocytoma on squash, three were odg grade 2 and one was anaplastic odg (grade 3) in hpe. one case diagnosed as low grade glioma (odg) on squash cytology showed mini-gemistocytes. this was diagnosed as an anaplastic odg on hpe. two cases were incorrectly diagnosed as central neurocytoma and lymphoma respectively on squash. conclusion: the absence of properly defined intra-operative squash cytological features and sparse literature, leads to inter-observer variability during diagnosis of odgs on squash smear alone. the discerning nuclear features of an odg on squash smear are also lost with increase in grade and thus, further pose a diagnostic dilemma. mini-gemistocytes are a helpful clue for anaplasia on squash smear.   abstract 22 free neuropathol 3:14:26 low prevalence of braf v600e mutations in pleomorphic xanthoastrocytoma ranjani j1, gandham ej2, beno d1, pai r1, balakrishan r3, jasper a4, gowri m5, moorthy rk2, chacko ag2, chacko g1 1 department of pathology, christian medical college, vellore, india 2 department of neurological sciences, christian medical college, vellore, india 3 department of radiation therapy, christian medical college, vellore, india 4 department of radiology, christian medical college, vellore, india 5 department of biostastics, christian medical college, vellore, india background: the prevalence of braf v600e mutations in pleomorphic xanthoastrocytomas (pxa) and anaplastic pxas (apxa) varies from 60 to 80%. there are conflicting reports on the prognostic relevance of this mutation in pxas. objectives: to study the prevalence of braf v600e mutation in patients with pleomorphic xanthoastrocytoma (pxa) and correlate this with the outcome. materials and methods: this retrospective study included 33 patients with a diagnosis of pxa/apxa operated between 2007 and 2020. the demographic and clinico-radiological data were obtained retrospectively from the electronic database. the biopsies were reviewed and the samples were assessed for the presence of braf v600e mutation, using droplet-digital polymerase chain reaction. the histological grade and braf v600e mutational status were correlated with progression-free survival (pfs) and overall survival (os). results: there were 20 patients in the pxa group and 13 patients in the apxa group. braf v600e mutation was seen in 40 % (8/20) of the pxa and 15 % (2/13) of the apxa cases. recurrence was seen in 7/13 (55%) apxas but none in the pxa group recurred at a mean follow-up of 45months. the overall survival was significantly better in pxas compared to the apxas (p=0.02). brafv600e mutated tumours had a better os as compared to wild type tumours but this did not reach statistical significance. (p=0.364). conclusion: braf v600e mutations were seen in only 30% of pxa/apxas limiting its usefulness as a diagnostic marker. braf v600e mutant tumours had a better overall survival, however, this was not statistically significant.   abstract 23 free neuropathol 3:14:27 unusual primary intracranial sarcomas – ewing’s sarcoma and synovial sarcoma rallabandi hima bindu1, meenakshi swain1, rahul lath2, subodh raju2 1 department of histopathology, apollo hospital, hyderabad, india 2 department of neurosurgery, apollo hospital, hyderabad, india background: primary sarcomas of brain are rare – hence two cases of primary intracranial sarcomas of brain are being presented. 1. synovial sarcoma is an aggressive soft tissue sarcoma and intracranial occurrence is rare. it is characterized by a unique chromosomal translocation t(x;18) (p11.2; q11.2). 2. primary intracranial ewing sarcoma is also rare. it is characterized by chromosomal translocation t(11;22) (q24;q12) . very few cases, of primary intracranial synovial sarcoma and primary intracranial ewing’s sarcoma have been reported in the literature. objectives: to present two unusual sarcomas of brain and emphasize on the diagnostic challenges. material and methods: case reports of two primary intracranial sarcomas results: case 1 – 31 years old male on imaging was found to have left frontal sol. histopathological examination with immune stains, suggested a synovial sarcoma. fish studies done at two laboratories were negative for syt-ssx2. later the translocation was detected by rtpcr, confirming the histological diagnosis. case 2 –twenty-year male came with complaints of headache since 3 months. mri – revealed heterogeneously enhancing left tentorium based sol. histopathological examination with immune stains showed features of primitive neuroectodermal tumor. fish studies revealed ewsr 1 gene re-arrangement with the final diagnosis of ppnet. conclusion: sarcomas though rare can be seen as primary intracranial tumors, hence awareness of these entities with appropriate molecular studies is important for making the correct diagnosis.   abstract 24 free neuropathol 3:14:28 relevance of the 4-variable risk stratification model in cns solitary fibrous tumours hemanth kumar r1, poonkodi m1, moorthy rk2, chacko ag2, joseph bv2, rajesh b3, chacko g1 1 department of pathology, christian medical college, vellore, india 2 department of neurosurgery, christian medical college, vellore, india 3 department of radiation therapy, christian medical college, vellore, india background: solitary fibrous tumour/hemangiopericytomas (sft/hpc) of the cns are graded based on their morphological phenotype and mitotic activity. in contrast, sfts arising in the non-meningeal sites are prognosticated using the 4-variable risk stratification model (described in who classification of soft tissue and bone tumours, 2020) which has been observed to be an improvement over the traditional benign/malignant distinction. objectives: to determine pertinence of the 4-variable risk stratification model in meningeal sft/hpcs. materials and methods: this retrospective study identified 53 patients operated between 2014 and 2021 and diagnosed as sft/hpc on histopathology. follow up data was available for 27/53 patients. the cases were sorted into low, intermediate and high risk groups as per the 4-variable risk stratification model. the results were correlated with the follow-up data, including the presence or absence of distance metastasis. results: one tumour was diagnosed as sft, who grade i, while 6 tumours and 20 tumours were diagnosed as hpc, who grade ii and iii, respectively. all 27 tumours were immunopositive for stat6. in accordance with the 4-variable risk stratification model, 19 (70.4%) patients fell in to the low risk group, while 8 (29.6%) fell into the intermediate risk group. local recurrence was observed in 3 patients, while distant metastasis was seen in 3 patients (lung (1/3), l5-s1 spine (1/3) and submental soft tissue (1/3)). all patients with local recurrence or distant metastasis were diagnosed with hpc, who cns grade iii. among the patients with metastasis, two belonged to low risk group and one to intermediate risk group (as per the 4-variable risk stratification model). conclusion: despite the limited sample size, it is observed that the 4-variable risk stratification model validated in non-meningeal sfts did not prognosticate the meningeal counterparts appropriately. it is therefore unlikely that this model might replace the original grading described in who classification of cns tumours, 2016.   abstract 25 free neuropathol 3:14:29 oropharyngeal psammomatous melanotic schwannoma, non-syndromic an unusual tumour at an unusual site poonam elhence1, rashim sharma1, divya aggarwal1, balamurugan t1, ravindra shukla2, amit goyal3 1 department of pathology, all india institute of medical sciences, jodhpur, india 2 department of endocrinology, all india institute of medical sciences, jodhpur, india 3 department of otorhinolaryngology, all india institute of medical sciences, jodhpur, india background: psammomatous melanotic schwannoma is a rare tumour of uncertain histogenesis and indeterminate biologic behaviour with known association with carney’s syndrome. objectives: to present a rare case of an oropharyngeal psammomatous melanotic schwannoma which has no syndromic association presently. material and methods: case report of oropharyngeal sammomatous melanotic schwannoma results: a 25-year-old female presented with complaints of a swelling in her oral cavity for about ten years, gradually increasing in size and causing difficulty in swallowing. a clinical diagnosis of hemangioma was given. the swelling was excised and sent for histopathological evaluation. a circumscribed reddish to brownish black mass measuring 5x4x3.5cms was received. the cut surface showed reddish-brown to haemorrhagic areas and foci of calcification. on microscopic examination, a diagnosis of psammomatous melanotic schwannoma was given. the patient was not found to have any associated feature of carney’s syndrome on clinical examination. she had no significant personal or family history. the patient is on regular follow up and is doing well three years post-surgery. conclusion: psammomatous melanotic schwannoma is a rare tumour of uncertain biologic potential. a knowledge of this entity is helpful for correct diagnosis and appropriate patient management with long-term follow-up in view of potential malignant transformation.   abstract 26 free neuropathol 3:14:30 silent corticogonadotroph adenoma (scga): a silent monster shalini suman1, swati mahajan1, mohd sulaiman2, vaishali suri1, deepak aggrawal2, sharma mc1 1 department of pathology, all india institute of medical sciences, new delhi, india 2 department of neurosurgery, all india institute of medical sciences, new delhi, india introduction: pituitary adenomas are usually benign tumors which are classified based on differentiated cell type origin. silent corticogonadotroph adenoma are rare, benign but aggressively growing biochemically silent adenoma subtype showing rare characteristics of bilineage differentiation of both corticotroph and gonadotroph. till now there is only a single study published in literature about this entity and further studies need to be done. objectives: to report a case of silent corticogonadotroph adenoma material and methods: case report of silent corticogonadotroph adenoma results: a 25year old male presented with complaints of multiple episodes of headache and vomiting since 3 weeks and bilateral loss of vision since 5-6 years. ncct brain showed large, well developed, extra-axial sellar and supra sellar predominantly solid lesion with peripheral cystic component measuring 6.3x2.7x5.3cm. histomorphological analysis showed a tumor with features of pituitary adenoma. immunohistochemistry for the hormonal profile showed diffuse positivity for acth, whereas tumor cells were negative for lh, fsh, prl, gh and tsh. in addition, the tumor cells were diffusely positive for lmwck with mib1 labelling index of 2%. hormonal profile suggested a corticotroph adenoma. however, ihc for transcription factors showed contradictory results with tumors cells positive for sf1 and gata3 while negative for tpit and pit1. integrating clinical, morphological and immunohistochemistry findings a finally diagnosis of silent corticogonadotroph adenoma was rendered. conclusion: due to the rarity of this lesion, scga may not be considered as a differentials while working up the case. the diagnosis of this subtype emphasizes increased postoperative surveillance for earlier detection of recurrences and hypopituitarism thereby reducing morbidity and improving quality of life in these patients.   abstract 27 free neuropathol 3:14:31 molecular profiling: a key to clinical and histological enigma in an ambiguous case priyanka singh1, iman dandapath1, swati mahajan1, satish verma2, ajay garg3, mehar c sharma1, vaishali suri1 1 department of pathology, all india institute of medical sciences, new delhi, 110029, india 2 department of neurosurgery, all india institute of medical sciences, new delhi, 110029, india 3 neuroradiodiagnosis, all india institute of medical sciences, new delhi, 110029, india background: the upcoming 2021 who classification of cns tumors highlights the importance of molecular diagnostics. objectives: molecular characterization of a tumor showing ambiguous histology and clinico-radiological picture. material and methods: a 27-year-old female presented with history of headache, vomiting and vision deterioration for a month with no motor/sensory deficits. on cect, there was a well-defined lobulated, solid-cystic lesion with calcification involving anterior corpus callosum and bilateral anterior frontal lobe. craniotomy and tumor excision were done. intraoperatively, it was soft and vascular tumor extending to the left lobe. a radio-clinical diagnosis of oligodendroglioma or high-grade glioma was rendered. results: h&e-stained sections showed a tumor comprising of round monomorphic cells with clear cytoplasm forming small nests and rosettes with extensive neuropil in the background. hyalinized blood vessels, rosenthal fibres and eosinophilic granular bodies were seen. there was an occasional focus of endothelial cell proliferation. no mitotic activity or necrosis was noted. immunohistochemically, the tumor cells were positive for olig2, map2, β-tubulin while negative for gfap, neu-n, nf, cd34, ema and ebp-50. neuropil was highlighted by synaptophysin. the tumor cells were negative for idhr132h, p53 and licam and showed retained atrx expression. based on these findings, a diagnosis of glioneuronal tumor, nec, who grade 1 was suggested. molecular analysis by fish, rt-pcr and sanger sequencing exhibited mapk pathway activation. conclusion: a cryptic case with deregulation of mapk pathway is highlighted. molecular characterization is essential in such unusual cases owing to availability of targeted therapy.   abstract 28 free neuropathol 3:14:32 pseudotumoral hemicerebellitis masquerading as lhermitte–duclos disease – a case report nufina t a1, alok mohan uppar2, jitender saini3, vani santosh1 1 department of neuropathology, nimhans, bangalore, india 2 department of neurosurgery, nimhans, bangalore, india 3 department of neuroimaging and interventional radiology, nimhans, bangalore, india background: pseudotumoral hemicerebellitis is an exceptionally rare presentation in which unilateral cerebellar involvement mimics a tumour. the aetiology is diverse including post vaccination and post infection. objectives: to report a rare case of pseudotumoral hemicerebellitis masquerading as lhermitte–duclos disease material and methods: case report of pseudotumoral hemicerebellitis masquerading as lhermitte–duclos disease. results: a 11 year old girl presented with headache and vomiting for 1 month, associated with right cerebellar signs. mri revealed a t2 hyperintense contrast enhancing right cerebellar lesion with diffusion restriction, and features of tiger stripe pattern suggestive of lhermitte–duclos disease. there were no neurocutaneous markers. she underwent craniotomy with biopsy of right cerebellar tissue which revealed an irregularly expanded cerebellar folia with florid lymphoplasmacytic infiltration admixed with histiocytes in the meninges and parenchyma. this was associated with marked reduction in the granule cell layer neurons and polyfocal destruction of folial architecture. there were no granulomas, parasites, viral inclusions, or collection of dysplastic neurons or neoplastic cells. special stains for fungi, acid fast tubercle bacilli and bacteria were negative. the patient did not have recent history of fever, vaccination or drug ingestion. extensive work up for infectious aetiology in both serum and csf proved to be negative. serum and csf panel for autoimmune encephalitis, paraneoplastic neuronal and nmo–mogsd antibodies were also negative. conclusion: pseudotumoral hemicerebellitis is the close differential diagnosis for lhermitte–duclos disease radiologically. it typically has a benign course, the main management comprising supportive measures, steroids or antivirals. better understanding and awareness of this rare entity would help in accurate presurgical diagnosis and patient management.   abstract 29 free neuropathol 3:14:33 anoctamin-5 muscular dystrophy: report of 2 cases with different phenotypes and genotypes from indian subcontinent bandana jassal1, swati mahajan1, aishwarya dhall1, alvee saluja2, mohammed faruq3, vaishali suri1, roopa rajan2, mehar chand sharma1 1 neuropathology laboratory, neurosciences centre, all india institute of medical sciences, new delhi, india 2 department of neurology, all india institute of medical sciences, new delhi, india 3 csir-institute of genomics and integrative biology (csir-igib), new delhi, india background: anoctaminopathies are a group of autosomal recessive skeletal muscle disorders with various clinical phenotypes, caused byanoctamin 5 (ano5) gene mutations and the abnormal expression of ano5 protein. patients with recessive mutations in ano5 present with variable symptoms ranging from asymptomatic hyperckemia and exercise-induced myalgia to proximal and/or distal muscle weakness. objectives: we describe the clinical, pathological, and molecular findings of two unrelated patients with ano5-related muscular dystrophy. material and methods: 96 histologically identified muscular dystrophy cases were subjected to next generation sequencing using a customized panel of 54 genes (iilumina design studio). results: two patients were diagnosed with ano5-related muscular dystrophy. one patient had a pathogenic homozygous mutation of c.1406g>a in exon 14 while the other patient had a novel heterozygous mutation of c.2141c>g in exon 19 of ano5 gene. both showed two different phenotypes (limb girdle muscular dystrophy 2l and miyoshi myopathy) and histomorphological pattern. muscle biopsy of one patient in addition showed amyloid deposition in the blood vessels walls. neurologic examination was unremarkable with insignificant family history. serum creatine kinase (ck) was elevated in both. conclusion: ano5-related muscular dystrophy is a heterogeneous disease with different clinical phenotypes as well as genotypes. all muscle biopsies with unclassified muscular dystrophies should be subjected to congo-red stain to look for amyloidosis. the results of this study further suggests that screening for ano5 gene should represent an early step in the diagnostic work-up of the patients with undiagnosed muscular dystrophy and persistent asymptomatic hyperckemia even when muscle biopsy is normal.   abstract 30 free neuropathol 3:14:34 dysferlinopathy in a cohort of north indian patients: clinical histopathological and mutational spectrum aishwarya dhall1, swati mahajan1, mohammed faruq2, pankaj pathak1, uzma shamim2, neena dhiman1, bandana jassal1, vaishali suri1, rohit bhatia3, mehar chand sharma1 1 neuropathology lab, neurosciences centre, all india institute of medical sciences, new delhi, india 2 csir institute of genomics and integrative biology, new delhi, india 3 department of neurology, all india institute of medical sciences, new delhi, india background: dysferlinopathy is a group of autosomal recessive muscular dystrophy caused by mutations in the dysferlin gene (dysf). it is the second most commonly reported lgmd subtype (27%) in india after gne myopathy (31%). phenotypic variants includes miyoshi myopathy (mm), limb-girdle muscular dystrophy (lgmd2b), and other atypical phenotypes, such as the proximo-distal phenotype and distal anterior compartment myopathy. objectives: to describe the clinical, histopathological and mutational spectrum of dysferlinopathy in a cohort of patients from northern india material and methods: 96 patients (2018-19) suspected of lgmd from non-related families underwent thorough phenotypic characterization followed by muscle histopathological analysis. these cases were subjected to next generation sequencing using a customized panel of 54 genes. results: eleven patients (6 male and 5 females) were diagnosed as dysferlinopathy amounting to a prevalence of 11.4% of lgmd in north indian population. eight patients presented with proximal lgmd2b, 2 with distal mm and 1 with proximo-distal phenotype. mean age of onset and diagnosis was 24.4 years and 36 years respectively. cpk was elevated in all and ranged from 540 to 13000 u/l. histomorphological analysis showed predominant dystrophic changes with necrotic-regeneration pattern and inflammation. immunohistochemistry revealed partial to complete loss of dysferlin in all except one case. sequencing reveal 9 novel and 6 known mutations including exonic (frameshift, stop gain and snvs) and splice variants. conclusion: a high proportion of novel mutations were identified in the dysf gene thus broadening the genetic spectrum of dysferlinopathy. no genotype-phenotype correlation existed suggesting that the clinical phenotype is determined not only by dysf variants but also likely through a complex interplay of environmental, epigenetic, and genetic factors.   abstract 31 free neuropathol 3:14:35 role of mitochondrial respiratory chain complexes in pathogenesis of temporal lobe epilepsy dhanya ck1, shwetha sd1, anita mahadevan1, srinivas bharath mm2, keshav prasad ts6, m.ravindranadh chowdary3, raghavendra k3, ajay asranna, arivazhagan a4, malla bhaskara rao4, jitendra saini5, rose dawn bharath5, sinha s3 1 department of neuropathology, nimhans, bangalore, india 2 department of clinical psychopharmacology and neurotoxicology nimhans, bangalore, india 3 department of neurology, nimhans, bangalore, india 4 department of neurosurgery, nimhans, bangalore, india 5 department of neuroimaging and interventional radiology, nimhans, bangalore, india 6 department of centre for systems biology and molecular medicine, yenepoya research centre, mangalore, india background: role of mitochondrial dysfunction in seizure generation comes from frequent occurrence of epilepsy in inherited mitochondrial disorders. less is known about its role in acquired epilepsies such as temporal lobe epilepsy (tle). targeting mitochondrial oxidative stress with antioxidant treatment may prove a useful adjuvant in the management of epilepsy. objective: to determine the role of mitochondrial dysfunction in the pathophysiology of tle by assessing mitochondrial function in the temporal lobe. materials and methods: mitochondria isolated from human temporal lobe samples, resected from patients with tle who underwent anterior temporal lobectomy (early-onset <10years of age, n=9 and late-onset >11years of age, n=9), compared with age matched normal controls (n=9) subjected to assays for malate dehydrogenase, succinate dehydrogenase, mitochondrial complexes i to iv and adp/atp ratio. results: mitochondrial respiratory complex assay data analysis revealed significant increase in complex i and iii activity in early and late onset tle compared to controls. the mitochondrial complex ii activity was higher in controls compared to early and late onset tle whereas complex iv had higher activity in controls compared to early onset. the atp/adp ratio was decreased indicating reduced bioenergetics. an elevated activity of malate dehydrogenase and succinate dehydrogenase was also observed. conclusion: the specific targeting of mitochondrial oxidative stress, dysfunction, and bioenergetics may have significant role in inducing epileptogenesis with respect to respiratory chain complexes and antioxidant treatment may prove to be a useful adjuvant in the epilepsy management.   abstract 32 free neuropathol 3:14:36 gliotic and destructive brain lesions associated with drug resistant epilepsy a clinicopathological study rajalakshmi poyuran1, ramshekhar n menon2, bejoy thomas2, george c. vilanilam2, ashalatha radhakrishnan2, ajith cherian2, mathew abraham2, kesavadas c2, sanjeev v thomas2, deepti narasimhaiah1 1 department of pathology, sree chitra tirunal institute for medical sciences and technology (sctimst), trivandrum, kerala, india 2 r madhavan nayar center for comprehensive epilepsy care, sree chitra tirunal institute for medical sciences and technology (sctimst), trivandrum, kerala, india background: destructive insults during brain development result in various radiologically-defined lesions which may lead to drug resistant epilepsy (dre). their histomorphology is rarely described. objectives: to describe the histopathological features of lesions resulting from destructive brain injuries/insults with dre. materials and methods: histopathological evaluation of surgical specimens of lesions attributable to destructive brain insults over 4 years’ period. results: study included 58 cases (32 males and 26 females) with 93.1% presenting with complex partial seizures and 67.2% having early life adverse events. age of onset ranged from day 12 to 18.6 years (mean=5.6 years) with mean age at surgery of 14.7 years (2-39 years). lesions frequently involved occipital lobe (51.7%) and often had multilobar involvement. histopathological abnormalities were categorised as: (1) gliosis and atrophy (n= 25) of cortex and/or white matter showing a combination of cortical abnormalities ranging from unlayered, 4-layered and nodular architecture to complete neuronal loss. (2) exclusive layer 4 neuronal loss and gliosis (n=6) without other cortical abnormalities. (3) fcd type iiid (n=9) in which there was cortical dyslamination (microcolumnar architecture in 8 and cortical layer 2 loss in 1) adjacent to gliotic and atrophic parenchyma. (4) non-specific changes (n=18). conclusions: histomorphological changes include a combination of cortical and white matter gliosis and multiple patterns of cortical dyslamination. exclusive layer 4 neuronal loss and gliosis may represent a specific subtype. fcd type iiid often takes the form of microcolumnar architecture in the adjacent uninvolved cortex.   abstract 33 free neuropathol 3:14:37 cytopathic changes of herpes zoster encephalitis in csf mimicking malignant cells praveen bk1, ashwani tandon1, e jayashankar1, nirendra k rai2, ujjawal khurana1, dinesh p asati3 1 department of pathology and lab medicine, all india institute of medical sciences, bhopal, india 2 department of neurology, all india institute of medical sciences, bhopal, india 3 department of dermatology and venereology, all india institute of medical sciences, bhopal, india background: herpes zoster is the reactivation of varicella zoster virus which remained latent in the dorsal root ganglia after the primary infection. only 0.1%–0.2% of patients with disseminated varicella-zoster have been reported to have manifest with encephalitis, that too in severely immune-compromised individuals. we are presenting a cytological challenging case of zoster meningitis mimicking malignancy. objectives: to substantiate large worrisome cells in csf due to cytopathic change masquerading as neoplastic cells. material and methods: case presentation of herpes zoster encephalitis results: a 72-year-old male presented with chest wall swelling and csf evaluation performed for his neurological symptoms viz. fever and episodes of focal clonic seizures of right upper limb and impaired awareness. mri brain with contrast was normal. csf was evaluated for cytology and biochemical parameters, displaying worrisome large pleomorphic singly scattered cells with few lymphocytes, plasma cells and neutrophils. further clinical workup was performed to substantiate large cells. on re-examining the patient, the chest lesions were multiple, painful vesicles with surrounding erythema. on review, csf cytology shows mature and transformed lymphocytes with viral cytopathic change. nucleomegaly, cytomegaly and bi/trinucleation were noted. the enlarge nuclei shows cowdry type a nuclear inclusion. csf proteins were 57mg/dl and glucose were 63mg/dl. csf pcr was positive for varicella zoster. conclusion: large worrisome cells in csf need proper substantiation as management of neoplastic vs infective etiology are significantly different. in our case we substantiate viral etiology by several tests and treated appropriately.   abstract 34 free neuropathol 3:14:38 resolving the diagnosis of hmsn with nerve histopathology and genetics shivani sharma1, yasha tc1, madhu nagappa2, govindaraj periyasamy3, sanjib sinha2, akhilesh shroti2, ramesh siram2, parayil s bindu2, arun b taly1 1 department of neuropathology, nimhans, bengaluru, india 2 department of neurology, nimhans, bengaluru, india 3 centre for dna fingerprinting and diagnostics, hyderabad, india background: atypical presentations of hereditary motor and sensory neuropathies (hmsn) pose a great diagnostic challenge and delay diagnosis by several years. objectives: to describe the diagnostic odyssey of three patients with hmsn. material and methods: case reports of hmsn with atypical presentations results: case 1: a 71-years-old gentleman developed progressive paraesthesias and weakness of distal limbs, and imbalance while walking, from 54 years of age. examination showed distal wasting, pes cavus, global hyporeflexia, and foot drop. nerve conduction studies (ncs) showed demyelinating neuropathy. nerve biopsy showed thinly myelinated fibers, regenerating clusters, ill-formed onion-bulbs and epineurial perivascular inflammation. he was treated on lines of chronic inflammatory demyelinating polyneuropathy (cidp) with multiple courses of intravenous immunoglobulin and steroids. his deficits progressively worsened. targeted sequencing revealed a novel heterozygous missense variation in mpz (c.223g>t, p.asp75tyr). case 2: a 54-years-old gentleman manifested with progressive hearing impairment, cramps, and asymmetric weakness and wasting of lower extremities from 34 years of age. he had pes cavus, bifacial weakness, bilateral sensory neural hearing impairment, global hyporeflexia and foot drop. nerve biopsy showed hypertrophic demyelinating neuropathy. the patient was treated with plasmapheresis, steroids, cyclophosphamide and azathioprine, despite which he continued to develop progressive deficits. targeted sequencing revealed a heterozygous six-base pair deletion in mpz (c.207_212delgcccga, p.pro70_glu71del) and a heterozygous missense variation in dnmt1 (c.1018g>a, p.a340t). case 3: a 38-years-old lady presented with paresthesias, weakness and wasting of both lower limbs and poorly controlled diabetes mellitus. onion bulbs of variable sizes were seen on the nerve biopsy. she was treated on lines of cidp. genetic testing revealed pmp22 duplication. conclusion: in view of heterogeneous onset, progression and severity, the diagnosis of hmsn may be delayed. nerve biopsy coupled with genetic testing can help in avoiding misdiagnosis and inadvertent treatment.   abstract 35 free neuropathol 3:14:39 understanding the er stress response to predict clinical outcome in focal cortical dysplasia patients madamanchi kishore1, madhamanchi pradeep1,2, sita jayalakshmi3, manas panigrahi3, anuja patil3, phanithi prakash babu1 1 department of biotechnology and bioinformatics, school of life sciences, university of hyderabad, hyderabad, telangana 500046, india 2 govt. degree college for men, srikakulam district, andhra pradesh 532001, india 3 department of neurology, krishna institute of medical sciences (kims), secunderabad, telangana, india background: focal cortical dysplasia (fcd) is a significant cause of drug-resistant epilepsy (dre). complete seizure-free outcomes are not observed even after surgery in some patients, but the reasons are still unclear. the epileptic patients were found to have severe endoplasmic (er) stress that led to cellular damage and even cell death. objectives: this study focused on understanding er stress in predicting the post-surgical seizure-free outcome in fcd patients. material and methods: magnetic resonance imaging (mri) and fluoro deoxy glucose positron emission tomography (fdg-pet) tests were used to find the lesion location. after surgery, the samples were snap-frozen with liquid nitrogen and stored at -80 0c. double immunofluorescence, hematoxylin & eosin staining, western blot, thioflavin-t, and h2o2 neutralization assays were performed. the post-surgical follow-up data was observed for more than three years to determine the clinical outcome. results: 10 (34.6%) patients were females in our patient cohort, and 16 (59%) were males. 14, 8, and 4 patients were fcd i, fcd iia, and fcd iib, respectively. the follow-up study was performed for more than three years to find the post-operative seizures reoccurrence. then we categorized the patients according to international league against epilepsy (ilae) guidelines. ten (38.4%), nine (34.6%) and seven (26.9%) patients were classified into class 1 (complete seizure-free), class 2 (less frequent seizures or auras) and class 3 (frequent seizures) categories. severe er stress was observed in class 2 and 3 patients, which further caused the accumulation of reactive oxygen species (ros) and protein aggregates. in addition to this, we observed upregulation of an apoptosis initiation marker chop/gadd54 in the brain samples of ilae classes 2 and 3 patient samples. conclusion: our data suggest that fcd patients belonging to ilae class 1 showed reduced protein aggregates and limited ros had better seizure-free outcomes than the rest. understanding the er stress response severity in resected clinical samples can help predict the possibility of post-surgical seizure-free outcomes and hint at the post-surgical anti-epileptic drug (aed) therapy.   abstract 36 free neuropathol 3:14:40 candida meningitis mimicking tuberculous meningitis in an immunocompetent patient diagnostic conundrum hema av1, aditi g1, nufina ta1, nandeesh bn1, rao s1, nashi s2, nagarathna c3, saini j4, santosh v1, mahadevan a1 1 departments of neuropathology, nimhans, bengaluru, india 2 departments of neurology, nimhans, bengaluru, india 3 departments of neuromicrobiology, nimhans, bengaluru, india 4 departments of neuroimaging & interventional radiolog3, nimhans, bengaluru, india background: fungal meningitis is often associated with immunocompromise due to primary/secondary immunodeficiency, hiv, prolonged antibiotic usage, indwelling catheters, intracranial shunts etc. candida is the fourth most common cause of nosocomial infections, and the most frequent cause of systemic opportunistic fungal infections, though cns involvement is rare, representing disseminated infection in severely immunocompromised patients. objectives: we present a rare case of candida meningitis occurring in a middle aged immunocompetent host diagnosed at autopsy. material and methods: case report of candida meningitis in an immunocompetent patient. results: a 43-year-old man without co-morbidities (diabetic/hypertension, hiv seronegative), presented with a history of altered sensorium and fever for 6 day. cranial mri revealed multiple ring enhancing lesions in putamen, caudate, medial basifrontal and cerebellum. repeated csf examination showed no bacterial/fungal growth. he was managed with anti-toxoplasma and anti-tubercular regimen, along with steroids. after seven days of admission patient succumbed. autopsy revealed thick yellowish white basal exudates in interpeduncular cisterns resembling tuberculous meningitis. several hemorrhagic necrotizing lesions were seen in left medial frontal, bilateral putamen, thalamus, right insula, bilateral hippocampus and cerebellum. histopathology revealed multiple necrotizing lesions, with dense vasculitis and clusters of pseudohyphal forms area with angio-invasion closely mimicking aspergillus spp on pas and gms stains. however, gram positivity of “hyphal” forms suggested candida and cultures from csf grew candida albicans. conclusion: candida albicans is a very rare cause of chronic meningitis, unsuspected in immunocompetent hosts. an antecedent bacterial meningitis, treated with broad-spectrum high dose antibiotics, anemia are high-risk factors. although rare, this has to be considered in the differential diagnosis of all atypical meningitis even in immunocompetent individuals and serological/pcr tests for fungal etiology should be included in panel of diagnostic tests.   abstract 37 free neuropathol 3:14:41 rare case of dorsally located multiple neurenteric-cyst without spinal dysraphism – neuropathology insights and systematic review krishna kumar singh1, rakesh mishra2, ashish gupta3, neeraj dhameja1, priyanka gupta1 1 department of pathology, institute of medical science, banaras hindu university, varanasi, india 2 department of neurosurgery, institute of medical science, banaras hindu university, varanasi, india 3 department of neurosurgery, all india institute of medical sciences, bhopal, india background: neurenteric-spinal-cysts (nc) are rare. most are isolated-ventral, and intradural-extramedullary. cervical location is commonest. reports of nc which are multiple, dorsally located, intramedullary and without spinal dysraphism are extremely rare. are they different embryologically/pathologically? objectives: we present a case (less than five are reported till now) of multiple dorsally located nc without spinal dysraphism, one of which was idem and the other was intramedullary. we also present a systematic review of all such cases with focus on neuropathology, ihc, and clinic-radiological-pathological correlation. material and methods: case: a 40 years old gentleman with back pain and lower limb weakness for three years underwent gross-total-resection of d10-d11 lesion and partial-resection of conus medullaris lesion with detethering. systematic review: as per prisma 2009 literature searched in pubmed, medline, web of science, scopus, cochrane, google scholar, scielo. neuropathology, surgery, demographics, and outcome extracted. quality of studies and descriptive statistics applied. results: gross: thick glistening white capsule with cheesy material. cytopathology: occasional cell clusters with cilia. histopathology: columnar ciliated and mucinous cells, pas+. ihc: ema+, ck7+, gfap-focal+. search yielded 1,788 citations. most were male. lumbar location was common for intramedullary and complete resection was not possible. most were ema+, ck7+, and focal-+-gfap. conclusions: multiple idem and intramedullary dorsally located nc without spinal dysraphism are extremely rare. most of these lesions have characteristic histopathology and ihc findings. it is important to differentiate it from arachnoid cyst for appropriate management. these lesions are distinct from the nc with spinal-dysraphism.   abstract 38 free neuropathol 3:14:42 efficacy of dimethyl fumarate in chronic constriction injury induced neuropathic pain in rats jagjit singh1*, manisha naithani2, shalinee rao3, shailendra handu1 1 department of pharmacology, all india institute of medical sciences (aiims), rishikesh, india 2 department of biochemistry, all india institute of medical sciences (aiims), rishikesh, india 3 department of pathology, all india institute of medical sciences (aiims), rishikesh, india background: dimethyl fumarate (dmf) has shown beneficial effects in multiple sclerosis. further, dmf has demonstrated promising results in experimental models of autoimmune neuropathy and chemotherapy induced peripheral neuropathy. objectives: to study the effect and mechanism of dmf in chronic constriction injury (cci) model of neuropathic pain in rats. material and methods:30 male wistar rats were divided into 5 groups of six rats each as follows: group i: sham + carboxymethyl cellulose (cmc) 0.5%, group ii: cci + cmc 0.5%, group iii: cci +gabapentin (75 mg/kg): group iv: cci + dmf (25 mg/kg). group v: cci + dmf (50 mg/kg). animals were anesthetized before surgery. cci was induced by silk ligature, applied 2 mm apart on sciatic nerve. dmf and gabapentin were administered daily from day 0 to day 28. pain threshold was assessed using thermal hyperalgesia, mechanical allodynia and cold allodynia responses on days 0, 7, 14 and 28. animals were sacrificed on day 28, using high dose of anaesthesia, and sciatic nerve was dissected for histopathological analysis (hematoxylin & eosin and luxul fast blue). plasma proinflammatory cytokine estimation was done for il-1β, il-6 and tnf-α. levels of p38 mapk and bdnf were quantified by elisa in the dorsal horn of the spinal cord. results: a significant increase in the pain threshold parameters was observed. histopathological analysis demonstrated that dmf significantly reduced the macrophage infiltration and myelin loss in the injured nerve. a significant decrease in proinflammatory cytokines, p38 mapk and bdnf were observed in the dmf treated animals. conclusion: the present study suggested that dmf reduces cci neuropathic pain by protecting the peripheral nerves and preventing the central sensitization.   abstract 39 free neuropathol 3:14:43 moya moya disease: an autopsy case study g. supriya1, megha s uppin1, sireeshayareeda2, afshan jabeen2 1 department of pathology, nizam’s institute of medical sciences, hyderabad, india 2 department of neurology, nizam’s institute of medical sciences, hyderabad, india background: moya moya disease is a rare chronic cerebrovascular disorder characterized by progressive narrowing of distal ica and proximal components of mca and aca leading to formation of collateral vessels which resembles a 'puff of smoke' on angiography. it is an uncommon cause of stroke in adults and children. objective: to illustrate the pathologic features of moya moya disease with the help of autopsy findings. material and methods: a 31 year diabetic patient presented with slurring of speech since 20 days associated with headache, moderate, non-throbbing type. this was followed by sudden onset weakness of right upper and lower limb. examination revealed right umn facial palsy with right hemiplegia. mri brain showed infarcts in left frontal white matter region with right supraclinoid ica and right aca narrowing. patient was diagnosed as stroke with possible cns vasculitis. he was treated with iv steroids, cyclophsophamide and antibiotics. his condition did not improve. he developed pneumonia and succumbed on day 18 of admission. results: a complete body autopsy was performed after informed consent. the brain showed extensive subarachnoid hemorrhage. the sections from bilateral ica and mca showed intimal prolifetaion of smooth muscle cells with reduplication of iel, irregular undulation of the internal elastic lamina and duplication. there was no evidence of vasculitis. bilateral lobar pneumonia was seen in lungs. discussion: sporadic mmd is common in east asian countries. intracerebral or ventricular hemorrhage is the catastrophic events leading to mortality. however, sah has been described rarely. the vascular morphology of this disease is also well characterized in this autopsy study which helps to rule out fibromuscular dysplasia, atherosclerosis and vasculitis. conclusion: mmd is an uncommon cause of stroke in adults. it needs to be differentiated from moya moya syndrome which can occur due to secondary causes. subarachnoid hemorrhage is an uncommon complication.   abstract 40 free neuropathol 3:14:44 hematolymphoid malignancies presenting with neurological manifestations and hand-mirror cells in peripheral blood: report of two cases sangeetha seshagiri k, mallithavana s, indira devi b*, nandeesh b n# departments of transfusion medicine and haematology, neurosurgery* and neuropathology#, nimhans, bengaluru, india background: hematolymphoid malignancies involve myeloid and lymphoid cell lineages and affect blood, bone marrow, lymph nodes and lymphatic system. clinically they may present with features suggestive of systemic involvement. cns involvement is not uncommon. these malignancies may present with handmirror cells in peripheral blood which can be the initial findings of such malignancies. objectives: this study aims to emphasize that the presence of hand-mirror cells should prompt appropriate investigations, workup, and management. material and methods: this study includes two patients, first, an adolescent male and second, a middle-aged female patient who separately presented with back pain and bilateral lower limb weakness. t results: the peripheral blood smears of both patients revealed atypical cells, hand-mirror cells and thrombocytopenia. imaging studies showed extradural spinal lesions in both cases. flow cytometric immunophenotyping of the first patient showed features suggestive of acute myeloid leukaemia. both underwent laminectomy. the histopathological study of the laminectomy specimen of the first patient showed features of myeloid sarcoma and that of the second patient showed diffuse b cell lymphoma. conclusions: hand-mirror cells can be seen in both malignant and non-malignant conditions. however, their presence may be the initial finding in peripheral blood that indicates underlying malignancy with or without symptomatology and leucocytosis. their presence should prompt the pathologist to recommend further evaluation for the diagnosis and management of hematolymphoid malignancies.   abstract 41 free neuropathol 3:14:45 mullerian choristoma as a cause of tethered cord syndrome: a case report in a 13-year-old worsening after the onset of menarche payal nitin pandya1, raju subodh1, swain meenakshi2, lath rahul1 1 department of neurosurgery, apollo hospitals, hyderabad, india 2 department of pathology, apollo hospitals, hyderabad, india background: mullerian choristoma as a cause for tethered cord syndrome is exceedingly rare with very few cases being reported in the literature. objectives: to document a rare cause of tethered cord syndrome material and methods: case report of a 13-year-old girl with spinal dysraphism who was operated in 2013 for lipomeningomyelocele and tethered cord. results: the patient now presented to us with back pain and leg pain for 6 months duration. mri of lumbosacral spine revealed an intra medullary cystic mass in the conus medullaris associated with haemorrhage in association with the residual lipoma. intraoperatively following excision of intramural lipoma, a hemorrhagic cystic intramedullary mass was encountered and excised. histopathology of the mass revealed a uterus like structure with endometrial lining and associated structure resembling fallopian tube. a diagnosis of mullerian choristoma was made. conclusion: mullerian choristoma is a rare cause of tethered cord syndrome.   abstract 42 free neuropathol 3:14:46 plurihormonal pit-1-positive adenoma: a short series deepti narasimhaiah1, kesavadas c2, prakash nair3, rajalakshmi poyuran1 1 departments of pathology, sree chitra tirunal institute for medical sciences and technology, trivandrum, india 2 departments imaging sciences and interventional radiology, sree chitra tirunal institute for medical sciences and technology, trivandrum, india 3 departments of neurosurgery, sree chitra tirunal institute for medical sciences and technology, trivandrum, india background: plurihormonal pit-1-positive adenoma is a newly described entity in the 2017 who classification of tumors of endocrine organs with an aggressive behaviour. objectives: a histopathological study of plurihormonal pit-1-positive adenomas diagnosed at our institute in last 5 years. materials and methods: this is a retrospective study. routine haematoxylin and eosin (h & e), immunohistochemistry for pituitary hormones, pituitary transcription factors (pit-1, tpit), mib-1 and p53 were performed in all cases. results: seven cases of plurihormonal pit-1-positive adenomas were diagnosed. the patient age ranged from 10-51 years (median: 35 years) with 4 males and 3 females. four adenomas presented with acromegaly and 3 with decreased vision. all adenomas, with the exception of one were primary. four adenomas were invasive and 3 were non invasive. on immunohistochemistry, the most extensively expressed hormones were gh and prl, followed by tsh-beta and alpha-subunit. acth was negative in all, except in one adenoma. pit-1 was diffusely expressed in all adenomas and tpit was negative. the mib-1 labelling index ranged from 1%-7% (median: 3%). all patients were treated with surgery and one patient received radiotherapy for residual disease. the follow-up period was 5-24 months (median: 13 months) and all patients were alive and free of disease at last follow-up. conclusions: 1. gh, prl, tsh-beta and alpha-subunit were co-expressed in all adenomas, with one adenoma also expressing acth. 2. pit-1 was diffusely expressed in all tumors. 3. there was almost equal distribution of invasive and non-invasive adenomas and median mib-1 labelling index was 3%.   abstract 43 free neuropathol 3:14:47 an interesting case of extradural tumour in a pediatric patient kavin devani1, batuk diyora1 1 department of neurosurgery, ltmgh, sion, mumbai, india background: ewing’s sarcoma is a highly malignant bone tumour, belonging to a family of small round blue cell tumors, derived from primordial bone marrowderived mesenchymal stem cell which typically affects the pelvis and the long bones of the lower extremities. it’s primary involvement of the skull is encountered in only 1% cases. objectives: to describe a rare extradural sarcoma in paediatric patient. material and methods: case report of a pediatric case with extradural tumour. results: we present a pediatric case with rapidly growing, painless swelling over the left temporal region, not compressible and fixed to the scalp and underlying bone. patient underwent gross total resection of the tumour with adjuvant chemotherapy and radiation. he had a favourable outcome without any neurological deficit. no local or systemic recurrence was found at 12 months postoperatively. conclusion: intracranial ewing sarcoma/ppnet is a rare tumor with nonspecific clinical presentation and radiological findings. they are locally invasive. gross total excision with adjuvant chemoradiation is the mainstay of treatment.   abstract 44 free neuropathol 3:14:48 case report of rare extra axial cerebellopontine angle medulloblastoma, a meningioma mimicker: caution advised abhishek chowdhury1, poonkodi m1, aniketh shenoy2, shilpa rao1, nishanth sadashiva2, yasha tc1 1 department of neuropathology, nimhans, bangalore, india 2 department of neurosurgery, nimhans, bangalore, india background: medulloblastoma, the most common malignant paediatric brain tumour, presents as a posterior fossa intra-axial mass. it infrequently affects adults forming less than 1% of intracranial tumours. very rarely, it is found in extra-axial locations. to the best of our knowledge, less than 50 cases of extra-axial medulloblastomas have been reported in literature, majority in cerebellopontine (cp) angle, affecting mainly adults and very few with dural attachment. objectives: to present a rare case of extra-axial tentorial medulloblastoma in an adult with radiological features of a meningioma expanding the differential diagnoses of lesions with dural attachment in the cp angle materials and methods: case report of a cp angle tumour results: a 40-year-old man presented with headache, imbalance and recent onset vomiting. mri revealed an extra-axial lesion in left cp angle arising from the inferior tentorium cerebellum, with dural tail, radiologically considered as meningioma. the patient underwent excision of lesion, and dural attachment was noted intraoperatively with no cerebellar involvement. histopathology revealed a classic medulloblastoma with stromal desmoplasia, and immunoprofile consistent with shh-activated, p53 wildtype molecular subtype: beta catenin (-), gab1(+), yap1 focal (+), p53 negative, otx2(-) conclusion: medulloblastoma rarely presents in the cp angle with tentorial attachment leading to its omission in the differential diagnoses. available literature cites that wntand shh-activated subtypes are seen in extra axial medulloblastoma. this report highlights the importance of its consideration in atypical posterior fossa extra-axial lesions. strong suspicion can lead to early detection, prognostication and appropriate management.   abstract 45 free neuropathol 3:14:49 potpourri of five cases of rare central nervous system tumors with review of literature thamilselvi r1, megala c1, prem parkash a2 1 department of pathology, vinayaga mission kirupananda variyar medical college, salem, india 2 department of neurosurgery, sims chellum hospital, salem, india background: the overall annual incidence rate of all brain tumours are 7 per 100 000 population. meningioma is the most common brain tumor, accounting for about 30 percent of them. here we are presenting 5 unusual & rare cases of cns tumours. objectives: to study the clinicomorphological & radiological features of unusual central nervous tumors (cns) & its association with any systemic diseases. materials and methods: case reports of 5 cases results: 1. a 5 years old female child presented with a swelling in the frontal region. 2. 35-year-old woman was presented with leg weakness, numbness, seizures and headache for 18 months. 3. 41 years old male who presented with a 9 months history of headache, double vision & leg weakness. 4. a 50 years old male presented with headache & visual disturbances of 6 months. 5. 11 years old child was presented with bilateral nasal obstruction & discharge for one year. h/o headache for 7 months. radiological imaging was done & patients were underwent tumor resection & submitted for histopathological examination. conclusion: herewith discussing one case of langerhans cell histiocytosis (lch), one case of chordoid meningioma, one case of chordoma, one case of primary lymphoma & one case of pituitary tumor. hence, we have to correlate with clinical, radiological, histopathological & immunohistochemical markers for final diagnosis and thereby providing therapeutic implication. follow up & further treatment is needed to prevent recurrence.   abstract 46 free neuropathol 3:14:50 primary yolk sac tumor of cerebellar vermis: a case report divya aggarwal1, poonam elhence1, suryanarayanan bhaskar2, deepak jha2, vikas janu2, sarbesh tiwari3 1 department of pathology and lab medicine, aiims jodhpur, india 2 department of neurosurgery, aiims jodhpur, india 3 department diagnostic and interventional radiology, aiims jodhpur, india background: extragonadal germ cell tumors are rare and usually occur along midline of body. primary yolk sac tumor (yst) of brain is rare, however occurs usually in pineal and suprasellar regions. we present a rare case of primary cerebellar vermis yst. objectives: to report a rare case of cerebellar yolk sac tumour. material and methods: case report of biopsy proven yolk sac tumour of the cerebellum. results: a 3-year old male presented with ataxia, vomiting and decreased feeding which the parents noted around 15-20 days back. contrast-enhanced mri revealed a left cerebellar lesion involving vermis, with perilesional edema which was isointense on t1 and t2 hyperintense. intense contrast enhancement was noted. radiological possibilities suggested were medulloblastoma and atypical teratoid/rhabdoid tumor. intra-operative frozen section was sent and the smears showed markedly pleomorphic cells in small glands, papillae and dispersed singly. a diagnosis of germ cell tumor was rendered. definitive sections showed a tumor with cells arranged in a myriad of histological patterns, including reticular microcystic pattern, glands and solid areas. other classical features seen were presence of pas positive intracytoplasmic hyaline globules and numerous schiller duval bodies. a diagnosis of yolk sac tumor was rendered and the child was started on chemotherapy 2 weeks back. no mediastinal or testicular lesions were identified. conclusion: primary yst of cerebellar vermis is a rare entity. a knowledge of such entities is helpful in timely diagnosis and appropriate patient management.   abstract 47 free neuropathol 3:14:51 a diagnostic dilemma astroblastoma kuldeep singh khangarot1, surabhi tyagi1 1 department of pathology, mahatma gandhi medical college & hospital, jaipur, india background: astroblastoma is an extremely rare cns tumor, accounting for 0.45 to 2.8% of all neuroglial tumors. it was first described by bailey and cushing in 1926 and further characterized by bailey and bucy in 1930.according to published data there is slight female preponderance and bimodal age distribution with one peak between 5-10 years and other between 21-30 years. objectives: to study a rare case of astroblastoma. material and methods: after diagnosing on h&e correlation with the radiological investigation was done. subsequently immunomarker study and molecular workup by fish was done. results: on h&e differential diagnosis of i) well differentiated astroblastoma ii) papillary ependymoma iii) atypical choroid plexus papilloma /ca was given and further workup was advised. ihc marker and molecular information by fish assay confirms the diagnosis of well differentiated astroblastoma. conclusion: astroblastoma is a very rare primary brain tumor. its diagnosis is often challenging because of the astroblastic aspects that can be found in astrocytic tumors in ependymoma and in non-neuroepithelial tumors. the low incidence rate makes it difficult to conduct studies to examine tumor characteristics. the exact histogenesis of astroblastoma is controversial. patient with astroblastoma should be treated with curative intent.   abstract 48 free neuropathol 3:14:52 meningioangiomatosis: a rare cause of refractory temporal lobe epilepsy manasa gajula1, megha uppin1, sujata patnaik2, rajesh alugolu3, mudumba vijaya saradhi3 1 department of pathology, nizam’s institute of medical sciences, hyderabad, india 2 department of radiology, nizam’s institute of medical sciences, hyderabad, india 3 department of neurosurgery, nizam’s institute of medical sciences, hyderabad, india background: meningioangiomatosis is a rare meningovascular malformation or hamartomatous lesion responsible for refractory seizures. radiographic appearance can be highly variable and histopathology is necessary for confirmed diagnosis. objectives: to describe a case of meningioangiomatosis materials & methods: a 27-year male, with no co morbidities presented with seizures since 15 years. he was treated with antiepileptic agents however there was no change in seizure pattern. the frequency of seizures increased from 4-5 episodes/month to 8 -10 episodes/month. the eeg showed abnormal record with diffuse slowing of left temporal epileptiform focus. mri brain – focal fairly smooth expansion of left squamous temporal bone with slight sclerosis of inner cortical region. he underwent left temporal lobectomy and hippocampectomy results: the temporal neocortex showed a well circumscribed mass of 3x2cm with white fibrous appearance on cut section. the histopathology of the mass showed a lesion comprised of multiple vascular channels surrounded by bland spindle cells. these cells have oval to elongated nuclei with intranuclear inclusions. multiple psammoma bodies were identified. there was no atypia. the cells showed immunoexpression for ema and vimentin. hippocampus showed loss of neurons in ca1, ca3 and ca4 regions with dispersion and bilayering of granular neurons of dentate gyrus. conclusion: meningioangiomatosis is a very rare hamartomatous lesion associated with epilepsy. the preoperative consideration is important as prognosis with surgical resection is good.   abstract 49 free neuropathol 3:14:53 isolated cerebral rosai dorfman disease with granulomatous angiitis shinde sweety1, shenoy asha2 1 department of pathology, b.y.l nair hospital, mumbai, india 1 department of pathology, k.e.m hospital, mumbai, india background: rosai dorfman is a non-dendritic, non-langerhans histiocytic disorder. isolated intracranial involvement without lymphadenopathy is uncommon. only one report in brain and lung respectively documented a coexistent granulomatous angiitis. objectives: case report of an uncommon association between two rare cerebral entities. material and methods: a 30-year-old male presented with seizures, limb weakness and violent behaviour since 3 months. there was no fever, lymphadenopathy or hepatosplenomegaly. complete hemogram, esr, hepatic and renal function tests were normal. serology was negative for retrovirus and autoantibodies. on radioimaging, parietotemporal lobe showed a large mass 8.6 x 7.4 x 3.2 cm. it was hypointense on t1w1, hypointense on t2w1/ flair and heterogenously contrast enhancing. mr spectroscopy showed reversed naa/cr and increased cho/cr ratio suggestive of primary cns lymphoma, glioblastoma and tumefactive demyelination. subtotal resection was done. results: histopathology revealed sheets of mature lymphocytes, plasma cells, foamy histiocytes showing emperipolesis, touton giant cells and florid granulomatous angiitis with resultant coagulative necrosis. there was absence of eosinophils, langerhans cells or atypical lymphoid cells. the differentials included lymphomatoid granulomatosis, tuberculosis, tumefactive demyelination and wegener’s granulomatosis. tumor cells were positive for cd68 and s100, while negative for cd1a, eber and igg4. fungal and mycobacterial stains were negative. thus, rosai dorfman disease with coexistent granulomatous angiitis was diagnosed. on follow up for six months, there was no recurrence or new lesions. conclusion: only two cases in literature (cerebral and pulmonary) show granulomatous angiitis associated with emperipoletic histiocytic tumefaction. an overlap between igg4 related disease and rosai dorfman is also postulated.   abstract 50 free neuropathol 3:14:54 twin tales of anaplastic ependymoma with extensive vacuolation/signet cell change and lipomatous differentiation rashim sharma1, balamurugan thirunavukkarasu1, sudeep khera1, poonam abhay elhence1, deepak jha2, taruna yadav3 1 department of pathology and lab medicine, aiims jodhpur, india 2 department of neurosurgery, aiims jodhpur, india 3 department diagnostic and interventional radiology, aiims jodhpur, india background: ependymomas are circumscribed glial tumours with unique morphology. the diagnosis is relatively straightforward in the majority of the cases. however, there are scenarios where the morphology is obscured leading to diagnostic difficulty. we present two such cases of supratentorial anaplastic ependymoma in pediatric age group with one showing large areas of vacuolated/ signet-ring change and another showing lipomatous differentiation. objectives: to describe uncommon histological features in ependymoma material and methods: case reports of two cases with histopathological description. results: case 1 is a 9-year-old male child with a well-defined intra-axial, lobulated solid cystic lesion (4.5x3.6cm) in the right parieto-temporal region. biopsy showed monomorphic tumour cells interspersed with large areas of vacuolated cytoplasm/signet ring change in large areas posing a diagnostic challenge. these cells showed strong and diffuse perinuclear cytoplasmic dot-like positivity for ema along with diffusely positive gfap. however, the tumour recurred within 11 months showing anaplastic morphology lacking the previous features. case 2 is a 3-year-old male child with similar presentation except in this case there was lipomatous change mimicking adipocytes in large areas posing diagnostic difficulty. conclusion: lipomatous change, vacuolation and signet ring change has been reported in ependymoma in few reports. the origin of vacuolar change is still debated. some reports state it as dilation of intracytoplasmic membrane and some as metaplasia. these changes have been observed in other tumours like neurocytomas, medulloblastomas, cerebellar and spinal cord astrocytomas. there can be confusion between clear cell ependymoma and areas showing oligdendroglial proliferation as both show cytoplasmic clearing. in tumours with extensive change, search for classical areas and immunohistochemistry can facilitate the diagnosis.   copyright: © 2022 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. aβ plaques feel free to add comments by clicking these icons on the sidebar free neuropathology 1:31 (2020) review aβ plaques lary c. walker department of neurology and yerkes national primate research center, emory university corresponding author: lary c. walker · department of neurology · emory university · 505 whitehead biomedical research building · 615 michael st · atlanta, ga 30322 · usa lary.walker@emory.edu submitted: 27 september 2020 accepted: 23 october 2020 copyedited by: bert m. verheijen published: 30 october 2020 https://doi.org/10.17879/freeneuropathology-2020-3025 keywords: alzheimer’s disease, amyloid, neuritic plaques, neurofibrillary tangles, senile plaques abstract aβ plaques are one of the two lesions in the brain that define the neuropathological diagnosis of alzheimer’s disease. plaques are highly diverse structures; many of them include massed, fibrillar polymers of the aβ protein referred to as aβ-amyloid, but some lack the defining features of amyloid. cellular elements in ‘classical’ plaques include abnormal neuronal processes and reactive glial cells, but these are not present in all plaques. plaques have been given various names since their discovery in 1892, including senile plaques, amyloid plaques, and neuritic plaques. however, with the identification in the 1980s of aβ as the obligatory and universal component of plaques, the term ‘aβ plaques’ has become a unifying term for these heterogeneous formations. tauopathy, the second essential lesion of the alzheimer’s disease diagnostic dyad, is downstream of aβ-proteopathy, but it is critically important for the manifestation of dementia. the etiologic link between aβ-proteopathy and tauopathy in alzheimer’s disease remains largely undefined. aβ plaques develop and propagate via the misfolding, self-assembly and spread of aβ by the prion-like mechanism of seeded protein aggregation. partially overlapping sets of risk factors and sequelae, including inflammation, genetic variations, and various environmental triggers have been linked to plaque development and idiopathic alzheimer’s disease, but no single factor has emerged as a requisite cause. the value of aβ plaques per se as therapeutic targets is uncertain; although some plaques are sites of focal gliosis and inflammation, the complexity of inflammatory biology presents challenges to glia-directed intervention. small, soluble, oligomeric assemblies of aβ are enriched in the vicinity of plaques, and these probably contribute to the toxic impact of aβ aggregation on the brain. measures designed to reduce the production or seeded self-assembly of aβ can impede the formation of aβ plaques and oligomers, along with their accompanying abnormalities; given the apparent long timecourse of the emergence, maturation and proliferation of aβ plaques in humans, such therapies are likely to be most effective when begun early in the pathogenic process, before significant damage has been done to the brain. since their discovery in the late 19th century, aβ plaques have, time and again, illuminated fundamental mechanisms driving neurodegeneration, and they should remain at the forefront of efforts to understand, and therefore treat, alzheimer’s disease. 1. aβ, amyloid, and alzheimer’s disease the most striking and yet still enigmatic pathologic features of alzheimer’s disease (ad) are lesions known for over a century as senile plaques microscopic anomalies in the parenchyma of the brain consisting of an abnormal accumulation of protein decorated by various molecules, and often including dystrophic neuronal processes and reactive glial cells (figure 1). although plaques are a frequent feature of the senescent brain and, when particularly numerous, an obligatory diagnostic marker of ad [1], the identity of the principal protein in the plaque core remained unknown until the 1980s. then, glenner and wong established a partial amino acid sequence of the protein in cerebral amyloid angiopathy (caa) from patients with ad and down syndrome [2, 3], and masters and beyreuther [4, 5] determined that the same protein is a key component of plaques. initially referred to as the β protein, a4, or β/a4, the protein now is commonly designated aβ [6]. collectively, these lesions are increasingly referred to as aβ plaques (see section 3). figure 1. 'classical' aβ (senile) plaques in the cortex of persons who had died with alzheimer's disease (ad). left, a plaque stained with the naoumenko-feigin silver method and periodic acid-schiff (pas) counterstain; an amyloid core (dark pink) is surrounded by profuse abnormal neurites (black). right, a plaque immunostained with antibody 4g8 to the aβ protein (brown) along with a nissl counterstain (blue); glial nuclei are visible in the region between the plaque core and outer corona, and within and surrounding the corona. bar = 20μm for both panels. 1.1 aβ aβ is a cleavage product of the aβ-precursor protein (app), a 695–770 amino acid, single membrane-spanning protein that is strongly expressed in the nervous system [7, 8]. aβ is generated mainly in endosomes, and its release into the extracellular space is influenced by synaptic activity [9]. to produce aβ, app is sequentially cleaved by the enzymes β-secretase [or β-amyloid cleaving enzyme (bace)] and γ-secretase [8], resulting in aβ proteins that are most often 40 or 42 amino acids in length (‘aβ40’ and ‘aβ42’), although many c-terminally and n-terminally variant and/or chemically modified aβ fragments also occur [7, 10-16]. different lengths of aβ can derive from their differential excision from app by secretases or from post-translational trimming of aβ by exopeptidases [10]. potential post-translational chemical modifications of aβ include pyroglutamylation, racemization, isomerization, oxidation, phosphorylation, n-homocysteinylation, nitration, and glycosylation [11, 17-19] (see also section 7, below). how post-translational modifications influence the process of protein aggregation in general remains poorly understood [20, 21]. aβ40 is the isoform of aβ that is most abundantly generated by neurons, but two c-terminal hydrophobic residues in aβ42 augment its tendency to self-assemble into amyloid [7, 22]. as a result, more plaques are immunoreactive for aβ42 than for aβ40 (figure 2), although the relative amounts of plaques stained for aβ40 and aβ42 vary. figure 2. adjacent cortical tissue sections from an ad patient, immunostained with antibodies r398 to aβ42 (top) and r361 to aβ40 (bottom). two of the plaques that are present in both sections are denoted by arrows. asterisks mark a blood vessel for reference. bar = 100μm. unlike plaques, cerebral aβ-amyloid angiopathy (aβ-caa) in large vessels is more consistently positive for aβ40, though aβ42 also is generally present [23]. the staining patterns of the two isoforms differ in capillary aβ-caa compared to large-vessel aβ-caa [24, 25], and in the vessel wall compared to the diffuse aβ that sometimes extends from the wall into the surrounding parenchyma (dyshoric amyloid angiopathy) [25, 26]. the mechanisms governing the ontogeny of plaques and aβ-caa also probably differ to some extent (see section 5.3). in addition to plaques and amyloid angiopathy, aβ multimerizes into a range of oligomeric species [27, 28] that can interact with cells and impair brain function [27, 29-35]. oligomers appear to be an important intermediate step in the assembly of polymeric amyloid of all types [20]. comparison of subjects expressing ad-type dementia to nondemented subjects with high aβ plaque pathology, the amount of oligomeric aβ correlates more strongly with cognitive decline than does the number of plaques per se [36]. experimental studies indicate that aβ plaques include abundant oligomers [36, 37], and that some plaques shed toxic oligomers into the surrounding parenchyma [37-39]. aβ42-oligomers have been shown to arise from secondary nucleation on aβ-amyloid fibrils during protein aggregation, directly linking them to the process of amyloidogenesis [34]. at least some aβ-oligomers are particularly potent seeds for the formation of aβ plaques [40, 41], although whether there are seed-active oligomers that differ from toxic oligomers, as has been found for prions [42], is unknown. the relationship between aβ-oligomers and the diverse plaque types [31, 33, 38] in the human brain e.g., dense-core vs diffuse also is an issue that remains incompletely defined. indeed, owing to their dynamicity and heterogeneity, the analysis of oligomers as they occur in living systems is technically challenging [20] (see also benilova et al. [43] for a critique of oligomers as toxic agents). regardless of the relative contribution of aβ-oligomers and amyloid fibrils to disease, both of these multimeric states denote the presence of an abnormal condition in the brain, i.e., the misfolding and accumulation of the aβ protein. aβ has assumed a prominent position in alzheimer research because all identified risk factors for ad increase its quantity and/or tendency to aggregate [33, 44, 45]. most notably, mutations in app and the presenilins (components of the γ-secretase complex) [22] are the only known autosomal dominant causes of ad, and a superfluous app gene due to trisomic chromosome 21 in down syndrome frequently leads to early-onset ad [35, 46, 47]. furthermore, a rare mutation that substitutes a threonine for alanine (a673t) at position 2 of aβ lowers both the production of aβ [48] and its propensity to aggregate [49]; this mutation is associated with a reduced risk of manifesting ad [48] and possibly parenchymal plaques as well [50]. contrariwise, when a valine replaces alanine at position 2 (a673v), aβ generation is increased, and the protein is more prone to aggregate, resulting in an autosomal recessive form of ad [51]. thus, there is little doubt that aβ is intimately involved in the pathogenesis of ad, although many questions remain about how plaques per se participate in the neurodegenerative process. 1.2 amyloid a persistent source of misunderstanding regarding the role of aβ in ad is the common use of the generic term ‘amyloid’ to refer to the protein aβ. in pathology, amyloid refers to ‘mainly extracellular tissue deposits of protein fibrils, recognized by certain properties, such as green-yellow birefringence after staining with congo red’ [6] (for historical considerations of amyloid, see [20, 52-55], and for more on the definition of amyloid see [20, 56, 57]. amyloid can arise from over 30 different proteins in various parts of the body in different human diseases [6, 58]. hence, ‘aβ’ the molecule and ‘amyloid’ the fibrillar mass are not synonymous. aβ refers exclusively to the protein that, when aggregated into distinctive fibrils, constitutes the specific type of amyloid that most commonly accumulates in the aging brain. the formation of amyloid involves the misfolding and self-assembly of a particular protein into filamentous structures with a distinctive cross-β architecture that is stabilized by a ‘steric zipper’ molecular motif [20, 59]. the misfolded protein has two notable characteristics that contribute to its amyloidogenicity: 1) it compels unfolded molecules of the same protein to similarly misfold by means of permissive templating [60]; and 2) the β-sheets in separate molecules hydrogen-bond to one another to form stable, filiform polymers with the β-sheets oriented perpendicular to the long axis of the polymer [59]. in this way, the misshapen proteins both corrupt and capture like proteins, which stack into protofilaments that wind together to build long, non-branching fibrils that typically range from ~6 to 13 nm in diameter [54]. these fibrils are characteristic of amyloid in general [6]. despite their shared cross-β backbone and similar appearance by conventional transmission electron microscopy, however, amyloid fibrils are polymorphic at the molecular level [20, 61-68]. although amyloid was long defined as an exclusively extracellular substance [69-71], it is now recognized to occur intracellularly as certain types of inclusion [6, 20]. the tau protein that polymerizes into neurofibrillary tangles the second mandatory pathologic hallmark of ad (figure 3) has attributes of amyloid [72]. thus, the two lesions that characterize ad pathologically plaques and tangles arise from two different proteins aβ and tau both of which can misfold and self-assemble into amyloid. figure 3. an aβ plaque (brown) alongside intracellular tau tangles (purple) in the cortex of an ad patient. combined polyclonal antibodies r398+r361 to aβ40+42 plus monoclonal antibody cp13 to hyperphosphorylated tau. bar = 20μm. despite genetic, biomarker and pathologic findings implicating aberrant aβ in the initiation of ad [9, 33, 44], tauopathy is more strongly correlated with cognitive decline than are plaques [73-78]. in the forebrain, tangles first appear in the medial temporal lobe [79, 80], but the dementia of ad is fully apparent only when tauopathy becomes severe in much of the neocortex [1, 81], a process that is facilitated by the presence of aβ pathology [9] (see also [82]). the precise nature of the mechanistic link between aβ-proteopathy and tauopathy in ad, however, remains a critical unsolved problem [45, 83, 84]. 2. the discovery and early exploration of plaques the late 19th and early 20th centuries saw a profusion of new staining methods that selectively revealed various elements in cells and tissues [85]. accordingly, the original depictions of plaques reflected what was disclosed by histologic stains and viewed with the light microscope. in 1892, paul blocq and georges marinesco at the salpêtrière hospital in paris reported microscopic ‘amas ronds’ (‘round clusters’, or ‘round heaps’) or ‘nodules de sclérose névroglique’ (‘nodules of neuroglial sclerosis’) in the brains of older epileptic patients [86].1 this report is generally considered to be the first unambiguous identification of plaques in the senescent brain [81, 87]. in 1898, emil redlich published evidence linking plaques to dementia [88]; in the brains of three elderly subjects, two of whom had died with clinically confirmed dementia, redlich described the structures as consisting of a core of uncertain substance along with surrounding astrocytes (‘spinnenzellen’) and their processes. because they resembled millet seeds under the microscope, he referred to these collective lesions as ‘miliary sclerosis’ (‘miliare sklerose’). notably, redlich also dubbed them ‘plaques’, a term that was expanded to ‘senile plaques’ by simchowicz in 1911 [89]. furthermore, redlich noted that some smaller lesions consisted of fine fibers with a cotton-like appearance [88], anticipating the use of the term ‘cotton-wool plaques’ to depict certain types of deposit today [90-94]. although alois alzheimer is often credited with instigating the burst of scientific analyses of plaques with his 1906 conference presentation in tübingen (published in 1907) [95], his report was brief, and plaques (‘miliary foci’) were only superficially mentioned.2 he did not issue his first detailed histopathologic examination of plaques until 1911 [96]. in fact, along with redlich [88], a good case can be made that oskar fischer deserves the credit for initiating the modern histopathologic analysis of dementia with a comprehensive series of reports published in 1907, 1910, and 1912 [87]. several other researchers contributed to the literature on plaques during this period, including, among others, miyake [97], lafora [98], bonfiglio [99], hübner [100], perusini [101], fuller [102], bielschowsky [103], barrett [104], simchowicz [105] and marinesco and minea [106] (see also christen [107] for a brief historical perspective on this period of research into what we now call ad). both alzheimer and fischer excelled in their analysis of plaques by implementing a silver-based staining method introduced by max bielschowsky [87] (see braak and braak [108] for a nice summary of the early development of silver stains).3 alzheimer did, however, correctly anticipate the evolution of neurology in his 1907 publication, in which he contended that the time had come to define neurologic diseases based on both their clinical and histologic characteristics [95].4 this view has a contemporary parallel in the call by an international group of experts for a biological, rather than syndromic, definition of ad [109]. furthermore, alzheimer noted in 1911 the prevailing technical inability to identify the substance in the plaque core: ‘... we have to consider the core of the plaque as an unorganized mass which emerges differently with different staining methods ... as perusini and fischer have already explained, we are not at present able to identify this mass with any of the substances known in pathological anatomy’ (translation from [110]). in addition, alzheimer highlighted the prominence of glial cells in the composition of plaques [96], a subject that has gained momentum in the 21st century, owing in part to the identification of compelling, glia-related genetic risk factors for ad [111-115] (see section 6.2). from the early 20th century on, researchers widely agreed that the main structural elements comprising plaques are abnormal neuronal processes, altered glial cells, and a central, disordered mass of unidentified material. in a 1929 review, macdonald critchley [116] wrote that the ‘modern conception of the plaque is that of a reactionary change directed against a specific metabolic process of a toxic nature’ (a description that, if we consider the material in the core to be the key toxic substance, resonates with leading 21st century concepts). many pioneering scientists attempted to explain the origin and nature of plaques based on their interpretations of static images in selectively stained tissue sections. not surprisingly, disagreement was common (see, e.g., [99], [101], [102], [117]). ferraro (1931) summarized this lack of consensus: ‘...one group of investigators favors the theory that [the plaque] originates from nerve cells, another that it originates from neuroglial elements, another from axis cylinders, and still another, from the intercellular ground substance’ [118]. soniat remarked in 1941 that ‘no less than twenty different concepts concerning their origin have appeared in the literature’ [119]. as late as 1960, liss wrote, citing three influential textbooks on pathology, that the ‘morphogenesis of senile plaques remains still an unsettled and controversial matter’ [120]. a crucial question, and a source of much of the discord among researchers, was the nature of the plaque core what does it consist of, how does it arise, what impact does it have, and what governs the proliferation of plaques in the brain? the answers to these questions would not begin to emerge for another half century. in fact, no compelling conceptual insights immediately followed the initial flurry of histopathological investigations of plaques, which, ultimately, were hampered by limitations in the available methods [119, 121]. beginning in the 1960s, theoretical and analytical advances accelerated; electron-microscopic studies showed that the mature plaque core consists of amyloid fibrils structurally similar to those in corporeal amyloidoses [122, 123], and quantitative analyses confirmed that plaque load in the brain is linked to dementia [124]. most important, however, was the molecular decipherment of aβ as the primary protein in cerebral amyloid by glenner and wong [2, 3] and masters and colleagues [4]. the genetic insights and technical tools resulting from this discovery ultimately established aβ as a critical player in the pathogenesis of ad, and the plaques that occur in normal aging and ad could, for the first time, be unified by a single, omnipresent component aberrant aβ. 1 ‘il existe de plus, disséminés dans les diverses couches de l’écorce, de petits amas ronds d’un diamètre de 60 µ environ, se distinguant du reste du tissu par une coloration beaucoup plus intense, à contours réguliers. ils apparaissent ainsi, parsemant discrètement le fond des préparations, d’une structure vaguement pointillée, ce pourquoi il est permis de considérer quelques-uns d’entre eux, au moins, comme de véritables nodules de sclérose névroglique (?).’ (question mark is in the original) 2 ‘über die ganze rinde zerstreut, besonders zahlreich in den oberen schichten, findet man miliare herdchen, welche durch einlagerung eines eigenartigen stoffes in die hirnrinde bedingt sind. er lässt sich schon ohne färbung erkennen, ist aber färbungen gegenüber sehr refractär.’ 3 many different silver stains have been developed to detect ad pathology. each method selectively reveals certain elements in the plaques, and they are sometimes considered to be less sensitive than is immunostaining with antibodies to aβ. some silver stains, however, are exquisitely sensitive even to small, diffuse aβ deposits, which have been recognized in ad tissue since the early 20th century (see, e.g., marinesco and minea [1912] [reference 106] and cowe, a. [1915] [reference 508]). note also figure 23. 4 ‘es gibt ganz zweifellos viel mehr psychische krankheiten, als sie unsere lehrbücher aufführen. in manchen solchen fällen wird dann eine spätere histologische untersuchung die besonderheit des falles feststellen lassen. dann werden wir aber auch allmählich dazu kommen, von den großen krankheitsgruppen unserer lehrbücher einzelne krankeiten (sic) klinisch abzuscheiden und jene selbst klinisch schärfer zu umgrenzen.’ 3. plaque nomenclature: the case for ‘aβ plaques’ the term ‘plaque’ (which historically has referred to a flat object such as a disk or tablet) was adopted by the medical community in the mid-to-late late 1800s to designate patch-like abnormalities such as atherosclerotic plaque or dental plaque.5 redlich [88] used the term to describe carmine-stained densities (‘intensiv gefärbten plaques’), and simchowicz [105] added the modifier ‘senile’ to denote their frequency in senescent brains, particularly in patients with senile dementia [89]. most plaques in the brain (unlike dental or atherosclerotic plaque) are not planar (one exception being the band-like subpial deposits [see figure 9]). of course, spheroidal plaques appear discoid in histologic sections, and their apparent size and composition are influenced by the plane through which they are cut (figure 4). figure 4. a neuritic aβ plaque in consecutive sections of the cortex from an ad patient; the core is evident in the left-hand image, whereas sections through the periphery (middle and right) reveal only neurites (black). serial sections may be required to unequivocally identify plaque types (a technical caveat noted by, among others, alzheimer [1911] [reference 96]). naoumenko-feigin (silver) and periodic-schiff stains. bar = 20 μm for all images. some of the designations for plaques derive from their staining characteristics. following divry’s discovery that certain plaques show amyloid-type birefringence after staining with the dye congo red [125], the terms ‘congophilic plaques’ or ‘amyloid plaques’ became common. the term ‘argyrophilic plaques’ also has been employed, owing to their detectability by various silver-based staining methods [81]. other labels such as ‘miliary plaques’, ‘drusen’,6 and ‘redlich-fischer plaques’ can be found in the earlier literature [116]. in 1972, wisniewski and terry introduced the term ‘neuritic plaques’ in recognition of the profusion of abnormal neuronal processes that invest many plaques. with the identification of the aβ protein in plaques, the term ‘aβ plaques’ is increasingly common. for the following reasons, ‘aβ plaques’ is recommended as the inclusive term that succinctly encompasses the multiplicity of these lesions under the umbrella of their shared feature aβ deposition:7 1) aβ is present in all of the plaques that are linked to ‘normal’ aging and ad, regardless of size, shape, aggregation state, location, or overall composition. 2) the term ‘senile’ is vague and arbitrary, and not all plaques occur in ‘senile’ humans. although aβ plaques become more common at older ages, they can emerge in the 4th decade of life or earlier, especially in people with some autosomal dominant forms of ad [126].8 3) plaques that are structurally similar to aβ plaques occur in other neurodegenerative disorders, yet these result from the misfolding and aggregation of different proteins. such plaque-forming proteins include the prion protein (prp) in certain spongiform encephalopathies [127, 128], the abri protein in familial british dementia [129, 130], and the adan protein in familial danish dementia [131, 132]. 4) not all aβ deposits incorporate abnormal neurites, which often are sparse or absent in diffuse plaques [133] including cotton-wool plaques [90, 93, 134] (see below). the term ‘neuritic plaques’ is suitable for the lesions that contain neurites, but these are only a subset of the entire family of aβ plaques. 5) the aβ in plaques does not always meet all of the criteria for amyloid [6] (see section 1.2). many diffuse aβ deposits in the aging brain do not show birefringence after staining with congo red. in addition, large, cotton-wool aβ plaques lacking amyloid cores are abundant in certain presenilin-1 mutant cases of autosomal dominant ad [90, 91, 93, 94, 134] and in some non-familial cases [92]. (the aβ in non-amyloid plaques from some presenilin-1 mutant cases is unusual in that it consists mostly of n-terminally truncated aβ [94], as do diffuse deposits in the cerebellum in ad [135] and down syndrome [135, 136]). the term ‘amyloid plaques’, like ‘neuritic plaques’, is appropriate for a subgroup of the lesions, but the universal constituent is aβ, whether it is in the form of amyloid or not; hence, more precise designations of plaque subtypes would be, for example, ‘aβ-amyloid plaques’ and ‘neuritic aβ plaques’. note that ‘diffuse plaques’ here refers to the fact that the aβ accumulation is ‘widely spread or scattered; not concentrated’ [137], without consideration of the nature of the aβ deposits, e.g., thread-like or punctate. ‘diffuse’ thus denotes only the characteristics of the aβ deposits, and not the dysmorphic neurites or any other component of the plaques. also, when analyzing aβ plaques histologically, it is useful to be cognizant of the plane of section, thickness of the tissue, and the limitations of a given staining protocol. plaques are 3-dimensional structures that, when large enough, are only partially captured in thin histologic sections (figure 4). furthermore, different stains recognize different components of plaques. consequently, a comprehensive assessment of plaques requires their full reconstruction and the application of suitable markers for potential components. in congruence with the trend to define ad according to its molecular underpinnings [109], defining the plaques that occur in aging and ad based on their principal proteinaceous component unambiguously distinguishes them from similar lesions in other disorders. in addition, this molecularly grounded moniker explicitly specifies the attribute that defines these plaques as unique pathologic entities: the misfolding and abnormal accumulation of the aβ protein. 5 ‘plaque (subject: medicine and health): any small patch or region of abnormal tissue within the body. see amyloid plaque, gliosis. [from french plaquer to plate, from middle dutch placken to beat metal].’ from: oxford dictionary of word origins: https://www.oxfordreference.com/view/10.1093/acref/9780199547920.001.0001/acref-9780199547920 6 note that ‘druse’ (‘geode’) differs from ‘drüse’ (with umlaut), which refers to a ‘gland’. 7 because ‘aβ’ and ‘plaque’ are both nouns, they could be connected by a hyphen (aβ-plaque). i have chosen not to include the hyphen (the ‘open form’) in order to simplify usage. in some cases (such as aβ-caa & aβ-oligomers), i have retained the hyphen for clarity. 8 w.h. mcmenemey opined in 1963: ‘...the structures first observed by blocq & marinesco (1892) and thought by them to be nodules of glial sclerosis were called by simchowicz (1910) ‘senile plaques’ an unfortunate choice of name for it has coloured our thinking for the past fifty years’ [reference 509]. 4. the anatomic distribution of aβ plaques 4.1 histology determination of the amino acid sequence of aβ [2-4] prompted the development of sensitive and specific antibodies that have facilitated the investigation of the anatomic localization, structural diversity, and biochemical composition of aβ deposits in the brain. aβ plaques become increasingly frequent as age advances [80, 138], but they are especially numerous in ad patients. the anatomic distribution of aβ plaques is variable, and it differs both among individuals and among brain regions in a given person [139-141] (figure 5). in general, association areas of the neocortex are more vulnerable and/or affected earlier than are primary motor and sensory areas [140]. aβ deposition is particularly profuse in the default mode network, an interconnected assemblage of brain regions that maintain vigorous metabolic activity when the brain is in an otherwise resting state [142]. the structure of aβ plaques is influenced in part by the architectonic characteristics of the areas in which they form [139, 143], but it is usual for several kinds of plaque to intermix within a given site (figure 6). in the neocortex, the laminar distribution of diverse aβ plaques can vary markedly [140] (figure 5). figure 5. variation in aβ deposition in adjacent cortical gyri from an ad patient. antibody 4g8, nissl counterstain. bar = 500μm. figure 6. variable morphology of aβ plaques in the cortex of an ad patient. classical dense-cored plaques with the core-space-corona pattern are in the upper left and lower right, and an irregular cloud of diffuse material is near the center, along with numerous very small patches. antibody 4g8; nissl counterstain. bar = 50μm. based on an analysis of human brains with different degrees of plaque accumulation, a spatiotemporal course of aβ plaque formation has been proposed [19, 144, 145]. there is general agreement that diffuse plaques are the earliest type to emerge, followed later by cored (amyloid) plaques [146]. according to thal and colleagues, in the first phase of the process, diffuse aβ plaques appear in the neo(iso)cortex; in the second phase, allocortex, the hippocampal formation and amygdala are affected; in the third phase, plaques arise in the basal ganglia and diencephalon; in the fourth phase they appear in the midbrain and medulla oblongata; and in the fifth phase, the pons and cerebellum are affected [19, 144, 145] (figure 7). these stages have been consolidated by serrano-pozo and colleagues [133] into an isocortical stage 1, allocortical/limbic stage 2, and subcortical stage 3. this general pattern of spread has been confirmed by a cross-sectional in vivo analysis of aβ-amyloid deposition profiles using florbetapir-pet imaging [147]. thus, in the end-stage of ad, most brain areas exhibit at least some aβ deposition. the spinal cord has been less studied; while it appears to be largely spared, plaques there have been reported in some instances [93, 148]. figure 7. the phases of aβ plaque distribution in the brain [references 19, 145]; illustration courtesy of dietmar thal, ku leuven. 4.2. in vivo imaging at the turn of the 21st century, the first imaging agents were introduced to detect amyloid in the living human brain via positron emission tomography [149, 150]. jorge barrio and colleagues introduced 2-(1-[[6-[(2-[18f]fluoroethyl)(methyl)amino]-2-naphthyl]]ethylidene) malononitrile ([18f]fddnp), which binds to both aβ-amyloid and tau tangles, and which has achieved some utility in diagnosing tauopathies [151-153]. a more aβ-selective ligand, developed by william klunk, chester mathis and colleagues, is 2-(4’-[11c]methylamino-phenyl)-6-hydroxybenzothiazole (pittsburgh compound-b [pib]) [149, 154]. derived from the chemical structure of the histologic staining agent thioflavin-t, pib crosses the blood-brain barrier and binds with high affinity and selectivity to aβ deposits in plaques and caa [154].9 pib (which is labeled with carbon-11), was followed by similar pet ligands labeled with fluorine-18 (a radiolabel with a longer half-life than carbon-11): florbetapir (amy-vid) [155, 156], florbetaben (neuraceq) [157], and flutemetamol (vizamyl) [158]. by assessing aβ-amyloid load in living subjects, these imaging agents have facilitated the differential diagnosis of ad and the longitudinal tracking of aβ accumulation. they are particularly sensitive in detecting dense-core aβ plaques, although they also bind to some extent to aβ-caa and diffuse aβ deposits [47, 159-161]. histochemical analysis of fluorescently labeled (‘clicked’) pib applied to ad tissue sections confirms the preference of pib at low concentration (100nm) for dense-core plaques [162]. interestingly, pib does not show significant high-affinity binding to aβ-amyloid deposits in aged nonhuman primates with substantial aβ burden [163], even though the amino acid sequence is identical to that of humans (see section 10). (note that binding of ligands can vary among humans; for example, a case of end-stage ad has been reported with extraordinarily high aβ load, a predominance of aβ40, and minimal high-affinity binding of pib [164]). since neither ad-like tauopathy nor dementia has been reported in nonhuman primates [165], comparative analysis of ligand binding could be useful in defining the variant molecular characteristics of aβ deposits and their relationship to disease phenotype (see sections 5.2 and 10). 9 in the 1920s, congo red was introduced as an in vivo diagnostic agent for non-cerebral amyloidosis. following intravenous injection, the rate at which congo red was cleared from the blood was thought to reflect amyloid burden in affected organs (the more amyloid to bind the dye, the more rapid its clearance from blood). for various reasons, the test never achieved widespread use (see buxbaum and linke [2012] [reference 52]). 5. the variety of aβ deposits 5.1 aβ plaques the histologic implementation of specific antibodies in the 1980s firmly established that aβ plaques in the brains of alzheimer patients comprise a remarkable variety of morphologies [143, 166-172]. several modern classification schemes have been proposed (e.g., [143, 166, 173-176]), and while there is not universal agreement on some of the terms, aβ plaques can be broadly categorized into amyloid plaques per se (with dense, congophilic cores), and a range of more loosely organized deposits of myriad sizes, shapes, densities and locations [133] (figures 5, 6, 8, 9). it is noteworthy that different genetic mutations can be associated with particular predominant plaque morphologies, as well as the presence of caa (see alzforum for a list of alzheimer-associated mutations (https://www.alzforum.org/mutations). note also that relatively few of the mutant forms of ad have been thoroughly scrutinized neuropathologically. within the general categories of plaque structure, the aβ-amyloid plaques are more or less spheroidal lesions that include ‘classical’ or ‘mature’ plaques and so-called ‘burned-out’ or ‘compact’ plaques [177, 178]. recently, a ‘coarse-grain’ plaque type with multiple small cores and a predominance of aβ40 has been described in advanced ad cases, often in association with apoe4 homozygosity and caa [179]. diffuse aβ plaques are much more numerous than are amyloid plaques in the alzheimeric brain [143] (figures 6, 8), and they span a range of compactness from vaguely aβ-immunoreactive, congo red-negative regions (e.g., ‘fleecy’ plaques [180]) to clusters of loose fibrillar material that sometimes are weakly congophilic [139, 166]. ultrastructurally, some of these diffuse deposits have been shown to include amyloid fibrils [181-183], whereas others do not [183], the latter possibly representing a pre-amyloid stage of aβ aggregation [139]. figure 8. small, often stellate aβ deposits in the cortex of an ad patient. some aβ accumulates within glial cells, most likely astrocytes (right). antibody 4g8; nissl counterstain. bars = 20μm. figure 9. band-like subpial aβ (left) in neocortical layer 1 and presubicular lake-like aβ (right) from two cases of ad. the subpial aβ can be discontinuous, confluent, or punctate. antibodies 4g8 (left) and 6e10 (right); nissl counterstain. bar = 100μm for both images. diffuse aβ plaques comprise very small, often stellate assemblies scattered about the parenchyma (figure 8), a sheet-like band of sometimes confluent, sometimes patchy material in the subpial cortex (figure 9), large ‘cotton-wool’ plaques, and very large ‘lake-like’ patches, including a distinctive cribriform deposit in the subicular complex [143, 171, 176, 184] (figure 9). abnormal neurites generally are absent or sparse in diffuse deposits [139], and this includes the cotton-wool plaques that are characteristic of some advanced ad cases [90-94, 134]. despite their abundance in the alzheimeric brain, very small diffuse deposits have received remarkably little scientific attention [175]. these probably correspond to the small (~2μm diameter) ‘sternchen’ which fischer in 1910 considered to be the first stage of plaque formation [185]. at least some of them appear to be related to astrocytes [175, 186] (figure 8), but the absence of systematic research on these ubiquitous lesions currently precludes meaningful consideration of their involvement in the proteopathic process. similarly, the immunoreactivity of some vestigial (extracellular) neurofibrillary tangles with antibodies to aβ [187-194] (figure 10) remains mechanistically undefined. figure 10. neurofibrillary tangles in the cortex of an ad patient immunostained with an antibody to aβ40. when present, this colocalization occurs mostly on extracellular ('ghost') tangles. nissl counterstain. bar = 50μm. certain types of aβ plaque are typical of the brain compartments in which they develop, e.g., in the cerebellum, basal ganglia, or different cortical regions and laminae (see [139]). in the white matter, distinctive granular accumulations of aβ [143] occur to varying degrees (figure 11). these clusters consist of fibrillar aβ lying outside of the axons, and they appear not to be associated with obvious tauopathy or other abnormalities of the axons themselves [143], although their functional significance is largely unexplored. the core-space-corona arrangement of aβ is a notable structural feature of classical aβ plaques that was noted in several early investigations (reviewed in [116, 119, 120]). these subdivisions of plaques have been given various designations in the early literature, for instance zentrum or kern, hof, ring, etc.10 in tissue that has been immunostained for aβ, classical aβ plaques have a condensed core of aβ-amyloid surrounded by an optically clear region with little aβ, and then an outer corona of more diffuse aβ [195] (see figure 1); the relatively clear intermediate space and the outer corona are occupied by neuronal and glial elements (which are considered in more detail in section 6). figure 11. aβ deposits in white matter of an ad patient comprise clusters of small puncta and filamentous bundles. left: light-micrograph of a cluster immunolabeled with antibody 4g8 (nissl counterstain). right, electron micrograph of a punctum immunolabeled with antibody 4g8 (black dots are gold particles bound to the secondary antibody). bars = 20μm (left) and 200nm (right). viewed in the electron microscope, aβ-amyloid fibrils in the plaque core are densely packed and often bundled to form a patchy matrix, and viable cellular processes there are largely absent. the more loosely organized aβ-amyloid sheaves in the space and corona interdigitate with cellular elements such as glial processes and neurites (figure 12; see also figures 18 and 20). embedded in the fibrillar meshwork of amyloid in plaques, various small, spherical particles can be seen (figure 13). the origin and significance of this material is obscure, but it could account for some of the non-aβ substances that have been detected in the cores of aβ plaques (see section 7). one possibility is that these vesicles originate from intracellular multivesicular bodies, which have been shown experimentally to be an important site of app/aβ biology [196-201]. in this regard, vesicular structures ranging from 50 to 300nm in diameter have been reported among the amyloid fibrils in a cell culture model of aβ amyloid deposition [202]. the center of the compact core in some aβ-amyloid plaques is refractory to aβ-immunostaining (figure 14), even though it is positive for the amyloid-selective dyes thioflavin-s and congo red [203]. ultrastructural analysis indicates that the material in the center of fully developed plaques often has a more granular, amorphous appearance (figure 13) than the obvious fibrils in the mantle of the core and in the periphery. classical aβ-amyloid plaques are often ascribed special relevance to neurodegeneration [1, 204], as they are much more likely to involve neuritic malformation and reactive gliosis than are the diffuse deposits [133]. in this regard, it is noteworthy that cognitively normal elderly subjects with abundant aβ plaques tend to have mostly diffuse plaques [1] with few neurites and little glial reactivity [139]. however, as noted above, there are rare cases of advanced ad in which classical plaques or dense-cored plaques are infrequent [90-93], suggesting that amyloid per se is not essential to the development of dementia. a similar situation holds for prion diseases, all of which are linked to the misfolding and self-assembly of prp [205, 206]; in some prionoses (such as gerstmann-sträussler-scheinker syndrome and new-variant creutzfeldt-jakob disease), prp-amyloid plaques can be numerous, whereas in others, little if any amyloid is present [127]. in these instances, oligomeric species of the proteins may have particular importance [20], although this has not been definitely established. figure 12. ultrastructure of fibrillar aβ in the plaque corona (left) and core (right) in an ad patient. bar = 500nm for both images. a small proportion of aβ-amyloid plaques lack the outer corona and have few or no neurites; these relatively plain structures have been thought to represent an end-stage in the evolution of plaques, and so were dubbed ‘burned out’ plaques [143, 178]. based on their apparent sequential appearance ance in the ad brain, a progression has been proposed in which plaques originate as diffuse (‘primitive’ or ‘immature’) deposits that evolve into classical (or ‘mature’) aβ plaques and then finally into burned-out plaques [143].11 while longitudinal studies in mouse models of cerebral aβ accumulation have begun to shed light on the time-course of plaque development (see section 10.2), the order of events in the human brain is still speculative [207]. figure 13. high-magnification electron micrograph of a portion of the core of an aβ-amyloid plaque in an ad patient. the fibrillarity of the material is less evident than in more peripheral zones. unidentified particles (2 are marked by arrows) of various sizes and densities are interspersed among the amyloid fibrils; these can be found both in the core and corona. bar = 200nm. figure 14. aβ plaque with an antibody-refractory central core in an ad patient. antibody 6e10; nissl counterstain. bar = 20μm. biochemical determination of the age of aβ deposits indicates that the amyloid core is older than the diffuse aβ in the corona and in diffuse plaques [208, 209]. armstrong [173] has suggested that the major plaque types mostly arise independently, rather than in an evolutionary progression. in any case, the transformation of diffuse plaques into compact amyloid might not be an inevitable occurrence; for instance, it appears that diffuse aβ deposits such as the lake-like cloud of aβ in the subicular complex (figure 9) do not progress into dense masses of amyloid, and this may be true also for cotton-wool plaques in ad cases with certain presenilin-1 mutations [90, 91, 93]. finally, it should be emphasized that the relative pathogenicity of the many different aβ plaque types in the aging human brain remains ambiguous. it is fairly certain that reactive gliosis/inflammation and the local disruption of neuronal processes in classical aβ plaques are deleterious to brain function (see section 6), but it is likely that oligomeric agents are the more directly injurious manifestation of misfolded proteins (see section 1.1). in fact, while the plaques themselves are indicative of a pathogenic molecular process, in and of themselves they may be relatively benign or even protective [210, 211], at least when inflammation and surrounding oligomers are negligible (see sections 1.1 and 6). 10 fischer [1910] [reference 185] referred to the central core as the ‘morgenstern’ (morning star), and described the structure of one type of plaque thusly: ‘auch hier ist ein zentraler morgenstern, aus dem mehr oder weniger lange büschel entspringen; der fädchenring ist ziemlich weit vom zentrum entfernt, so dass ein grosser hof entsteht, der von den strähnchen durchzogen wird’ 11 diffuse plaques have long been considered an early stage in plaque formation (see, e.g., critchley [1929] [reference 116]). 5.2 aβ strains in ad, the diverse morphological attributes of plaques might reflect, in addition to the local tissue organization, the variable truncation, folding, and molecular architecture of aβ [212, 213]. these variants are referred to as proteopathic strains, a biological concept that was adopted by the spongiform encephalopathy community to explain the alternative disease phenotypes resulting from prion infections [214, 215]. at the molecular level, the formation and propagation of aβ aggregates (as well as the proteins involved in several other proteopathies [216]), constitute a mechanism that is fundamentally similar to that of prions [217, 218] (see section 9). the capacity to spawn distinct strains is considered to be a shared property of proteins that are prone to misfolding and self-assembly [56, 59, 219]. in vitro, a given protein can create morphologically diverse amyloid fibrils under different environmental influences, for example temperature, ph, ionic strength, protein concentration [220, 221] and agitation [67, 222]. strain properties can be conveyed to newly forming amyloid fibrils; in vivo, it is thought that proteopathic strains undergo conformational selection by which the strain best suited to a given environment predominates [215, 220, 223, 224]. studies in genetically modified mouse models (which can be customized to make various types of aβ) can shed light on the factors that govern alternative plaque morphologies in the living brain [225]. the generation of aβ strains is influenced by characteristics of the aggregating aβ such as mutations, truncations and chemical modifications (see sections 1 and 7). aβ forms distinct structural strains in different subtypes of ad [226-231]. investigations of the molecular configuration of aβ fibrils in vitro have yielded insights into potential determinants of aβ strains (see, e.g., [228, 232-235]), but cryo-electron microscopic analysis of meningovascular aβ-amyloid indicates that aβ-caa fibrils formed in vivo, though polymorphic, differ in important ways from those formed in vitro [66]. a similar analytic comparison of aβ fibrils from plaques in the brain parenchyma and caa could help to explain the inconsistent co-presence of plaques and amyloid angiopathy in ad. 5.3 cerebral aβ-amyloid angiopathy (aβ-caa) several different proteins can form caa in different disorders, but aβ is the most common source of caa in the elderly [236]. aβ accumulates in the vascular wall and perivascular zone in cases of primary aβ-caa involving mutations in the gene for app [21, 237-240] and to varying degrees in nearly all cases of ad [241-244]. ad and aβ-caa share many genetic risk factors, and like aβ plaques, idiopathic aβ-caa sometimes is present in the nondemented elderly [240, 241]. caa is a significant risk factor for lobar hemorrhage [236, 245], particularly in individuals with hypertension [246]. in end-stage ad, the amount of aβ-caa varies widely, even in the presence of copious plaques [247], although the severity of aβ-caa tends to increase with increasing plaque load [21]. furthermore, in some instances, aβ-caa can emerge in the absence or near absence of aβ plaques, notably in an autosomal dominant form of aβ-caa known as hereditary cerebral hemorrhage with amyloidosis (dutch type) (hchwa-d) [239, 248, 249]. there is evidence for some diffuse parenchymal aβ deposition [250, 251] and cognitive decline [238, 252] in these cases, but the clinical phenotype probably reflects the vascular pathology more than an ad-like disorder in which plaques and tangles are abundant [253]. cognitive dysfunction [254-258] and neurodegenerative changes [259] also have been associated with idiopathic aβ-caa. in approximately 25% of end-stage ad patients, aβ-caa affecting large vessels is considered to be severe (arterioles are more often afflicted than are veins); capillary aβ-caa is less common, being severe in approximately 10% of cases [247]. in advanced aβ-caa, the amyloid often extends through the tunica adventitia and into the surrounding parenchyma, where it is pervaded by tau-immunoreactive abnormal neurites [25, 260, 261] (figure 15). for unknown reasons, in regions of the neocortex where capillary aβ-caa is focally abundant, aβ plaques are relatively scarce [25, 247, 262]. figure 15. fluorescence-immunolabeled dyshoric cerebral aβ-amyloid angiopathy (red; antibody r398) and tau-immunoreactive neurites (green; antibody cp13) in the cortex of an ad patient. bar = 50μm. in the early stages of large-vessel aβ-caa, aβ42 is more commonly present than is aβ40 [263], but in later stages aβ40 predominates [263, 264]. capillary aβ-caa, however, more often is positive for aβ42 than for aβ40 [263]. it has been suggested that the deposition of aβ in capillaries transpires by a different mechanism than that in large vessels and aβ plaques [25, 26]. quantitative spatial analysis has largely refuted the hypothesis that cerebral capillaries are the nidus of aβ plaque formation [265]. interestingly, ‘coarse-grain’ plaques, a special type of lesion (see section 5.1), are more common in cases with abundant aβ-caa, particularly capillary aβ-caa [179]. aβ-caa, like aβ-plaques, is associated with reactive gliosis and a perivascular inflammatory response [240, 260], although the presence of frank perivascular inflammation is inconsistent [25, 266]. aβ-amyloid plaques are occasionally confluent with aβ-caa (‘juxtavascular plaques’; figure 16), but the etiologic relationship between these merged lesions is uncertain. figure 16. juxtavascular aβ-plaque (arrow) in the cortex of an ad patient. antibody 4g8, nissl counterstain. bar = 20μm. various genetic, biochemical and pathophysiologic factors appear to influence how the misfolding and aggregation of the same protein aβ can lead to two different phenotypic presentations parenchymal plaques and vascular amyloid [21]. while many auxiliary molecules are present in both aβ plaques and aβ-caa, some are not shared by the two lesions [267]. thus, aβ-caa and aβ plaques likely result from at least partially distinct ontogenetic pathways [21] (in this regard, it is noteworthy that the disappearance of plaques in alzheimer patients immunized against aβ is accompanied by a [possibly transient] increase in aβ-caa, suggesting a transfer of aβ from the parenchyma to the walls of blood vessels [268]). for in depth reviews of caa, see [21, 236, 237, 240, 241, 260, 269]. 6. cellular components of aβ plaques the main cellular elements neuronal processes and glial cells in classical plaques were well-documented by pioneering investigators in the field (see [116]),12 although the nature of their involvement, and their functional relationship to the core, have been a persistent matter of speculation [207]. diffuse deposits of aβ mostly lack obvious changes in local neurons and glial cells, whereas these cells are conspicuously altered in classical aβ plaques. since classical plaques are especially numerous in most cases of late-stage ad, the associated abnormal neurites and activated glial cells probably contribute to the disturbance of brain function by the plaques [133]. 12 early descriptions of plaques included drawings that enabled the artist to clearly depict all cellular elements throughout the depth of the tissue sections in a way that photomicrography, still in its infancy, could not. the result was sometimes striking images that have been difficult to surpass in the century-plus since (see, e.g., the fine reproductions in defelipe [2010] [reference 510]). 6.1. abnormal neurites in advanced ad, many aβ plaques are decorated with an impressive profusion of dysmorphic neurites (figures 1, 4, 17). both axons and dendrites contribute neurites to plaques [207, 270]. although most swollen neurites have been reported to be axonal in origin [138, 178, 207], a quantitative analysis of plaques in humans using axonand dendrite-specific markers is needed to establish the relative involvement of these neuronal processes. tortuous, atypical neurites that are not spatially associated with plaques are fairly common in the aging brain [139], but neuritic pathology is particularly evident in many aβ-amyloid plaques. by disrupting the structure and trajectory of neuronal processes, aβ plaques are thought to interfere with the connectivity and network functionality of the brain [38]. figure 17. abnormal neurites associated with cortical aβ plaques in two ad patients. left: immunostain for neurofilament-h (antibody smi31) with a nissl counterstain; right, immunostain for a conformational epitope on tau filaments (antibody mc1). the presence of aberrant neurites that are immunoreactive for these antigens in plaques is variable. bar = 25μm (right) and 50μm (left). abnormal neurites are heterogeneous in size, shape and content. ultrastructurally, plaque-associated neurites may contain any of a number of inclusions, including, in addition to paired helical filaments, profuse mitochondria, various dense bodies, membranes, and multivesicular profiles [139, 178] (figure 18). the mitochondria appear to be in different stages of degeneration, and they have been hypothesized to be a source of the aβ-amyloid in plaques [207], as have multivesicular bodies [199, 271, 272]. the cytoskeleton is disrupted in swollen neurites [273], and studies of mouse models found that neuritic calcium (ca2+) homeostasis [274] and autophagy [275] are dysregulated in them. dickson [139] divided abnormal neurites into paired helical filament (phf)-type neurites, which are characteristic of advanced ad, and dystrophic-type neurites, which are relatively more frequent in the plaques found in aged, non-demented subjects (and in animal models, in which phfs per se are rare or absent [165]; see section 10). dickson also notes, however, that many neurites have the properties of both types, and that abnormal neurites tend to arise from axons or dendrites that just happen to be in the vicinity of the plaque [139]. this is likely to be a general rule for the presence of specific types of neurites in plaques, including those containing markers for diverse neurotransmitters (see below) and, e.g., the alpha-synuclein-positive neurites in aβ plaques that are sometimes co-morbid with synucleinopathy in lewy body disease [276]. figure 18. abnormal neurite (top) containing organelles /debris adjacent to fibrillar amyloid (bottom) in the plaque corona of a patient with ad. bar = 500nm. histochemically, lysosomal enzyme activity is pronounced in dystrophic neurites, as is histochemical reactivity for app and markers of degeneration such as chromogranin-a and ubiquitin [139]. the chemical variability of neurites may reflect, in addition to the neurons of origin, their stage of development and their response to injury or stress [277]. several early researchers, including fischer [117] and ramon y cajal (see [278]), thought that the swollen neurites in plaques represented an attempt by the neuronal processes to sprout. since then, multiple growth-promoting factors have been detected in these neurites [278, 279], and aβ deposits have been shown experimentally to induce axonal sprouting in the mouse brain [280]. considered as a whole, these observations indicate the presence of both degenerative and regenerative mechanisms in the aberrant neuronal processes that are associated with aβ plaques [133, 279]. analyses of aβ plaques in humans and aged nonhuman primates found that many different neurotransmitter systems contribute anomalous neurites to plaques [281-287], and that an individual plaque can contain neurites from multiple sources [288, 289]. these studies cast doubt on the hypothesis [290] that plaques emerge from the regression of neurites from a specific transmitter system, in particular the acetylcholinergic neurons of the basal forebrain [141]. rather, they highlight the probable role of a common catalyst (e.g., misfolded aβ and/or reactive glia) in driving neuritic dystrophy [139, 289, 291]. indeed, the influential model proposed by wisniewski and terry [178] (see also [81]) that posited neuritic abnormalities in general as the initial stage of plaque ontogeny now seems untenable, especially in light of genetic findings implicating aβ as the prime mover in the pathogenesis of ad [9, 22, 44, 45, 212]. even so, dysmorphic neurites do influence the pathologic plaque milieu [207], and it is possible that, by releasing aβ into the extracellular space, they contribute to the growth of plaques [272]. in addition, neuritic aβ plaques are generally more strongly associated with dementia than are diffuse plaques [1, 133, 204]. finally, the loss of synapses correlates strongly with the degree of dementia in ad [292-295]; synaptic pathology is especially evident in the immediate vicinity of aβ plaques (see [296, 297], possibly owing to increased oligomeric aβ in this region [297]. 6.2 glial cells of the many genetic risk factors for ad [298], two of the most potent variant genes apoe (apolipoprotein e) and trem2 (triggering receptor expressed on myeloid cells-2) are highly expressed in glial cells [115], as are several other ad-associated genes [299-301]. astrocytes and microglia are protean and interactive components of the homeostatic intrinsic immune system in the brain and spinal cord [299, 302, 303]. histologic, genetic, biochemical and physiological findings strongly implicate them in the pathogenesis of ad [111-115, 299, 303-310] (figure 19). microglia and astrocytes do not operate independently of one another, but rather jointly influence aβ processing and plaque biology [311, 312]. in the vicinity of aβ-amyloid, these glial cells together form a partially integrated ‘reactive glial net’ [313] that, while considered to be an attempt to shield nearby neurons from aβ aggregates [314], ultimately engenders a neurotoxic inflammatory microenvironment [313]. figure 19. reactive astrocytes (left; antibody to gfap) and microglia (right; antibody to iba1) in cortical aβ plaques of two ad patients. despite some overlap of the two cell types within plaques, astrocytic somata tend to be more peripherally located than are microglial somata. bar = 20μm for both panels. inflammation is both a risk factor for, and a result of, the deposition of aβ in the brain [45, 315]. the recruitment and activation of glial cells by aβ plaques has been likened to a local inflammatory reaction to a foreign body [138, 139], although glia contribute to the pathobiology of plaques in complex ways [299, 302, 305, 309, 312, 316-318]. mouse models have enabled a dynamic view of glial function and the general biology of plaques, whereas the genetic and physiologic analysis of glia in human ad is much less advanced [299]. even given the caveat that glia differ in humans compared to other species [316, 319, 320], mice have furnished unique insights into glial functionality in the living brain [304, 316, 321-324]. a growing literature underscores the ability of both microglia and astrocytes to adopt different physiologic states that influence how they contribute positively or negatively to ad (see, e.g., [299, 303, 306]). current views of glial cells thus emphasize their dual role in the pathobiology of ad: they participate in the clearance of aberrant aβ and other debris, but they also can secrete a variety of inflammationand cell-stress-related molecules [304, 325, 326]. much contemporary research seeks to define and disentangle these intricate and seemingly incompatible mechanisms. 6.2.1 microglia activated microglia are intimately associated with the fibrillar aβ in classical aβ plaques [139, 327-330] (figure 20). they occupy much of the space between the plaque core and outer corona, and their processes interdigitate with the bundles of amyloid [311, 327]. the discovery that loss of function mutations in trem2 are a strong risk factor for ad has heightened interest in the role of microglia in neurodegeneration [299, 306]. trem2 is a cell-surface immune receptor on many myeloid cells, including microglia, which exclusively express trem2 in the brain [306]. the production of trem2 is increased in ad [331], and it mediates the activation and responsiveness of microglia to aβ-amyloid plaques [332]. microglia have been thought to either phagocytose [139] or produce [311] multimeric aβ, and their functional variability makes both actions conceivable, depending on the circumstances. on the one hand, there is evidence that microglia normally impede the generation of aβ plaques; inhibition of microglial functionality in mice was found to increase plaque load [333, 334], and microglia contribute to the clearance of dense-core plaques following anti-aβ immunization therapy [335] (see also the analysis of immunized humans by nicoll and colleagues [336]). additionally, studies in mice indicate that trem2 signaling transforms homeostatic microglia into disease-associated microglia (dam), in which state they phagocytose aβ in plaques [306, 337]. impeding trem2 functionality in microglia reduces the binding of apoe to aβ-amyloid in plaques and augments the seeded propagation of aβ-amyloid [338]. (genetic knockout of trem2 also promotes the seeded aggregation and spread of tau in neuritic aβ plaques [339]). on the other hand, ultrastructural [311, 340, 341] and experimental [342] investigations have suggested that microglia can generate aβ-amyloid fibrils. in support of this hypothesis, sustained pharmacologic reduction of microglia significantly diminished aβ plaque load in a transgenic mouse model [343]. the ability of microglia to assume multiple phenotypic states underscores the complexity of their participation in the biology of aβ plaques [299, 300, 305, 344-346]; they contribute to normal brain homeostasis, but they also have injurious properties, particularly when activated [299, 300]. in mice, microglia have been found to exhibit a range of activation states, each of which involves the expression of distinct gene modules [299]. microglia become activated in the presence of aggregated aβ, and in this condition they can harm the brain both through the secretion of pro-inflammatory agents and the elimination of synapses [300]. to complicate matters further, a variety of microglial phenotypes are simultaneously present within the same brain [345], and the involvement of microglia in plaques differs as a function of age and disease stage [299]. finally, while the discovery of microglial risk factors for ad emerged from human genetic analyses [306], we know far more about microglia in rodent models than in human ad, and current evidence suggests that there are important differences that cannot be overlooked [299, 347-349]. these findings collectively highlight the challenges presented by microglia as therapeutic targets in ad. figure 20. electron micrographs of a microglial cell in an aβ-amyloid plaque of an ad patient. the white box in the image on the left denotes the region at higher magnification on the right. the fibrillar bundles of aβ interdigitate with the microglial soma. note that the microglial cytoplasm appears artifactually rarefied in this autopsy-derived tissue. bar = 500 nm (right), 2.8μm (left). 6.2.2 astrocytes in the vicinity of many aβ-amyloid plaques, astrocytes hypertrophy and increase their expression of glial fibrillary acidic protein (gfap) [316] (figure 19). the degree of astrocytic hypertrophy surrounding plaques, however, is inconsistent [317]. gfap expression is a reasonably reliable index of astrocytic reactivity, but gfap is not detectable in many healthy astrocytes, and its expression varies in different parts of the brain, in different animal species, and as a function of age.13 compared to microglia, astrocytic somata tend to localize more peripherally to the aggregated aβ in plaques [175, 302, 311, 313, 327, 330], whence their processes penetrate and to some extent encapsulate the plaques (figure 19). despite their tendency to partially segregate, astrocytes and microglia show some spatial overlap, and physical and chemical interactions between them help to define the inflammatory state of plaques [304]. activated astrocytes promote the inflammatory milieu around plaques through the generation of pro-inflammatory substances, including cytokines/chemokines, activation of the complement cascade, and reactive nitrogen and oxygen species [316]. as in the case of microglia, the role of astrocytes in neurodegeneration is complicated by their variable and sometimes paradoxical phenotypes [317]. in ad, astrocytes can both gain a toxic function and lose their normal physiologic function [316, 350, 351]. astrocytes have been shown experimentally to take up and degrade aβ [315]. they also are capable of expressing aβ [352], and astrocytes containing ample aβ are present in the alzheimeric brain [186, 316, 353, 354] (figure 8). in addition, the extent of peri-plaque reactive astrocytosis is positively correlated with cognitive status in aged subjects, and their abundance is reduced in persons expressing apolipoprotein e4, a major risk factor for ad [316]. in summary, research on microglia and astrocytes has disclosed the extraordinary malleability of these glial cells, the complexity of their involvement in plaques, and thus the attendant difficulties in targeting them therapeutically. interventions that modulate the activity of glia could either promote or hinder disease progression, depending on the state of the cells in different brain areas, their relative abundance, and the timing of therapeutic delivery in the protracted course of ad. nevertheless, the obvious importance of microglia and astrocytes in the pathobiology of ad justifies continued efforts to decipher the mechanisms by which they interact with aβ and with the other cellular components of plaques. for additional reviews of microglia and astrocytes in aging and ad, see [312, 355-357]. 13 the authors note that the findings should be interpreted cautiously in light of the pitfalls associated with histochemical methods (garwood et al. [2017] [reference 316]). this advice applies to histologic analyses in general, as methods and interpretations can vary among laboratories (e.g., alafuzoff et al. [2008] [reference 511]). 6.2.3 oligodendrocytes compared to microglia and astrocytes, oligodendrocytes have been less studied in ad [358]. their involvement in plaques has long been debated (see, e.g., the contrasting views of critchley [116] and ferraro [118]: ‘oligodendroglia apparently does not participate in the structure of plaques’ [critchley, 1929]; ‘it is certain, then, that both oligodendroglia and microglia cells are usual components of senile plaques’ [ferraro, 1931]). soniat contended that oligodendrocytes are not integral to the formation of plaques, but rather, when present, their presence is purely coincidental [119]. a recent analysis, however, has revealed oligodendrocyte progenitor cells in aβ plaques that become senescent and pro-inflammatory, in which state they are thought to augment the pathogenicity of aberrant aβ [359]. more work on oligodendrocytes in association with aβ plaques is clearly needed. 7. the broader biochemistry of aβ in plaques the number of molecules that have been linked in some way to aβ plaques is considerable (see, e.g., [139, 175, 278, 279, 360-364]), creating fertile ground for hypotheses on both the origin of plaques and the nature of ad. along with the many substances directly associated with neurons and glia, aggregated aβ itself is rich in accompanying molecules. amyloid p component is present in different types of amyloid throughout the body [6, 365], including aβ plaques [364, 366, 367]. other molecules that have been reported to directly co-localize with at least some aβ deposits include proteoglycans [6, 364, 368, 369], complement proteins [370-373], apolipoprotein e [374, 375], alpha-1 antichymotrypsin [376] and advanced glycation end products [377, 378], along with lipids, metal ions, reactive oxygen species and nucleic acids (see stewart and radford [364]). how aβ-linked substances might be involved in the pathobiology of plaques is attracting increasing attention. for instance, a study in mice found that aβ bound to nucleic acids acts as an immune signal, stimulating an antiviral response in microglia and astrocytes that instigates the complement-mediated elimination of local synapses [379]. the plaque-associated proteome can be interrogated by laser-microdissection of aβ plaques followed by mass-spectrometric analysis [380-385]. these studies have identified numerous proteins that are enriched in plaques, though whether they are directly associated with multimeric aβ or with the cellular constituents is sometimes undefined. it has been proposed that plaques mature through three biochemical stages within which the toxicity of the aggregates may differ; in stage 1, the aggregates lack both pyroglutamation at residue 3 (aβnp3e) and phosphorylation at residue 8 (pser8aβ); in stage 2, aβnp3e appears, and in stage 3, both aβnp3e and pser8aβ are present [19, 386]. post-translational chemical modifications of aβ can influence the aggregation of the protein along with the type of deposit that is formed in the brain [19, 387-391], but the mechanisms are, in many cases, still uncertain. 8. microbes and plaques the notion that microbes might participate in the genesis of plaques has been considered at least since the early 20th century.14 fischer likened mature plaques to actinomyces ‘drusen’, although he noted that they were negative for multiple bacterial stains [117]. critchley remarked in 1929 that the microbial origin hypothesis had failed to gain traction [116]. despite more recent claims that senile plaques in ad ‘are made up by spirochetes’ [392], there is still no credible evidence that aβ plaques are primarily collections of microbes or their remains. that said, there is fairly compelling evidence that certain microbial infections are risk factors for ad [393-395]. perhaps the best evidence indicates that some herpesviridae increase the probability of developing ad [394, 396, 397]. over 15 different microbes have been linked to ad by various researchers [398], but in many instances the findings are weak or contradictory (see, e.g., [399, 400]). in addition, it is important to distinguish cases of dementia in general (for which there are over 50 different causes [330]) from cases of dementia specifically due to the pathology of ad (as defined by jack and colleagues [109]). it is fair to say that no known infectious agent is universally and exclusively associated with ad [395], but it seems likely that any of several types of brain infection (including chronic infection and/or reactivation of resident microbes) can accelerate plaque formation and the pathogenesis of ad [394, 395, 401, 402]. in other words, at least in some instances the development of plaques may represent a non-specific response to various infectious organisms. aggregated aβ has antimicrobial properties [395, 403, 404], and some microbial antigens have been reported in aβ plaques [392, 405], but a systematic and comprehensive survey of microbial markers in different types of plaques and aβ-caa throughout the central nervous system has not been reported. app-transgenic mice raised in a germ-free environment develop some aβ plaques as they age, albeit fewer than mice raised in normal caging [406]. with the caveat that the mice strongly overexpress transgenic aβ, the findings suggest that infection is not required for plaque formation, but that it can trigger and/or accelerate the process. the role of infection in the causation of aβ plaques and as a risk factor for ad is an intriguing topic with potential implications for prevention and therapy, but supporting evidence for a specific role of specific microbes in pathogenesis is needed. for a critical consideration of the state of the field, see [393]. 14 i use the term ‘microbe’ here to include both conventional (living) microorganisms and viruses (but not prions). 9. the seeded induction of aβ plaque formation the prion paradigm has become the dominant mechanistic explanation for the aberrant self-assembly and propagation of misfolded proteins in the brain and elsewhere in the body [58, 205, 218, 407-409]. at the molecular level, the prion paradigm postulates that misfolded, β-sheet-rich proteins aggregate into oligomeric/polymeric assemblies that can induce protein molecules of the same type to adopt a similar conformation. in this condition, the proteins tend to stick together, with the assemblies often (but not always) amassing into amyloid deposits. in the prion diseases, misfolded prp self-assembles into highly stable multimers that are transmissible from one organism to another the first verified instance of an infectious protein particle (‘prion’) [410, 411]. human prion diseases also originate spontaneously or as a result of mutations in the gene for prp [412]. the pathological signature of the prion diseases varies considerably [127, 128], but, as in ad, the universal feature of prionopathies is the accumulation of an abnormally folded protein in this case prp in the nervous system. systematic studies in transgenic mouse models expressing human app have determined that aβ plaque formation is driven by a molecular process that is indistinguishable from the mechanism by which prions instigate disease [217, 218, 413-415] (figure 21). in this paradigm, brain extracts containing aggregated aβ are infused into the brains of susceptible mice, instigating aβ plaque development in a model-, doseand time-dependent fashion [218, 407]. analyses of seeded aggregation in experimental systems have demonstrated that aβ seeds share key properties with prions: 1) they are protein-only agents that are resistant to destruction by heat and formaldehyde; 2) they incite the formation of cerebral aβ plaques and aβ-amyloid angiopathy when introduced into the brain or into the periphery; 3) they exist in multiple sizes; and 4) they can fold into different molecular variants referred to as proteopathic strains [212, 213, 217, 218, 407] (see section 5.2). the strain-like properties of aβ deriving from different subtypes of ad can be partially transmitted to plaques via exogenous seeding in mouse models [227, 230]. figure 21. seeded aβ deposition in the hippocampal formation of a tg2576 app-transgenic mouse 5-months following unilateral injection of dilute ad brain extract into one hemisphere (left). the contralateral hippocampus in the same tissue section is on the right. antibody 4g8; bar = 100μm. these investigations highlight the prion-like seeded aggregation of aβ as the propulsive mechanism behind the formation of aβ plaques. since there is currently no evidence that ad or other cerebral proteopathies are infectious under ordinary circumstances [416, 417], it is likely that plaques ordinarily arise endogenously with the stochastic emergence, persistence and spread of aβ seeds. this process can be advanced by various environmental and endogenous risk factors that influence the likelihood that aβ will misfold and propagate in the brain [45]. under extraordinary circumstances, however, aβ plaques and aβ-caa can be instigated by exogenous aβ seeds in humans [417]. treatment of young people with growth hormone derived from cadaveric human pituitary glands, beginning in the late 1950s, resulted unexpectedly in the development of prion disease (creutzfeldt-jakob disease) many years later [418, 419]. the apparent cause was the presence of infectious prions in the preparations, probably because the large batches of pituitaries that were homogenized for extraction of growth hormone contained some glands from decedents with prion disease. researchers in england later found that both aβ plaques and aβ-caa were much more common in human growth hormone-treated subjects than in non-treated controls [420]. furthermore, aβ deposition was precipitated both in growth hormone recipients dying with [420] or without [421] creutzfeldt-jakob disease. an increase in aβ-proteopathy also has been reported in a subset of people who had received cadaveric dura mater transplants [422, 423]. the most parsimonious explanation for these findings is that some batches of therapeutic growth hormone and dura mater were tainted by aβ seeds that were present in the tissues taken from donors with ad (or incipient ad) [417]. this possibility is reinforced by the demonstrable presence of aggregated aβ in some pituitary glands [424] and dura mater [425] from ad patients. furthermore, aβ was detected in archival samples of cadaveric human growth hormone [426], and stored hormone was shown to stimulate cerebral aβ deposition when injected intracerebrally into app-transgenic mice [427]. interestingly, tauopathy was not apparent in most of these cases (even though some abnormal tau is present in alzheimeric pituitaries), and no recipients of cadaveric growth hormone or dura mater have yet been found to develop full-blown ad. whether this will happen as the subjects age further remains to be determined. 10. aβ plaques in nonhuman species 10.1 native aβ plaques naturally occurring aβ plaques and/or aβ-caa have been identified in aged animals of many species, including such diverse creatures as woodpeckers [428], bears [429-431], dogs [432-435], cats [436], camels [437] wolverines [438], and all species of nonhuman primate examined to date [165, 435, 439]. the mammalian mainstays of experimental biology rats and mice do not normally manifest plaques in old age, possibly owing to 3 amino acid differences in the n-terminal segment of aβ that render the protein less likely to aggregate [440, 441]. most research on native aβ plaques in nonhuman species has focused on primates ranging from prosimians to monkeys and apes [439], work that has yielded insights into the pathobiology of the lesions [178, 439, 442-444]. nonhuman primates express aβ with the same sequence of amino acids as in humans, and both diffuse and dense-core aβ plaques can be abundant in aged primates [439] (figure 22). some of the plaques include reactive glial cells and dysmorphic neurites [444]. mass-spectrometry has shown that post-translational modifications of aβ are similar in humans and squirrel monkeys (saimiri sciureus), and by elisa, the amount of aβ in the nonhuman primate brain sometimes exceeds that in humans with ad [163]. despite the presence of copious aberrant aβ, no nonhuman species has yet been found to exhibit the full clinicopathologic phenotype of ad as it occurs in humans [165]. specifically, a dementia-like condition has not yet been identified, and tauopathy, though often present, is generally mild, even in the presence of profuse aβ plaques. for unknown reasons, aβ-caa, especially capillary aβ-caa, is more commonly present in nonhuman primates than in humans [439, 445, 446]. although congophilic aβ plaques occur, human-like classical plaques with an aβ core, space, and outer corona (see figure 1) are rare, if they can be found at all, in prosimians and monkeys (we cannot yet rule out such lesions in great apes, as relatively few have been examined in advanced old age). surprisingly, there is little high-affinity binding of the aβ-amyloid-imaging agent pittsburgh compound-b (pib) to aβ plaques in nonhuman primates, suggesting biochemical and/or conformational differences in the protein between humans and other primates [447]. it is necessary to determine how differences in lifespan and environmental and genetic risk factors might influence the apparent species-specificity of ad and the aβ-deposition phenotype. however, current evidence suggests that, despite similarities in the sequence, expression, modification, and deposition of aβ, nonhuman primates lack the permissive connection between aβ-proteopathy and tauopathy that is critical to the occurrence of ad in humans [165]. clarifying the nature of this naturally occurring interruption of the aβ cascade in nonhuman species could reveal new pathogenic pathways for therapeutic intervention in ad. figure 22. aβ deposition (left) in the superior temporal gyrus and a neuritic plaque (right) in the hippocampal formation of two aged rhesus monkeys (macaca mulatta; 35 years and ~30 years, respectively). left: antibody 82e1 to the n-terminal segment of aβ, nissl counterstain; right: antibody 06-17 to phosphorylated neurofilaments. bars = 200μm (left) and 25μm (right). the maximum known lifespan of rhesus monkeys is 44 years (see stonebarger et al. [2020] [reference 512]). figure 23. aβ plaques in an aged (28 months) tg2576 app-transgenic mouse. diffuse deposits are black, and some dense deposits have a golden core (one in the frontal cortex is magnified at right). campbell-gallyas silver stain. bars = 1mm (left) and 50μm (right). 10.2 aβ plaques in genetically modified animals studies of naturally occurring plaques in various species have shed some light on the lesions, but there was no practical model in which plaques could be experimentally investigated until the mid-1990s. then, transgenic mice were introduced that overexpress human app with genetic mutations linked to ad [448-450]. with age, these app-transgenic mice deposit copious aβ in the brain (figure 23). they were followed by a wealth of additional models in various mouse (and later rat) strains with diverse genetic alterations, transgene expression levels, and the expression or deletion of interacting molecules [451-453]; see alzforum for a list of rodent models of ad-like pathology: https://www.alzforum.org/research-models/alzheimers-disease). not surprisingly, the sundry genetically modified animals exhibit many plaque (and caa) phenotypes. no genetically modified rodent, including those with multiple modifications, has manifested fully ad-like aβ plaques. as in nonhuman primates, the core-space-corona type of plaque is not typical of the transgenic rodent models. the plaques do, however, share several key features with those in humans; they exhibit a range of morphologies, many have bona fide amyloid cores, and they are invested by aberrant neurites and glial cells [452]. tau abnormalities occur, but human-like neurofibrillary tangles have not yet been generated in rodents. nonvertebrate transgenic animals such as fruit flies (drosophila melanogaster) [454-456] and roundworms (caenorhabditis elegans) [457, 458] have been developed to study the pathobiology of aβ. these models can be useful for analyzing molecular mechanisms and for studying the early-stage efficacy and toxicity of investigational agents, but no nonvertebrate model has yet generated aβ plaques that remotely resemble those in humans. it is difficult to overstate the impact that the introduction of genetically modified animals has had on the course of research on the mechanisms underlying plaque formation and ad. for example, transgenic rodents were used to establish the prion paradigm as the pre-eminent theory of plaque ontogeny and spread [217, 218, 407] (see section 9), they are being used to probe the role of glial cells and neuritic dystrophy in plaque pathophysiology (see section 6), and they are a vital tool in the preclinical testing of new therapeutic and diagnostic strategies [452, 459-463]. for instance, whereas the first evidence that aβ plaques and aβ-caa could be targeted by anti-aβ antibodies in the living brain came from experiments in nonhuman primates [464], genetically modified mice enabled the development of aβ-immunization as a strategy for the prevention or treatment of ad [465]. the application of longitudinal, in vivo-imaging studies in murine models has facilitated unique insights into the dynamics of aβ plaques and their cellular constituents (e.g., [323, 324, 466-469]), as well as the response of plaques and aβ-caa to therapeutic intervention [451, 452, 470]. currently, genetically modified nonhuman primates are being created with the hope that they will more completely recapitulate a human-like ad phenotype [471, 472], but no histopathologic findings have yet been reported. despite the limitation that genetically modified animals do not yet fully recapitulate ad, they will continue to play an important part in deciphering the pathobiology of aβ plaques. 11. conclusions: aβ plaques as a therapeutic objective aβ plaques are an obligatory component of the pathobiology of ad, and as such, strategies to reduce or neutralize plaques intersect with general strategies to prevent or treat ad. however, the value of plaques, in and of themselves, as therapeutic targets is uncertain. there is little question that aβ plaques, especially in their more elaborate states, are deleterious to brain tissue; they disrupt neuronal processes and synapses, they can be a source of harmful aβ-oligomers, and local glial cells create a toxic inflammatory environment. therapeutically targeting plaques thus presents both opportunity and obstacles. first, given the long, pre-symptomatic emergence and proliferation of aβ plaques (and neurofibrillary tangles) in the brain [109, 473], as well as evidence of extensive brain damage by the time dementia sets in, early prevention is likely to be the most effective strategy for subduing ad [474, 475]. the promise of prevention is underscored by the protective effects of the a673t mutation in app, which diminishes aβ production throughout life and lowers the risk of ad [48]. since the most effective preventive protocol should be initiated years, and possibly decades, before the predicted onset of dementia, testing for long-term safety and efficacy will be challenging. additionally, it is not known when, in the course of life, therapy must begin to effectively prevent or delay ad. second, it is possible that some, if not most, of the direct toxic influence of the aβ is mediated by oligomeric aβ rather than by fibrillar amyloid per se. evidence that aβ plaques can serve as a source of oligomers (section 1.1) argues that some benefit can be achieved by reducing plaque burden and thus the accompanying oligomers. it is encouraging that several of the more promising antibodies currently in clinical trials for ad show activity against oligomeric aβ [475, 476]. a recent study in mice indicates that the short-term neutralization of oligomeric aβ seeds early in life diminishes plaque formation as the animals age [477]. however, whether mitigating the production, seeding potential or toxicity of oligomeric aβ will be beneficial in humans, either as a preventive or as a treatment for discernible dementia, remains to be determined. it is also important to consider the possibility that treatments that block aβ-amyloid fibril assembly, or that disassemble plaques, might inadvertently increase the presence of toxic oligomers. third, the inability of anti-aβ immunotherapies to substantially impede dementia in symptomatic subjects, even when aβ plaques are reduced in number [336, 476, 478, 479], suggests that the dis-integration of the cerebral connectome caused by plaques and tangles is pronounced and largely irreversible once dementia commences [9, 474]. furthermore, tauopathy is an essential contributor to dementia that itself progresses by a prion-like mechanism [480, 481] that may be at least partly independent of aβ-proteopathy [336]. whether the alternative approach of lowering the inflammatory state associated with plaques will meaningfully improve behavior at this later stage of disease also has not been established. in short, once aβ-amyloid plaques and tauopathy become widespread, especially in neocortical regions [1], removing the plaques is unlikely to significantly reverse the course of dementia. even so, there is evidence that a reduction of tauopathy [336], and possibly some cognitive benefit, can be achieved in symptomatic patients by anti-aβ immunotherapy [476, 482-484]. indeed, active immunization with an1792, which targets both aβ plaques and oligomers, resulted in a long-term decrease in all components of the plaques aggregated aβ, aberrant neurites, tauopathy, and focal gliopathy along with improved indices of ‘neuronal health’ [336]. thus, notwithstanding the mostly disheartening outcome of therapeutic trials to date, current preclinical and clinical data indicate that the right anti-aβ treatment, at the right time, has a good chance of delaying or preventing ad. finally, it is imperative to remain vigilant to the impact of environmental factors and the microbiome [485, 486] on the risk of developing ad. for instance, if specific microbes are convincingly found to increase the risk of plaque formation and ad, early immunization against this infectious agent could be an effective preventive measure. it is important also to consider the possibility that interactions among genetic, microbiomic and/or environmental influences could raise disease risk well above the additive impact of individual risk factors. the search for disease-modifying therapies for ad is a broad and rapidly evolving endeavor that has been extensively reviewed (see, e.g., [9, 33, 476, 479, 487-491]. in addition to small molecules, we have entered a phase in medicine in which biologics such as antibodies [492, 493] and nucleic acid-based agents [494, 495] have unprecedented potential to treat neurodegenerative diseases. for well over a century, aβ plaques have been recognized as an important correlate of dementia in the aging brain. revealing the mechanisms by which plaques arise, proliferate, and interact with molecular and cellular elements in the nervous system will continue to yield insights into both the ontogeny and treatment of ad. 12. methods tissue samples were collected from human subjects with end-stage ad (figures 1-6, 8-20) and from aged nonhuman species with cerebral aβ deposition (figures 21-23). postmortem collection of samples by the emory university goizueta alzheimer’s disease research center brain bank was approved by the emory institutional review board. tissues from mice and monkeys were collected at emory’s yerkes national primate research center in accordance with federal and institutional guidelines for the humane care and use of experimental animals. the yerkes center is fully accredited by aaalac international. 12.1 immunohistochemistry for light-microscopy, tissue blocks were embedded, sectioned at 8-10 μm thickness, and mounted onto glass slides for staining. the following antibodies were used for immunohistochemistry: 4g8, mouse monoclonal antibody from covance (princeton, nj) raised against residues 17-24 of aβ peptide, with an epitope at residues 18-22 [496]; 6e10, mouse monoclonal antibody from covance raised against residues 1-16 of the aβ peptide, with an epitope at residues 3-8 [496]; rabbit polyclonal antibodies r361 and r398, kindly provided by dr. pankaj mehta (institute for basic research on developmental disabilities, staten island, ny), were raised against synthetic aβ32-40 and aβ33-42, respectively [497]; 82e1, mouse monoclonal antibody raised against residues 1-16 of synthetic aβ [498], from ibl (gunma, japan); cp13, mouse monoclonal antibody kindly provided by dr. peter davies (feinstein institutes for medical research, manhasset, ny), was raised against a synthetic peptide representing the region around phosphorylated serine residue 202 on the tau protein [499]; mc1, mouse monoclonal antibody, also from dr. davies, was raised against alz50-immunopurified paired helical filaments and then epitope-mapped to similar conformation-specific regions as alz50 [500]; anti-gfap, purified immunoglobulin fraction of rabbit antiserum from dako (carpinteria, ca) (catalog no. z0334), raised against gfap isolated from cow spinal cord and purified by solid-phase absorption with human and cow serum proteins; anti-iba1, rabbit polyclonal antibody from wako (osaka, japan), raised against a synthetic peptide corresponding to the c-terminus of ionized calcium-binding adapter molecule 1 (iba1), a 17-kda protein that is specifically expressed in macrophages/microglia and is upregulated during the activation of these cells [501, 502]; smi-31, mouse monoclonal antibody raised against a phosphorylated epitope on the neurofilament heavy subunit (nf-h) [503] from biolegend (san diego, ca); and 06-17, mouse monoclonal antibody to a phosphorylated epitope shared by the heavy and medium kda neurofilament polypeptides (generous gift of drs. ludwig and nancy sternberger, university of maryland, baltimore) [504, 505]. vectastain elite kits (vector laboratories, burlingame, ca) were used for abc-based immunodetection of antigen-antibody complexes according to the manufacturer’s instructions, with diaminobenzidine (dab) as coloring agent for images in figures 1, 2, 5, 6, 8-11, 14, 16, 17, 19, 21, & 22. in most cases, a nissl counterstain was applied after immunostaining, as noted. for dual fluorescence immunostaining (figure 15), the section was incubated in mouse monoclonal antibody cp13 (diluted in 2% normal goat serum) overnight at 4°c, rinsed, and then incubated for 90 minutes in cy2-conjugated anti-mouse secondary antibody (green; jackson labs, west grove, pa). the section was rinsed thoroughly, incubated overnight in diluted rabbit polyclonal antibody r398 at 4°c, rinsed, and placed for 90 minutes in rhodamine-red-x goat anti-rabbit secondary antibody (jackson labs). for dual immunostaining by standard transillumination light microscopy (figure 3), antibodies were sequentially applied as described above except that the polyclonal anti-aβ antibodies r398+r361 were colored with dab (brown), and the anti-tau monoclonal antibody cp13 was colored with vip (purple; vector laboratories). non-immune mouse igg or rabbit sera were used in place of the primary antibodies as negative controls. tissues shown in figures 1 (left) and 4 were stained with the naoumenko-feigin silver stain [506] followed by a periodic acid-schiff (pas) counterstain. figure 23 was stained with the campbell-gallyas silver stain [507]. light-microscopic photomicrographs were taken with a leica dmlb or dmls microscope (wetzlar, germany) and a spot flex (diagnostic instruments, sterling heights, mi) or moticam 5+ (motic, hong kong) digital camera. 12.2 electron microscopy for conventional ultrastructural analysis (figures 12, 13, 18, 20), small blocks of cortex were sub-dissected from larger, autopsy-derived tissue blocks that had been immersion-fixed in 10% neutral buffered formalin. the tissue samples were washed in phosphate buffer (0.1m, ph 7.4) and immersed in osmium tetroxide (1% in phosphate buffer) for 20 minutes. they were then rinsed in phosphate buffer and dehydrated in a graded series of ethanol and propylene oxide. uranyl acetate (1%) was added to the 70% ethanol (35 minute immersion) to improve contrast in the electron microscope. the sections were then embedded in epoxy resin (durcupan acm; fluka, ft. washington, pa) on microscope slides and heated for 48 hours at 60°c. areas of interest were selected, excised from the slide and glued onto resin blocks. ultrathin sections were cut with a leica ultracut t2 (nussloch, germany), collected onto single-slot copper grids, and stained with lead citrate. for immunogold em (figure 11, right), sections were preincubated in pbs containing 5% nonfat dry milk and then washed in tris-buffered saline (tbs)-gelatin buffer (0.02 m tris, 0.15 m nacl, 1 μl/ml fish gelatin, ph 7.6) to block nonspecific sites. sections were then incubated for 48 hours at 4°c with antibody 4g8 diluted in pbs-bsa, rinsed in tbs-gelatin, and incubated for 2 hours at room temperature in gold-conjugated goat anti-mouse fab’ fragments (dilution 1:100; nanogold [nanoprobes inc., yaphank, ny]). gold particles were silver-enhanced with the hq silver kit (nanoprobes). the tissue was then embedded and cut as described above. thin sections were examined with a zeiss em10-c electron microscope (oberkochen, germany) and digital images were captured using a dual view camera (gatan inc., pleasanton, ca). acknowledgements i gratefully acknowledge enlightening discussions with mathias jucker, dietmar thal, marla gearing, harry levine, rebecca rosen, amaryllis cintron, eric heuer, david lynn, yury chernoff, rolf warzok, silke vogelgesang, sanjeev gumber, linda cork, allan levey and james lah. hailian xiao, jean-françois paré, and jeromy dooyema provided expert technical assistance. portions of this work were supported by the metlife foundation, cart foundation, alexander von humboldt 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are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. neurooncology: 2023 update feel free to add comments by clicking these icons on the sidebar free neuropathology 4:4 (2023) review neurooncology: 2023 update michel mittelbronn1-6 national center of pathology (ncp), laboratoire national de santé (lns), dudelange, luxembourg luxembourg center of neuropathology (lcnp), luxembourg department of oncology (donc), luxembourg institute of health (lih), luxembourg, luxembourg department of life sciences and medicine, university of luxembourg, esch sur alzette, luxembourg luxembourg centre for systems biomedicine (lcsb), university of luxembourg, esch-sur-alzette, luxembourg faculty of science, technology and medicine (fstm), university of luxembourg, esch-sur-alzette, luxembourg corresponding author: michel mittelbronn · national center of pathology (ncp) and luxembourg center of neuropathology (lcnp) · laboratoire national de santé (lns) · 1, rue louis rech · 3555 dudelange · luxembourg · luxembourg michel.mittelbronn@lns.etat.lu submitted: 12 february 2023 accepted: 14 march 2023 copyedited by: vanessa goodwill published: 20 march 2023 https://doi.org/10.17879/freeneuropathology-2023-4692 keywords: neuro-oncology, neuropathology, brain tumors, glioblastoma, brain metastasis, meningioma abstract this article presents some of the author’s neuropathological highlights in the field on neuro-oncology research encountered in 2022. major advances were made in the development of more precise, faster, easier, less invasive and unbiased diagnostic tools ranging from immunohistochemical prediction of 1p/19q loss in diffuse glioma, methylation analyses in csf samples, molecular profiling for cns lymphoma, proteomic analyses of recurrent glioblastoma, integrated molecular diagnostics for better stratification in meningioma, intraoperative profiling making use of raman effect or methylation analysis, to finally, the assessment of histological slides by means of machine learning for the prediction of molecular tumor features. in addition, as the discovery of a new tumor entity may also be a highlight for the neuropathology community, the newly described high-grade glioma with pleomorphic and pseudopapillary features (hpap) has been selected for this article. regarding new innovative treatment approaches, a drug screening platform for brain metastasis is presented. although diagnostic speed and precision is steadily increasing, clinical prognosis for patients with malignant tumors affecting the nervous system remains largely unchanged over the last decade, therefore future neuro-oncological research focus should be put on how the amazing developments presented in this article can be more sustainably applied to positively impact patient prognosis. introduction for this top ten series regarding the neuro-oncological highlights of the year 2022, the author aimed at selecting pioneering work with a direct link to neuropathology. apart from previously read papers, the author was inspired by discussions with colleagues and finally screened hundreds of articles limited to the year 2022 in pubmed. in addition, the author “googled” by using the key words “neurooncology” or “neuropathology” in combination with different tumor entity names or laboratory techniques, as it has been shown that for some questions in the biomedical field, google scholar search may help to retrieve up to twice more relevant articles than a classic pubmed search or information that is otherwise almost unretrievable [1, 2]. apart from a high number of pure clinical or neuroradiological papers or case reports that did not directly touch the field of neuropathology and were therefore excluded from this highlight list, the year 2022 can (maybe once again) be summarized under the motto “profiling, profiling, profiling” frequently in combination with machine learning approaches to increase diagnostic velocity and precision. on the other hand, research work with promising treatment approaches making use of the new molecular data was relatively poor being in line with an almost unchanged prognosis for the majority of patients with malignant brain tumors. certainly, more time is needed to decipher precisely which of these multitudes of new potential therapeutic targets, that nowadays can be detected almost in real-time, will make it into future clinical application. for single topics, the author co-selected two studies due to their similarity or complementarity. the author’s “charts of 2022” in neurooncology reads as follows (not meant in a hierarchical manner but rather in a progression from neuropathological core work to early clinical application): new brain tumor entity: high-grade glioma with pleomorphic and pseudopapillary features (hpap) [3]. prediction of 1p/19q status by immunohistochemistry in idh-mutant glioma [4]. prediction of molecular tumor features from histology by machine learning [5]. histomolecular grading for meningioma [6]. molecular profiling of cns lymphoma [7]. proteomic landscape of primary and recurrent glioblastoma [8]. methylome-based brain tumor diagnostics in csf liquid biopsies [9]. intraoperative diagnostics for brain tumors based on raman effect [10, 11]. intraoperative molecular tumor profiling [12, 13]. drug screening platform for brain metastasis [14]. the author invites you on a joint journey across some of the most recent neurooncological developments touching the field of neuropathology. of course, this selection is highly subjective, however, the author aimed at selecting very diverse studies from different neurooncological subfields so that the majority of the readership of free neuropathology may at least partly be able to share the author’s enthusiasm about the presented studies. 1. new brain tumor entity: high-grade glioma with pleomorphic and pseudopapillary features (hpap) [3] even after the neuropathological community has molecularly profiled a huge amount of brain tumor cases worldwide, while also mainly using the same brain tumor methylation classifier, we still struggle from time to time with providing precise diagnoses for particular cerebral tumors. pratt et al. made use of the brain tumor methylation classifier [https://www.molecularneuropathology.org/mnp based on 15] combined with a next generation sequencing approach using both an amplicon-based brain tumor specific panel [16], as well as the commercial trusight oncology 500 panel (tso 500, illumina, san diego, usa) [3]. with this approach, the authors were able to classify 31 previously non-classifiable brain tumors (median patient age was 46.5 years), mainly but not exclusively localized in cortical areas, with highly diverse morphological features, including those of pleomorphic xanthoastrocytoma (pxa), astroblastoma, ependymoma, polymorphous neuroepithelial tumor of the young (plnty), as well as idh wildtype glioblastoma. one important morphological feature was the mainly non-infiltrative growth pattern observed in hpap. in the brain methylation classifier, these brain tumors did not cluster with any of the known methylation classes in v11.4, however were closest to pxa, mn1-altered astroblastoma and plnty. apart from several frequently encountered mutations in these brain tumors, such as tp53, rb1, nf1 or less frequently nf2 or braf-v600e expression, a frequent chromosomal aneuploidy was observed, with a very high rate of a monosomy 13, detected in 28 of 31 cases analyzed. of note, mgmt promoter methylation was only detected in 1 patient, and a cdkn2a/b deletion in 2 patients of this cohort. as a working entity name, the authors suggested “high-grade glioma with pleomorphic and pseudopapillary features (hpap)”. the authors proposed 2 molecular subtypes (a and b) without any specific associated genetic or morphological features being associated with those subclasses, however significantly older patients in subtype a as compared to b. although some of the histologically described morphological features rather indicated a high-grade glioma, the overall survival was not significantly different from pilocytic astrocytoma, pxa and high-grade astrocytoma with piloid features, however, was significantly better than the group of idh wildtype glioblastomas, therefore, molecularly suggesting a biologically less malignant tumor entity. as the histological picture is highly variable and patient survival not impaired by morphological features of atypia or anaplasia, the authors did not provide definite diagnostic criteria, however, recommended to base diagnosis on the following features: a) no match with a known brain tumor methylation class in the v11.4 (or more recently also v12.5) classifier, b) monosomy of chromosome 13 and c) lack of cdkn2a/b loss. given the mutational constellation (e.g. braf or rb1 mutations), some of the hpap patients might be eligible for targeted treatment approaches (for comprehensive summary of the hpap characteristics, please see table 1). table 1: histopathological and molecular features of high-grade glioma with pleomorphic and pseudopapillary features (hpap) histological features very heterogenous, mainly non-infiltrative comprising features of pleomorphic xanthoastrocytoma (pxa), astroblastoma, anaplastic ependymoma, polymorphous neuroepithelial tumor of the young (plnty) and glioblastoma. methylation classifier (v11.4 and v12.5)* no match mutations tp53 (57%), rb1 (26%), nf1 (26%), nf2 (14%), braf-v600e (12%), cdkn2a/b (6%) mgmt promoter methylation mainly unmethylated (97%) chromosomal alterations frequent aneuploidy, loss of chromosome 13 (90%), otherwise losses of chr. 3, 6, 10-15, 17, 18, 22; gains of chr. 4q, 5, 7, 19 *v11.4 was indicated by the authors, however, also the most recent brain tumor methylation classifier version v12.5 does not yet recognize this tumor entity. 2. prediction of 1p/19q status by immunohistochemistry in idh-mutant glioma [4] nowadays, comprehensive molecular profiling is equal or (for some tumor types) ever superior to histopathology, the previous gold standard. however, broader molecular testing is far from being available in all health care systems across the world. therefore, it is not only important to promote cutting-edge technology, but also to come up with simple, fast and cheap analytic tools that can be applied in all types of socio-economic systems. felix et al. started with a mass spectrometric approach to evaluate the most differentially expressed proteins in a series of more than 100 frozen and formalin-fixed, paraffin-embedded diffuse glioma samples with known idh and 1p/19q status [4]. out of a larger set of differentially regulated candidate proteins that could have been used as targets for subsequent immunohistochemical (ihc) analyses, the authors selected the ones for which best staining results were obtained, namely vimentin for non-1p/19q-codeleted astrocytomas and hip1r (huntingtin interacting protein 1 related) for 1p/19q-codeleted oligodendrogliomas. with this simple immunohistochemistry-based approach, the authors were able to predict 1p/19q status in diffuse gliomas with high accuracy (figure 1). this approach could therefore also be applied in laboratories without state-of-the art molecular pathological pipelines or in less developed countries without broad accessibility to modern molecular testing methods. figure 1: algorithm for a 2-step immunohistochemical prediction of the 1p/19q status in idh-mutant gliomas (positivity of tumor cells is evaluated). for each staining, four different expression levels were defined. hip1r staining was considered positive if expression was observed in the cytoplasm or at the cell membrane. scores h0 to h3 were defined as follows: h0: less than 10% of tumor cells are weakly positive; h1: 10%-75% of tumor cells are positive, however, less than 30% are strongly positive; h2: more than 50% of the tumor cells are positive, and more than 30% are strongly positive, however, still showing unstained spaces between the tumor cells. the score h2 is also given if the tumor was only moderately positive, however, approximately 100% of the cells showed moderate immunoreactivity in a densely packed manner; h3: approximately 100% of the tumor cells are strongly positive in a diffuse and densely packed manner, with only blood vessels being spared. for the assessment of vimentin (scores v0 to v3 were given), staining of blood vessels was excluded, however, no distinction could be made between reactive and neoplastic glial cells. v0: less than 10% of tumor cells are positive (however, blood vessels and reactive astrocytes could be positive); v1: 10%-20% of tumor cells or tumor cell processes are positive; v2: 20%-70% of the tumor cells with many tumor cell processes are positively labeled, however still 30% or more of the tumor cells are negative; v3: more than 70% of the tumor cells are strongly positive, thereby the tumors exhibit a strong diffuse staining pattern. after this individual assessment, the comparison between hip1r and vimentin staining was done as shown in figure 1. 3. prediction of molecular tumor features from histology by machine learning [5] while molecular analyses in neuropathology are rapidly progressing with regard to speed of analysis and precision of unbiased diagnosis, being always up to a perfect state-of-the-art level cannot currently be guaranteed worldwide. therefore, machine-learning-based analyses requiring a good microscopic detection system together with powerful computers could be an alternative if prediction would be highly precise. the search for good prediction of molecular properties of tumors from histological images is currently a rapidly progressing research field. liechty et al. aimed for predicting idh mutation from histological slides [5]. the authors compared the prediction of a machine learning (ml) system with the prediction of expert neuropathologists’ examination, however, also analyzed if a synergistic approach of ml together with the neuropathological assessment could further enhance the predictive power. for this purpose, they used 801 idh mutant and wildtype gliomas from the tcga database, which were split into a training and a validation set (tcga research network: https://www.cancer.gov/tcga). afterwards, an institutional cohort of 174 idh mutant and wildtype gliomas was assessed. the neuropathologists were allowed to provide a semiquantitative score between 0 and 1 (with 0.5 for lowest certainty of tumor being either idh mutant or wildtype), while convolutional neural network (cnn) analyses were applied in ml assessments. no ml approach performed better than the neuropathologists in this task, however, reached similarly good results. creating an average score of the assessment of two neuropathologists was superior than the prediction of each individual neuropathologist alone. the combination of the prediction of one neuropathologist together with the ml assessment further increased correct prediction as compared to ml or a neuropathologist alone. of note, the hybrid model of ml and one neuropathologist was similarly precise as compared to the combined score derived from two neuropathologists. within this relatively restricted cohort of gliomas, ml approaches were able to perform similarly well as trained neuropathologists. taking into account that the training of a neuropathologist takes several years while ml-based analyses only took a very short time to train; these findings reflect the high potential of ml approaches in diagnostic pathology including neuropathology. this is all the more promising as initial ml training and validation has been performed on an external image set in which potential differences with regard to histology, for example in slide thickness or staining intensities, could have led to improper recognition of histomorphological features. therefore, computer-assisted diagnostic procedures could be a way to improve diagnostic precision, especially for smaller hospitals or pathological institutes where review by two neuropathologists may not be feasible. one should still consider the current result with caution, however, as still both ml and neuropathologists mixed up idh wildtype and mutant glioma to a similar extent, clearly indicating limits of predicting molecular features by means of histological analyses only. this task may even be more challenging in small round blue cell tumors or sarcomas, tumor types in which morphological features often correlate even less with the final integrated molecular diagnoses. 4. histomolecular grading for meningioma [6] besides the classic grading of brain tumors according to the who classification of central nervous system (cns) tumors, several multicentric studies aimed to more precisely stratify brain tumor patients with regard to their risk of recurrence. in a previous study, it was shown that combining histomorphological assessment of meningioma with methylation analyses led to inverted recurrence risk in some cases, namely that some meningiomas classified as who grade i (now cns who grade 1) displayed a higher recurrence risk when applying the meningioma methylation classifier, as compared to cases initially diagnosed as who grade 2 tumors according to histomorphological criteria [17]. along this line, driver et al. aimed at providing a model for more precise prediction of the clinical behavior of meningiomas going beyond the classic histological grading system [6]. this paper has already been briefly presented in the “neurooncology: 2022 update” series, however, given the importance of the study and the urgent need for an alignment between different grading schemes, the author of this year’s edition would like to stress the study in more detail again [18]. the authors analyzed a large variety of clinical (gross total or subtotal resection assessed by mri analyses, progression-free survival), histomorphological (including who grade, features of atypia, proliferation and mitotic index) and molecular features (copy number variation, as well as partially targeted mutational profiling and methylation analyses). by correlating all those features with progression-free survival of meningioma patients, the authors finally proposed a new 3-tiered grading scheme taking into account the mitotic count, loss of distinct chromosomal arms or entire chromosomes, or loss of cdkn2a/b. a maximum of 11 points could be obtained (9 for high-molecular risk alterations and 2 for mitotic count), leading to a) grade 1 for 0-1 point, b) grade 2 for 2-3 points and c) grade 3 if 4 or more points were obtained (figure 2). with this approach, more than 30% of tumors were reclassified as compared to the previously applied who grade. of note, in meningiomas with an integrated tumor score of 3, recurrences could not even be significantly delayed by a gross total resection, which is otherwise a positive factor for longer progression free survival intervals. to avoid confusion between different institutional grading schemes in the future [see also a competing approach here: 19], the who should bring different multicenter study groups together and refine the current grading schemes, as the high discrepancies between histomorphological assessment and different integrated scores with regard to patient prognosis has the potential to have a considerably negative impact on patient care. this seems to be all the more important as – in contrast to other chapters in the current who classification – the meningioma chapter is still relatively defensive with regard to molecular markers impacting patient prognosis. figure 2: algorithm for integrated grading for recurrence risk prediction in meningioma (mitotic index defined as mitoses per 10 high-power fields). 5. molecular profiling of cns lymphoma [7] primary diffuse large b-cell lymphomas of the cns (cns-dlbcl) are the most frequent primary cns tumors of haematolymphoid origin sharing multiple morphological and molecular features with both its systemic non-cns and/or secondary cns counterparts [20]. as the origin of cns-dlbcl is still not entirely clear and also some of the very recent, promising molecular tools such as dna methylation-based analyses could not fully distinguish cns-dlbcl from its non-cns counterpart, a deeper understanding of the molecular pedigree of cns-dlbcl is urgently needed. radke et al. attended to this challenge by studying 51 cns-dlbcl using transcriptomic and whole genomic analyses and comparing this data set to one derived from 75 lymphomas originating from outside the cns, the latter including both follicular and dlbc lymphomas [7]. the study could confirm myd88 (l265p) and cd79b mutations, as well as frequently biallelic cdkn2a loss as very early tumor drivers in cns-dlbcl. other frequently encountered alterations comprised activating mutations of bcr signaling and structural variants of igh, igl and igk as well as losses of chromosome 6p. notably the alteration of chromosome 6p where the human leukocyte antigen (hla) complex is located may lead to an immune escape from cytotoxic t cells due to limited neoantigen presentation [21]. in addition to their findings on dna level, radke et al. could also provide a robust transcriptional signature obtained by rna sequencing that was able to distinguish cns-dlbcl from its non-cns counterparts. they detected higher rna expression levels of loci associated with aberrant somatic hypermutation phenotypes, although it still remains to be determined what is chicken and what is egg in this association. the cns-dlbcl group also displayed a higher tert expression as compared to the group of non-cns lymphomas, however, without being associated with increased telomere content. of note, ebv-positive cns-dlbcl did not share many of the classic mutational hotspots apart from igh and hla-drb locus. it remains to be determined how this important data can be transferred into the daily diagnostic routine for a fast and precise differentiation between a primary cns-dlbcl, a secondary dlbcl of the cns, or primary non-cns dlbcl. this would be all the more important as depending on the method – if immunohistochemical phenotyping or gene expressing profiling is applied – cns-dlbcl may either be classified as of non-germinal center b-cell like (non-gcb) or late germinal-center exit b-cell origin. table 2: molecular differences between cns diffuse large b-cell lymphoma (dlbcl) and its non-cns counterpart. molecular feature non-cns dlbcl primary cns dlbcl myd88 (l265p) mutation rare frequent cd79b mutation rare frequent biallelic loss of cdkn2a rare frequent number of single nucleotide variants (snv) lower higher number of insertions or deletions (indel) lower higher expression of ig constant genes higher lower expression of ighm lower higher tert expression lower higher 6. proteomic landscape of primary and recurrent glioblastoma [8] while many current brain tumor studies analyze molecular changes at genomic, epigenomic or transcriptomic levels, large-scale proteomic approaches are largely underrepresented. as malignant primary and secondary brain tumors display a detrimental prognosis despite maximum treatment, detection of potential treatment targets at protein level is key for innovative future treatment approaches. this is especially important for clinical situations in which patients suffer from recurrent tumors with potentially new tumor-related pathways that might be newly activated in the context of treatment resistance. buehler et al. address this topic by performing proteomic profiling of primary and recurrent glioblastomas deriving from 42 patients of two different clinical cohorts [8]. of note, the applied proteomic analysis is equally suitable for frozen and formalin-fixed, paraffin-embedded (ffpe) tissues, the latter facilitating its applicability in diagnostic pathology [22]. in general, the proteomes of the initial glioblastomas as compared to their recurrent counterparts were highly similar, however, several proteins were significantly enriched in recurrences, including, among others, brain enriched myelin associated protein 1 (bcas1), inverted formin 2 (inf2), calcium-calmodulin-dependent protein kinase 2 (camk2), phosphoribosyl pyrophosphate synthetase 2 (prps2) and f-box only protein 2 (fbxo2). these results show that the method is able to detect tumor progression-associated changes in ffpe tissue. the authors proceeded in validating an overexpression for bcas2, inf2 and fbxo2 in recurrent glioblastoma by means of immunohistochemistry and rna sequencing. regarding the clinical impact of those findings, significantly lower expression levels for inf2 and fbxo2 were found in glioblastoma patients with longer survival rates, therefore indicating that both factors might be implicated in tumor progression or treatment-resistance mechanisms. using a murine approach, the authors could validate the survival benefit in fbxo2 knockout conditions, as well as showing a reduced tumor cell growth and invasive behavior in organotypic brain tumor slice cultures. while the authors focus more on addressing the role of fbxo2 activation as a driver for glioblastoma progression and recurrence at the end of their paper, i would rather stress the potential of the method for personalized medicine approaches in the future. taking into account a) the horrible clinical prognosis of glioblastoma patients, b) the poor treatment options at the time of glioblastoma recurrence and c) that data derived from large-scale dna or rna analyses do not unequivocally predict the functional proteomic landscape, deciphering active tumor-related pathways by proteomic approaches in a close to routine pathological setting could be a great asset especially for recurrent tumors as compared to their primary counterparts. it would be of interest to run proteomic analyses much more frequently in parallel to large-scale genetic screenings, which could help to support decision making in molecular tumor boards. 7. methylome-based brain tumor diagnostics in csf liquid biopsies [9] since neurosurgical interventions are among the most invasive medical procedures, the threshold to perform a brain biopsy is high. avoiding biopsies for brain tumor types in which no subsequent resection would be performed (e.g. lymphomas) or that would significantly benefit from maximum resection would be highly desirable. with their approach to diagnose brain tumors by methylation analyses from cerebrospinal fluid (csf) samples, zuccato et al. pave the way for a less invasive, still highly reliable liquid-based brain tumor classification [9]. in this pioneering approach necessitating only very small amounts of csf, the authors analyzed 57 brain metastases, glioblastomas and central nervous system lymphomas. a largely similar amount of cell-free dna was obtained from csf samples of all 3 tumor classes. the methylation profile significantly correlated between csf and tumor samples in all 3 tumor entities. furthermore, the methylation analyses could convincingly distinguish between brain metastases, glioblastoma and cns lymphoma. the methylation profiles of cns lymphomas were highly discriminatory. therefore, perioperative morbidity and mortality could be considerably reduced in the future if such a csf based methylation approach would be implemented in the routine diagnostic set-up before the final decision to perform gross resection is made. the prediction from methylation data in the present study was superior as compared to previously published data using plasma samples, which could be related to the closer proximity of brain tumors to the sampled csf and the distribution of cell-free dna in less volume with less liquid turnover as compared to plasma samples [23, 24]. in addition to the very promising findings regarding initial diagnostics of a primary tumor, this approach has also a high potential to track treatment responses or detect recurrent tumor cell growth. the strong correlation between primary tumors and csf samples might also be useful to differentiate a primary brain tumor from metastasis in patients suffering from different cancer entities at the same time. 8. intraoperative diagnostics for brain tumors based on raman effect [10, 11] intraoperative brain tumor diagnostics necessitate an availability of experienced neuropathologists, however, multiple smaller centers have only one or none of this rather rare “species”. furthermore, diagnostic quality and precision may be suboptimal and frequently show less than 70% complete concordance with final neuropathological diagnoses [25]. therefore, the central intraoperative questions are rather if the neurosurgeon correctly hit the lesion or if enough material was obtained for further microscopic and molecular diagnostics. taking all those arguments into account, there is an obvious need for faster, more unbiased, less work intensive and, ideally, automated intraoperative diagnostic procedures. it was shown that label-free, stimulated raman based histology (srh) assessed by machine learning algorithms was able to provide an intraoperative diagnosis almost in real-time without showing less diagnostic precision as compared to experienced pathologists [26]. this method is based on the raman scattering effect, namely, that the energy level of light can be altered upon interaction with molecular bonds, changes that can be visualized spectrophotometrically only [for comprehensive overview, please see 27]. when this method iscombined with a microscopic device, a spatial resolution between 0.5 and 1µm is achieved. reinecke et al. applied srh on a larger series of intraoperative neuro-oncological cases and created an automated diagnostic pipeline based on convolutional neural network analyses, revealing a high diagnostic accuracy as compared to an independently performed neuropathological assessment [11]. furthermore, a high consistency between randomly chosen areas on tumor samples was observed. taking into account that decision from this automated diagnostic pipeline took less than 5 minutes in total, this approach seems to be highly promising to replace intraoperative neuropathological intervention in the medium term. as especially pediatric brain tumors are nowadays much more specifically classified according to molecular pathological markers, microscopic diagnostics during surgery is frequently of limited value if intraoperative statement is, for example, “small round blue cell tumor” or “suspicion of low-grade glioma”. if a fast unbiased intraoperative method would be able to distinguish normal from tumor tissue and high from lower grade brain tumors in addition to a good estimation of the tumor cell content, this would considerably decrease the need for neuropathology expertise during the surgical procedure. in particular, the presence or absence of tumoris strongly needed information during the surgical procedure, as the extent of resection is one the most important predictors for progression free survival in pediatric brain tumors [28] jabarkheel et al. investigated the suitability of raman spectroscopy for pediatric brain tumor cases [10]. with their approach, jabarkheel et al. were able to correctly distinguish tumor tissue from normal tissue in 89.9%; furthermore, the more complicated task of differentiating low-grade tumors from normal brain tissue was mastered in 86.2%. although this development may impact neuropathology as a gold standard in intraoperative brain tumor diagnostics (figure 3), neuropathologists are still key for the further development of these approaches as well-functioning machine learning pipelines need an excellent prior histological and molecular annotation. figure 3: potential intraoperative applications of raman effect-based techniques combined with machine learning tools. raman spectroscopy is a label-free, non-disruptive method based on an inelastic scattering effect of photons allowing for the quantitative molecular assessment of biological samples resulting in different spectra. stimulated raman histology (srh) is a variant of raman spectroscopy allowing for the creation of hematoxylin and eosin (he) slide mimics based on quantification of distinct molecules (mainly lipids, proteins and nucleic acids). 9. intraoperative molecular tumor profiling [12, 13] molecular diagnostics becomes more and more important not only for precise patient stratification but also for targeted therapy strategies [29]. especially in the field of cns neoplasms, some entities can nowadays only be unequivocally diagnosed by methylation profiling [20]. along this line, technical developments and applications related to molecular tumor testing need to take into account diagnostic precision, speed and costs. as the method of the year 2022, nature methods has chosen long-read sequencing, with single molecule real-time (smrt) from pacific biosciences and nanopore sequencing (nps) from oxford nanopore technologies being the most frequently used commercially available applications [30]. while accuracy seems to be only slightly superior in smrt as compared to nps, the latter allows for longer average read lengths (approximately 100kb for ultra-long reads) as compared to smrt. it is therefore no surprise that those techniques could also have an impact on diagnostic strategies in neuropathological tumor diagnostics. using nanopore sequencing techniques, patel et al. introduce a diagnostic pipeline entitled rapid-cns2 allowing for testing copy-number, mutational and methylation profiling in parallel [13]. with this approach, considerably simplified library preparations at lower costs are possible with highly similar diagnostic precision as compared to standard methods (figure 4). discrepancies in isolated cases were related to a low tumor cell content, therefore, a careful standard microscopic neuropathological assessment is mandatory during the workflow. a pure sequencing time of 24 hours was sufficient to conclude cases with a comprehensive neuropathological diagnosis, however, this pipeline is currently restricted in use for cryo-preserved tissue only. in a similar nanopore sequencing approach, however, focusing on methylation profiling without having the full molecular profile for important selected mutations, kuschel et al. performed a combined retroand prospective study arriving at a total turnaround time of only 21 hours from sample reception to report of the diagnoses [12]. of note, as few as 1000 randomly analyzed cpgs were sufficient for highly meaningful methylation classification. multiple initiatives are ongoing, pushing even further towards direct intraoperative molecular tumor profiling [31]. as final treatment decisions according to standard treatment guidelines are often made only several days after the operation in multidisciplinary tumor boards, one could question the necessity of speeding up intraoperative diagnostic assessments. however, as multiple innovative, targeted anti-tumor treatment approaches such as the application of t cells harboring chimeric antigen receptors (car-t cells) or oncolytic viruses would necessitate a follow-up neurosurgical intervention, e.g. for the placement of a catheter after the exact molecular definition of potential targets, intraoperative tumor diagnostics could overcome those obstacles. figure 4: scheme for fast, precise and unbiased molecular diagnostics using nanopore sequencing, suitable for mutational, transcriptome and methylome analyses. 10. drug screening platform for brain metastasis [14] brain metastases constitute the group of the most frequent and malignant brain tumors that may affect around 20-25% of all cancer patients and are usually associated with a very poor prognosis [32, 33]. although brain metastases comprise a very heterogenous group regarding their cellular origin, the site of metastasis as well as the number of metastatic spots, in many clinical conditions no entityor target-specific treatment is available. furthermore, even though both primary and secondary tumors are more and more profiled by means of molecular diagnostics, potential molecular targets might be no longer successfully druggable as the development of resistance mechanisms may negatively impact targeted treatment. to overcome these drawbacks, drug screening platforms are emerging that may allow for deciphering the best drugs specifically applicable for individual brain metastasis in a preclinical or clinical setting (figure 5). zhu et al. developed a murine model giving rise to brain metastasis upon tumor cell injection in the left heart ventricle [14]. from those animals, organotypic slice cultures of brain metastases were performed and treated with a drug-library consisting of 114 drugs, that were either fda-approved or are currently in use in clinical trials. the use of slice culture has the advantage that tumor cells can grow in their associated microenvironment which is key for both the metastasizing process, but may also play an important role in treatment resistance. with this so-called medium-throughput drug-screening platform (metplatform), they were able to identify a blood-barrier permeable hsp90 inhibitor as a promising drug for the treatment of brain metastases. in a follow-up experiment, the authors successfully treated patient-derived organotypic cultures deriving from various different cancer types. while the paper was focusing more on elucidating the underlying mechanisms of hsp90 inhibition in brain metastasis treatment, targeting hsp90 will certainly once again not be suitable as a “one fits all” in future brain metastasis treatment. what could be much more important in the future, is to make use of metplatform or other similar drug-testing pipelines as a clinical “avatar”, as the authors called their approach. with this, medium or even high-throughput drug libraries could be applied on either primary tumor slices or tumor-derived organoids with the goal to individually decipher the most promising treatment strategies for this most detrimental clinical situation. although slice culture-based approaches seem to excellently reflect the microenvironmental composition of the tumor in situ, the high variability between slices as well as the potentially limited availability of consecutive slices may negatively impact a homogenous readout when using larger drug libraries. this potential drawback could be potentially overcome by creating ex-vivo spheroids as an alternative “avatar”, that have already shown their clinical suitability in single patients with metastatic tumors [34]. figure 5: potential implementation of personalized drug-screening approaches in the clinical setting. discussion this overview regarding key developments of the year 2022 in the field of neuro-oncology (with a link to neuropathology) clearly shows that scientific findings are very closely linked to technical developments. nanopore sequencing has been proposed as one of the methods of the year 2022 [30] by nature methods as it allows for faster, unbiased, precise diagnostics having the advantage of assessing mutations, transcriptome and methylome by using only one device at relatively low costs. it is, therefore, no wonder that multiple initiatives have started to apply such techniques seeing a potential to provide intraoperative molecular diagnostics [12, 13]. in some clinical disciplines such as gynecological oncology [35], more and more molecular tests are commercialized in a highly protected manner, especially if it comes to prognostic prediction for distinct treatment regimens. in contrast, the neuropathology community – at least until now – has managed to keep complex diagnostic tests running within the scientific community for a long time without commercialization [15]. those initiatives, mainly based on excellent clinical data, neuropathological annotations and research-driven modeling and machine learning, were mostly financed by governmental support or funding organizations and have frequently proven their high value for the healthcare system, e.g. by dramatically changing the most recent who classification [20]. it remains to be determined how those approaches can sustainably be kept running, for example by hosting joint platforms openly accessible on university servers. another potential risk is the fact that several machine learning approaches are based on data mainly deriving from a specific platform (e.g. illumina infinium methylationepic array ®) and would need new validation when new techniques for the same application are coming on the market (e.g. changing from array-based methylome to nanopore sequencing). this could even affect the who classification as several tumor entities are currently essentially defined by their methylation profile, such as high-grade astrocytoma with piloid features, astroblastoma with mn1 alteration, diffuse glioneuronal tumour with oligodendroglioma-like features and nuclear clusters (dgonc) or papillary glioneuronal tumor, while for many others the methylation profile is included as a desirable diagnostic criterion [20]. the use of joint worldwide platforms also has the advantage to discover much faster new, rare tumor entities as it has been successfully shown in 2022 with high-grade glioma with pleomorphic and pseudopapillary features (hpap), an entity that would otherwise have been associated with a wrong prognostic prediction if diagnosis would have been based on classic microscopic assessment only [3]. along this line, the who has an important role in balancing between the clinical needs in underdeveloped countries and the amazing advances that especially come up in first world academic centers in the field of molecular tumor profiling. it is therefore as important to continue with finding simple diagnostic tools for exact prognostic or therapeutic prediction as presented for the prediction of the 1p/19q status in idh-mutant glioma by an easy 2-step ihc-based approach [4]. if machine learning algorithms for microscopic (neuro-)pathological assessments would be as powerful as shown for molecular pathology or radiology [36], the latter now even aiming for quantitative analyses, new histology-based classification algorithms would then allow for a very cheap assessment of he slides in underdeveloped countries. therefore, the study of liechty et al. has also been selected as a highlight as it shows that machine learning-based diagnostics may be similarly powerful as a (neuro-)pathologist [5]. such an approach could be helpful not only for countries where no neuropathologists are available, but also for smaller health care centers, in which a 4 (or more) eye principle is not possible. the fact that “artificial intelligence” never gets tired and is able to quickly capture information from huge databases, a task for which an experienced pathologist would need decades, may be very worrying for classic “histomorphologists”. this might be even more the case when machine-learning pipelines will be more directly fed with clinical, radiological and molecular data. nowadays, it is already very clear that morphological classification alone is significantly inferior to approaches that also take into account molecular pathological data [6]. as previously shown for several joint initiatives in molecular profiling, it would be highly beneficial if prognostic prediction algorithms would be cross-validated with the goal to constantly improve, instead of having several competing grading systems [6, 17]. similarly, joint efforts would of course be necessary for other ongoing initiatives that have been presented in this manuscript, such as methylome-based brain tumor diagnostics in csf liquid biopsies [9], molecular profiling for entities that are not yet broadly covered in openly accessible classification pipelines, [7] or more recently upcoming techniques such as close to real-time diagnostic approaches based on the raman effect [10, 11]. while most profiling studies published in 2022 rather focused on “faster, deeper, broader, more precise”, really new concepts were very infrequently encountered. one very interesting approach is based on a deciphering the proteomic landscape in recurrent glioblastoma as compared to its primary counterpart [8]. this method would allow for a personalized treatment addressing potentially newly activated tumor pathways emerging during treatment resistance and recurrence. focusing on the tumor-associated proteome, one may get more real functional information about the tumor cells as compared to transcriptome, methylome or mutational analyses. finally, it is of high importance to come up with new, more personalized treatment approaches, especially for patients suffering from recurrent malignant brain tumors with a detrimental prognosis for which no really helpful established treatment schemes are available. a promising approach is the use of drug screening libraries which may be very useful to detect potential drugs that cannot easily be predicted by deep sequencing analyses [14]. as the author put the brain tumor cell(s) in the center of this “top ten” series, several high impact papers that focused on cells of or interaction mechanisms within the tumor microenvironment unfortunately did not make it on this list. an amazing new concept about how glioblastoma cells are hijacking neuronal activity for invasion has been presented, however, it remains to be determined to which degree this is important in humans and if this mechanism may serve as a potential future treatment target [37]. another fascinating study showed in a murine model that a loss of mhc-ii on blood-borne myeloid cells leads to dysfunctional cd8-positive cytotoxic t cells and therefore impaired tumor control while functional mhc-ii-restricted antigen presentation was associated with improved tumor control [38]. although the authors could show an inverse correlation of mhc-ii expression with dysfunctional cd8-positive t cell phenotypes, this finding was only provided in a very small cohort of human glioblastoma samples and needs further corroboration. in conclusion, neurooncological research in 2022 provided a huge number of excellent studies that considerably improved speed and precision in classifying tumors of the central nervous system, detected new tumor entities and provided promising approaches for more individualized treatment approaches. there is nothing else for it but to hope that at least some of those approaches finally make it into a therapeutic application thereby helping to improve the still detrimental prognosis of patients with malignant brain tumors. acknowledgements mm would like to thank the luxembourg national research fond (fnr) for the support (fnr pearl p16/bm/11192868 grant). the figures were prepared using https://biorender.com figure creation tool. single images using for the figures were freely available tagged with creative common license. declaration of interests the author declares to not have any competing interests. references 1. falagas me, pitsouni ei, malietzis ga, pappas g. comparison of pubmed, scopus, web of science, and google scholar: strengths and weaknesses. faseb j. 2008 feb;22(2):338-42. https://doi.org/10.1096/fj.07-9492lsf. epub 2007 sep 20. pmid: 17884971. 2. shariff sz, bejaimal sa, sontrop jm, iansavichus av, haynes rb, weir ma, garg ax. retrieving clinical evidence: a comparison of pubmed and google scholar for quick clinical searches. j med internet res. 2013 aug 15;15(8):e164. https://doi.org/10.2196/jmir.2624. pmid: 23948488. 3. pratt d, abdullaev z, papanicolau-sengos a, ketchum c, panneer 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dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. medulloblastoma and cowden syndrome: further evidence of an association feel free to add comments by clicking these icons on the sidebar free neuropathology 3:1 (2022) case report medulloblastoma and cowden syndrome: further evidence of an association steffen albrecht1,*, barbara miedzybrodzki2, laura palma3,4, van hung nguyen1, roy w.r. dudley5, torsten pietsch6, tobias goschzik6, nada jabado3,7,8, catherine goudie8,9, william d. foulkes3,10,11,12 1 department of pathology, mcgill university, montreal, qc, canada 2 division of dermatology, department of pediatrics, mcgill university health centre, montreal children's hospital, montreal, qc, canada 3 department of human genetics, mcgill university, montreal, qc, canada 4 division of medical genetics, department of specialized medicine, mcgill university health centre, montreal, qc, canada 5 division of neurosurgery, department of pediatric surgery, montreal children's hospital, mcgill university health centre, montreal, qc, canada 6 department of neuropathology, dgnn brain tumor reference center, university of bonn medical center, bonn, germany 7 department of pediatrics, mcgill university, montreal, qc, canada 8 the research institute of the mcgill university health centre, child health and human development program, montreal, qc, canada 9 division of hematology-oncology, montreal children's hospital, department of pediatrics, mcgill university, montreal, qc, canada 10 cancer axis, lady davis institute, the jewish general hospital, montreal, qc, canada 11 cancer research program, the research institute of the mcgill university health centre, montreal, qc, canada 12 gerald bronfman department of oncology, mcgill university, montreal, qc, canada * retired since aug. 2021 corresponding author: steffen albrecht · 511-244 sherbrooke street east · montreal, qc · h2x 1e1 · canada steffen.albrecht@hotmail.com submitted: 02 december 2021 accepted: 02 january 2022 copyedited by: jeffrey nirschl published: 11 january 2022 https://doi.org/10.17879/freeneuropathology-2022-3684 keywords: medulloblastoma, cowden syndrome, pten abstract cowden syndrome (cs) is an autosomal dominant hamartoma and tumor predisposition syndrome caused by heterozygous pathogenic germline variants in pten in most affected individuals. major features include macrocrania, multiple facial tricholemmomas, acral and oral keratoses and papillomas, as well as mammary, non-medullary thyroid, renal, and endometrial carcinomas. lhermitte-duclos disease (ldd), or dysplastic gangliocytoma of the cerebellum, is the typical brain tumor associated with cs; the lifetime risk for ldd in cs patients has been estimated to be as high as 30%. in contrast, medulloblastoma is much rarer in cs, with only 4 reported cases in the literature. we report a 5th such patient. all 5 patients were diagnosed between 1 and 2 years of age and not all showed the pathognomonic clinical stigmata of cs at the time of their medulloblastoma diagnosis. where detailed information was available, the medulloblastoma was of the shh-subtype, in keeping with the observation that in sporadic medulloblastomas, pten-alterations are usually encountered in the shh-subtype. medulloblastomas can be associated with several tumor-predisposition syndromes and of the 4 medulloblastoma subtypes, shh-medulloblastomas in children have the highest prevalence of predisposing germline variants (approx. 40%). cs should be added to the list of shh-medulloblastoma-associated syndromes. germline analysis of pten should be performed in infants with shh-medulloblastomas, regardless of their clinical phenotype, especially if they do not carry pathogenic germline variants in ptch1 or sufu, the most commonly altered predisposing genes in this age-group. in addition, these cases show that cs has a biphasic brain tumor distribution, both in regards to the age of onset and the tumor type: a small number of cs patients develop a medulloblastoma in infancy while many more develop ldd in adulthood. case history the patient was born at term after an uneventful pregnancy; however, she was noted to have pronounced frontal bossing and macrocrania, (head circumference > 98th percentile). she presented at age 15 months because of rapidly increasing head circumference. mri showed an extra-axial posterior fossa mass within the cisterna magna, measuring 5.3 x 4.4 x 3.2 cm. the tumor was resected; post-operative mri confirmed gross total resection. histologically, it was a medulloblastoma with extensive nodularity (mben). she was treated according to the ccg-99703 protocol, which consists of 3 cycles of induction chemotherapy followed by 3 cycles of marrow-ablative consolidation chemotherapy with autologous stem-cell rescue [cohen et al. 2015]; however, she only received 2 of the latter due to hematological toxicity. during follow-up, some keratotic papules on the fingers and buttocks were noted and therefore gorlin syndrome (nevoid basal cell carcinoma syndrome; online mendelian inheritance in man (omim) #109400) was suspected clinically. some of these cutaneous lesions were biopsied but consisted of non-specific keratoses or warts; there were no basal carcinomas. initial germline genetic testing at age 2 consisted of deletion analysis and sanger sequencing of ptch1, which were negative. sanger sequencing of sufu was done at age 5 and was negative. at age 8, a firm papule was noted on the neck; histologically, it was a sclerotic fibroma. (a similar lesion was also noted on the left thigh, but not excised.) at age 9, four palmar pits were noted on the right hand. although the combination of palmar pits, mben, and macrocrania was suggestive of gorlin syndrome, sclerotic fibroma has been associated with cowden syndrome (cs; omim #158350) [kieselova et al. 2017]. therefore, next-generation germline sequencing of ptch1, ptch2, sufu, and pten was performed. a previously reported pathogenic variant in pten, c.388c>t, p.r130* (cosv64288463), was identified. no variants were found in the other genes. parental studies were negative, suggesting most likely a de novo mutation, although gonadal mosaicism in one of the parents cannot be ruled out. at age 10, a posterior fossa dural arterio-venous fistula (davf) was suspected on a routine follow-up mri and confirmed by cerebral angiogram to be a borden type 1 davf draining into an ectopic pouch of the right transverse sinus; it was fed by branches of the right and left external carotid arteries and the right posterior cerebral artery. although hemorrhage from intracranial avfs has been reported in patients with cs [prats-sánchez et al. 2016], borden type 1 davfs have a very low risk of hemorrhage [gandhi et al. 2012] and the lesion was completely asymptomatic. it was therefore not treated. it was no longer seen on repeat imaging a year later, suggesting spontaneous involution-resolution. at age 12, multiple thyroid nodules were noted bilaterally on ultrasound; fine needle aspiration cytology was consistent with benign follicular nodules. now age 13 (i.e., 12 years post-diagnosis), the patient is alive and well without evidence of recurrent medulloblastoma. she does not have facial or oral lesions. figure 1. representative images of the cerebellar tumor’s histology and immunophenotype. individual images are labelled with the respective stain. on low power, the tumor is biphasic and distinctly nodular. most of the tumor is made up of neurocytic to gangliocytic cells (high power inset) that are strongly neun immunoreactive and embedded in a synaptophysin-positive neuropil-like matrix that contains bundles of neurites. the highly cellular desmoplastic component shows some staining for gfap and is strongly positive for gab1 and yap1. ki-67 labelling is seen almost exclusively in the desmoplastic component. both components lack expression of pten, which is retained in endothelial cells. clicking the figure will lead you to the full virtual slides. pathology representative images of the cerebellar tumor are shown in figure 1. the tumor was biphasic and had a distinctly nodular growth pattern. most of the tumor consisted of large confluent islands of isomorphic cells with a neurocytic to gangliocytic appearance, embedded in a finely fibrillary neuropil-like matrix. these cells were strongly positive for neun and the neuropil stained intensely for synaptophysin and showed bundles of neurites on a neurofilament immunostain. there was virtually no ki-67 labelling in this component and it was negative for yap1 and gab1. the remainder of the tumor was made up of a very cellular "small blue cell" component, which was rich in reticulin fibers, had a high mitotic rate with approximately 50% ki-67 labelling, and focal immunoreactivity for gfap along with strong staining for yap1 and gab1. the features were typical of an mben, shh-activated. immunostaining for pten and genetic analysis of the tumor were performed after the pten germline variant was identified. expression of pten was retained in vessels but lost in both tumor components. the tumor carried the pten p.r130* variant, as expected. in addition, using a truseq custom amplicon (illumina) for 13 genes frequently mutated in shh-medulloblastomas [goschzik et al. 2021], a known pathogenic variant in smo, c.1234c>t, p.l412f (cosv50824425), was identified, which was confirmed by sanger sequencing. molecular inversion probe technology was used to perform high-resolution genome-wide copy number analysis as described previously [wang et al. 2012]. it showed a stable genome with copy-neutral allelic loss on chromosome arm 10q. there was no amplification of myc or mycn. discussion cowden syndrome (omim #158350) is an autosomal dominant hamartoma and tumor predisposition syndrome caused by heterozygous pathogenic germline variants in pten in about 80% of affected individuals [yehia & eng 2001/2021]. major features include macrocrania, multiple facial tricholemmomas, acral and oral keratoses and papillomas, as well as mammary, non-medullary thyroid, renal, and endometrial carcinomas [yehia & eng 2001/2021]. lhermitte-duclos disease (ldd), or dysplastic gangliocytoma of the cerebellum, is the typical brain tumor associated with cs; in fact, in adults, ldd is considered pathognomonic of cs [yehia & eng 2001/2021]. conversely, the lifetime risk for ldd in cs patients has been reported to be as high as 30% [riegert-johnson et al. 2010]. in contrast, the association between cs and medulloblastoma is very rare. in a study of 914 children and adolescents with cancer, including 227 with medulloblastoma, no pten germline variants were identified [gröbner et al. 2018]. similarly, among 1022 medulloblastoma patients screened for germline variants in 110 cancer predisposition genes, only one pathogenic variant was reported in pten [waszak et al. 2018]. in a clinical cohort of 368 patients with cowden syndrome and pathogenic pten germline variants (including 98 patients under the age of 18 years) recruited and followed prospectively by an international consortium between 2000 and 2010, no cases of medulloblastoma were reported [tan et al. 2012]. on the other hand, including this case, five patients with cs and medulloblastoma have now been reported in the literature; they are summarized in table 1. patient 1 was reported before the genetic defect in cs was known [bagan et al. 1989], hence the diagnosis of cs is based on the clinical findings [eng 2000]; he has since been lost to follow-up (j.v. bagan, personal communication). the four other patients carried a pathogenic germline variant in pten. given the rarity of cs, with an estimated prevalence of 1 in 200,000 [yehia & eng 2001/2021], this is unlikely to be a coincidence. table 1: patients with cowden syndrome and medulloblastoma. case sex/age at diagnosis (case id if applicable) mb subtype (histology if known) germline pten variant additional variants and/or other genetic changes in tumor status reference               1 m/2 y (case 2) na na na ned at 19 y bagan et al. 1989               2 m/2 y ns c.697c>t, p.r233* nd ned at 14 y patini et al. 2016               3 f/1 y (icgc_mb217) shh c.856dela, p.t286pfs*5 loss of heterozygosity ns waszak et al. 2018               4 f/14 mo shh (dn¶) c.18dup, p.e7rfs*4 sufu c.412delinscc, p.a138pfs*32 kdr c.787c>a, p.p263t ned at 5½ y¶ tolonen et al. 2020               5 f/15 mo shh (mben) c.388c>t, p.r130* smo c.1234c>t, p.l412f copy-neutral allelic loss on 10q ned at 13 y current report abbreviations: dn, desmoplastic nodular; mb, medulloblastoma; mben, medulloblastoma with extensive nodularity; mo, month; na, not applicable; nd, not done; ned, no evidence of disease; ns, not stated; y, year ¶ r. niinimäki, personal communication interestingly, in the three cases in which a detailed analysis of the medulloblastoma was performed, it was an shh-activated medulloblastoma. two of them also had an additional pathogenic variant in a component of the shh-pathway and 2 showed allelic loss involving pten/10q. this mirrors findings in sporadic medulloblastomas, where mutations or deletions of pten are more frequent in shh-medulloblastomas than in other medulloblastoma subtypes [northcott et al. 2012]. furthermore, in mouse models of shh-medulloblastomas driven either by inactivation of ptch1 [metcalfe et al. 2013] or activation of smo [castellino et al. 2010], additional loss of pten changed the tumor histology from tumors resembling classic medulloblastomas to tumors resembling mben. using similarity network fusion applied to genome-wide dna methylation and gene expression data, shh-medulloblastomas can be divided into 4 distinct subtypes [cavalli et al. 2017]; we did not perform this type of analysis on our patient’s tumor and based on the published data, neither did the authors of the two other cases where the tumor was analyzed (cases 3 and 4). brain-specific suppression of pten in mice produces cerebellar lesions resembling ldd, but not medulloblastomas [backman et al. 2001; kwon et al. 2001]. in human shh-medulloblastomas, loss of pten almost never occurs in isolation. in a detailed analysis of 196 sporadic shh-medulloblastomas [skowron et al. 2021, figure 2a], nine had a deletion and/or mutation of pten. eight of these tumors had additional, often multiple, genetic alterations, typically involving a gene of the shh-pathway, loss of 9q (which deletes ptch1), loss of 10q (which deletes pten and sufu), or mutations and fusions in other genes. only one tumor had an isolated variant in pten, but the analyses may not have captured all possible genetic alterations (p. skowron and m. taylor, personal communications). these findings indicate that in most instances, loss of pten alone is insufficient to produce medulloblastomas. shh-activated medulloblastomas are thought to arise from granule cell precursors that form the external granular layer (egl) of the cerebellar cortex; their proliferation and inward migration to form the internal granule cell layer is driven by purkinje cell-derived shh [tamayo-orreo & charron 2019]. in humans, the egl persists after birth. it starts to involute by the 2nd to 4th post-natal month and usually has disappeared by 1 year, although rare cases of persistence until 2 years have been reported [friede 1989]. in a patient with cs, all granule cell precursors in the egl are haploinsufficient for pten, but this is not sufficient to produce a medulloblastoma. however, if one of these cells acquires additional alterations of the type described by skowron et al. [skowron et al. 2021], it can give rise to an shh-medulloblastoma. this would be a rare event, resulting in a low frequency of medulloblastomas in cs patients. furthermore, given the rapid disappearance of the egl after birth, the "window of opportunity" for the development of a medulloblastoma in cs is brief and explains why they only occur in infants in these patients. cs may be under-recognized in medulloblastoma patients. while 90% of patients with cs will have some features of cs by age 20 [yehia & eng 2002/2021], infants and young children may not show some of the pathognomonic features, such as multiple facial tricholemmomas, and present instead with non-specific findings, like macrocrania, autism, or developmental delay [busa et al. 2015]. among the 5 patients with cs and medulloblastoma, patient 1 had oral papillomas at the time of his medulloblastoma diagnosis [bagan et al. 1989]. patient 2 developed gastro-intestinal polyps after his medulloblastoma and sought medical attention for oral and cutaneous lesions at age 14, at which time the diagnosis of cs was made [palatini et al. 2016]. no clinical details are available on patient 3 [waszak et al. 2018]. patient 4 showed only macrocrania at the time her medulloblastoma was diagnosed [tolonen et al. 2020] and still does not have other stigmata of cs (r. niinimäki, personal communication). in our patient, sequencing of pten was only undertaken at age 9, after the diagnosis of the sclerotic fibroma. thus, in some of these young patients, the diagnosis of cs was not clinically obvious at the time they presented with a medulloblastoma; we therefore suspect that cs may be underdiagnosed in medulloblastoma patients. a high prevalence of tumor predisposition syndromes in children with shh-medulloblastomas, especially infants, has long been known. for instance, in a single-institution series of 82 medulloblastoma patients, 6 of 12 patients with an mben had a genetic syndrome (5 gorlin, 1 fragile-x); 5 of these 6 were less than 24 months of age [garrè et al. 2009]. in the aforementioned series of 1022 medulloblastomas, predisposing germline variants in apc, brca2, palpb2, ptch1, sufu, and tp53 occurred in 6% of patients overall, and in 20% of those with shh-medulloblastomas [waszak et al. 2018]. a further analysis by the same group [waszak et al. 2020] identified in addition pathogenic germline variants in elp1 in 14% of pediatric patients with shh-medulloblastomas; all in all, 77 of 202 children (38%) with an shh-medulloblastoma had a predisposing germline variant in one of 7 genes. here, we present evidence that cs, too, can be associated with shh-medulloblastomas in infants. therefore, pten should be analyzed in these patients, regardless of their clinical phenotype, especially if they do not carry pathogenic germline variants in ptch1 or sufu, the most commonly altered predisposing genes in infant shh-medulloblastomas [waszak et al. 2020]. whether cs also predisposes to other types of medulloblastoma (i.e., wnt, group 3, or group 4) remains to be seen. in addition, these cases show that cowden syndrome has a biphasic brain tumor distribution, both in regards to the age of onset and the tumor type: a small number of cowden patients develop a medulloblastoma in infancy while many more develop ldd in adulthood. acknowledgements we thank j.v. bagan, r. niinimäki, s. pfister, p. skowron, and m.d. taylor for kindly providing additional information on previously published cases. w. foulkes receives funding from the canadian institutes of health research (grant fdn-148390) and so does n. jabado (grant mop-286756 and fdn-154307). references backman sa et al. deletion of pten in mouse brain causes seizures, ataxia and defects in soma size resembling lhermitte-duclos disease. nat genet 29: 396–403, 2001. https://doi.org/10.1038/ng782 bagan jv et al. cowden syndrome: clinical and pathological consideration in two new cases. j oral maxillofac surg 47: 291–4, 1989. 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https://doi.org/10.1038/s41586-020-2164-5 yehia l, eng c. pten hamartoma tumor syndrome. 2001 nov 29 [updated 2021 feb 11]. in: adam mp, ardinger hh, pagon ra, et al., editors. genereviews® [internet]. seattle (wa): university of washington, seattle; 1993-2021. copyright: © 2022 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. neuropathology and epilepsy surgery – 2024 update feel free to add comments by clicking these icons on the sidebar free neuropathology 5:8 (2024) review neuropathology and epilepsy surgery – 2024 update ingmar blümcke department of neuropathology, university hospital erlangen, germany corresponding author: ingmar blümcke · department of neuropathology · university hospital erlangen · schwabachanlage 6 · erlangen · germany bluemcke@uk-erlangen.de submitted: 05 february 2024 accepted: 11 march 2024 copyedited by: joão gama published: 22 march 2024 https://doi.org/10.17879/freeneuropathology-2024-5347 keywords: brain, histopathology, seizure, hippocampus, neocortex, dysplasia, tumor abstract neuropathology-based studies in neurosurgically resected brain tissue obtained from carefully examined patients with focal epilepsies remain a treasure box for excellent insights into human neuroscience, including avenues to better understand the neurobiology of human brain organization and neuronal hyperexcitability at the cellular level including glio-neuronal interaction. it also allows to translate results from animal models in order to develop personalized treatment strategies in the near future. a nice example of this is the discovery of a new disease entity in 2017, termed mild malformation of cortical development with oligodendroglial hyperplasia in epilepsy or moghe, in the frontal lobe of young children with intractable seizures. in 2021, a brain somatic missense mutation of the galactose transporter slc35a2 leading to altered glycosylation of lipoproteins in the golgi apparatus was detected in 50 % of moghe samples. in 2023, the first clinical trial evaluated galactose supplementation in patients with histopathologically confirmed moghe carrying brain somatic slc35a2 mutations that were not seizure free after surgery. the promising results of this pilot trial are an example of personalized medicine in the arena of epileptology. besides this, neuropathological studies of epilepsy samples have revealed many other fascinating results for the main disease categories in focal epilepsies, such as the first deep-learning based classifier for focal cortical dysplasia, or the genomic landscape of cortical malformations showing new candidate genes such as ptpn11, which is associated with ganglioglioma and adverse clinical outcome. this update will also ask why common pathogenic variants accumulate in certain brain regions, e.g., mtor in the frontal lobe, and braf in the temporal lobe. finally, i will highlight the ongoing discussion addressing commonalities between temporal lobe epilepsy and alzheimer's disease, the impact of adult neurogenesis and gliogenesis for the initiation and progression of temporal lobe seizures in the human brain as well as the immunopathogenesis of glutamic acid decarboxylase antibody associated temporal lobe epilepsy as a meaningful disease entity. this review will update the reader on some of these fascinating publications from 2022 and 2023 which were selected carefully, yet subjectively, by the author. introduction this review is a continuation of my 2022 update of interesting research papers and topics covering the search terms "neuropathology" and "epilepsy surgery" from 2021 [9], now discussing the 10 most interesting topics and/or papers published in 2022 and 2023. these search terms produced about 150 hits in pubmed in each year. the 'subjective' selection by the author is not an attempt at including all published papers or topics of the field and over this time period. rather, i hope to highlight active areas of clinical and basic research with the potential to advance our concepts and understanding of disease pathomechanisms, e.g. how does a given 'molecular' pathomechanism translate into a lesional and epileptic phenotype. i will start with last year's "missing topic", i.e., the lack of published articles using artificial intelligence (ai) based algorithms to support or supplement histopathology diagnosis of epilepsy-associated brain lesions. this chapter will also enlighten the discussion about the substitution of histopathologists by ai-driven technology as well as ongoing technical obstacles to successfully develop the field by histopathology experts rather than by industry-driven incentives. the first update to the internationally renowned fcd classification scheme, originally published in 2011 under the direction of the international league against epilepsy (ilae) and since then applied in epilepsy surgery units around the world as well as in research studies (cumulative citation index of > 1,199 in the clarivate web-of-science database as of january 2024), represents probably one of the most important papers published in the time period under review. the updated fcd classification has been already previewed in my 2022 update, however [9]. the 2022 update emphasized the impact of brain somatic mutations in lesional epilepsies such as focal cortical dysplasia (fcd). three international and collaborative research teams studied a series of 807 human brain tissues to unravel the genomic landscape of focal (lesional) epilepsies, which will be subsumed under item #2. these studies independently identified new epilepsy-related candidate genes, such as ptpn11, which belongs to the map-kinase signaling pathway and is well known from noonan syndrome, a common rasopathy in children characterized by short height, congenital heart disease and often epilepsy, amongst many other clinical features. interestingly, ptpn11 variants are most commonly detectable in temporal lobe epilepsy (item #3) and ganglioglioma (gg) with adverse clinical outcome, respectively (item #6). the fcd update from 2022 also introduced a new disease entity, i.e. moghe, which was a topic of ongoing research in 2022 and 2023. herein, i will review the clinical, genetic and histopathological assessments in a large, international cohort of 47 patients with moghe and slc35a2 mutations (item #4). a first trial assessing d-galactose supplementation in moghe patients with slc35a2 mutations not being seizure free after surgery is the consequential next important step in the translation of our tissue research towards the implementation of personalized medicine and will be covered in the same chapter. the frequent localization of fcd ilae type 2 in the frontal lobe and of leat in the temporal lobe is another question not yet fully understood in the current literature. however, a recent brain article tackled this question and delivered a fascinating hypothesis supported by the allan brain atlas describing region-specific expression patterns of the mtor protein to explain the impact of either loss-of-function or activating gene mutations in their associated signaling pathways (see also item #3). temporal lobe epilepsy remains the most common form of focal epilepsy in adults and thus raises continuous interest. in the last four chapters, i will discuss a series of publications reporting expression of hyperphosphorylated tau in the epileptic condition as compared to neurodegenerative disorders (chapter #7). the impact of altered neurogenesis and gliogenesis in the dentate gyrus of the human hippocampus represents another hot topic (#8) as will the impact of hippocampal innate inflammatory gliosis (#9). these papers may redirect our research focus and interest on glial cells as important modulators if not initiators of epileptogenesis in the temporal lobe. my final topic addresses neuroinflammation and autoimmunity. the lymphocytic and microglial response to inflammatory pathomechanisms will always remain an important topic in the realm of focal epilepsy, and i do hope that my update will stimulate the readers' interest in this field. table 1: top 10 publications and topics to be discussed in this update topic title major reference(s) #1 ai-based classification of focal cortical dysplasia (fcd) [43] [24] #2 the genomic landscape of epileptogenic brain lesions [30] [13] [7] #3 a new hypothesis about regional mtor gene variant accumulation in the frontal lobe and map-kinase gene variant accumulation in the temporal lobe [26] [31] #4 new disease entities: i. moghe [6] [1] #5 new disease entities: ii. encephaloceles [16] [4] #6 what did we learn in 2022 and 2023 about low-grade, epilepsy-associated brain tumors (leat) [21] [40] [35] #7 tauopathies in mesial temporal lobe epilepsy (mtle) [46] [14] [3] #8 'hippocampal innate inflammatory gliosis only' in pharmacoresistant temporal lobe epilepsy (tle) [19] #9 altered adult neurogenesis and gliogenesis in patients with mesial tle [2] #10 autoimmune encephalitis and new-onset refractory status epilepticus (norse) [41] [8] [20] topic 1: which topic was missing in the 2022 update? ai-supplemented applications for routine histopathology review and diagnosis may be seen as the next era in neuropathology. however, ai has not been used in the arena of epilepsy surgery yet and this was identified as a missing topic in my 2022 update review article [9]. such a deep-learning based classifier for fcd was published in 2023 [43] and claimed to recognize most of the diagnostic entities defined in the 2022 fcd classification update [33]. the resnet18 deep-learning algorithm was trained with extraordinarily large 1000 x 1000 µm digital tiles automatically extracted from whole slide images (wsi) of 414 manually annotated, h&e stained of fcd1a, fcd2a, fcd2b, mmcd and moghe samples (figure 1). these were similarly processed to a series of 198 postmortem tissue blocks from 59 patients without neurological disorders including homotypic frontal, temporal and occipital areas and heterotypic brodmann areas 4 and 17, entorhinal cortex and dentate gyrus. overall, the algorithm's accuracy was specified as 98.8 % (f1 score = 0.82) in detecting 25 different anatomical regions and fcd categories. the classifier was also made temporarily available as an open access web application. figure 1: evaluation and visualization of a complete wsi slide obtained from epilepsy surgery figure 1. a: this is an example of the dl-based fcd classifier output as published by vorndran et al. a digital slide (visible in grey in the background) was automatically segmented into a grid of tiles each measuring 1000 x 1000 µm. all tiles reaching the predefined confidence threshold (herein = > 0.9) can be visualized by a color-index map, e.g., purple or yellow. summary values were also given on the upper left corner of the output screen. b: a blurring image filter is applied for the final output image hiding all information shown in a. please note that the user can choose which of the available and trained fcd categories should be displayed. in this example, it is only fcd1a and white matter (wm). the sample was taken from a patient with histopathologically classified fcd1a [22]. c: a validation case example of a wsi obtained from a patient with histopathologically confirmed fcd2b and genetically confirmed pathogenic mtor mosaicism. the lesion was detected and colored in dark blue for areas classified as fcd2b, light blue for neocortex associated with fcd2b, yellow for white matter associated with fcd2b. however, there were also areas recognized by the algorithm as fcd2a (x light purple) and moghe (y pink). the algorithm's confidence score for these regions was low, however (< 0.415, 0.562, respectively). such regions should then be reviewed at the light microscope by the responsible histopathologist. in this case, moghe (y) was not confirmed histopathologically by the authors. d: a validation case example of a wsi obtained from a patient with histopathologically confirmed moghe with slc35a2 mutation. z points to a region with fcd1a as confirmed by the authors. modified from [43] with permission from the author. such digital slide suite projects will pave the way towards ai-supported histopathology diagnosis in years to come pending the clarification and resolution of ongoing obstacles in the field of whole slide imaging (wsi). firstly, many parts of the world may not have access to a fast enough internet to upload such large digital wsi files, which can easily exceed multiple gbyte (the range was described as 0.47-5.72 gbyte per wsi in the current study). secondly, a uniform laboratory standard of section thickness in the range of 4-5 µm and standardized h&e staining protocols must be applied to be compatible with the trained dataset of the ai algorithm. thirdly and unfortunately most challenging, vendors of different wsi scanners implement their unique file formats [27]. there are open access wsi reading applications such as open.slide which can help to read and import different formats into an ai algorithm. however, any technical update of the file format or reading header by the vendor requires prompt adaptation by software stewards for any independent application. a solution to this problem would be the use of internationally defined dicom standards, which already exists for wsi [27]. however, this has not been implemented by wsi vendors with the intention to control ai based histopathology slide suits as a walled garden laboratory solution wherein the wsi equipment and laboratory software must be purchased from the same company. in contrast, these applications should be openly available for research and managed by experts in the field to allow broad access to this technology irrespective of any country's or researcher's socio-economic background. another open question remains, if ai-based histopathology classifier will replace the pathology expert in the near future. my personal belief is that ai will not replace the physician as the legal accountability of a signed pathology report for clinical patient management remains so high. instead, such tools should help to prescreen routine cases. the application may then suggest a preselection of regions of interest, pending on an individually chosen level of the algorithm's detection sensitivity and which can be adapted by each reviewer (see figure 2). each reviewer must also determine if technical pitfalls have compromised the results, e.g., wrinkles, dust particles or other coloring errors, etc. these decisions will always remain with the responsible physician and cannot be replaced easily by any ai. however, this discussion will be ongoing in the community and my hope is that many world renowned experts will share their expertise in ai-based slide suites and classifier tools and which can be used open access around the world, in particular in those regions where training and experience in less broadly available. figure 2: new genotype-phenotype associations in epileptogenic brain lesions figure 2. a-b: two patients with a complex malformation of cortical development including heterotopias (a) and polymicrogyria (b) revealed brain somatic mutations in nras. c: a novel association was shown in another patient for a large chromosomal 1q duplication that included akt3 and type fcd2a with hyaline astrocytic inclusions. figure modified from open-access paper published under a creative commons license [30], with permission from the authors. just to mention in this chapter that machine learning and ai tools were recognized and increasingly published already in the arena of epileptology [25], e.g., when virtual digital brain models can help to map a brain network in a given patient with epilepsy [24], when neuroimaging algorithms help to better identify structural brain lesions [44], or when predicting seizure outcome after epilepsy surgery in order to avoid unnecessary invasive diagnostic procedures for the patient [17]. notwithstanding, the histopathology diagnosis often remains an important predictor in many of these ai-based applications. topic 2: deciphering the genomic landscape of epileptogenic brain lesions the search for genetic factors in lesional focal epilepsies is ongoing and pushed our knowledge of brain somatic mutations, chromosomal aberrations and epigenetic factors to a next level. i will review herein a series of three publications from 2022 and 2023 which comprehensively study brain tissues obtained from a total of 807 patients using different bioinformatic approaches. chung et al. reported a multidisciplinary team effort including the fcd neurogenetics consortium and the brain somatic mosaicism network [13]. they retrieved 283 resected brain samples of patients with clinically and/or histopathologically validated malformations of cortical development (mcd) and identified a total of 69 mutated candidate genes. they had access to whole exome sequencing and targeted-amplicon sequencing platforms, and included in utero electroporation of the developing mouse brain to test the effect of their newly discovered genetic variants. finally, single-nucleus rna sequencing was employed to learn about the spatial gene expression patterns and enriched cell populations which may help to better understand the underlying pathomechanisms of mcd. the most commonly affected genes were, however, the already well-known candidates mtor, pik3ca, slc35a2, tsc2, akt3 and braf. cheung et al. comprehensively studied their dataset from various angles. firstly, their analysis of genes mutated in mcd highlighted four affected gene networks. the largest and most prominent cluster pointed to the well-known mtor-map kinase pathway, in addition to a calcium dynamics cluster, a synapse cluster and a cluster aligned to gene expression. then, the newly identified gene variants of praga, grin2c and klhl22 were electroporated into the developing mouse brain and revealed functional (migration) defects resembling that of a cortical malformation. thirdly, their genotype-phenotype correlation highlighted an association of fcd ilae type 2b and 2a with mtor rather than map-kinase pathways. this association has been now consistently reported [23] and will be further discussed in the chapter below. finally, they studied single-nucleus spatial transcriptomics of mutated genes obtained from patient samples versus controls, which indicated a critical role in excitatory neurogenic cell pools during brain development, and further supported the neurobiological basis for enhanced neuronal hyperexcitability in the epileptogenic brain. of note, all slc35a2 mutated cases were histopathologically classified as fcd 1 and the braf cases as fcd 3. this is likely a histopathology-disagreement error frequently encountered in the histopathology work-up of epilepsy surgery tissue samples and this discussion will be continued below. in fact, re-review of published cases carrying a slc35a2 gene mutation were frequently re-classified as moghe [6, 10, 12]. the braf mutations described by cheung and colleagues most likely represents ganglioglioma tumor cell infiltration into surrounding brain tissue, which is often regarded as an associated fcd ilae type 3b. as another example for disagreement in histopathology diagnosis, however, a recently published paper claimed a new diagnostic entity with braf mutated cells invading/infiltrating the hippocampus [29]. such cases have been seen also in our routine practice but regarded rather as remnants of a ganglioglioma infiltrating the hippocampus. cheung and coworkers also briefly mention ptpn11 as new lesional epilepsy gene. two other studies have made the same observation but at a much larger frequency of affected samples and diagnostic emphasis. in the second paper, lópez-rivera and coworkers examined a total of 474 new samples not previously reported in other studies [30]. the surgical tissues were selected from four different centers in germany and the us. histopathology diagnosis covering the spectrum of mcd (n = 223), leat (n = 154) and hippocampal sclerosis (n = 97) were included. they reported nineteen genes of interest and reported 153 samples carrying at least one single nucleotide variant, copy number variant or loss of heterozygosity affecting 31.4 % of the entire cohort, 7.2 % of hs, 31.8 % of mcd and 51.9 % of leat. these numbers remain low despite their effort to use deep sequencing rates at a mean depth of > 350 x. a novel association was histopathologically established for ptpn11 with low-grade epilepsy-associated brain tumors (ganglioglioma), nras with a complex mcd including polymicrogyria, and akt3 with fcd 2a and hyaline astrocytic inclusions (figure 2). in addition, chromosomal losses for germline variants recognized for tsc2, depdc5 and pten were most compatible with the previously proposed second hit hypothesis to also play a role in the etiopathology of mcd [36]. the authors then concluded to design mcd-specific panels for clinical routine testing of surgical samples to support clinical diagnosis but also enable targeted treatment in those patients that failed successful postsurgical seizure control. a third, smaller study from the us enrolled surgical brain tissue samples obtained from 50 children and used whole exome and rna sequencing [7]. overall, 56 % of their patients could be explained by genetic findings, including the slc35a2 gene and mtor pathway genes in mcd, and map-kinase pathway genes in tumors. amongst the map-kinase pathway genes, the authors were the first to report ptpn11, which was detected in two samples including fcd iiia and iiid. they also reported slc35a2 genotypes in two patients with the histopathology diagnosis of fcd ic, but these histopathology phenotype-genotype correlations may not stand an independent second review. as mentioned above, an independent re-review of previously published slc35a2 altered samples identified the 'new disease entity' of moghe to be the underlying pathology rather than any fcd or mmcd subtypes [6, 12]. the same conclusion is drawn from the paper of carmen barba reviewing 47 patients with moghe as discussed below (#4). topic 3: a new hypothesis about regional mtor gene variant accumulation in the frontal lobe and map-kinase gene variant accumulation in the temporal lobe the genomic landscape of focal lesional epilepsy gets better recognized as large enough patient series are examined. however, it remained a hitherto open question why some lesions and their associated genetic alterations occur overwhelmingly frequent in certain brain localizations. as a prominent example, fcd ilae type 2 lesions carry constitutively activating mtor pathway gene variants and occur most frequently in the frontal lobe, same as do slc35a2 altered moghe. in contrast, gg and other low-grade epilepsy associated brain tumors (leat) overwhelmingly often carry activating map-ras-raf kinase signaling pathway gene variants (e.g., braf or ptpn11) and occur predominantly in the temporal lobe. a recent brain publication from macdonald-laurs and coworkers offered an intriguing answer to that question [31]. by studying the clinical, pathologic and genetic landscape of their cohort of 85 patients with fcd at the bottom-of-sulcus (bosd), which is a peculiar fcd type 2 variant specifically highlighted also in the international fcd classification update of 2022, they reported that 62 % of their cohort presented in the frontal lobe, 18 % in the parietal lobe, and 14 % in the insula, whereas the temporal and occipital lobes were rarely affected (5 % and 1 %, respectively). only pathogenic mtor pathway gene variants were detected and occurred in 60 % of their patient cohort. then, they compared the regional frequency of the bosd with the mtor protein expression pattern published in the open access online allen human brain atlas (figure 3). intriguingly, mtor is less dominantly expressed in the frontal lobe compared to temporal or occipital lobes suggesting that a gain-of-function alteration of mtor signaling, e.g., mtor activating mutations or inactivating depdc5 mutations/losses, would have had a much higher impact in the frontal lobe where these genes are not naturally highly expressed. notwithstanding, this hypothesis needs to be studied further. an intriguing study would be to search for pathogenic mutations in mtor or map-kinase pathway genes in other brain regions not affected by a pathology lesion of the same patients or in the general population. if one can prove their presence it could well support the functional impact in generating a structural lesion only when their signal gets abnormally high during the neurodevelopmental process. figure 3: brain expression of mtor suggests regional vulnerability for fcd 2 figure 3. schaefer parcellation of the lateral and medial surface of the left hemisphere showing (a) the number of bottom-of-sulcus-dysplasia (bosd) with at least 10 % overlap with each parcel, and (b) relative mtor expression from the allen human brain atlas. note that privileged regions of bold associated with low expression of mtor. figure modified from open-access paper published under a creative commons license [31], with permission from the authors. a same regional accumulation of activating mutations of the map-kinase pathway, such braf or ptpn11 have been identified in the temporal lobe, as recently published by khoshkhoo and coworkers [26]. they studied 105 individuals with mesial temporal lobe epilepsy (mtle) compared to 30 neurotypical controls. they detected 11 pathogenic somatic variants in hippocampal tissue neurosurgically resected in their patient cohort but none in their controls. these variants included ptpn11, sos1, kras, braf and nf1, all of which are predicted to constitutively activate the map-kinase signaling pathway and which might contribute to the pathogenesis of sporadically occurring, drug-resistant mtle. in other words, not only map-kinase associated low-grade brain tumors but also hippocampal sclerosis accumulate map-kinase activating pathogens and may contribute to its high epileptogenic susceptibility. topic 4: new diagnostic disease entities: i. moghe the clinico-pathologic diagnosis of moghe as a new disease entity in patients with focal, drug-resistant early onset (frontal lobe) epilepsy was first described in 2017 [37]. since then, scientific and clinical interest has continuously grown towards a better understanding of moghe. in 2018, a novel brain somatic loss-of-function mutation of the galactose-transporter slc35a2 gene was identified in epilepsy surgery brain tissues [38, 45]. this was confirmed in 2019 [5] and all three publications assigned the slc35a2 mutation to variable histopathology phenotypes, such as mmcd, fcd type 1 or non-lesional samples. two papers concluded in 2021, however, that the slc35a2 mutation is specifically associated with moghe and found in about 50 % of reported patients [10, 12]. this enduring uncertainty stimulated an international consortium of 13 epilepsy centers around the world led by dr. barba to review the neuropathology, clinical features, and postsurgical outcome in a total of 47 patients previously described to carry brain somatic slc35a2 mutations [6]. the microscopic re-review of all available surgical specimens identified moghe in 94 % of their patient series, leaving very limited room to assign the slc35a2 mutation to any other disease or fcd entity. as a matter of fact, the diagnosis was inconclusive in 3 patients because of insufficient surgical sampling (6 %). twenty-nine cases were previously published with a diagnosis of fcd type 1, mmcd or non-lesional, which needs reconsideration and which also highlights the difficulty to achieve agreement in the histopathological diagnosis of epilepsy surgery brain samples as was already discussed above. interestingly, they observed two clinical phenotypes of moghe. the majority of their patients (n = 39; 83 %) had early epileptic encephalopathy with a seizure onset range from 3 month to 3.5 years and severe to moderate intellectual disability. the second and smaller cohort of eight patients (17 %) could be characterized as drug-resistant focal epilepsy of young to adolescent (later) onset associated with almost normal cognitive function. another interesting observation was the lack of any hot spot mutation across the slc35a2 gene (figure 4). all mutations were predominantly distributed across the nine transmembrane domains. finally, there was no association between the genetic variant or variant allelic frequency (vaf), which ranged from 1.4 % 52.6 % in all samples (mean vaf = 17 %), with the clinical phenotype or the postsurgical outcome. indeed, seizure freedom was achieved in 30 patients (63.8 %, engel class i), and favorable outcome was significantly associated with shorter disease duration and complete resection. these numbers dramatically changed from the first published results in 2017, where only 33 % of patients were reported to achieve postsurgical seizure control. partial resection due to unknown lesion boundaries were the most likely explanation in this early study. the challenge to anatomically define moghe's lesion borders remains actually, which may open possible avenues for preor perioperative online sequencing methodologies using the real-time, direct sequencing technology, e.g. see [42], from intraoperative specimens or depth electrodes obtained from presumptive lesion borders. it is yet unknown, however, if the low variant frequency in these lesions and lack of hot spot mutations can be resolved by the technology. figure 4: membrane topology modeling of currently known slc35a2 mutations in moghe figure 4. schematic representation of variants reported by [6]. novel variants are labeled in red, variants already described are labeled in black. membrane topology was predicted using the protter online tool41 (p78381-slc35a2_human). figure collected from open-access paper published under a creative commons license, with permission from the authors. in 2023, another small study described five new patients with moghe confirming the predominance of frontal lobe epilepsy and epileptic encephalopathy phenotypes in patients histopathologically diagnosed as moghe [18]. however, this study has not applied genetic testing for brain somatic mutations. as reported above, all patients responded favorably to extensive frontal lobe resections, despite widespread epileptic activity recorded by surface and intracranial eeg preand postoperatively. the authors concluded, therefore, that an epileptic encephalopathy phenotype in the first years of life should not discourage a surgical treatment option. an additional recently published paper from a european consortium of epilepsy surgery centers led by dr. angel aledo-serano from madrid, spain sheds further light on moghe. twelve patients with histopathologically confirmed moghe were included in their postsurgical clinical trial attempting to test the benefit of d-galactose supplementation when patients were not seizure free after surgery. so far, research evidence reached the conclusion that at least 50 % of moghe patients suffer from a brain somatic loss-of-function mutation in the galactose transporter gene slc35a2, which delivers galactose into the endoplasmic reticulum and golgi apparatus, thereby glycosylating sphingolipids into functionally active protein epitopes. one therapeutic option to circumvent the lack of slc35a2 protein would be to push alternative transporter pathways by offering vast amounts of galactose substrate to the patient. this treatment strategy was tested in 2020 in young children with the germline variant of slc35a2 deficiency, i.e. slc35a2-congenital glycosylation disorder characterized by epileptic encephalopathy, developmental disability, growth deficiency, dysmorphism as well as high mortality rates within the first years of life [47]. their hypothesis was that galactose supplementation partially overcomes the golgi udp-galactose deficiency and improves galactosylation [47]. in addition, oral galactose supplementation was well tolerated showing promise as dietary therapy. aledo-serano aimed, therefore, to evaluate the effects of d-galactose supplementation also in patients with histopathologically confirmed moghe, with uncontrolled seizures or cognitive impairment and epileptiform activity at the eeg after epilepsy surgery as inclusion criteria to their clinical trial (nct04833322). patients were orally supplemented with d-galactose for 6 months in doses up to 1.5 g/kg/day and monitored for seizure frequency including 24-h video-eeg recording, cognition and behavioral scores, before and six months after treatment. positive response was defined by more than 50 % improvement of seizure frequency and/or cognition and behavior. twelve patients aged 5-28 years were included from three different centers. neurosurgical tissue samples were available in all patients and revealed a brain somatic variant in slc35a2 in six patients (not-present in the blood). after 6 months of supplementation, d-galactose was well tolerated with just two patients presenting abdominal discomfort, solved after dose spacing or reduction. there was a 50 % reduction or higher of seizure frequency in 3/6 patients, with an improvement at eeg in 2/5 patients and a global responder rate of 6/6 in slc35a2-positive patients. one patient became seizure-free. an improvement of cognitive/behavioral features was also observed. in contrast, only three out of nine responders carried the slc35a2 wildtype gene and all non-responders were slc35a2 wildtype. their results suggested that supplementation with d-galactose in patients with moghe is safe and well tolerated and, although the efficacy data warranted larger studies, it might build a rationale for precision medicine after epilepsy surgery. topic 5: new diagnostic disease entities: ii. encephaloceles encephaloceles (enc) are protrusions of the neocortex and its meninges expanding into the diploic space of the skull bone (figure 5), i.e. the medullary cavity, leaving the outer skull table intact [4]. enc can occur in any location, most commonly in the frontal or parietal region [4]. they can develop congenitally, as will be discussed herein, which needs to be separated from a traumatic, neoplastic, inflammatory or iatrogenic origin. increased intracranial hypertension, e.g., empty sella or optic nerve sheath dilatation, female gender and obesity have been repetitively reported as risk factor for enc when occurring without a clear etiology. in patients with drug-resistant temporal lobe epilepsy (tle), enc are increasingly recognized in the temporo-polar and/or basal region of the brain. these are likely congenital enc and can vary in size from mm to cm, vary in number up to more than 10 in a focal region and affect either one or both hemispheres. very small enc are often seen only during surgery as they escape the current resolution scale of clinical routine mri. a recent paper from di giacomo and coworkers looked at electro-clinical, neuroimaging and histopathology features of their series of 12 patients with enc [16]. all patients were retrospectively identified by reviewing 3t mri scans. there were no exact numbers of enc given per patient and data from their patient table suggested one mri-visible enc was present in either one (n = 6 patients) or in both hemispheres (n = 6 patients). however, eight patients underwent neurosurgical resection of only one hemisphere following a careful neurophysiological video-eeg assessment of the seizure origin. all patients were postoperatively seizure free (engel class i). the surgical tissue specimens allowed for a systematic histopathological analysis, which revealed focal distortion of layer i, white matter extending into the neocortex and/or an altered gyral profile. considering the difficulty to anatomically reconstruct such small protrusions in a surgical sample, their analysis comprehensively describe enc as a common histopathology entity to be recognized and diagnosed within the lesional spectrum of epilepsy surgery. i found their description of a focal distortion of neocortical layer i quite interesting, as it reliably describes for the first time a disease condition with horizontally oriented cortical layer abnormalities compatible with fcd ilae type 1b. the fcd 1b subtype is neither well studied nor characterized yet and some people even question their existence due to the lack of a characteristic and consistent clinico-pathological phenotype. nevertheless, the authors did not claim such a fcd classification and we need to await further studies to better understand its likely neurodevelopmental origin and pathogenesis. a genetic or epigenetic characterization would also be helpful to assess their origin and specificity as new disease entity. notwithstanding, the clinical history available in their presented patient cohort may add to the disease description as a majority revealed mri positive intracranial hypertension (9 out of 12 patients), later onset of seizures compared to a control group of 26 patients with non-enc related tle, and more frequent psychiatric comorbidity. figure 5: intraoperative snapshot demonstrating multiple encephaloceles in the human temporal lobe figure 5. the intraoperative vision angle is directed towards the basis of the temporal pole in a 23-year old patient with left tle. there were several larger and smaller holes in the skull bone as indicated by the arrows, some of which were too small to be preoperatively detected by mri. all clefts were occupied by cortical brain tissue protrusions. the exact histopathology and/or genetic nature of these encephaloceles need further clarification. image not previously published and kindly provided prof. s. brander, neurosurgery dept. at klinikum fürth, germany. topic 6: what did we learn in 2022 and 2023 about leat patients suffering from low-grade epilepsy-associated brain tumors (leat) have a high propensity to become seizure and drug free after epilepsy surgery [28]. this is a promising observation factoring into every day's patient management and counseling. the question remains, however, why up to 30 % of patients with leat do not achieve sufficient seizure control postsurgically. unfortunately, this measure is not recognized in the who brain tumor classification scheme and does, therefore, not play a role in our current neurooncology-focused literature of low-grade brain tumors. this contrasts a recently published work from hoffmann and coworker presenting their series of 72 leat samples submitted to whole exome sequencing [21]. the same patients were presented also in the work of lópez-rivera described above (see topic #2). there were eight patient samples, in which the aforementioned ptpn11 gene was altered, mostly by a gain of the long arm of chromosome 12. these eight patients also showed chromosomal gains affecting other genes of the map-kinase pathway, e.g., shoc2, braf, kras, nf1 or fgfr4, representing a much more complex genotype than usually seen in the group of gg. another seven patients showed a similarly complex genotype including several genes of the map-kinase and mtor signaling pathway (figure 6). compared to their group of 29 patients with a gg and a braf v600e mutation, the group of gg with a complex genotype had a much lower seizure control two years after surgery (38 %). this contrasted the 85 % of successful postsurgical seizure control in the braf altered group of gg. histopathology analysis of this cohort revealed additional features of what the authors describe as atypical features in gg (analogue who cns grade 2), e.g., subarachnoidal tumor spread of predominantly astroglial and dysplastic neuronal phenotypes, as well as a consistent combination of cd34 and p16 immunoreactivity. finally, a new unsupervised dna methylation cluster analysis was presented including more tumor samples from previously published cohorts readily separating between gg, dysembryoplastic neuroepithelial tumors (dnt), low-grade glioma myb altered, and pleomorphic xanthoastrocytoma (pxa). there was a fifth cluster, however, including all of the ptpn11 altered gg and even more tumor samples, some of which also had an adverse postsurgical outcome (one patient died from sudep as seizures could not be controlled). figure 6: oncoplot of wes and cnv detection in ganglioglioma with clinically adverse outcome figure 6. columns 1-44 represent patient samples submitted to wes. these data were intentionally classified into three groups of patients: gg with complex genetic variants (cases 1-15), gg with non-complex genetic variants (cases 16-44), and gg without any detected genetic variants (cases 45-72, not shown). diagnosis: histopathology review confirmed the diagnosis of ganglioglioma (green tiles) in 43 cases. lack of seizure freedom (red tiles) was explicable by incomplete resection only in cases #9, #42, and #43 (greyish tiles). outcome data was missing in cases #15 and #18 (white tiles). figure collected from open-access paper published under a creative commons license, with permission from the authors. these results prompted the authors to suggest that the who panel may use a different strategy when classifying leat, at least reconsidering a grading scale which re-introduces who cns grade 2. as a reminder, previous editions of the who brain tumor classification included even anaplastic variants of gg. along these lines, reinhard and coworkers recently published their study addressing an internationally collected cohort of 54 anaplastic ganglioglioma, often including epilepsy, tumor relapse and second surgeries in the patient's histories, by using the existing heidelberg dna methylation classifier [35]. the majority of tumors designated as anaplastic gangliogliomas in the original histopathology report resolved into other who diagnoses, most commonly pxa (30 %) and glioblastoma, idh wildtype (20 %). only ten of their tumor samples were not assignable to a cns who diagnosis. it remains to be shown, however, if atypic or anaplastic ganglioglioma do not exist at all as a category defined also by dna methylation. the work of hoffmann et al. approved such an atypic category, but using a different bioinformatic programming pipeline [21]. it is the availability of enough and clinically well annotated tumor samples covering the entire landscape of leat that will make the difference. the issue of a lower tumor cell content in leat represents an additional challenge to be also addressed in any dna methylation brain tumor classifier. these conflicting data may also pave the way towards new approaches to classify brain tumors, e.g., using the real-time, direct sequencing technology [42], and that a single histopathology consultant may need to apply different systems to obtain a sound picture of the underlying tumor diagnosis. that clinical considerations play a major role in leat and that individual approaches in developing dna methylation classifier can also be concluded from a paper of stone and coworkers [40]. they asked the question if dna methylation is helpful to synergize the classification of leat into histopathology and neuroimaging similar subgroups and constructed a methylation-based support vector machine model for their prediction. they extended a previously published series now including 83 leat samples and comprising glio-neuronal tumors of two major phenotypes, i.e., gg and dnt [39]. these tumor entities indeed segregated into two groups. at the histopathological level, prominent astrocytic or oligodendrocyte-like components, dysplastic neurons or a specific glioneuronal element were important discriminators between both groups. at the neuroradiological level, the location, margin definition, enhancement and t2 flair-rim sign were successful discriminators. in a validation round their ai-based algorithm classified 22/23 samples correctly. the authors concluded the importance and superiority of an integrated diagnostic approach using histopathology, molecular data but also clinical information as our current diagnostic criteria inadequately reflect glioneuronal tumor biology and leave a proportion unresolvable. these recent papers continue to pose questions how to reliably classify and grade leat, and agree on the terminology use. continued disagreement will affect any interpretation of published research and also obstructs a better prediction of their clinical behavior beyond being regarded as benign who cns grade 1. topic 7: tauopathies in mesial temporal lobe epilepsy there is increasing research interest in tauopathies related to temporal lobe epilepsy. based on the original observations of abnormal tau phosphorylation in drug-resistant epilepsy [48] that aggregates into neuropil threads (nt) or neurofibrillary tangles (nft) in brain biopsies surgically resected from patients with drug-resistant epilepsies, there is an ongoing discussion about precocious aging in tle patients as a result from similar pathomechanisms observed in alzheimer's disease. this claim was not only spectacular at that time but may bridge the open question of ongoing and aggravating memory decline in drug-resistant tle patients. however, available research data does not fully support the hypothesis anymore as will be shown in a small compilation of three recently published papers. aroor and coworkers addressed the possibility that cognitive decline in epilepsy may be associated with mechanisms typical of alzheimer's disease (ad) [3]. they determined the abundance of phospho-tau and aβ proteins in association with cognitive function in 12 cases of drug-resistant epilepsy. indeed, they could observe a robust presence of tauphospho-related nt (ser202/thr205) and nft pathology, as well as aβ deposits, and phosphos6 (phosphorylation of s6 at ser240/244 or ser235/236 indicating an activation of the mtor complex) in the epileptic samples. they found no significant correlation, however, between tauphospho (thr205; thr181), aβ, or mtor markers with cognitive scores. these findings still support the existence of hyperphosphorylated tau protein and aβ deposits in patients with drug-refractory tle, but their relation to cognitive decline remains uncertain and requires further investigation. concepcion and coworkers studied the progressive dysregulation of tau phosphorylation in a rat pilocarpine status epilepticus (se) model of tle [14]. they measured tau expression at two months and four months after se. total tau levels were modestly reduced compared to naïve controls at two months post-se, but there was no significant reduction in s202/t205 phosphorylation. in the whole hippocampal formation from four month post-se rats, total tau expression had reverted to normal, but there was a significant reduction in ca1 and ca3 for s202/t205 tau phosphorylation. no change in phosphorylation was seen at the t181 and t231 tau loci. in somatosensory cortex, outside of the seizure onset zone, no changes in tau expression or phosphorylation were seen at the later time point. the authors concluded that total tau expression and phosphorylation did not show hyperphosphorylation at the three ad canonical tau loci tested in their animal model of tle. instead, the s202/t205 locus showed progressive dephosphorylation. this suggested that changes in tau expression may play a different role in tle than in ad. further studies are needed, therefore, to understand how such tau changes may impact neuronal excitability in chronic epilepsy. witt and coworkers approached this interesting topic from a neuropsychology standpoint claiming that the etiology of cognitive deficits in epilepsy is really multifactorial, the neuropathology of mtle rather multifaceted and beyond that of justifying the study of hyperphosphorylated tau [46]. indeed, the bidirectionality between epilepsy and dementia is of high interest, also from the epileptological perspective. however, there are many etiologies in tle that were associated with cognitive decline in epilepsy, as histopathology findings in mtle samples reported hippocampal sclerosis, dysplastic lesions, and neurodevelopmental neoplasm as major disease categories. last but not least, anti-seizure medication can also have adverse effects on cognition. these authors concluded that the neuropsychology and neuropathology of mtle is actually more complex than recently postulated [49]. the suggested model that ad-tle represent a specific disease subtype needs further validation to confirm the role of hyperphosphorylated tau in epilepsy patients with and without ad considering age and age at epilepsy onset as potential moderator variables. topic 8: 'hippocampal innate inflammatory gliosis only' in pharmacoresistant tle hippocampal sclerosis (hs) remains the most common structural brain lesion of all focal epilepsies and is the hallmark of temporal lobe epilepsy (tle). histopathology patterns vary, however, in this cohort of affected individuals and the ilae has developed a 3-tiered classification scheme based on the pattern of segmental neuronal cell loss in the ca regions [11]. there has always been a fourth category of 'no-hs, gliosis only', however. this category represented almost 20 % of the entire cohort of tle patients with surgical resection of the hippocampus and clinical histories were described as slightly different from the hs cohort, i.e. later seizure onset and later age at surgery, respectively. grote and coworkers have addressed this challenging category of 'no-hs, gliosis only' with a comprehensive and innovative clinico-pathological and molecular study design [19]. they reviewed a cohort of 627 patients with drug-resistant tle submitted to hippocampal resection. seventy samples showed 'no-hs, gliosis only' at the histopathology examination (11 %). indeed, a careful review of the clinical charts confirmed a significantly later onset of seizures when compared to hs (16.3 vs. 12.2 years, p < 0.005), and a significantly worse rate of seizure freedom after surgery (43 % vs. 68 %, p = 0.0001). more interestingly, however, was their rna sequencing analysis comparing no-hs vs. hs samples, which revealed a distinct transcriptional program visible only in the no-hs group and which suggested an innate inflammatory response of reactive astrocytes. notwithstanding, reactive astrocytes are the cellular correlate of 'gliosis only', histopathologically characterized by a diffuse astrogliosis pattern lacking restricted segmental focality and which is, therefore, poorly controllable by surgery. poorer postsurgical outcome in this cohort should alert any physician, therefore, when red flags occur during the presurgical evaluation, e.g., clinical and demographic features such as later disease onset, negative mri findings or bilateral epileptogenicity patterns (figure 7). a more adequate treatment algorithm should rather address the innate inflammatory tissue response, but these may not be readily available yet. however, the same group has published their results of fingolimod treatment in an experimental tle model targeting the innate inflammatory gliosis in 2019 [34]. fingolimod targets sphingosine-phosphate receptors (s1prs) and revealed robust anti-convulsive activity in kainate-induced se mice. in addition, it had neuroprotective and anti-gliotic effects and reduced cytotoxic t cell infiltrates suggesting repurposing of fingolimod (previously known as suitable drug in multiple sclerosis treatment) as novel therapeutic option in focal epilepsies. it is hoped, therefore, that patients with no-hs can be identified early on during the presurgical evaluation phase to carefully consider all therapeutic options. figure 7: graphical summary of the differences between hippocampal sclerosis and innate inflammatory 'gliosis only' (i²go) figure 7. the authors claim that i²go constitutes a distinct mtle syndrome with characteristic clinical and pathological features. i²go is less amenable by surgery and bears a greater hazard for postoperative neuropsychological deterioration. 'gliosis only' is the neuropathological hallmark of i²go and shows a unique transcriptional signature marked by an astrocyte-mediated chronic inflammation pattern. figure collected from open-access paper published under a creative commons license, with permission from the authors. topic 9: altered adult neurogenesis and gliogenesis in patients with mesial tle the impact of gliogenesis and reactive gliosis in epileptogenic human tissue has also been addressed in a seminal paper published by ammothumkandy and coworkers [2]. they used immunofluorescence microscopy in human surgical hippocampus obtained from 19 mtle patients, performed neuronal stem-cell cultures and multi-electrode array recordings of ex vivo hippocampal slices to address the hitherto open question if and how aberrant neurogenesis and gliogenesis play a role in seizure initiation and progression. this has been repetitively reported in rodent animal models but needs further confirmation in the human disease condition. as reported in previous studies (see below), the authors confirmed a decline of neurogenesis in tle patients when their disease had a longer duration. these data were obtained from fixed tissue specimens as well as in vitro from neural stem cell cultures. colocalization of dcx+ and prox1+ immunoreactivity was used to recognize immature dentate granule cells and by comparing biopsy tissue with age-matched postmortem controls. in contrast, gliogenesis remained persistently high even in the chronic epileptic condition. immature astroglia were characterized by dcx+/prox1and gfap+ immunoreactivity compared to their mature counterpart being only gfap+ immunoreactive. 400 µm thick slices of the human hippocampus were then used for multi-electrode array recordings followed by immunofluorescence stainings of the aforementioned cell marker sets. based on these findings they classified tissue regions as active or non-active. neurogenesis was convincingly proven only in neurophysiologically non-active tissues but not in tissue with epileptiform activity. they concluded from this set of experiments that newborn neurons do not contribute to epileptiform activity at chronic stages of mtle, which was further corroborated by low levels of immediate early gene expression in immature neurons. this situation was different when looking at immature astroglia, which were present in all mtle cases, but their location and activity was very much associated with epileptiform activity. specifically, their results indicated that immature astrocytes can migrate towards neuronal activity, e.g., the hilus, or directly contribute to generating seizures. their results suggest an intriguing hypothesis that immature astroglia play a role as 'lossy capacitor' in initiating coordinated epileptiform activity. astrocytes would usually prevent excessive neurotransmission and limit neuronal hyperactivity by soaking calcium intracellularly and then activate inhibitory gabaergic interneurons [15, 32]. once immature astroglia are unable to maintain extracellular ionic homeostasis, functional alterations in their activity lead to aberrant modulation of the network. as mentioned in the chapter above, these results offer a new avenue in rather targeting reactive astroglia, e.g., immature astroglia as described by ammothumkandy or those described above as reactive astroglia with an innate inflammatory response. it needs to be shown, however, if these astroglial populations are similar to each other or present a new cell type specific to the disease condition. topic 10: autoimmune encephalitis and norse in my final chapter, i would like to highlight a paper addressing the impact of autoimmunity to the pathogenesis of tle. tle is one of the syndromes linked to autoantibodies, such as anti-n-methyl-d-aspartate (nmda), anti-leucine-rich glioma-inactivated protein 1 (gli1), or glutamic acid decarboxylase (gad). tröscher and coworker specifically addressed the latter syndrome of gad-tle, in which the immunopathogenesis remains enigmatic [41]. they reviewed imaging, serum and cerebral spinal fluid analysis and brain tissue in a consecutive series of 15 patients with gad-tle, including immunohistochemistry, multiplex fluorescent microscopy and transcriptomic analysis for inflammatory mediators and neuronal degeneration. in nine of their patients, they observed medio-temporal swelling and t2 signal increase within the first 6 years after the onset of symptoms, and all developed unilateral or bilateral hippocampal sclerosis. early csf analysis < 6 years after onset revealed intrathecal synthesis of iggs. there were also high numbers of plasma cells in surgical brain tissue but no evidence for complement-mediated tissue damage. in contrast, there was a dense infiltration of cd8+ cytotoxic t cells, some of which had a specific resident memory t cell phenotype. cytotoxic granzyme b-positive t cells attached to neurons were also recognized at the microscopic level. their data convincingly showed that patients with gad-tle go through an early encephalitic stage (< 6 years) with cd8+ cytotoxic t lymphocytes mediated, antigen-driven neuronal cell loss but without signs of complement-mediated cell death. subsequently, patients enter an apparently immunological inactive or low-active stage with ongoing seizures, probably caused by the structural damage to the temporal lobe. the observation of such an early stage of tissue damage is clinically most helpful to understand why immunotherapies usually do not lead to seizure freedom. a review from bien and bauer further highlighted what neuropathology teaches us about autoimmune encephalitis, e.g., nmda and gli1, autoimmune associated epilepsies including rasmussen, paraneoplastic or gad-tle [8]. the authors argue in favor of a research-driven neuropathology approach to better understand these diseases. it is so important to receive morphological as well as temporal information over disease periods, and which can best be acquired by studying the affected brain tissue. molecular technique will then broaden and support these data. last but not least is a comprehensive neuropathology description of new-onset refractory status epilepticus (norse), including its subtype with a preceding febrile illness also known as fires (febrile infection-related epilepsy syndrome). these conditions represent a most severe emergency condition in epileptology, e.g. status epilepticus (se). indeed, the prognosis of norse has a high rate of mortality. as a matter of fact, most cases of norse remain inexplicable despite extensive clinical evaluations, eeg studies, neuroimaging and laboratory testing. as mentioned above, neuropathology evaluations conducted in biopsies or autopsy tissue are helpful, therefore, to better identify an etiology in norse or fires. hanin and coworkers collected published data from 64 cases of norse and fires, including 66 neuropathology tissue samples: 37 biopsies, 18 autopsies, 7 epilepsy surgery samples, 4 not otherwise specified [20]. in general, se associated neuropathology findings include neuronal cell loss in the hippocampus, cerebellum and thalamus associated with reactive gliosis and often also microglial activation. same findings can be observed in norse and fires, with the addition of perivascular t-cell infiltration in a number of cases. these lymphocytic infiltrates are similar to those described above, mainly being cytotoxic t cells. they have been confirmed by the neuropathological examination in 16 % of published cases and helped to solve the etiology. these histopathology findings also triggered a different therapeutic approach, including a high dose pulse of steroids or even tacrolimus, although this treatment had not resolved the symptoms. meanwhile, autoimmune encephalitis has been identified as most frequent cause of norse and fires, and which call for more studies to better identify the molecular target for a successful therapy. thus, this series of 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19: 2697-2706. https://doi.org/10.1002/alz.12943 copyright: © 2024 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. golgi localization of sars-cov-2 spike protein and interaction with furin in cerebral covid-19 microangiopathy: a clue to the central nervous system involvement? feel free to add comments by clicking these icons on the sidebar free neuropathology 4:1 (2023) original paper golgi localization of sars-cov-2 spike protein and interaction with furin in cerebral covid-19 microangiopathy: a clue to the central nervous system involvement? susana boluda1,2, karima mokhtari1,2, bruno mégarbane3, djillali annane4, bertrand mathon5, albert cao6, clovis adam7, alexandre androuin2, franck bielle1,2, guy brochier1,8, frédéric charlotte9, lydia chougar10, khalid hamid el hachimi2,11, marc eloit12, stéphane haïk1,2, dominique hervé13, amal kasri2, valentin leducq14, stéphane lehéricy10, etienne levavasseur1,2, christian lobsiger2, geoffroy lorin de la grandmaison15, isabelle malet14, isabelle malissin3, stéphane marot14, serge marty2, philippe pérot12, isabelle plu1,2, annick prigent2, lev stimmer2, marie-claude potier2, anne-geneviève marcelin14, benoît delatour2, charles duyckaerts1,2, danielle seilhean1,2 1department of neuropathology, pitié-salpêtrière hospital, ap-hp sorbonne university, paris, france 2institut du cerveau paris brain institute icm, inserm u1127, cnrs umr7225, aphp, sorbonne university, pitié-salpêtrière hospital, paris, france 3department of medical and toxicological critical care, lariboisière hospital, ap-hp, paris university, inserm umrs-1144, paris, france 4department of critical care, raymond poincaré hospital, boulevard raymond poincaré, aphp, paris-saclay university, inserm u1173, garches, france 5department of neurosurgery, pitié-salpêtrière hospital, ap-hp sorbonne university, paris, france 6department of neurology, neuro-icu, pitié-salpêtrière hospital, ap-hp sorbonne university, paris, france 7department of pathology, bicêtre hospital, ap-hp, paris saclay university, le kremlin-bicêtre, france 8institut de myologie, pitié-salpêtrière hospital, paris, france 9department of pathology, pitié-salpêtrière hospital, ap-hp sorbonne university, paris, france 10department of neuroimaging, pitié-salpètrière hospital, ap-hp sorbonne university, paris, france 11ecole pratique des hautes etudes (ephe), paris sciences et lettres (psl) university, paris, france 12institut pasteur, pathogen discovery laboratory, paris, france 13department of neurology, lariboisière hospital, ap-hp nordparis university, paris, france 14department of virology, pitié-salpêtrière hospital, ap-hp sorbonne university, inserm 1136, institut pierre louis d’epidémiologie et de santé publique (iplesp), paris, france 15department of forensic medicine and pathology, raymond poincaré hospital, ap-hp, paris saclay university, garches, france corresponding author: danielle seilhean · department of neuropathology · pitié-salpêtrière hospital · ap-hp sorbonne university · paris · france danielle.seilhean@gmail.com additional resources and electronic supplementary material: supplementary material submitted: 04 january 2023 accepted: 06 february 2023 copyedited by: georg haase published: 10 february 2023 keywords: covid-19, neuro-covid, microangiopathy, furin, blood-brain barrier (bbb) https://doi.org/10.17879/freeneuropathology-2023-4584 abstract in a neuropathological series of 20 covid-19 cases, we analyzed six cases (three biopsies and three autopsies) with multiple foci predominantly affecting the white matter as shown by mri. the cases presented with microhemorrhages evocative of small artery diseases. this covid-19 associated cerebral microangiopathy (ccm) was characterized by perivascular changes: arterioles were surrounded by vacuolized tissue, clustered macrophages, large axonal swellings and a crown arrangement of aquaporin-4 immunoreactivity. there was evidence of blood-brain-barrier leakage. fibrinoid necrosis, vascular occlusion, perivascular cuffing and demyelination were absent. while no viral particle or viral rna was found in the brain, the sars-cov-2 spike protein was detected in the golgi apparatus of brain endothelial cells where it closely associated with furin, a host protease known to play a key role in virus replication. endothelial cells in culture were not permissive to sars-cov-2 replication. the distribution of the spike protein in brain endothelial cells differed from that observed in pneumocytes. in the latter, the diffuse cytoplasmic labeling suggested a complete replication cycle with viral release, notably through the lysosomal pathway. in contrast, in cerebral endothelial cells the excretion cycle was blocked in the golgi apparatus. interruption of the excretion cycle could explain the difficulty of sars-cov-2 to infect endothelial cells in vitro and to produce viral rna in the brain. specific metabolism of the virus in brain endothelial cells could weaken the cell walls and eventually lead to the characteristic lesions of covid-19 associated cerebral microangiopathy. furin as a modulator of vascular permeability could provide some clues for the control of late effects of microangiopathy. introduction many questions remain open regarding the frequent and diverse involvement of the central nervous system (cns) by sars-cov-2 [11, 27, 49]. inflammatory infiltrates have been found, especially in the brainstem [28], and have been taken as evidence of encephalitis [28, 54]. nevertheless, in most cases, inflammation is inconspicuous or even absent [10, 15, 47]. in some cases, direct neuroinvasion by the virus has been suggested or observed. the virus may invade the brain through the olfactory mucosa and the nerve processes that cross the cribriform plate [13, 32]. bitemporal necrosis found at ct scan and mri, similar to the one seen in herpetic encephalitis, has been reported [34, 36, 43]. the presence of the virus genome has been demonstrated in the olfactory mucosa in a subset of patients (21 among 40 patients for matschke et al.[28], four among 20 for serrano et al.[47]). although rarely, the sars-cov-2 virus genome has also been observed in various regions of the cns including the olfactory bulb, brainstem, temporal cortex, amygdala and leptomeninges [28, 44, 47]. even though the possibility of direct neuroinvasion has been confirmed by some experi-mental data [50], the viral genome turned out to be difficult to detect in the human cns [47, 49], except in a recent study that found viral genome throughout the brain and other organs by droplet digital polymerase chain reaction (ddpcr)[52]. multiple lesions evocative of acute disseminated encephalomyelitis (adem) have been described [37, 45]. anti-aquaporin-4 circulating antibodies have been found as in neuromyelitis optica [8]. this observation emphasizes the role of secondary immune mechanisms and downplays the direct impact of the virus in the cns. large vessel occlusions occur especially in young covid-19 patients [26, 40]. in some cases, the lesions also involve the brain microvasculature. these cases of covid-19 associated cerebral microangiopathy (ccm) present with multiple, often hemorrhagic lesions, evocative of small artery disease [6, 7, 22]. histopathological studies show micro-infarcts, the mechanism of which is still under debate [16, 18, 21, 24]. in the present paper, six cases of ccm were histopathologically examined and the mechanisms of the lesions were investigated. while these cases did not display viral genomes or viral particles detectable by molecular techniques, they showed the presence of spike protein (sp) in the endothelial vessels. sp protein was consistently associated to furin. furin is a serine protease involved in the metabolism of the spike protein (sp) of sars-cov-2. it cleaves sp and plays a key role in virus entry and viral protein biogenesis [17, 20, 39, 48]. we investigated the subcellular localization of furin and sp in the lesions of ccm. material and methods patients the study received approval from the sorbonne university ethic committee (cer-202028). twenty cases of acute covid-19 infection collected during 2020, corresponding to the first two waves of the pandemic, were analyzed: 13 cases in the neuropathology department of pitié-salpêtrière hospital (ten autopsies and three cerebral biopsies); seven others in the pathology department of bicêtre hospital, after autopsy at raymond poincaré hospital, garches. a magnetic resonance imaging (mri) was performed either ante-mortem or post-mortem in all the pitié-salpêtrière cases and ante-mortem in one patient from bicêtre hospital. for those cases in bicêtre hospital without mri, inclusion was based on histopathological criteria. the three biopsied cases (patients # 1 to 3, table 1, fig.1) and two autopsied cases (patients # 4 and 5), presented with multifocal hypersignals in the deep white matter, evocative of microbleeds. microbleeds were seen in patient # 6 at histopathological examination (table 1, fig. 1, supplementary table). fig 1. cerebral microangiopathy with multiple microhemorrhagic foci. magnetic resonance imaging. case # 1 (a d), case # 2 (e h), case # 3 (i l) and case # 4 (m p). multi-territory recent ischemic lesions with reduced apparent diffusion coefficient were detected on dwi diffusion-weighted (left column a, e, i, m) and flair images (middle left column, b, f, j, n), with frequent contrast enhancement on t1-weighted images (middle right column, c, g, k, o). susceptibility-weighted images showed numerous microbleeds in cortico-subcortical areas and deep hemispheric white matter (swi, right column, d, h, l, p). brain biopsies. case # 1 (q-s), case # 2 (t-v), case # 3 (w-y). (q) sma (red)-cd163 (brown) double labeling showing a macrophagic infiltrate around a vessel; (r) mbp (brown)-nf (red) with hematoxylin (blue) counterstain showing no demyelination but bleeding; (s) a few cd8+ lymphocytes are observed within the microhemorrhagic foci (q, r, s : scale bars = 80 μm). needle biopsy sample showing focal lesions in the white matter: (t) he-luxol fast blue; (u) cd163 for macrophages (t, u: scale bar = 400 µm). (v) mbp (brown)-nf (red) with hematoxylin (blue) counterstain showing an area rich in macrophages without accumulation of myelin debris (scale bar = 40 µm). (w) foci of vacuolated tissue around a vessel (h&e, scale bar = 80 μm). (x) axonal damage was severe within the foci with numerous axonal swelling and vacuolization; (y) at the periphery of the lesion occasional glial nuclear alterations (arrow), and apoptotic nuclei (arrow head) were noticed (x, y: h&e, scale bars = 20 µm). table 1: clinical data bmi: body-mass index; csf: cerebrospinal fluid. regarding the remaining autopsied cases, three presented with deep venous thrombosis and the others did not show cns involvement on mri. in total, six cases of covid-19 infection with white matter anomalies were analyzed in the present study. the clinical histories of the selected patients showed similarities and are summarized in table 1. the age of the patients ranged from 37 to 77 years (mean: 54.8 years). all of them were males, except for patient # 3. they all presented at least one risk factor: obesity, hypertension or both, but none of them was diabetic. patient # 2 was under immunosuppressive treatment as he had a kidney transplantation. seven to ten days after the first covid-19 symptoms, the patients were admitted to the hospital with acute respiratory distress syndrome. diagnosis was made by detecting sars-cov-2 rna with real time-polymerase chain reaction (rt-pcr) in samples from nasopharyngeal swabs. the patients were sedated, mechanically ventilated and received corticosteroids and supportive care, which improved their respiratory conditions seven to 33 days later. despite sedative drug discontinuation, awakening was unsatisfactory: the patients opened their eyes spontaneously but they did not respond to simple orders. brainstem reflexes were preserved. skew eye deviation and left internuclear ophtalmoplegia were observed in patient # 2. no alternative metabolic or drug-related causes were found to explain impaired consciousness. patients # 2 and 6 were treated with extracorporeal membrane oxygenation (ecmo). multiple punctate lesions predominating in the white matter were seen in patients # 1 to 5, for whom mri was available (fig. 1, a-p). the therapeutic management of patients # 1 to 3 has been described in a previous work [4]. control samples autopsy samples from 16 cases with various conditions were used as controls. they included samples from normal brains (n=4), covid-19 patients without brain lesions (n=4), cases with acute disseminated encephalomyelitis (adem, n=1) or multiple sclerosis (n=1) and cases with vascular lesions of various causes: watershed infarcts (n=2), post ecmo hypoxemia (n=2), endovascular lymphoma (n=1), amyloid microangiopathy thal type 1 (n=2), in which recent hemorrhages and ischemic lesions were seen. immunohistochemistry for spike protein and furin was done in the frontal white matter, striatum and brainstem in all cases. biopsy and autopsy tissue processing the autopsies were authorized according to french current regulation and the use of samples for research was authorized by the next of kin. the samples were collected and stored according to the covid tissue bank (covitis) protocol, approved by the national biomedical agency and the french ministry of research (agence de la biomédecine, pfs 20-008; french ministry of research dc2020-4022). the protocol includes sampling of the brain, lungs, heart, trachea, lymph nodes, tonsils, spleen, liver, pancreas, guts, bladder, kidneys, skin, muscles, nerves and bone marrow. each sample was prepared in three ways: 1) fixed in formaldehyde and paraffin embedded (ffpe); 2) snap frozen in liquid nitrogen cooled isopentane for cryostat sections; 3) directly frozen at -80 °c for molecular biology. in cases # 5 and 6 the frozen extra-cerebral samples were limited to the lung. for brain sampling, the cerebral hemispheres were separated immediately after removal by a section across the corpus callosum; the sagittal section was extended to the brainstem and the cerebellum. the right hemi-brain was sliced into coronal sections. samples from the olfactory bulb and piriform gyrus, frontal, temporal, occipital and hippocampal cortices, corona radiata white matter, striatum, thalamus, amygdala, cerebellum, mesencephalon, pons, medulla oblongata and leptomeninges were fast-frozen at -80°c. nearby samples were fixed for 15 days in buffered 4% formaldehyde, then immersed one hour in formic acid (a systematic measure in the laboratory to decrease the infectivity of a potential prion disease), before paraffin embedding, cutting and staining. the left hemi-brain was immersed for one month in buffered 4% formaldehyde and the same regions as in the right hemi-brain were sampled. after one-hour immersion in a 90% formic acid solution, the samples were embedded in paraffin and cut at a thickness of 3 µm. the biopsy samples were fixed for eight hours in 4% formaldehyde, directly embedded in paraffin without a formic acid pretreatment and cut at a thickness of 4 µm. in cases # 1 to 4, brain tissue was fast frozen at -80°c for next generation sequencing (ngs). staining and immunohistochemistry (ihc) tissue sections from the biopsy samples and selected regions of the autopsy cases were stained with hematoxylin-eosin (h&e), perls’ method, orcein, masson’s trichrome, martius scarlet blue, periodic acid schiff (pas), gram’s and grocott’s technique. in addition, sections from selected regions of the autopsy cases were stained with h&e combined with luxol fast blue. some sections were immunostained with a ventana benchmark stainer (rochetm). the biotinylated secondary antibody was included in the detection kit (ventana medical systems basic dab detection kit 250-001). diaminobenzidine (dab), occasionally combined with alkaline phosphatase (alp) for double labelling, was used as a chromogen. the pretreatment and the antibodies used for ihc are listed in table 2. table 2: immunohistochemical methods cc1, a proprietary high ph (=8) buffer at 95°c; cc2, a proprietary ph (=6) buffer; fa: formic acid; rt: room temperature. multispectral microscopy ffpe sections from the frontal cortex (case # 4) were doubly immunostained against sars-cov-2 sp (dab chromogenic substrate) and giantin (fast red fr chromogen). to separate dab and fr signals, the sections were analyzed with the mantratm multispectral imaging station and the inform software (akoya biosciences). dab and fr signals were each associated with a pseudo-color in order to distinguish sp and giantin immunolabelling. confocal microscopy to determine the intracellular localization of sp protein in the cells and its interaction with furin, ffpe sections from the frontal cortex of cases # 4, 5 and 6 and from the lung (case # 4) were immunostained with the sp antibody (revealed with secondary antibody coupled with alexa fluor-af488), and doubly labelled with antibodies against bip, giantin, furin, tgn46 or cathepsin b (table 2), revealed with secondary antibody coupled with alexa fluor-af555. the sections were counterstained with dapi. distribution and colocalization of immunofluorescent signals were analyzed with a confocal microscope (leica sp8 dls). electron microscopy in patient # 4, 1-mm3 samples from altered white matter were fixed immediately with glutaraldehyde (2.5%, ph 7.4), post fixed with osmium tetroxide (2%), dehydrated and embedded in resin. ultra-thin sections were obtained at different depths from three blocks and stained with uranyl acetate and lead citrate. the grids were observed using a hitachi ht 7700 electron microscope (elexiencetm, verrières-le-buisson, france) operating at 100kv. molecular sars-cov-2 analyses various morphological methods (in situ hybridization-ish, rnascope) and non-morphological methods (rt-pcr, ngs) were used to detect sars-cov-2 virus rna in the tissues. rt – pcr (real time polymerase chain reaction) to detect viral rna in cases # 1 to 3, samples snap-frozen at -80°c were used. in case # 4, for which abundance of tissue was available, samples snap-frozen at -80°c from nine regions of the central nervous system and from multiple extra cerebral tissues were tested (table 3). total rna was extracted with the rneasy mini kit (qiagentm), after a mechanical disruption with tissuelyserlt (quiagentm). total rna samples were treated by dnase to avoid residual genomic dna contamination. rt-pcr was subsequently performed using the realstar sars-cov-2 rt-pcr kit (altona diagnosticstm) to estimate the sars-cov-2 viral load. table 3: post mortem rt pcr autopsy case # 4 next generation sequencing (ngs) ngs-based transcriptomic analysis to search for viral transcripts was used in tissue samples from four cases (cases # 1 to 4) as previously described [42]. this technique is agnostic and provides sequences of the human transcriptome, possibly mixed with exogenous sequences of pathogens. those are identified by comparison with the sequences of known microbes. in addition, ngs-based target capture was used to increase the sensitivity towards sars-cov-2 rna (twist biosciencestm, san francisco, usa). ish (in situ hybridization) to explore further the presence of virus in various tissues, ish was performed as previously described [42] on ffpe sections of lung and frontal lobe in case # 4. viewrna ish tissue assay kit 2-plex (thermo fisher scientifictm) was used with a cocktail of custom branched dna (bdna) probes targeting six segments of known opening reading frames (orfs) for covid-19 wuhan-hu-1 strain based on genbank mn909847 and a mix of control probes targeting human gapdh, actb and ppi transcripts. the oligonucleotide probes were conjugated to alkaline phosphatase. chromogens were fast red (for sars-cov-2 probe) and fast blue (for the control probes). rnascope in another attempt to detect viral rna in the autopsy samples fixed for 15 days, ffpe rna ish was performed using the rnascope tm sars-cov-2 probes for the s gene encoding sp (catalogue #848561, advanced cell diagnosticstm) and the rnascope tm 2.5 hd reagent kit-browntm (catalogue #322300). rna integrity in tissue samples was controlled by a preliminary step using the rnascope tm positive control probe hs-ubc (catalogue #310041). all experiments were carried out manually according to the manufacturer’s instructions. cell culture to improve our understanding of the interaction of the virus with endothelial cells and furin, cultures of four cell lines were used: human endothelial cells derived from induced pluripotent stem cells (ipsc-d-ecs), a human cerebral microvessel endothelial cell line (hcmec/d3), a human umbilical vein endothelial cell line (huvec) and a primate kidney cell line (vero e6 cells). the control ipsc-d-ecs line (welcome trust sanger institute, hipsci, cell line name: bhipsci0914i-zerv_8 catalogue number: 77650327) was differentiated into endothelial cells from an in vitro monolayer following a previously developed protocol [41]. after differentiation, the ipsc-d-ecs were purified using fluorescence activated cell sorting (facs). cells were incubated with either alexa fluor 488-conjugated cd31 (pecam1) antibody for 30 minutes, or isotype-matched secondary antibody in the absence of primary antibody serving as negative control. the purified ipsc-d-ecs were expanded in endothelial growth medium-2 (egm-2 bullet kit, #cc86 3202, lonza tm) and characterized by immunofluorescence for the expression of the endothelial markers cd31/pecam1, cd144/ve-cadherin and von willebrand factor. the hcmec/d3 was kindly donated by p. o. couraud from the cochin institute, université de paris, france. it was derived from microvessels obtained from tissue excised during surgery for epilepsy. the cells were immortalized by lentiviral transduction with the catalytic subunit of human telomerase (htert) and sv40 large t antigen [56]. these cells, which express a brain endothelium phenotype, were cultured as previously described [55]. ipsc-d-ecs and hcmec/d3 were grown on surfaces coated with matrigel (corningtm #354277) or type i collagen (corningtm, #354236: collagen i, rat tail natural 100mg), respectively. the cells were cultured at 37°c with 5% co2 in egm-2 medium, supplemented with 0.1% penicillin-streptomycin (p/s; gibcotm). huvec cells (a gift from the lab of pr. yong chen) were grown at 37°c with 5% co2 in egmtm -2 bulletkittm medium with 0.1% p/s. at passage 7 the cells were seeded on 12-well plate on coverslips (12 mm). vero e6 cells (atcc no. crl-1586) correspond to a cell line isolated from kidney epithelial cells of african green monkey. the cells have lost the type i interferon gene cluster and are particularly sensitive to viral infection. they were used as controls for the in vitro infection by sars-cov-2 viruses. the line was cultured in dulbecco’s modified eagle’s medium (dmem), supplemented with 10% fetal bovine serum (fbs), 1% non-essential amino-acids and 1% p/s (gibcotm). in vitro infection of ipsc-d-ec and hcmec/d3 cell line ipsc-d-ecs at passage 2 (p2) or hcmec/d3 cell line at p33 were seeded on coated glass coverslips. at 50 % of confluence, the ipsc-d-ec or hcmec/d3 were incubated with 0.1, 0.5 and 1 multiplicities of infection (moi). the supernatants and the cells were harvested at 48, 72 and 96 hours. viral rna (gene e) was quantified by rt-pcr in the supernatant and in the intra-cellular compartment. vero e6 cells were used as controls. cell fixation and immunofluorescence the cells were fixed with phosphate buffer saline (pbs)-4% paraformaldehyde (electron microscopy sciences) at room temperature, then quenched for 10 minutes with 50 mm pbs-nh4cl. for permeabilization, cells were incubated in pbs supplemented with 2 g/l bovine serum albumin (bsa) and 0.5 g/l saponin for 20 minutes at room temperature. the antibodies were diluted in the permeabilization buffer. the cells were immunostained with the polyclonal rabbit anti-sp antibody (abcam, #ab272504, 1:300) for one hour in a humid chamber at room temperature. after washes with pbs, the cells were incubated with alexa fluor-555 anti-rabbit secondary antibody (1:500) and co-stained with dapi (vector laboratories, 1:10000). slides were mounted in vectashieldtm antifade medium and observed with a nikon a1r hd25 confocal microscope with resonant scanning at the x60 objective. cell incubation with sp peptides huvec cells were incubated with recombinant sp s1 full length peptide (val16-arg685 ; prosci #10-300) and control rbd peptide (arg319-phe541 ; prosci #10-303), as described in nuovo et al. [38]. in brief, cells were incubated with 70ng/ml of s1 and rbd peptide for 48 hours. cell fixation was performed with paraformaldehyde 4% for 10 min at room temperature. for permeabilization, cells were incubated in pbs supplemented with bsa 2g/l and saponin 0.5g/l for 20 min at room temperature. rabbit anti -s1 (prosci 9083) and anti rbd (prosci 9087) were incubated in the permeabilization buffer. the secondary antibody was an anti-rabbit conjugated to alexa fluor 568. coverslips were mounted in vectashield antifade mounting medium. results microangiopathy seen as microhemorrhages of small arteries is a common trait in a subset of sars-cov-2 infected patients. as mentioned previously, radiological findings shared common features in the cases for whom mri was available (fig. 1, a-p). the same was true for neuropathology. in the biopsy samples, multiple well-delineated foci of cd163 positive macrophages were observed in the white matter (fig. 1. q, t, u, v). macrophages were clustered around small caliber blood vessels (fig. 1. q, r, w). they were not laden with luxophilic, myelin basic protein (mbp) positive debris, nor was there myelino-axonal dissociation (fig. 1. r, v). cd3 cd8 positive t lymphocytes were scattered among the macrophages and around the foci (fig 1. s ). in the foci, numerous and large axonal swellings were seen and white matter was vacuolized (fig. 1. x). at the periphery of the lesions, apoptotic nuclei and occasional glial nuclear alterations, reminiscent of creutzfeldt-peters cells, were seen (fig. 1. y). some recent blood extravasation (fig. 1. r) and a few siderophages were observed. there was no fibrinoid necrosis or inflammatory cells seen within the vessel walls. we did not observe vascular occlusion, lymphocytic perivascular cuffing or neuronophagia. ihc against the proteins of herpes simplex virus 1, cytomegalovirus, varicella-zoster virus, polyomavirus, measles and toxoplasma gondii was negative, as was negative ish with the epstein-barr virus eber probe. the lesions observed on the biopsies were more thoroughly studied in the autopsy cases that provided more material. macroscopic examination of the three autopsied brains was characterized by multiple hemorrhages scattered in the deep and subcortical white matter. in case # 4 these were predominant in the supramarginal gyrus, first and second temporal gyri, where they were particularly numerous, and also in the pons and cerebellum. there was a 4 mm hemorrhage in the left occipital pole. in cases # 5 and # 6 the hemorrhages were predominant in the frontal lobe with additional bleeds in the striatum (case # 5) and in the pons (case # 6). microscopic examination of the autopsied cases showed similar lesions as those seen in the biopsies. the most conspicuous lesions were small foci rich in cd163 positive macrophages centered by a small caliber arteriole (fig. 2. a, b, c) associated with swollen axons and vacuolization of the surrounding white matter (fig. 2. d, e). as in the biopsies, there was no lymphocytic perivascular cuffing, demyelination, vascular occlusion or fibrinoid necrosis. compared to biopsy cases, perivascular bleeding was more frequent and of different age (fig. 2. f, g). fibrinogen and igg ihc were positive in the lesions, evidencing blood-brain barrier leakage (fig. 2. h). large reactive astrocytes formed aquaporin-4 (aqp4) positive rings around the lesions, contrasting with a low immunoreactivity in the normal looking white matter (fig. 2. i, j). aβ ihc was negative indicating absence of amyloid angiopathy. in summary, the lesions of the white matter can be described as ccm. fig 2. case # 4 : neuropathology. (a) small foci of necrosis in the white matter were centered by a small caliber blood vessel. h&e (a : scale bar = 40 µm). (b and c) double labelling of sma (in red) and of cd163 (in brown) labeled the compact wall of an arteriole surrounded by macrophages (b: scale bar = 80 µm; c: scale bar = 40 µm). (d and e) within the foci, swollen axons were numerous (d: he; scale bar = 20 µm), associated with a vacuolization of the cerebral tissue (e: he-luxol; scale bar = 80 µm). (f) some lesions were centered by a recent hemorrhage (f: he-luxol; scale bar = 80 µm). (g) perls positive siderophages indicated older bleedings (g: scale bar = 80 µm). (h) fibrinogen immunoreactivity was evidence of blood brain barrier leakage (h: scale bar = 80 µm). (i) aqp4 immunoreactivity showed positive rings (arrows) in the subcortical and deep white matter (i: scale bar = 5 mm). (j) large reactive astrocytes formed aqp4 positive rings around the lesions (arrows), contrasting with the depletion of aqp4 in the surrounding white matter * (j: scale bar = 400 µm). for the detection of sars-cov-2, two different antibodies were used, yielding similar results in the biopsied and autopsied cases. within the foci, the hemorrhage and cellular debris interfered with a thorough analysis of endothelial cells. however at a short distance from the hemorrhagic foci, both antibodies labelled numerous inclusions in endothelial cells of apparently spared vessels (fig. 3. a, b). most positive vessels where observed in the frontal, parietal and temporal white matter, although some labeling was observed in the brain stem and striatum in autopsied cases. with the monoclonal anti-n antibody, immunoreactivity was predominant in endothelial cells, with a diffuse endothelial labelling and para-nuclear enhancement (fig. 3. a). with the monoclonal antibody directed against sp (fig. 3. b), the labelling was paranuclear in a topography suggestive of the golgi apparatus. in the cortical gray matter, a few sp positive dots were also seen in the endothelial cells, however, no sign of microangiopathy was detected. thus, ccm appeared to be confined to the white matter. in the olfactory bulb, sp immunoreactivity was detected in the endothelial cells only (fig. 3. c), as in the other regions of the nervous system. in the choroid plexus, around 20% of the villi’s epithelial cells contained sp (fig. 3. d). the intracellular localization was similar to what was previously seen. no sp was observed in the covid-19 cases that did not develop brain vascular lesions. fig 3. localization of sars-cov-2 in brain tissue (case # 4). (a and b) sars-cov-2 was detected in the endothelial cells with two different antibodies (a, b: scale bars = 20 µm). with the anti-n antibody from clinisciencetm the immunoreactivity was predominant in endothelial cells, with a diffuse endothelial labelling and paranuclear enhancement (a, arrowhead). with the abcamtm anti-sp antibody, immunoreactivity was limited to the endothelial cells with paranuclear inclusions (b, arrowheads). inset: higher magnification of cells indicated by arrow heads. (c) immunoreactivity limited to paranuclear inclusions was also found with the abcamtm anti-sp antibody in the endothelial cells of the olfactory bulb (c, arrowheads) and (d) in the epithelial cells of the choroid plexus (d, arrowheads) (c, d: scale bars = 8 µm). anti-giantin (e) and anti-furin (f) immunoreactivity showed a paranuclear distribution in cerebral endothelial cells (e, f: scale bar = 20 µm). insets : higher magnification of cells indicated by arrow heads. ace2 immunoreactivity was present in brain endothelial cells (g: scale bar = 8 µm). sp was detected in blood vessels labeled with a cd147 receptor antibody (h: scale bar = 8 µm). absence of viral genome or viral particles in brain parenchyma different molecular and histological techniques were used to detect viral genome or viral particles of sars-cov-2, all of them with negative results. rt-pcr was negative in the three brain biopsy samples (cases # 1 to 3). in case # 4, rt-pcr was positive in the lungs, mediastinal and cervical lymph nodes, and spleen, but negative elsewhere, including the different brain samples analyzed (table 3). no viral sequence was found by agnostic next generation sequencing (ngs) in the three biopsy samples (cases # 1 to 3). regarding autopsy case # 4, sars-cov-2 sequence was not detected in any of the brain samples tested (frontal, temporal and parietal lobes). instead, one read of hsv-1 was identified in the frontal lobe. both sars-cov-2 and hsv-1 viral sequences were found in the lung, spleen, lymph node and tongue. in situ hybridization (ish) for sars-cov-2 was negative in the brain in case # 4, but positive in the lung where large metaplastic pneumocytes were positive (supplementary material). lack of detectable viral genome in brain tissue was confirmed with high-amplification and target-specific ish (rnascope technology, data not shown). electron microscopy examination of white matter tissue from case # 4 did not reveal the presence of viral particles. instead, many clathrin coated particles were identified (data not shown). sp immunocytochemistry was positive in infected vero e6 cells (fig.4. b) but negative in both ipsc-d-ecs and hcmec/d3 infected cell lines (fig. 4. b). in addition, sars-cov-2 rt-pcrs on samples from the cell culture supernatant and from the intracellular compartment of endothelial cells derived from control ipsc-d-ecs, showed no amplification of the viral rna either in the cells (fig. 4. c) or in the supernatant (fig. 4. d) at the three moi done. furthermore, after incubation of huvec cells with recombinant sp s1 full length peptide and control rbd peptide, no staining was detected using anti-s1 and anti-rbd antibodies (data not shown), although control vero e6 cells, infected with sars-cov-2 virus, showed immunoreactivity for both anti-s1 and anti-rbd antibodies. these results suggest that endothelial cells are refractory to sars-cov-2 in vitro infection. fig. 4. human endothelial and vero e6 cells. (a) furin immunoreactivity colocalizes with gm130 immunoreactivity in human ipsc-derived endothelial cells (ipsc-dec). (b) vero e6 and human endothelial cells (ipsc-d-ecs and hcmec/d3) were infected with sars-cov-2 (moi 0.1, 0.5, 1) for 48, 72 and 96 hours. sp was detected in vero e6 but not in infected human endothelial cells. the not infected condition is a negative control for the infection (scale bar in a and b = 10μm). (c,d) the rt-pcrs were determined over time (48, 72 and 96 hours post-infection) in (c) cell extract or (d) cell supernatant of ipsc derived endothelial cells infected or not with sars-cov-2. no evidence of viral rna amplification was observed. sp is localized in the golgi apparatus and is associated with furin as mentioned above, sp immunoreactivity was paranuclear suggesting a localization of the viral particles in the golgi apparatus. this was confirmed by giantin and tgn46 immunoreactivity (fig 3. e & 5. a). giantin is a protein of the cis-media cisternae and tgn46 is found in the trans-golgi network. both proteins were partly colocalized with sp in brain endothelial cells (fig. 5 a). by contrast, bip, a protein of the endoplasmic reticulum and cathepsin b, a protein of the lysosomal pathway, did not show colocalization with sp in the brain endothelial cells (fig. 5 a). the ihc labelling of furin, which is also a golgi protein that can migrate into the lysosomal system [29], was detected in the same paranuclear localization as sp in patients (fig. 3. f) and colocalization of the two proteins was confirmed by confocal microscopy (fig. 5 a). we then looked for furin immunoreactivity in cases with vascular lesions due to ecmo, amyloid angiopathy or intravascular lymphoma and could not detect furin expression. this ruled out furin expression as a non specific reaction of the endothelium. after infection of ipsc-d-ecs and hcmec/d3 cell lines we observed that furin was mainly localized in the golgi apparatus and in cytosolic vesicles (fig. 4. a). this localization was the same as previously reported [29]. we also investigated the association of ace2 and cd147/basigin in the vascular lesions of sars-cov-2 cases with ccm. ace2 and cd147/basigin have both been considered receptors of sars-cov-2. ace2 immunoreactivity was seen in most endothelial cells (fig. 3. g) whereas cd147/basigin was expressed at the luminal surface of endothelial cells in some vessels. double labeling showed that sp immunoreactivity was present in cd147/basigin positive vessels and in furin positive endothelial cells (fig 3. h). the distribution of sp was different between brain and lung. while in the brain sp was compact and had a paranuclear localization limited to the endothelial cells, it was spread as small particles in the cytoplasm of type ii pneumocytes of the lung (fig. 5 a, b). in the brain, sp colocalized with the golgi proteins: giantin, furin, and tgn46 (fig. 5. a), while in the lung, colocalization was seen with tgn46 and cathepsin b (fig 5. b). fig 5. sp localizes to the golgi apparatus and differs in its distribution between brain and lung. (a) spike protein colocalizes with giantin, furin and tgn46 in the endothelial cells of brain vessels. first row: bip protein is seen dispersed throughout the cytoplasm of the endothelial cell and does not colocalize with sp. second row: giantin immunoreactivity is paranuclear similarly to sp and is found partially colocalizing with sp. third row: furin is seen in a paranuclear location in endothelial cells and colocalizes with sp. fourth row: tgn46, which localizes next to the nucleus in the endothelial cell, colocalizes with sp. fifth row: cathepsin b is seen dispersed throughout the cytoplasm of the endothelial cells with no colocalization with sp. (first, third and fourth row: case # 4; second and fifth row: case # 6; scale bars in all rows = 5μm). (b) spike protein colocalizes with tgn46 and cathepsin b in type ii pneumocytes. first row: bip protein is dispersed throughout the cytoplasm of type ii pneumocytes and does not colocalize with sp. second row: giantin is distributed diffusely in type ii pneumocytes and does not colocalize with sp. third row: furin is seen as fine granular positivity and is dispersed in the cytoplasm of type ii pneumocytes. it does not colocalize with sp. fourth row: tgn46 is seen as fine granular diffuse positivity and partially colocalizes with sp. fifth row: cathepsin b is dispersed throughout the cytoplasm of the endothelial cells and colocalizes partially with sp. (all rows: case # 4) (scale bars in all rows = 20μm). discussion in six covid-19 cases with neurological symptoms, multiple microvascular lesions were found in the white matter. they were made of foci of vacuolized tissue with red blood cells, macrophages, axonal swellings, a few t lymphocytes and perivascular fibrinogen deposits. the vessels were not occluded and the alteration of perivascular tissue was different from acute necrosis such as seen in recent lacunae. perivascular cuffing commonly seen in encephalitides was absent. the small number of lymphocytes was in contrast with the abundance of macrophage infiltration. the high number of macrophages and the absence of polymorphonuclear leukocytes suggest that the lesions occurred at least several days earlier. the absence of myelino-axonal dissociation and of myelin laden macrophages suggested that demyelination was not involved. the nuclear abnormalities in the glial cells observed in the biopsy cases could not be related to a cytopathogenic effect since no viral presence in these cells could be detected with the techniques used. on the other hand, these astrocytes evoke creutzfeldt-peters cells, initially described in multiple sclerosis but present, in a non-specific way, in a wide spectrum of cerebral diseases including anoxic encephalopathy [51]. the pattern of aqp4 ihc was different from that described in demyelinating diseases such as multiple sclerosis, neuromyelitis optica (nmo) or adem [33]. in our cases, aqp4 was strongly expressed by astrocytes at the periphery of the foci. vacuolization of the perivascular tissue and fibrinogen extravasation were evidence of a blood-brain barrier leakage. in addition to the observations made by lee et al. [24, 25] (macrophages, few lymphocytes, blood-brain barrier leakage), we stress the presence of axonal injury and astrocytic alterations in the foci. arterial damage could be explained by critical care in itself [23]. however these lesions are particularly frequent in covid-19 patients and may occur in the absence of severe cardiovascular failure or extracorporeal membrane oxygenation (ecmo) [6]. in addition, the presence of sp in the endothelial cell in our patients argues for the specificity of the lesions. despite the use of different techniques (rt-pcr, nested-pcr, agnostic and targeted ngs, in situ hybridization, electron microscopy) sars-cov-2 viral sequences or particles were not detected in the brain parenchyma, in agreement with previous observations [12, 24, 53]. we could, however, show the presence of viral proteins in the endothelial cells, as meinhardt et al. who used the same antibody (abcam, ab272420; 1:100) as us [32]. we could confirm this result with another antibody against the nucleocapsid (cliniscience). we found an association between the presence of significant sp immunoreactivity in the endothelial cells and the tissue damage. the detection of sp was not limited to the most injured vessels (whose wall was too altered to analyze the endothelial cells) but to the vessels around the regions in which the lesions were observed. these anomalies could be interpreted as a preparation of the vessel for a more severe damage. our study was limited by the small number of autopsies obtained, that restrained the access to more representative cases of a relatively rare event. when only biopsy was available, the material was not sufficient to reproduce all the techniques. nevertheless our results suggest that the role of sars-cov-2 sp in microangiopathy, described in animal models [38], could apply to humans. the discrepancy between the presence of sp and nucleocapsid immunoreactivity and the absence of viral genomes suggests that rna levels were too low at this stage to be detected, while sp and nucleocapsid protein immunoreactivity have persisted after the rna degradation, several weeks after acute infection. the detection of these viral proteins in the endothelium and epithelial cells of the choroid plexus suggests that the virus could enter the cns via the vessels rather than through nerve processes. sp could be transported in the bloodstream, cross the blood brain barrier [46] and enter endothelial cells by endocytosis [38]. nevertheless, the hypothesis of an isolated protein transport seems implausible given the presence not only of sp but also of nucleocapsid protein in the endothelial cells. in addition, our attempts to incubate recombinant sp s1 full length peptide with huvec cells did not lead to internalization of the protein. in cell culture, neither the virus nor the sp could not penetrate the endothelial cells, in accordance with recent data from the literature [1, 30, 35], suggesting that endothelial cells are refractory to sars-cov-2 in vitro infection. both cd147/basigin and ace2 were expressed in the ipsc-d-ec and hcmec/d3 lines underlining that resistance to infection was not due to the absence of these receptors. we are aware that our models do not fully account for the brain endothelial cell behavior towards sars-cov-2 infection, since they were not specific for this type of cells and did not reproduce the complex structure and function of the blood-brain barrier. however, we propose that the vascular abnormalities we observed are related to peculiarities of pathways followed by the virus once it has entered a cerebral endothelial cell, precluding its subsequent replication. the paranuclear topography of sp1 and its association with giantin and tgn46 are evidence that sp molecules were located in the golgi apparatus. the golgi and trans-golgi immunoreactivity was in strong contrast with that observed in the lung where there was colocalization between sp not only with tgn46 but also with cathepsin b, a lysosomal marker, suggesting that sp follows a complete cycle of replication in the lung. thus, it appears that the metabolism of sp is different in brain endothelial cells and in pneumocytes. in the latter, the virions egress from the cell by a lysosomal pathway [14]. in the former, sp accumulates in the golgi and trans-golgi network. the cause of the differential use of these pathways and their impact on pathology remains to be further explored. since the start of the present study, several papers have proposed mechanisms leading to sars-cov-2 specific vascular lesions. sars-cov-2 protein (mpro) could interact with the host protein nuclear factor nf-κb essential modulator (nemo), which is part of the signaling cascade that regulates the transcription of several genes, some of which are involved in inflammation [57]. other authors have identified an sp (spike)-triggered transcriptional re-sponse associated with extracellular matrix reorganization and tgf-β signaling [3]. we have found colocalization of sp immunoreactivity with furin protease in the trans-golgi network. our observations suggest that sp, in association with furin and possibly other molecules, may play a role in the development of vascular pathologies. among the genomic features that differentiate sars-cov-2 from the other members of the cov family, the insertion of four amino acid residues (prra) at the junction of the s1 and s2 subunits of sp has generated a cleavage site for furin and other proteases [9]. furin inactivation has been shown to decrease sars-cov-2 infectivity, virus production and cytopathic effect [5, 19]. besides its role in sars-cov-2 infectivity, furin is highly expressed in vascular endothelial cells and modulates endothelial permeability [29]. furin has been considered as a possible therapeutic target [19]. inhibition of furin may indeed have a protective effect against hypoxia and blood brain barrier disruption [2, 31]. these observations not only shed light on the neurological lesions observed in some covid-19 patients in the acute phase but may provide insight into the mechanism of later onset dysfunction. a network-based approach linked cerebral microvascular lesions to late cognitive impairment after the covid-19 episode, suggesting that microangiopathy could have long-term consequences [58]. in conclusion, we have defined additional histological characteristics of ccm associated with leakage of the blood brain barrier: foci of vacuolized white matter, axonal swellings, aqp4 redistribution in astrocytes. we detected the presence of sp in the trans-golgi network associated with furin protease. the accumulation of sp in the golgi of endothelial cells was in contrast with the aspect of infected pneumocytes in which sp and nucleocapsid protein immunoreactivity was spread in the whole cell body. the difference in the metabolism of the virus in the two cell types explain the differences in pathogenic effect. in the brain, the tight association between sp and furin suggests that their interaction is involved in the pathogenesis of covid associated microangiopathy. the late effects of the microangiopathy are not known. as furin plays a role in the modulation of vascular permeability, our findings may provide further arguments in the control of late effects of microangiopathy. acknowlegments we thank the patients and their family. the authors thank the cohort covid-19 neurosciences (coco neurosciences) study supported by aphp and funded by the generous support of the federation internationale de l’automobile (fia), the fia foundation and donors of paris brain institute – icm. the authors thank the coco-neurosciences study group for their participation to data collection and pierre rufat for his valuable contribution. the research leading to these results has received funding from sorbonne university (pathocov project) and from the program “investissements d’avenir” anr-10iaihu-06. we thank the staff of the raymond escourolle neuropathology laboratory for their commitment into the project. parts of the histological work-analysis were performed on the histomics and icm.quant platforms of the paris brain institute (icm), we thank all technical staff involved. this work benefited from equipment and services from the celis cell culture core facility (paris brain institute), a platform receiving support from anr-10-iaihu-06 and anr-11-inbs-0011-neuratris. we thank for targeted ngs sequencing the biomics platform, c2rt, institut pasteur, paris, france, supported by france génomique (anr-10-inbs-09-09), ibisa and the illumina covid-19 projects’ offer. references 1. ahmetaj-shala b, peacock tp, baillon l, swann oc, gashaw h, barclay ws, mitchell ja (2020) resistance of endothelial cells to sars-cov-2 infection in vitro. biorxiv: 2020.2011.2008.372581. https://doi.org/10.1101/2020.11.08.372581 2. baumann j, huang sf, gassmann m, tsao cc, ogunshola oo (2019) furin inhibition prevents hypoxic and tgfbeta-mediated 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https://doi.org/10.1186/s13195-021-00850-3 copyright: © 2023 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. pathological perspectives in pilocytic astrocytomas: extent of resection as the sole critical factor for recurrence-free survival, and the challenge of evaluating conclusions derived from limited data feel free to add comments by clicking these icons on the sidebar free neuropathology 4:17 (2023) original paper pathological perspectives in pilocytic astrocytomas: extent of resection as the sole critical factor for recurrence-free survival, and the challenge of evaluating conclusions derived from limited data ibrahim kulac1, irem yenidogan2, banu oflaz sozmen2,3, arzu baygul4, soonmee cha5, melike pekmezci6, tarik tihan6 department of pathology, koc university school of medicine, istanbul, turkey department of pediatrics, koc university school of medicine, istanbul, turkey division of pediatric hematology and oncology, koc university school of medicine, istanbul, turkey department of biostatistics, koc university school of medicine, istanbul, turkey department of radiology, university of california, san francisco, ca, usa department of pathology, university of california, san francisco, ca, usa corresponding author: tarik tihan · department of pathology · division of neuropathology · university of california · san francisco · california · usa tarik.tihan@ucsf.edu submitted: 17 september 2023 accepted: 09 october 2023 copyedited by: lauren walker published: 20 october 2023 https://doi.org/10.17879/freeneuropathology-2023-5116 keywords: astrocytoma, entity, circumscribed gliomas, glioma, juvenile pilocytic astrocytoma, pilocytic astrocytoma, piloid, pilomyxoid, tumor type abstract introduction: pilocytic astrocytoma (pa) is one of the most common primary intracranial neoplasms in childhood with an overall favorable prognosis. despite decades of experience, there are still diagnostic and treatment challenges and unresolved issues regarding risk factors associated with recurrence, most often due to conclusions of publications with limited data. we analyzed 499 patients with pa diagnosed in a single institution over 30 years in order to provide answers to some of the unresolved issues. materials and methods: we identified pilocytic astrocytomas diagnosed at the university of california, san francisco, between 1989 and 2019, confirmed the diagnoses using the who 2021 essential and desirable criteria, and performed a retrospective review of the demographic and clinical features of the patients and the radiological, pathologic and molecular features of the tumors. results: among the patients identified from pathology archives, 499 cases fulfilled the inclusion criteria. median age at presentation was 12 years (range 3.5 months – 73 years) and the median follow-up was 78.5 months. tumors were predominantly located in the posterior fossa (52.6%). there were six deaths, but there were confounding factors that prevented a clear association of death to tumor progression. extent of resection was the only significant factor for recurrence-free survival. recurrence-free survival time was 321.0 months for gross total resection, compared to 160.9 months for subtotal resection (log rank, p <0.001). conclusion: multivariate analysis was able to identify extent of resection as the only significant variable to influence recurrence-free survival. we did not find a statistically significant association between age, nf1 status, tumor location, molecular alterations, and outcome. smaller series with apparently significant results may have suffered from limited sample size, limited variables, acceptance of univariate analysis findings as well as a larger p value for biological significance. pa still remains a predominantly surgical disease and every attempt should be made to achieve gross total resection since this appears to be the most reliable predictor of recurrence-free survival. introduction pilocytic astrocytoma (pa) is a circumscribed astrocytoma with classic histologic features such as biphasic compact and loose growth patterns, piloid cytology, and low proliferative activity, with or without rosenthal fibers and/or eosinophilic granular bodies [1]. the essential criteria adopted by the world health organization (who) 2021 also defines pa as a “piloid astrocytic neoplasm with a solitary mapk pathway alteration, such as kiaa1549::braf fusion” [2]. over the last century, beginning with the first use of the word “pilocytic” [3] who classification schemes defined pa as a clinically, radiologically, pathologically, and most recently, a molecularly distinct entity. typical radiological and histological features are presented in figure 1. figure 1. a cystic tumor with mural nodule in posterior fossa on axial t1 post-contrast (a) and t2 (b) weighted mri images. an intraoperative smear slide of a pa case with plenty of piloid cells distributed on a glial background (c). hematoxylin & eosin image of a demonstrative pa case with an overall fibrillary appearance (d), closer view with plenty of rosenthal fibers, classical finding in pas (e). myxoid, loose areas are another common finding in pa (f). linear glomeruloid vascular structures are commonly observed (g) along with hyalinized, thick vessels (h). pa can be observed at any age with a reported incidence rate of approximately 0.84 per 100,000 [4-6], and has a favorable outcome [5]. despite being the most common glioma among the pediatric population, overall rarity of this neoplasm makes it an orphan disease as well as a “chronic disease” affecting patients and families for many years, creating challenges in its management [7-11]. coding of pas in tumor registries has been problematic in recent years due to their designation as “malignant” in some countries. this change was made in order to capture these tumors in cancer registries [12]. consequently, incidence of benign or malignant pediatric brain tumors show significant variations over time in epidemiological analyses [13]. we are not sure whether designating pas as “malignant” in order to capture them in registries is a good idea, or whether this practice should be abandoned to bring more clarity and uniformity to the issue worldwide, but risk losing their identification in cancer statistics. pa is most commonly located in the posterior fossa, specifically in cerebellar hemispheres. other common locations include hypothalamic/chiasmatic region and the optic nerve, the latter being particularly common in the setting of neurofibromatosis 1 syndrome [14]. thalamic, cerebral, and spinal tumors are distinctly less common [5]. some authors suggested that there were prognostic differences among pas in different locations that could not be explained simply by differences in clinical variables [15-17]. it is not clear whether different locations are associated with different outcomes beyond access to gross total resection, and whether tumor location is associated with different driver mutations and treatment response. in addition to location, some studies suggest that age is important determinant of outcome, and adult and pediatric pas do not have similar prognoses [18-21]. pas can have diverse morphological features, some of which may result in misclassification of some tumors as diffuse gliomas. some pas with the so-called “diffuse pilocytic pattern” may be easily misdiagnosed as diffuse gliomas [22]. conversely, other entities may also be misclassified as pa and some high-grade tumors with remote resemblance to pa may be erroneously classified as tumors with piloid features or simply anaplastic pa [23]. high-grade histological features in otherwise typical pas are also problematic and the current who classification scheme did not find sufficient evidence to define an anaplastic subtype [24]. however, there is clear evidence that rare pas behave aggressively and some of these tumors may be justifiably designated as high-grade gliomas [25]. on the other hand, some worrisome histological features in pa have not been associated with adverse prognosis [26-28]. additional studies and observations are needed to validate a high-grade subtype of pa and its definition. currently, the only recognized subtype of pa is the pilomyxoid astrocytoma (pma), that appears to be more aggressive compared to typical pas [29]. however, in the recent editions of the who classification of central nervous system (cns) tumors, the working groups decided that the data for recognizing this subtype as more aggressive were not sufficient to designate a specific who grade for pma [15, 29, 30]. further studies are needed for the pilomyxoid tumors to allow their grading and prognostication. recent genomic studies outline the molecular landscape of pas as tumors with a very stable genome, carrying less than 0.1 mutations per megabase [31, 32]. mitogen activated protein kinase (mapk) pathway is the most commonly affected pathway in pas. the most common genetic alteration is the braf internal tandem duplication resulting with a fusion of a neighboring gene, kiaa1549. other alterations of the mapk pathway, such as other braf fusions, mutations in braf, ras or nf1 were also reported [2, 33-39]. while the type of mapk alterations has not been associated with different outcomes, it is not clear whether the cumulative effect of multiple genetic alterations may lead to a more aggressive behavior in pas. some studies implicated molecular alterations such as loss of cdkn2a, gain of chromosome 7 and loss of chromosome 17q as being associated with worse outcome, but none of these studies were validated in large series, prospective trials or by more than one group [25, 40]. several clinical and biological markers reportedly influence prognosis in patients with pa, but these parameters, with the exception of extent of resection, have not been consistently found as independent variables [26-28, 41]. we also suggested that access to healthcare is a key factor in the outcome of patients with pa, most likely unrelated to tumor biology [42]. in this retrospective study, we report our experience with 499 pas diagnosed and treated in a single institution over the last three decades in order to bring clarity to some of the uncertainties mentioned above, and to further demonstrate the need for collaborative studies and larger series to better understand factors associated with outcome. material and methods patient selection all patients diagnosed as pa or pma between 1989 and 2019, were retrieved from the pathology archives of the department of pathology. the inclusion criteria were as follows: 1all patients diagnosed and/or treated at our institution between 1989 and 2019, and 2sufficient clinical information to include into the database, and 3sufficient pathology material available for review and diagnosis, and 4the diagnosis of pa or pma upon review of the available material. the database search included a series of keywords for final diagnoses, and the search was conducted at two separate occasions, which yielded nearly identical results with a rare exception due to delay in the database registry. clinical and radiological information were obtained from the hospital electronic information systems or from the patient charts for older cases. all relevant clinical information necessary for the purposes of our analyses were collected in an anonymized fashion and a research database was created. radiological reports were also reviewed to ensure that radiological impression was consistent with pa. extent of resection was obtained from the operative reports and was reported as either gross total resection (gtr) or subtotal resection (str). any level of resection less than gtr was considered str. recurrence was defined as worsening of clinical symptoms attributed to tumor growth. recurrence-free survival (rfs) was calculated between the date of the initial surgery and the first record of clinical worsening in the chart. overall survival was calculated as the time between the initial surgery and death due to tumor, and all others were censored. the cut-off for the follow-up time was january 2022. patients who could not be identified in the system by that date were considered lost to follow-up. this study was approved by the university of california san francisco institutional review board (irb approval no. chr 10-01252). pathological evaluation all available pathology material from all cases were reviewed by two of the authors (ik, tt) and one of the authors (mp) contributed to the pathological review process for the recent cases during the last decade. pa and pma diagnoses were based on the essential and desirable criteria proposed by the who 2021 classification scheme. pertinent histopathological features were recorded. immunohistochemical studies were performed as a part of the routine work-up of cases to establish diagnosis and further characterize the histological features. braf v600 mutation analysis by real time pcr a total of 222 patient samples were submitted to braf v600e mutational analysis by real time pcr as a part of their routine diagnosis. real time pcr was performed according to published methodology at the clinical cancer genomics laboratory that operates under a clia license. appropriate controls and quality assurance parameters have been established at this laboratory (https://genomics.ucsf.edu/content/braf-mutation-testing-including-v600e). braf duplication / kiaa1549::braf fusion analysis in limited number of cases, braf duplication only was investigated via fluorescence in-situ hybridization (fish) as previously described as a part of the patients’ routine diagnostic work-up at the clinical cancer genomics laboratory (https://genomics.ucsf.edu/content/braf-rearrangement-fish). analysis of molecular alterations and copy number variations (cnv) capture-based next-generation dna sequencing (ngs) was performed at the ucsf clinical cancer genomics laboratory (also referred as ucsf500 ngs assay) in 106 patients as a part of their routine diagnostic work-up according to protocols described previously [43]. further information on the specifics of the ucsf500 ngs platform is available at the ccgl website (https://genomics.ucsf.edu/content/ucsf-500-cancer-gene-panel-test-ucsf500-uc500). statistical analysis statistical analysis was performed using the ibm spss statistics for windows (ibm corp., version 28.0, released 2021, armonk, ny: ibm corp). the normality of continuous variables was investigated by shapiro-wilk’s test. descriptive statistics were presented using mean, standard deviation, median and interquartile range. categorical variables were expressed by using frequencies (n) and percentages (%). to compare categorical variables chi-square test (or fisher exact test/yates continuity correction as appropriate) was used. the recurrence-free survival was evaluated by kaplan-meier method and the median survival times were compared by log rank test. the associations between clinicopathologic features and the recurrence free survival were evaluated by cox regression model. the cut off for statistical significance was set as p<0.001. results demographic and clinical features among a total of 528 patients identified from the database with the diagnosis of pa or pma between 1989-2019, 499 patients fulfilled the inclusion criteria and were included in this study. mean age at the diagnosis was 15.5 years (range: 3.4 months-72 years) and the median age was 12 years. there were 276 females (55.3%) and 223 males (44.7%). the patients were grouped into three age categories; 286 patients were <14 years old, 77 patients were between 14-21 years old, and 136 cases were >21 years old. the median follow-up was 78.5 months (1 – 580 months) and 47 (9.4%) patients had a follow-up period less than 12 months. in addition, 178 patients had >5 years and 114 patients had >10 years of follow-up. non-surgical treatment data were available on approximately half (n=298) of the cases and among them, 82 patients have received chemotherapy and 63 have received radiotherapy. six patients died during the follow-up period, but detailed analysis of their disease course could not directly establish cause of death as being due to tumor. all six patients initially underwent subtotal resections, received radiotherapy and multiple chemotherapy regimens, and had significant comorbidities (see table 1). in most cases, there was no clear tumor growth, and the disease course was complicated by additional factors. table 1. clinical details of deceased patients id patient age at sx location treatment comorbidities molecular findings os case# 29 5 years cerebral 4 surgeries wbrt multiple chemotherapies hemiparesis. seizures. panhypopituitarism radiation-associated necrosis*. stable tumor in last neuroimaging study n/a 24 yr case# 49 1.3 years hypothalamic/ chiasmatic 2 surgeries multiple chemotherapies external beam radiation 11 surgical procedures radiation-associated necrosis*. seizures. hydrocephalus. secondary infection-sepsis. n/a 11 mo case# 86 49 years posterior fossa 2 surgeries external beam radiation multiple chemotherapies radiation-associated necrosis*. hemiparesis hydrocephalus. siadh. stable tumor in last neuroimaging study n/a 2 yr case #177 39 years spinal cord 2 surgeries radiation treatment multiple chemotherapies radiation associated necrosis*. paraplegia neurogenic bladder, recurrent uti. papillary urothelial carcinoma. stable tumor in last neuroimaging study. n/a 14 yr case# 249 4 years hypothalamic/ chiasmatic 2 surgeries radiation treatment multiple chemotherapies multiple vp shunting hydrocephalus. radiation-associated necrosis*. seizures. cerebral infarction. siadh. dysphagia and aspiration pneumonia braf::kiaa1549 11 yr case #454 35 years posterior fossa 3 surgeries radiation treatments (3) multiple chemotherapies multiple shunt revisions neurofibromatosis 1. aspiration pneumonia multiple neurofibromas. radiation-associated necrosis*, cerebellum. multiple flair abnormalities in the cerebral hemispheres. no tumor growth reported following third surgery on radioimaging nf1 loss somatic/ germline cdkn2a homozygous deletion in recurrent tumor 6 yr • radiation-associated necrosis was confirmed on radioimaging as well as pathological studies of the recurrent tumors • wbrtwhole brain radiation; vp: ventriculoperitoneal; siadh: syndrome of inappropriate adh secretion; uti: urinary tract infection; os: overall survival. tumor recurrence information was available for 321 patients, of which 109 suffered at least one recurrence (34%, 109 of 321 cases). major demographic and clinical features are presented in table 2. tumor localization among the cases where the localization information was available (n=494), 259 (52.6%) were in the posterior fossa, 208 (42.3%) were supratentorial (includes hemispheric, hypothalamic/chiasmatic, intraventricular and optic nerve tumors), and 25 (5.1%) were in the spinal cord. two patients had multifocal tumors at diagnosis, and the exact information on location was not available for 5 patients. table 2. major demographic and clinical features of patients variable groups n % age n:499 0-14 14-21 >21 286 77 136 57.3% 15.4% 27.3% sex n:499 male female 276 223 55.3% 44.7% extent of resection n:499 str gtr unknown 235 169 95 47.1% 33.9% 19.0% tumor localization n:499 infratentorial supratentorial spinal multifocal unknown 259 208 25 2 5 51.9% 41.6% 05.0% 0.5% 01.0% treatment n:499 chemotherapy    yes    no    unknown radiotherapy    yes    no    unknown 82 216 201 63 235 201 16.4% 43.3% 40.3% 12.6% 47.1% 40.3% outcome n:499 no evidence of disease alive with disease lost, to follow up died of other causes 80 62 351 6 16.0% 12.4% 70.3% 01.2% recurrence n:499 yes no unknown 109 212 178 21.8% 42.5% 35.7% second recurrence n:109 yes no unknown 20 58 31 18.3% 53.2% 28.5% n: total number of patients; str: subtotal resection; gtr: gross total resection. second recurrence is reported for 109 patients who had documented recurrences. among 208 supratentorial tumors, 108 (51.9%) were in the cerebral hemispheres, 79 (38%) were in the hypothalamic/chiasmatic region, 6 (2.9%) were intraventricular, and 15 (7.2%) were involving the optic nerve. while there was a suggestion that posterior fossa tumors were less likely to recur compared with hypothalamic/chiasmatic tumors, there was no statistical significance (p=0.043) partly due to the small number of cases for each location category when controlled for other variables. even after regrouping cases into “hypothalamo-suprachiasmatic+brainstem" and "other locations", we did not find statistically significant correlation with recurrence rates (p=0.004), or with rfs. location was not found to be significant on multivariate analysis, suggesting that it is a dependent variable. hypothalamic/midline tumors are difficult to reach and harder to remove completely or there may be additional confounding factors and our dataset may not be large enough to tease out these specific factors, so that a significance may be observed in larger studies with longer follow-up. there was no statistically significant difference in rfs between infratentorial and supratentorial tumors, with supratentorial tumors having a minimally higher recurrence rate on univariate analysis that disappeared on multivariate analysis. histological and molecular/genetic features we identified 6 patients with pma, and 6 other cases with histological evidence of anaplasia on pathological evaluation. the remaining 487 demonstrated histological features diagnostic of pa, occasionally aided by immunohistochemical evaluation. it was not possible to make a statistical analysis due to limited numbers of cases with unusual histological features. likewise, a meaningful analysis of rfs for pma and tumors with anaplastic histology could not be made. salient features of pmas and tumors with anaplastic histology are presented on tables 3 and 4, respectively. histological features were critical for the recognition of pa, but their analysis did not reveal statistically meaningful associations (data not shown). table 3. clinical and pathological features of patients with tumors showing anaplastic histology   age/sex localization surgery type diagnosis mutational profile initial pathology/ history chr loss chr gains recurrence/ time to rec (months) case# 226 42/f hemispheric gross total resection pilocytic astrocytoma with anaplastic features braf::kiaa1549 fusion, cdkn2a loss, atrx and nf1 mutations history of pa, grade 1 (12 years ago) 2q, 8p, 9q, 10p, 11p, 14q 2p, 3q, 7q yes / 146 case# 384 46/m posterior fossa subtotal resection pilocytic astrocytoma with anaplastic features fgfr1, ptpn11 mutations, cdkn2a/b loss, atrx mutation, pik3r1 mutation history of a posterior fossa tumor at childhood with unknown histology 2q, 8p, 9p, 10q, 13q, 22q 8q yes / 6 case# 454 35/f posterior fossa subtotal resection pilocytic astrocytoma with anaplastic features nf1 and chek2 mutations, cdkn2a/b loss (in recurrent tumor) history of pa, grade 1 (4 years ago) 1, 4p, 5q, 9p, 10q, 13q, 16p 2p, 4p, 12q, 22q yes / 35 case# 362 27/f posterior fossa subtotal resection pilocytic astrocytoma with anaplastic features germline nf1 mutation, atrx mutation, cdkn2a/b loss history of pa, grade 1 (22 years ago) none none lost fu / no information case# 488 54/m posterior fossa subtotal resection pilocytic astrocytoma with anaplastic features cdk4 and gab2 amplification, tp53 mutation history of a posterior fossa tumor with unknown histology 5q, 10q, 18p, 20p 4, 8p, 8q, 9q, 12p, 16, 17q, 18q, 21q lost fu / no information case# 347 73/m hemispheric subtotal resection pilocytic astrocytoma with anaplastic features n/a concurrent pa morphology. history of unknown brain tumor.     lost fu / no information table 4. clinical and pathological features of patients with pilomyxoid astrocytoma   age/sex localization surgery type mutational profile chr loss chr gains recurrence/ time to rec (months) case# 237 1.5/m hypothalamic-suprachiasmatic subtotal resection braf::kiaa1549 fusion none none yes / 77 case# 315 <1/f hypothalamic-suprachiasmatic no information braf::kiaa1549 fusion none none no case# 345 6/f hypothalamic-suprachiasmatic subtotal resection kras p.q61l none none lost fu / no information case# 366 9.1/f posterior fossa gross total resection braf::kiaa1549 fusion none 11 lost fu / no information case# 379 8.3/f multifocal subtotal resection braf p.599dup none 5, 6, 7, 8, 11, 12, 14, 16, 18, 20 yes / 1 case# 382 8.5/f hypothalamic-suprachiasmatic subtotal resection fgfr1 and ptpn11 mutations none 8, 12 no because of the retrospective nature of this study and due to the long period of study, the type of molecular testing significantly varied within the group. tumors from earlier years were tested either with sanger sequencing for braf mutations or with fish for braf duplication (n=191). in addition, 106 more recent cases were analyzed with a ucsf 500 ngs platform. twenty-four of the cases had confirmed germline nf1 mutation (while nf1 mutations were detected in 5 additional tumors, the ucsf500 ngs platform was conducted solely on tumor tissue in these cases, making it impossible to determine whether these mutations were germline or somatic.) one of the tumors harbored both fgfr1 and nf1 mutations. the distribution of mutations is displayed in table 5. table 5. mutations in 199 pilocytic astrocytomas mutation type n (%) braf fusion/duplication 114 (57.3%) braf v600 mutations 36 (18.1%) nf1 alterations (germline + somatic) 29 (14.6%) fgfr1 alterations 8 (4%) raf1 alterations 3 (1.5%) kras alterations 2 (1%) sos1 mutation 1 (%0.5) cdk4 & gab2 amplification 1 (%0.5) fgfr1 + nf1 alterations 1 (%0.5) no alterations identified 4 (2%) total 199 (100%) among 199 cases that had molecular analysis performed, the majority (n=150, 75.4%) had alterations in braf gene (either p.v600 mutations, internal tandem duplication and or kiaa1549::braf fusion); and the majority of the rest had alterations in other components of mapk pathway. because of the small number of tumors in other groups, it was not possible to make a statistical analysis between the type of mutation and rfs. we analyzed whether tumors with distinct genetic alterations cluster in particular locations. as anticipated, braf alterations were the most frequent type of mutations in all locations. however, braf alterations were more frequent in posterior fossa tumors (81%) in comparison to supratentorial tumors (69.4%; p<0.001). notably, fgfr1 mutations were primarily observed in supratentorial tumors, with seven out of eight cases located in the hemispheric, hypothalamic-suprachiasmatic, or intraventricular regions. although the number of cases was limited, all seven cases with mutations in other components of the mapk pathway (kras, raf1, sos1) were supratentorial. table 6 shows the distribution of mutations across tumor locations. table 6. distribution of specific mutations across tumor locations mutation type tumor location   supratentorial infratentorial spinal total braf alterations 59 81 9 149 fgfr1 alterations 7 1 0 8 nf1 alterations (inc. germline nf1) 10 16 3 29 other mapk alterations 7 1 0 8 no abnormality found 2 1 1 4 homozygous loss of cdkn2a has been reported as one of the poor prognostic factors in the literature [44]. among 106 patients where this information was available, cdkn2a homozygous loss occurred in only 4 patients. histologically, all four tumors had anaplastic histologic features, typical mapk driver mutations (nf1 mutation in two cases; fgfr1 mutation in, one case; kiaa1549::braf fusion in one case), additional molecular changes (atrx mutations in three cases) and multiple chromosomal gains and losses (see table 3). all four patients had suffered recurrences with a median time to recurrence of 35.5 months (6, 35, 35, 146 months). in 102 tumors without cdkn2a alterations (102/106), 26 of the tumors recurred with a median rfs of 32 months (1 269 months). chromosomal copy number variation (cnv) analysis was done in 106 cases and showed at least one cnv in 39 tumors (36.8%). nineteen tumors had whole chromosomal gains only, while 27 tumors had either gains or losses. in 12 tumors, partial losses or gains were recorded. statistical analysis did not show a significant effect of the presence of copy number alteration at any degree on recurrence or rfs (p=0.816, figure 2). there was no association between any type of genetic alteration (braf fusion, fgfr1 alteration, etc.) with the presence or extent of copy number loss or gain. figure 2. recurrence-free survival in patients with pilocytic astrocytoma, stratified by presence of copy number variations (cnv: copy number variation). we have also analyzed impact of chromosomal losses and gains separately. partial and/or whole chromosomal loss was detected in 23 patients; of which, 8 was whole chromosomal loss. partial and/or whole chromosomal gain was observed in 34 patients; of which 23 was whole chromosomal gains. cases with any degree of chromosomal loss had a median rfs of 63 months whereas, it was 54 months for cases without any chromosomal losses. on the other hand, cases with chromosomal gain of any size had a median rfs of 63 months, whereas it was 54 months for cases without any chromosomal gains. no significant influence of whole chromosomal losses or gains was seen on recurrence, rfs and underlying molecular features of the tumor as well. previous studies proposed whole chromosomal gains as an age-associated alteration without an impact on outcome [45]. there were 14 cases with whole chromosomal gains with no other cnv. thus, we excluded these cases and conducted the analysis on the remaining 25. the results showed no statistically significant difference between cases with or without cnv (excluding whole chromosomal gains). impact of age on recurrence-free survival the recurrence rates for patients younger than 14 years, between 14 and 21 years, and 21 years and over were 40.9%, 34.6%, and 20.4%, respectively. although the recurrence rates are higher in the youngest age group, this difference was not statistically significant. (p=0.003). among patients who underwent subtotal resection, patients older than 21 years at diagnosis showed higher recurrence rates (p<0.001), while there was no difference between age groups among patients who underwent gtr. we further investigated the confounding factors on the difference in str group and included tumor location; even after including tumor location as a variable, patients >21 years of age showed a significantly higher recurrence rate (p<0.001). impact of extent of resection on recurrence-free survival one-hundred-sixty-nine (33.9%) patients underwent gtr and 235 (47.1%) had str. the data on the extent of surgery was missing in 95 (19.0%) cases. recurrence rates were 8.5% and 20% for patients who underwent gtr and str, respectively. the mean rfs was 321 months for patients who underwent gtr (ci: 292.3 – 350.1), and 160.9 months (ci:124.5 – 197.4) for patients who underwent str. the patients with gtr have a significantly longer recurrence-free survival (rfs) compared to those with str (log rank p <0.001, figure 3). the median survival probability for overall survival could not be calculated due to absence of effect of extension of surgery on overall survival. figure 3. recurrence-free survival in patients with pilocytic astrocytoma, stratified by the extent of surgical resection (gtr: gross total resection, str: subtotal resection). impact of chemotherapy and/or radiotherapy on recurrence-free survival among 81 patients who received chemotherapy, 77 had a prior str. patients who received chemotherapy demonstrated a shorter median rfs (141 vs 253 months). however, we think that this finding could be potentially skewed due to selection bias, as the patients with clinically more aggressive appearing tumors are more likely to receive chemotherapy. a great majority of cases (97.5%) received chemotherapy had subtotal resections, which strongly supports our hypothesis on selection bias. thus, the observed difference in rfs may not be entirely surprising. a similar pattern was also observed among patients treated with radiotherapy (151 vs 243 months). review of patients with neurofibromatosis 1 there were 24 confirmed neurofibromatosis type 1 patients (11 female and 13 male), which comprise 4.8% of the entire cohort. mean age at diagnosis was 19.5 years (3.1 – 42.7). majority of the tumors (n=16) were in the posterior fossa, and four tumors were in the hypothalamic-suprachiasmatic region. out of 15 patients with sufficient clinical data, three experienced recurrences. one patient died during follow-up period due to other causes (for details see table 1, case#454). two cases had anaplastic features on histology, and the remaining 22 had classical pa morphology. none of the nf1-associated cases had mutations in other genes activating mapk pathway (braf, fgfr1, etc.). review of patients with >5-year follow-up given pas grow slowly and sufficiently long follow-up times are crucial for a realistic assessment of outcome, we performed a subgroup analysis of the patients who have more than 5 years of follow-up (n=178). mean follow up time in this subgroup was 263.3 months; median follow up time was 252 months. out of the 164 patients in this group for whom we had adequate recurrence data, 68 experienced a recurrence. the type of surgery remained one of the strongest factors that determined the rate of recurrence and rfs, as cases with str recurred more often and earlier (figure 4). sixty-one percent of the patients with str experienced recurrence at least once whereas the recurrence rate was 11% for patients with gtr (p<0.001). we were not able to show an association between the underlying genetic alterations or tumor location and recurrence rate or rfs. the number of cases in each group was not sufficient to perform an analysis of rfs for cases with and without cnv. figure 4. subgroup analysis of recurrence-free survival in patients with pilocytic astrocytoma followed for >60 months, stratified by the extent of surgical resection (gtr: gross total resection, str: subtotal resection). after conducting a multivariate analysis of rfs considering the age groups, extent of resection, tumor location, nf1 status, and treatment modalities, we found that only the extent of resection had a statistically significant association with rfs (table 7). table 7. multivariate analysis of factors for recurrence free survival probability   univariate multivariate variable significance hr %95 ci significance hr %95 ci type of surgery    gtr vs str   <0.001   6.28   3.35 – 11.77   <0.001   6.23   3.23 – 11.99   location    infratentorial vs supratentorial    infratentorial vs spinal 0.049 0.027 0.097   1.58 1.97   1.05 – 2.36 0.88 – 4.40 0.957 0.925 0.811   0.98 1.11   0.63 – 1.51 0.46 – 2.67   age    0-14 vs 14-21    0-14 vs >21 0.082 0.564 0.026   0.855 0.554   0.50 – 1.46 0.33 – 0.93 0.094 0.337 0.036   0.76 0.56   0.44 – 1.33 0.33 – 0.96   sex 0.266 1.25 0.85 – 1.83       discussion multivariate analysis of outcome did not reveal any significant association with clinical and pathological variables except for extent of resection. despite the large number of patients and follow-up time that is considerably longer than most studies in the literature, age, nf1 status, tumor location, adjuvant treatment and molecular alterations did not significantly influence rfs. these findings are at odds with some of the studies that suggest differences in outcome between adult and pediatric patients or between germline nf1 and wildtype tumors [46-48]. some suggested that adult cases have a worse prognosis than pediatric counterparts [18-21] while others demonstrated a very favorable prognosis regardless of age [18, 49]. it is well recognized that older age is associated with a higher incidence of deaths compared to younger populations in patients with tumors. this is often attributed to accelerated epigenetic age and the simple effect on age on overall survival as opposed to factors directly associated with tumor progression [50, 51]. in our series, we have separated cases into three age groups and although recurrence rates increased with age, this difference was not significant. even in subgroup analysis of cases with gtr and str, age did not stand up as a significant factor. we were not able to assess pa specific overall survival because we observed no pa-related fatality. it has been noted that location could be an independent prognostic variable and tumors located in the posterior fossa generally have more favorable outcomes [26, 52, 53]. this was presumed to be associated with surgical access to tumor and ability to remove completely, and therefore tumors located in regions difficult to access specifically the brain stem and hypothalamo-suprachiasmatic region were associated with worse outcomes [20, 54]. one particular study shows that while tumor location initially appears to be a strong prognosticator, its significance diminishes when considering the extent of the resection [55]. our analysis failed to reveal a statistically significant relationship between location and recurrence rate or rfs, despite re-categorizing cases into narrower, and later, two broad groups "hypothalamo-suprachiasmatic + brainstem" and "other locations". the discrepancies between our findings and those in other studies may be due to the acceptance of univariate calculations as significant, even though they may not hold up on multivariate analyses when all pertinent variables, especially extent of resection, are considered. another problem is the immediate acceptance of p<0.05 as a significant cut-off to determine biologically meaningful differences [56-58]. yet, a series of 499 patients may still be insufficient to accurately determine the small but significant effect of some variables on outcome. one critical issue in our study is the absence of patients who died due to their disease progression and presence of rare deaths due to other factors. this could still reflect a limitation of sampling, since earlier studies reported occasional deaths due to disease progression in pa [59, 60]. the reported deaths may obviously be associated with confounding factors (such as radiation treatment or pathological misclassification) or with the assumption that the death of a patient is always a consequence of disease progression. our data is sourced from a single referral center which minimizes certain confounding factors, allowing for a more streamlined analysis. however, the data are not associated with an epidemiologically relevant catchment area and may have selection bias due to the fact that our institution is a major national and international referral center. therefore, our findings need further validation by large, and multi-institutional and epidemiologically relevant studies. influence of histological features on the course of the disease has always been a debate in the literature as well as the criteria for histological anaplasia. who defines pa with histologic features of anaplasia as pa with brisk mitotic activity with or without necrosis. while there exist a handful of studies suggesting an association between histological anaplasia in pa and unfavorable outcome [61, 62], our findings failed to provide substantial evidence to support or refute these claims. in our series, we had only 6 cases (among 499) with histologic features of anaplasia, a sample size insufficient for meaningful comparison. these six patients were adults, with a mean age of 46.2, ranging from 27 to 72 years old. four tumors were located in posterior fossa whereas two were hemispheric. of these cases, five tumors were analyzed by ucsf500 ngs platform and four of them revealed a cdkn2a loss. as stated in the result section, all four cases with cdkn2a loss had histological features of anaplasia. although there seems to be an association between cdkn2a loss and histological features of anaplasia, small number of cases prevent a meaningful and statistically significant conclusion. in our series, we identified 6 cases of pma, which suggests that the prevalence of pma among pilocytic tumors is only around 1%, also supported by earlier studies [29, 63]. the overall ratio and diagnostic criteria of pma and pa with anaplastic features have been quite variable, and some published studies reported a rate of 8-20% of pma among their pa cases [63-65]. this may reflect the challenges of making the diagnosis or the variations in histological criteria for pma. we believe that pma is typically a tumor of young age and hypothalamic/chiasmatic location with monomorphous histological features and deviations from this typical spectrum should be interpreted with caution [63, 65]. this could be the reason some studies did not identify significant prognostic differences between tumors designated as pma and pa [64, 66]. molecular studies have consistently demonstrated activation of the mapk pathway in all cases of pa. the most prevalent genetic alteration involves kinase domain duplication and fusion of the braf gene with kiaa1549. other alterations seen in pa are brafv600e mutations, fgfr1 alterations, nf1 alterations and also rare alterations in other components of mapk pathway. fgfr1 alterations are highly enriched in supratentorial tumors, but spinal and cerebellar tumors also showed this molecular alteration, as observed in previous series [33, 67]. many reports showed evidence that molecular features such as tert promoter mutation, cdkn2a loss, tp53 mutation, or chromosomal copy number alterations may be associated with adverse clinical outcome [68-71]. in our series, we were unable to identify a meaningful association between molecular alterations and rfs. whole or partial chromosome copy number alterations could not be correlated with outcome either. this was partially due to small numbers and partly due to short follow-up times for tumors with comprehensive molecular analysis since such analyses began only in recent years. analysis of impact of molecular alterations including copy number variations are more likely to provide meaningful results in future studies with longer follow-up terms and larger number of patients. while molecular alterations help in establishing the diagnosis in pa, their association with location and outcome remain tenuous and require larger and prospective studies for definitive conclusions. one other point of discussion that has been a matter of contention is the decision to use the term “tumor type” instead of “tumor entity” by who 2021 classification. while this seems like a reasonable change prima facia due to the desire to classify tumors similar to animal and plant kingdom taxonomy, this approach entirely misses the point that tumors do not fit into neat categories of species and genera that could be easily placed in a taxonomical framework similar to animals and plants. the major objection to the use of tumor “type” is that a diagnostic entity such as pa may not be composed of a single tumor “type” but rather is a group of tumors that have sufficiently similar clinicopathological (including genetic) features to constitute a meaningful group of disorders from the perspective of the treating physician. attempting to create “types” with every advancing bit of information would not result in the same clinically meaningful group of diseases. this is demonstrated in our study that despite their excellent outcome, tumors classified as pa harbor sufficiently different genetic alterations, radiological and pathological features that are not sufficient enough to consider them as belonging to a different entity but may arguably imply different tumor “types” or “subtypes”. in the opinion of the authors, attempting to classify tumors by placing rigid types and subtypes akin to genera, and species designations for animal and plant kingdoms may underestimate the biological diversity of pathological processes as opposed to evolutionary biology. this approach also ignores the principal necessity of trying to classify tumors to be able to manage the patients successfully. from that perspective, the tumors in this series that we believe belong to pa as an entity, have excellent long-term survival and may be considered “chronic diseases” and managed accordingly [72, 73]. our study is in agreement with many of the prior studies highlighting the extent of resection as the key determinant of recurrence in pa [16, 53, 55, 74-76]. pa still remains a predominantly surgical disease due to the importance of extent of resection and every attempt should be made to achieve gtr for maximum benefit [77, 78]. but, there is yet much to be learned about pa, especially the biological implication of the histological features of anaplasia and additional molecular abnormalities. we were unable to identify any association between these variables and outcome, most likely because we began analyzing some of these molecular alterations only recently and sufficient time has not 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(ii) new / emerging diseases; (iii) advances in understanding of disease etiology and pathogenesis; (iv) diagnostic advances; and (v) therapeutic advances. within this general framework, the individual disease entities that are discussed in more detail include neuromuscular complications of covid-19 (another look at the topic first covered in the 2021 and 2022 reviews), dnajb4-associated myopathy, nmnat2-deficient hereditary axonal neuropathy, guillain-barré syndrome, sporadic inclusion body myositis, and amyotrophic lateral sclerosis. in addition, the review highlights a few other advances (including new insights into mechanisms of fiber maturation during muscle regeneration and fiber rebuilding following reinnervation, improved genetic testing methods for facioscapulohumeral and myotonic muscular dystrophies, and the use of sarm1 inhibitors to block wallerian degeneration) that will be of significant interest for clinicians and researchers who specialize in neuromuscular disease. abbreviations ad autosomal dominant, adpr adenosine diphosphate ribose, aidp acute inflammatory demyelinating polyneuropathy, als amyotrophic lateral sclerosis, aman acute motor axonal neuropathy, amsan acute multifocal sensorimotor neuropathy, ar autosomal recessive, cnm centronuclear myopathy, cox cytochrome c oxidase, covid-19 coronavirus disease 2019, dm dystrophia myotonica (myotonic dystrophy), emg electromyography, fshd facioscapulohumeral dystrophy, gbs guillain-barré syndrome, iefnd intraep-idermal nerve fiber density, ivig intravenous immunoglobulin, lmn lower motor neuron, mac membrane attack complex, mfn2 mitofusin 2, mhc-i major histocompatibility complex i, mnd motor neuron disease, myhc myosin heavy chain, ncs nerve conduction studies, nor node(s) of ranvier, pasc post-acute sequalae of sars-cov-2 infection, pm-mito polymyositis with mitochondrial abnormalities, sc schwann cell, sfn small fiber neuropathy, sibm sporadic inclusion body myositis, str short tandem repeat, xlmtm x-linked myotubular myopathy in this annual update, i will briefly describe ten neuromuscular field advances from 2022 that i consider to be most important and/or interesting; as in the prior updates (margeta, 2020, 2021, 2022), these advances will be grouped into different “discovery clusters” and listed in no particular order. advances in fundamental neuromuscular biology with implications for neuromuscular disease 1. regulation of the neonatal-adult maturation checkpoint during skeletal muscle regeneration in the past two reviews (margeta, 2021, 2022), i described the crosstalk between macrophages and satellite cells (muscle stem cells) that initiates skeletal muscle repair. in this review, the focus will be on a later stage of myofiber regeneration, which recapitulates muscle development and involves myofiber growth and maturation from a fetal to the adult state. in mammals, there are seven distinct isoforms of myosin heavy chain (myhc; the major contractile component of thick filaments) – two isoforms that are normally expressed only during development [embryonic (myhc-emb / mhc3) and neonatal (myhc-neo / mhc8)] and four isoforms that are expressed in mature muscle fibers [one slow isoform (mhc-slow / mhc7; type 1 fibers) and three fast isoforms: myhc-2a / mhc2 (type 2a fibers), myhc-2x / mhc1 (type 2x fibers) and myhc-2b / mhc4 (type 2b fibers)] (schiaffino et al., 2015). during muscle repair, regenerated myofibers mature over ~14 days by recapitulating developmental sequence of myhc expression: newly formed fibers first express myhc-emb, followed by myhc-neo, and finally one of the adult myhc isoforms. [this aspect of the regeneration process is diagnostically useful: in adult muscle, immunohistochemistry for developmental myhc isoforms can be used to detect regenerating fibers [(sewry et al., 2021) and fig. 1.]. figure 1. regenerating muscle fibers express developmental myosin heavy chain isoforms, myhc-emb (mhc3) and/or myhc-neo (mhc8). dual immunohistochemical stain for myhc-emb (red) and myhc-neo (brown), here performed on cryosections, can be used to detect regenerating fibers at different stages of maturation. (a) in normal adult skeletal muscle, there is no staining for either myhc-emb or myhc-neo. (b) a case of immune-mediated necrotizing myopathy (imnm) shows frequent regenerating fibers, most of which are in the early stages of maturation and express only myhc-emb (an example is highlighted by the green arrow). a few regenerating fibers are in the late stages of maturation and express only myhc-neo (an example is highlighted by the orange arrow), while several regenerating fibers are transitioning from an early to a late maturation stage and co-express both myhc-emb and myhc-neo (dark orange sarcoplasmic staining; an example is highlighted by the black arrow). (c) a case of sporadic inclusion body myositis (sibm) shows abundant regenerating fibers spanning the entire spectrum of maturation. (d) in dermatomyositis (dm), both early and late regenerating fibers are mainly found in the perifascicular areas. scale bars: a and d, 200 µm; b and c, 50 µm. wang and co-authors have now elucidated molecular and cellular mechanisms that regulate the neonatal / adult transition checkpoint, a key step in this maturation sequence (wang et al., 2022). using a myotoxin-induced model of skeletal muscle injury, they showed that expression of mitofusin 2 (mfn2; a mitochondrial membrane protein that initiates mitochondrial fusion and is induced in activated satellite cells) is required for maturation of newly formed myofibers: in mfn2-deficient mice, myotoxic muscle injury led to formation of early (centrally nucleated, mhc3-expressing) regenerating fibers, but these fibers were smaller than regenerating myofibers in wild-type (wt) mice and ultimately arrested their maturation at the neonatal (mhc8-expressing) developmental stage. this maturation defect was not due to a metabolic abnormality in satellite cells; rather, mfn2 deletion led to nuclear translocation of transcription factor nfatc2 (nuclear factor of activated t cells 2) and increased expression of hif1α (hypoxia-induced factor 1α), ultimately resulting in the lack of repressive methylation marks at the mhc8 genetic locus, which are required for the neonatal / adult fate transition. in wt animals, expression of hif1α protein was induced following muscle injury, but its level returned to the preinjury baseline within 5 days; in mfn2-null animals, hif1α levels remained elevated 14 days post-injury. importantly, sustained hif1α signaling was both necessary and sufficient for the arrest of regenerating myofibers at the neonatal stage: treatment of mfn2-/mice with px-478 (a chemical compound that reduces hif1α levels) for 14 days, starting at 14 days after muscle injury, enabled mfn2-/regenerating myofibers to escape the maturation arrest, adopt the adult fate, and grow closer to a normal adult fiber size. the same rescue could be achieved through genetic deletion of hif1α or through inhibition of h3k27 demethylases; conversely, the effect of mfn2 deletion could be replicated by genetic manipulation of vhl (von-hippel lindau tumor suppressor) signaling, which leads to hif1α stabilization independent of mfn2 signaling. finally, the authors used an ischemic model of muscle injury to show that in wt mice, repressive methylation marks at the mhc8 locus started appearing on day 9 (coincidental with reperfusion), followed by fiber growth and transition to the adult fate on day 10, and complete downregulation of mhc8 expression by day 12. in contrast, myofiber maturation was significantly accelerated in animals lacking hif1α, with completely differentiated fibers lacking any mhc8 expression present on day 8. taken together, these data indicate that mfn2/hif1α signaling regulates myofiber fate specification through the epigenetic control of myhc-neo / mhc8 expression, and that this checkpoint enables synchronous development of all skeletal muscle tissue elements. however, it should be noted that all experiments in this study were done on the mouse tibialis anterior muscle, which is largely composed of type 2 fibers; thus, it remains to be shown whether a similar maturation checkpoint also affects type 1 fibers. what is the relevance of this regulatory pathway for human disease? while that question needs to be investigated in much greater detail in the future, initial experiments performed by wang and co-authors offer some tantalizing clues. because maturation-arrested regenerating fibers are reminiscent of atrophic, centrally nucleated fibers seen in severe centronuclear myopathies (cnms), they evaluated biopsies from infantile patients with cnm 1 (caused by mutations in dynamin 2) and cnm 2 / x-linked myotubular myopathy (xlmtm, caused by mutations in myotubularin 1); in both diseases, they detected myofibers strongly positive for mhc8 as well as nuclear nfatc2 and sarcoplasmic carbonic anhydrase 3 (a hif1α target). while mhc3+ and mhc8+ positive fibers can be seen in any myopathy that involves significant fiber necrosis (fig. 1), they are generally not very numerous in congenital myopathies, raising the possibility that the mfn2/hif1α-controlled maturation checkpoint is specifically dysregulated in cnms. interestingly, a different 2022 study demonstrated that epigenetic alterations are a conserved feature of xlmtm and that histone deacetylase inhibition is a promising therapeutic strategy for this severe muscle disease (volpatti et al., 2022). while altered muscle development is one of the epigenetically modified pathways uncovered by volpatti et al., it remains to be shown whether changes in the methylation of the mhc8 genetic locus are part of the xlmtm epigenetic disease signature. interestingly, autosomal dominant mutations in mfn2 cause axonal charcot-marie-tooth disease type 2a (cmt 2a) but are not known to produce a significant myopathic phenotype. it is possible that cmt 2a patients exhibit atypical muscle development and/or aberrant response to muscle injury that contributes to their weakness but has been masked by their peripheral neuropathy; that needs to be explored by future studies. in addition, it needs to be investigated whether pharmaceutical treatments that turn off this maturation checkpoint can accelerate muscle repair following injury and/or prove beneficial for any muscle disease characterized by significant fiber necrosis and regeneration. newly defined / emerging neuromuscular diseases 2. neuromuscular complications of covid-19: post-acute sequelae of sars-cov-2 infection (“long covid”) syndrome covid-19, the novel infectious disease caused by sars-cov-2, primarily targets the respiratory system but also affects many other tissues and organs, including the pns. in the past two reviews (margeta, 2021, 2022), i discussed the emerging understanding of acute neuromuscular complications of covid-19; in this review, i will summarize what is currently known about neuromuscular involvement in the post-acute sequelae of sars-cov-2 infection (pasc) syndrome (also known as “post-covid conditions” or “long covid”). the cdc (centers for disease control and prevention, a branch of the united states government) defines pasc as continuation of covid-19 symptoms – or the emergence of new symptoms – four weeks or longer after initial sars-cov-2 infection; given this broad definition and the continuously changing spectrum of viral variants, the true prevalence of pasc is not known, but the current estimates range from 5-30%. while pasc can include a wide array of symptoms, some of the most common and particularly debilitating concerns (including fatigue / exertional intolerance, dysautonomia, and sensory symptoms such as paresthesias and numbness) suggest that the pns is affected. two small retrospective studies published in 2022 explored whether autonomic and sensory symptoms experienced by pasc patients can be attributed to small fiber neuropathy (sfn). the first study included 13 patients (8 women and 5 men) who had acute covid-19 infection in march-april 2020 and developed paresthesias 0-2 months later; the patients were evaluated by electromyography / nerve conduction studies (emg/ncs) and two punch skin biopsies (from distal leg and proximal thigh) to determine intraepidermal nerve fiber density (iefnd) (abrams et al., 2022). of these 13 patients, only one had severe covid, and none had a history of prior neurologic symptoms or evidence of large fiber polyneuropathy on emg/ncs. seven of 13 patients also reported orthostatic discomfort; four of these seven patients underwent autonomic function testing, with abnormal findings reported in three. reduced iefnd was seen in 46% patients (6 of 13; all female), all of whom had clinical findings consistent with sfn (mainly a decreased pinprick sensation in distal legs); the biopsy findings were also more severe distally. in contrast, most patients with normal biopsy results showed no objective sensory abnormalities on neurologic exam. the second study enrolled 17 patients (11 women and 6 men) who developed covid-19 between february 2020 and january 2021 and presented with new neuropathy symptoms 0.1 to 14.9 months following covid onset; again, only 1 of these 17 patients had a severe covid infection (oaklander et al., 2022). in this cohort, 16.7% of electrodiagnostic studies showed abnormal findings, while 62.5% of lower leg skin biopsies and 50% of upper thigh biopsies showed reduced iefnd. importantly, in both studies the clinicopathologic evaluation was performed with a significant delay due to pandemic-related disruptions in health care delivery; thus, it is possible that partial or complete small fiber recovery occurred in some patients in the interval between the symptom onset and assessment, leading to false-negative test results. both studies should be considered preliminary given their small size and confounding by referral bias; however, the reported findings are very similar and – if conformed in larger cohorts – suggest that the pasc-associated peripheral neuropathy reflects small nerve fiber injury. the underlying disease mechanisms also remain to be determined; however, a delay between covid infection and neurologic symptom onset in all but one of these 30 pa-tients, along with therapeutic effectiveness of the repeated ivig treatment in 5 patients who received it, suggest that the pasc-associated sfn is due to post-infectious dysregulation of the immune system. persistent fatigue is experienced by 50-70% of pasc patients, making it one of the most common pasc symptoms. however, it has been challenging to determine whether this relatively subjective and nonspecific clinical symptom is caused by functional response of muscle tissue to circulating inflammatory mediators, microcirculatory abnormalities, or an underlying skeletal myopathy. to start addressing this question, hejbøl and colleagues evaluated 16 patients (13 women and 3 men) with chronic post-covid fatigue; none of the patients had been severely ill with covid or hospitalized in intensive care unit, and clinicopathologic evaluation for myopathy (which included quantitative emg and skeletal muscle biopsy) occurred 5-14 months following acute infection (hejbøl et al., 2022). among these 16 patients with pasc fatigue, 69% also had paresthesias, but none had abnormal deep tendon reflexes or altered vibratory sensation, and ncs were normal in all 16. in addition to the fatigue, 81% of study patients had myalgia, 50% objective muscle weakness, and 75% myopathic findings on emg; serologic studies were generally unremarkable, except for one patient with a marginally elevated creatine kinase level and another with elevated tif1-γ antibody titer but normal creatine kinase level. remarkably, muscle biopsies from all 16 patients showed histologic and/or ultrastructural muscle fiber pathology; the findings were variable, but included prominent nucleoli and non-selective fiber atrophy (suggestive of fiber regeneration), basal lamina reduplication, myofibrillar disorganization, and mitochondrial abnormalities (fig. 2). in addition, inflammation and/or mhc-i upregulation was present in 62% of biopsies, while capillary abnormalities (including basal lamina reduplication and capillary loss/degeneration) were detected in 75% of biopsies. finally, one patient showed a loss of non-myelinated, small caliber axons in the intramuscular nerve twigs. the spectrum of findings seen in these specimens is very broad and does not point to a specific disease mechanism; however, both mitochondrial and capillary pathology could provide an explanation for fatigue, while basal lamina reduplication could be due to increased tgfβ (transforming growth factor β) levels, which often accompany chronic low-grade inflammation. like the two sfn studies described above, this study has several important limitations, including small cohort size, lack of patients from later stages of the pandemic (who were infected by sars-cov-2 variants other than the ancestral strain), and lack of a control group (i.e., patients with covid-19 who did not develop fatigue or other pasc symptoms); nonetheless, the findings suggest that muscle biopsy is the most sensitive test for diagnosis of the pasc-associated myopathy. figure 2. myopathy as a cause of fatigue in long-term post-covid-19 symptoms: histopathological changes in muscle fibers. (a, b) muscle fiber atrophy. (a) atrophic fiber and (b) folds in the basal lamina, both in patient 9. (c–f) muscle fiber damage indicated by basal lamina proliferation. (c) basal lamina duplication (arrow) in patient 16. (d, e) multiple myofiber basal lamina (d) and (e) higher magnification showing continuity between the different layers (arrows) in patient 14. (f) from patient 6, showing aggregation of nuclei, and multiple layers of myofiber basal lamina (arrow). (g–i) myofibrillary disorganization: (g) streaming of z-bands in patient 14, (h) disorganized myofibrils in patient 13, and (i) myofibril disorganization and a cytoplasmic body in patient 1. (j–l) mitochondrial changes: (j) a cytochrome c oxidase-negative fiber (blue) and (k) subsarcolemmal accumulation of structurally abnormal mitochondria from patient 8, and (l) subsarcolemmal accumulation of ultrastructural normal-appearing mitochondria in patient 9. scale bars: k = 500 nm; c, e, h, and l = 1 μm; a, b, d, f, g, and i = 2 μm; j = 100 μm. [this figure and its legend are reproduced without modifications from (hejbøl et al., 2022); this use is permitted under the creative commons attribution 4.0 cc-by-nc-nd international license.] collectively, these three studies provide initial evidence that neuromuscular symptoms commonly seen in pasc patients are associated with – and likely attributable to – structural abnormalities of peripheral nerves and skeletal muscles; however, these preliminary findings should be replicated in larger and temporally more homogenous cohorts of pasc patients. furthermore, additional work is required to establish whether similar nerve and muscle abnormalities are detectable in patients with myalgic encephalomyelitis / chronic fatigue syndrome, which shares many clinical features with the pasc syndrome. 3. dnajb4-associated myopathy: a new chaperonopathy molecular chaperones play a key role in the maintenance of protein homeostasis by facilitating folding of newly synthetized proteins and degradation of misfolded or damaged / unfolded proteins; the latter role is particularly critical in skeletal muscle, where mechanical stress leads to constant damage to sarcomeric proteins and where misfolded protein aggregates cannot be diluted through cell division. as a result, although chaperones and co-chaperones are ubiquitously expressed proteins, skeletal myopathy is generally the main (and often only) clinical manifestation of many chaperonopathies. dnaj/hsp40 co-chaperones determine the specificity of the chaperone/client interaction and are highly evolutionarily conserved from yeast to humans; mutations in several dnaj co-chaperones from the b family (dnajb2, dnajb5 and dnajb6) cause neuromuscular disease, but prior to 2022 no human disease has been linked to dnajb4. dnajb6 is a close homolog of dnajb4; it localizes to the z-disc and, together with hspb8 and bag3, plays a critical role in the z-disc maintenance via chaperone-assisted selective autophagy. autosomal dominant mutations in dnajb6 lead to two distinct clinical phenotypes, limb-girdle muscular dystrophy d1 and distal myopathy, both of which are histologically characterized by myofibrillar disorganization, protein aggregates, and rimmed vacuoles (sarparanta et al., 2020). last year, two separate research groups showed that mutations in dnajb4 cause a similar but distinct skeletal myopathy that is inherited in either autosomal recessive (ar; weihl et al., 2023) or autosomal dominant (ad; inoue et al., 2023) manner. weihl and co-authors used a reverse genetic approach to identify four patients from three different families who carry autosomal recessive, loss-of-function mutations in dnajb4 that either severely abrogate protein expression (c.856a>t; p.lys286ter and c.785t>c; p.leu262ser) or lead to expression of a non-functional protein (c.74g>a; p.arg25gln). clinically, these patients presented in the first decades of life with spinal rigidity and severe diaphragmatic weakness that led to early-onset respiratory failure. in contrast, inoue and colleagues used a more traditional forward genetic approach: they identified a single family with an autosomal dominant, slowly progressive myopathy characterized by onset in the 3rd to 5th decade, asymmetric thumb and grip weakness, symmetric distal weakness, respiratory failure, scoliosis, and ultimately loss of ambulation, and showed that a heterozygous missense variant of dnajb4 (c.270t>a; p.phe90leu) segregated with this clinical phenotype. muscle pathology was similar in both ad and ar forms of dnajb4 disease, with fiber size variation, rubbed out fiber centers on oxidative stains, rimmed vacuoles in atrophic fibers, and very large central inclusions / protein aggregates (up to 30-40 sarcomeres in length) in non-atrophic fibers that stained red on modified gomori trichrome, had an amorphous “woolly” or granulofilamentous appearance on em, and were immunopositive positive for p62, hsp70, desmin, myotilin, filamin c, and (in the ad form of disease) for dnajb4. ultrastructurally, there was also widespread degeneration of sarcomeres, but z-discs were relatively spared, with no definite z-band widening or streaming. interestingly, inoue and co-authors showed that this pathology is more prominent in type 1 muscle fibers, providing explanation for the preferential clinical involvement of muscles that are type 1 fiber-predominant (soleus, diaphragm, paraspinals, as well as thenar and hypothenar muscles). impressively, both groups generated model mice that recapitulated key aspects of human dnajb4 disease, including its unique myopathologic features and preferential involvement of type 1 muscle fibers; this was true both for knockout dnajb4-/mice (which model ar human disease; created independently by both groups) and for dnajb4f90l/+ knock-in mice (which model ad human disease; created by inoue et al.), suggesting that the f90l dnajb4 mutation has a dominant negative effect. of course, a lot remains to be discovered about this new disease. for example, it remains to be established why type 1 muscle fibers are more vulnerable to dnajb4 deficiency than type 2 fibers. in addition, it is not entirely clear why there are significant clinicopathologic differences between dnajb6 and dnajb4 diseases given the large degree of structural homology between these two proteins. one clue that has emerged from the work of inoue et al. is a subtle difference in the subcellular localization of these two proteins: while dnajb4 is localized to the close vicinity of the z-disc, dnajb6 directly colocalizes with this structure. future studies will be necessary to delineate the functional differences between these two co-chaperones, particularly with respect to their roles in chaperone-assisted selective autophagy, and in formation and/or degradation of rna stress granules. 4. nmnat2-deficient, sarm1-dependent hereditary axonal neuropathy (“sarmopathy”) wallerian degeneration is a highly regulated, active process of axon destruction; it is typically seen following acute mechanical or ischemic injury, but can also be caused by toxins, nutritional deficiencies, immune-mediated injury (further discussed in advance #5), and genetic alterations. regardless of the underlying etiology, wallerian degeneration is triggered by nad+ hydrolase sarm1 (sterile alpha and toll/interleukin-1 receptor motif-containing 1), the activity of which is regulated by the axonal nmn/nad+ ratio (osterloh et al., 2012; figley et al., 2021). under normal conditions, the activity of sarm1 (which is often described as “the central executioner of a cell-autonomous axon degeneration program”) is kept in check by a highly labile nad+-generating enzyme nmnat2 (nicotinamide mononucleotide adenylyltransferase 2); nmnat2 is trafficked from the soma into the axon, where it catalyzes synthesis of nad+ using nmn and atp as substrates (conforti et al., 2014). axonal injury disrupts axon transport of nmnat2, resulting in the buildup of nmn and depletion of nad+ distal to the injury site; this increases the nmn/nad+ ratio and activates sarm1, further depleting nad+ levels and causing metabolic crisis that ultimately leads to axon fragmentation. not surprisingly, deletion of nmnat2 in mouse models leads to perinatal lethality; however, mice lacking both nmnat2 and sarm1 are viable and protected against axonal degeneration. in humans, nmnat2 deficiency is very rare; however, homozygous r232q nmnat2 variant (which causes severe reduction in nmnat2 activity) has been linked to fetal akinesia deformation sequence (lukacs et al., 2019), while homozygous t94m nmnat2 variant (which reduces nmnat2 thermal stability) causes mild sensorimotor axonal polyneuropathy with erythromelalgia (huppke et al., 2019). (erythromelalgia is a syndrome that consists of sporadic attacks of burning pain, redness, and swelling, typically affecting the lower extremities; it is most often caused by gain-of-function mutations in scn9a, which encodes sodium channel nav1.7.) in their tour-de-force study published last year, dingwall and coauthors expanded the spectrum of human nmnat2-associated disease by showing that heterozygous nmnat2 loss-of-function mutations cause severe hereditary axonal neuropathy through a sarm1-dependent, macrophage-mediated non-cell autonomous pathway; consequently, the authors have termed this new disease “sarmopathy” (dingwall et al., 2022). the two affected brothers described by dingwall et al. share a very unique clinical phenotype that includes (i) repeated episodes of acute multifocal sensorimotor axonal neuropathy (amsan) that mimic guillain-barré syndrome (gbs), are typically triggered by acute infection, and include severe pain, erythromelalgia, flaccid quadriparesis, and respiratory failure; and (ii) chronic axonal motor neuropathy that progresses between these acute episodes and leads to distal weakness, scoliosis, and wheelchair dependence in the third decade of life. the patients carry compound heterozygous nmnat2 variants (the previously identified pathogenic variant c.695g>a; p.arg232gln / r232q and a novel variant c.292g>a; p.val98met / v98m); each of these variants was inherited from one of the patients’ asymptomatic parents and was shown to markedly reduce nmnat2 enzymatic activity. to further study this new hereditary neuropathy, the authors generated nmnat2v98m/r232q model mice. like the two patients, these model mice developed an age-dependent, motor-predominant axonal polyneuropathy that led to severe hindlimb wasting by 9-12 months of age and was characterized by a severe loss of axons in mixed and motor nerves (fig. 3a-b); in contrast, sensory nerves were largely spared (fig. 3c), as were motor neuron cell bodies in the ventral horn of the spinal cord. unlike the two patients, the model mice did not develop amsan episodes or hypersensitivity to pain, possibly because they were housed in a pathogen-free animal facility and thereby spared from acute infections. as expected, the axonal neuropathy phenotype in model mice was rescued by simultaneous deletion of sarm1 (fig. 3d-f), demonstrating that sarm1 activation was necessary for axon degeneration; a similar, but slightly less effective rescue was achieved by transgenic expression of a dominant negative variant of sarm1. more surprisingly, the sarm1 effect in this mouse model was not entirely cell-autonomous; instead, it was at least in part mediated by macrophage activation, and could therefore be blocked by macrophage depletion (via monoclonal antibody against colony stimulating factor 1 receptor). importantly, macrophage depletion was effective not only when treatment was started at birth, but also when it was started at 4 months of age (by which point mice already developed significant neuropathy); this suggests that blocking ongoing axonal degeneration can tip the balance toward axonal regeneration and functional recovery. figure 3. nmnat2 variants cause progressive axon loss in mice in a sarm1-dependent manner. (a–c) representative images of sciatic (a), femoral (b), and sural (c) nerves in 9–12-month-old nmnat2v98m/r232q (n = 9) or wt (n = 5) mice. percent axonal area/total nerve area are indicated to the right (n = 4–11 mice per age cohort, per genotype). (d–f) representative images of sciatic (d), femoral (e), and sural (f) nerves in 9–12-month-old nmnat2v98m/r232q; sarm1-ko mice. percent axonal area/total nerve area is calculated below each corresponding nerve (n = 3–11 mice per age cohort, per genotype). statistical significance was determined by 2-way anova with multiple comparisons. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. scale bars, 50 μm. [this figure and its legend were adopted from figures 3 (a-c) and 5 (d-f) in (dingwall et al., 2022); this use is permitted under the creative commons attribution 4.0 cc-by international license.] as always, many questions remain to be answered. in particular, it is not clear why acute attacks in human patients involve both sensory and motor nerves, while chronic neuropathy predominantly affects motor nerves in humans as well as mice. [preferential involvement of motor axons in the mouse model can be attributed to differences in macrophage activation between sensory and motor nerves; however, given that increased sarm1 activity is observed in both nerve types, it is not clear what leads to these nerve-specific differences in macrophage activation. it is also possible that motor neurons are particularly vulnerable to sarm1 activation; in agreement with that possibility, hypermorphic sarm1 variants were found to be enriched in patients with amyotrophic lateral sclerosis (als); (gilley et al., 2021; bloom et al., 2022)]. moreover, it is not understood why nmnat2 deficiency primarily affects peripheral nerves in both mice and humans when sarm1 acts as the central regulator of wallerian degeneration in both cns and pns; a plausible explanation that needs experimental confirmation is that longer length of pns axons makes them more vulnerable to nmnat2 deficiency. finally, the prodegenerative effect of macrophage activation in the nmnat2v98m/r232q mouse model was entirely unexpected, not only because it was previously thought that sarm1 acts in an entirely cell-autonomous manner, but also because of a large body of prior research that demonstrated a key role of macrophages in axon regeneration [as discussed in advance #1 of my last update (margeta, 2022)]. these discrepancies suggest that macrophage effects on axonal degeneration are complex and context-dependent, and raise the possibility that macrophage depletion therapies could be either beneficial or detrimental, depending on the specific clinical scenario. one intriguing possibility, which needs to be addressed by future studies, is that macrophage activation promotes degeneration of damaged but still viable axons in chronic axonal neuropathies, while promoting axonal regeneration following severe insults (such as nerve trauma or ischemia) that result in catastrophic axonal injury. advances in understanding of etiology and pathogenesis of neuromuscular diseases 5. axonal injury in guillain-barré syndrome starts at the nodes of ranvier gbs is an autoimmune disorder of the peripheral nerves and spinal roots that is typically triggered by a preceding infection or vaccination; nerve injury is the result of autoantibody-induced complement fixation and macrophage activation. while gbs is traditionally considered to be a demyelinating disease (also known as acute inflammatory demyelinating polyneuropathy / aidp), it can also manifest as a primary acute motor or sensorimotor axonal neuropathy (aman / amsan). bystander axonal injury can also develop in the aidp variant of gbs, secondary to schwann cell (sc) injury. regardless of the disease variant, the long-term severity of gbs is ultimately determined by the extent of axon loss, which is less reversible than the myelin loss; however, the mechanisms that underlie the primary and secondary axon injury in gbs have been difficult to dissect because the key autoantibody targets (such as gm1 gangliosides) are often expressed by both neurons and scs. to circumvent this challenge, a research group in scotland created model mice that selectively express gm1 ganglioside on either sc or neuronal membranes; the peripheral nerves of these mice were then exposed to monoclonal anti-gm1 antibodies and normal human serum (as a source of complement) to trigger acute or subacute nerve injury, either ex vivo (in the triangularis sterni nerve-muscle preparations) or in vivo (mcgonigal et al., 2022). in both mouse models, early injury [following a short (4 h) treatment with anti-gm1 antibodies and complement] occurred at the nodes of ranvier (nor); this is concordant with the neuropathologic studies of patients with gbs, which demonstrated nodal autoantibody and complement deposition (hafer-macko et al., 1996a; hafer-macko et al., 1996b) along with alterations of the nodal ultrastructure (griffin et al., 1996; vallat et al., 2020). in neuronal gbs model mice (which express gm1 selectively in neurons), formation of the membrane attack complex (mac) pores in the nodal axolemma led to calpain-mediated cleavage of adaptor protein ankyrin-g, dispersion and disappearance of the nodal sodium channel clusters, and conduction failure; however, there were no structural changes in the paranodal sc loops and no disruption of the cell adhesion complexes that form “transverse bands” at the axo-glial interface. in glial gbs model mice (which express gm1 selectively in scs), injury started with the mac-induced distortion of the paranodal sc loops and calpain-mediated cleavage of the sc scaffolding protein ankyrin-b, leading to disruption of transverse bands, dispersion of the sodium channel clusters, and conduction failure (fig. 4). thus, the ultimate functional consequence – acute conduction failure – was the same in both mouse models, although the underlying disease mechanisms and associated structural changes were different. acute conduction failure – which is seen in many human gbs patients in addition to these experimental disease models – is often temporary and reversible; however, it can also lead to subsequent axonal transection / wallerian degeneration. indeed, extended (20 h) treatment with anti-gm1 antibodies and complement in vivo led to a loss of the distal motor axon integrity in glial mice, indicative of secondary / bystander axonal degeneration due to nodal sc injury. (the authors have yet to perform similar extended treatment studies in their neuronal mice.) taken together, these results point to nor as the site of initial injury in both neuronal/axonal and glial/demyelinating forms of gbs. [interestingly, nor is also the main site of injury in autoimmune nodopathies, which are caused by antibodies that directly target glial or neuronal cell-adhesion molecules expressed in the nodal membranes; clinically, autoimmune nodopathies show significant overlap with gbs, but are typically more severe and resistant to ivig immunomodulatory therapy (martin-aguilar et al., 2022).] figure 4. ultrastructural evaluation of diaphragms from in vivo gbs models. neuronal and glial mice were dosed i.p. with 50 mg/kg anti-gm1 ab followed 16 hours later with 30 μl/g normal human serum (nhs) (injury) or nhs only (control). (a) a normal paranode from glial control tissue. this image is also representative of the neuronal control nor (not shown). (b) higher magnification of boxed region from a shows tight junctions (large arrowhead) between the paranodal loops, and transverse bands (tbs, small arrowheads) at the axo-glial junction between the axon and paranodal loops. (c) injured glial nors show severely disrupted paranodal loop organization compared with control. (d) magnification of boxed area from c, shows tbs are present between the paranodal loops and axon at the juxtaparanodal-proximal paranode (above black line); however, they are absent at the node-proximal border (above white line, right of asterisk). (e) injured neuronal nors show no architectural disruption. (f) neuronal control motor nerve terminal (mnt) displays normal architecture and contains synaptic vesicles (black arrows). (g) disturbance to the injured neuronal mnt includes an absence of neurofilament, synaptic vesicles, and the formation of dense or vacuolated mitochondria (white arrows). results are representative of analysis from 8–10 nors per mouse (n = 3/genotype/treatment). [this figure and its legend were adopted from figure 6 in (mcgonigal et al., 2022); this use is permitted under the creative commons attribution 4.0 cc-by international license.] what is the relevance of these findings for human gbs? since gm1 gangliosides (and other ganglioside antigens) are expressed by neurons as well as scs, both of these mechanisms are likely in play in human disease, and which mechanism dominates in any given gbs patient likely depends on a combination of factors, including antibody titer / specificity and intrinsic host factors that shape disease vulnerability. moreover, these findings have potential diagnostic implications: taken together with the 2020 ultrastructural study of peripheral nerves from patients with gbs and other nodo-paranodopathies (vallat et al., 2020), which showed ultrastructural nodal pathology similar to ultrastructural abnormalities seen in glial gbs mice (fig. 4), they suggest that electron microscopic evaluation of nor could help delineate immune-mediated from ischemic and other causes of axonal injury, and should therefore be included in the nerve biopsy workup whenever an obvious cause of axon degeneration (such as vasculitis) is not detected in a nerve biopsy. 6. target formation is indicative of muscle fiber reinnervation target fibers are one of the classic diagnostic features of a neurogenic change secondary to lower motor neuron (lmn) injury. targets are most commonly seen in type 1 fibers, are best visualized on oxidative stains, and in the cross-sectional view consist of several distinct concentric layers / zones – typically a central pale area devoid of mitochondrial activity and reminiscent of a core, an intermediate zone that shows a decreased but not entirely absent mitochondrial activity, and an outer zone that resembles the rest of the myofiber and often shows an increase in mitochondrial activity (fig. 5a-b). ultrastructurally, the central zone contains the z-disc material and is devoid of glycogen and mitochondria, the intermediate zone shows disorganized myofilaments with mild z-line abnormalities, while the outer zone contains ultrastructurally normal myofilaments. similar formations that have a less distinctively layered appearance are called “targetoid” and can be more difficult to distinguish from cores if they are not accompanied by true targets and/or other diagnostic features of neurogenic change. however, targets are not present in all muscle biopsies with neuropathic changes, and can be seen both in small angulated and normally sized fibers. as a result, it has been unclear whether target formation reflects denervation [as originally proposed (engel, 1961)] or reinnervation [as suggested by subsequent animal model studies (de reuck et al., 1977)]. a recent proteomic study done by the kley lab sheds a new light on this old question, providing compelling evidence that target formation reflects myofibril assembly that occurs following reinnervation (krause et al., 2022). figure 5. target fibers. (a) nadh-tr stain shows two type 1 fibers with central target formations; on myosin heavy chain immunohistochemistry, these fibers were positive for myhc-slow (mhc7; not shown). the same microscopic field, captured on consecutive cryosections, is shown in all main figure panels; target formations are highlighted by arrowheads. (b) the multilayered appearance of target formations (arrowheads), with the central pale zone, dark mitochondria-rich outer rim, and an intermediate zone in between, is more obvious on sdh histochemistry. (c) desmin immunohistochemistry highlights the inner and intermediate zones of target formations, where new myofibrils are assembled; in some cases, desmin staining is most prominent in the intermediate zone (main panel), while in others it is equally prominent in both zones (inset). (orange line, inner zone boundary; green line, intermediate zone boundary; black line, outer zone boundary.) (d). lc3 immunohistochemistry highlights autophagosomes, a key component of the quality-control machinery responsible for degradation of misassembled or damaged myofibrils; in some target formations, lc3 staining is most prominent in the central zone (main panel), while in others it is most prominent in the intermediate zone (inset). different patterns of target formation staining observed with desmin and lc3 immunostains could reflect different stages in myofibril assembly following fiber reinnervation. scale bar, 50 µm. to investigate the molecular composition of targets, krause and co-authors employed a non-biased, hypothesis-free proteomic strategy: they used 20 muscle biopsies from patients with neurogenic muscle atrophy to micro-dissect targets and analogously sized portions of uninvolved type 1 fibers, and then compared the protein composition of both samples via label-free mass spectroscopy. out of 1026 proteins identified in these samples, 55 proteins were overrepresented and 40 proteins underrepresented in targets compared to the control regions of type 1 fibers. unsurprisingly, the underrepresented proteins were mainly mitochondrial proteins. the overrepresented proteins were more interesting and could be classified into four main groups based on their subcellular localization and/or function: (i) z-disc and actin dynamics (15 proteins, including filamin c and desmin), (ii) myosins and myosin-associated proteins (10), (iii) protein biosynthesis (10, including ribosomal proteins), and (iv) molecular chaperones (5, including alpha(b)-crystallin and bag3); the remaining 15 proteins were classified as “other.” the key mass spectroscopy findings were then validated by immunofluorescent staining and showed zonal pattern of expression, with filamin c, other z-disc proteins, and chaperones in the central zone, desmin mainly in the intermediate zone (fig. 5c), and myosins in both zones. the model that emerged from these proteomic findings suggests that the central and intermediate zones are the sites of new myofibril synthesis; premyofibrils are formed in the central zone, and then assembled and linked with the existing myofibrils in the intermediate zone. chaperones and other components of the machinery involved in chaperone-assisted selective autophagy (such as autophagosomal protein lc3; fig. 5d) are enriched in both zones, providing a quality-control mechanism that ensures that nascent myofibrils are either properly built and assembled or immediately degraded. while this study represents a major advance in our understanding of the reinnervation-induced myofiber rebuilding mechanisms, some aspects of this process still remain to be elucidated. for example, it is not clear why targets are mainly seen in type 1 fibers. does the same process occur in a less structured / morphologically less distinctive way in type 2 fibers, or are reinnervated type 2 fibers rebuilt through entirely different molecular mechanisms? furthermore, it remains to be established how the motor neuron axon and muscle fiber interact to orchestrate this highly organized process of new myofibril formation. 7. sporadic inclusion body myositis revisited: what comes first, inflammation or myodegeneration? sporadic inclusion body myositis (sibm) primarily affects people older than 50 and has distinctive clinicopathologic features that differentiate it from other idiopathic inflammatory myopathies; clinically, there is predominant involvement of quadriceps and finger flexor muscles, while pathologically, there is endomysial inflammation accompanied by diffuse mhc-i upregulation and invasion of intact muscle fibers by cytotoxic cd8+ t lymphocytes and macrophages, along with mitochondrial abnormalities, chronic myopathic features resembling muscular dystrophy, rimmed vacuoles, and p62and tdp-43-positive protein aggregates. however, pathogenesis of sibm remains poorly understood. in particular, the role of inflammation has remained controversial: although sibm muscle biopsies typically show prominent inflammatory infiltrates, the disease does not respond to immunosuppression (or any other therapy) and generally leads to wheelchair dependence ~15 years following the diagnosis. several important studies published in 2019 [and discussed in the first paper in this review series (margeta, 2020)] suggested that the lack of response to immunomodulatory therapy is due to involvement of terminally differentiated effector memory t cells (so-called temra cells), which are positive for klrg1 (killer cell lectin-like receptor g1; a marker of t cell exhaustion) and resistant to steroid-induced apoptosis (greenberg et al., 2019; knauss et al., 2019). several important follow-up studies were published in 2022, prompting me to revisit this important topic in the current review. our understanding of sibm pathogenesis has been limited by the lack of a disease model that replicates both inflammatory and myodegenerative features of this disease. to circumvent this barrier, britson and colleagues developed a xenograft model of sibm, in which human muscle biopsy tissue is transplanted into immunodeficient mice; following transplantation, transacted mature muscle fibers degenerate and are replaced by newly formed fibers generated from satellite cells that were harnessed along with the graft (britson et al., 2022). remarkably, in sibm xenografts – but not in the xenografts generated from relatively normal muscle biopsies or from biopsies showing other types of myositis – regenerated myofibers showed typical features of sibm, including mhc-i positivity, rimmed vacuoles, loss of nuclear tdp-43 expression, and mitochondrial abnormalities (cox deficiency). in addition, sibm xenografts contained donor-derived oligoclonal cd8+klrg1+ temra cells that were enriched compared to the source sibm biopsies. finally, ~50% of sibm xenografts (but only 3% of control xenografts) showed expression of cryptic exons in tdp-43-regulated mrnas, indicative of functional tdp-43 deficiency. (tdp-43 is an rna-binding protein that maintains the health of the transcriptome by repressing incorporation of non-conserved cryptic exons during pre-mrna splicing; cryptic exon expression in the tdp-43-regulated, muscle-specific mrna transcripts was 84% sensitive and 99% specific marker of sibm in the cohort of 119 myositis patients enrolled in this study). having developed the xenograft sibm model, britson et al. used to it to evaluate whether treatment with monoclonal anti-cd3 antibody okt3 would improve various aspects of sibm pathology. the okt3 treatment, which almost completely depleted t cells and significantly reduced the number of klrg1-positive cells in sibm xenografts after 4 months of weekly infusions, led to a significant decrease in the extent of mhc-i upregulation; however, it had no significant effect on myodegenerative sibm features (cox-negative fibers, rimmed vacuoles, and p62-positive protein aggregates). in addition, there was no change in the aberrant expression of cryptic exons. these results quite definitively demonstrate that myodegenerative pathology is intrinsic to sibm muscle and can persist independent of sustained inflammation or circulating factors, providing an additional explanation for refractoriness of sibm to standard immunosuppressive treatments, but also diminishing hope that therapies that directly target klrg1-positive temra cells (which are already in development) will significantly benefit sibm patients. however, it is less clear what these findings mean for our understanding of sibm pathogenesis: while the authors of this study favor a model in which tdp-43 dysfunction is an early event in the disease progression that then drives aberrant mrna splicing, neoantigen expression, and secondary inflammation, it is equally possible that chronic inflammation within the human xenograft donor prior to the biopsy (and/or within the early xenograft, prior to the okt treatment) leads to genetic and/or epigenetic alterations in satellite cells, which then recapitulate tdp-43 dysfunction and myodegenerative changes in newly formed muscle fibers. additional work will be required to distinguish between these two models of sibm pathogenesis; in particular, it should be established whether aberrant expression of tdp-43-regulated cryptic exons is also present in hereditary inclusion body myopathies, which show sibm-like myodegenerative features but lack inflammation. interestingly, the results of another 2022 paper provide support for inflammation as an early event in sibm disease progression (kleefeld et al., 2022). in their cross-sectional study, kleefeld and colleagues examined the similarities and differences between polymyositis with mitochondrial pathology (pm-mito) and sibm. in contrast to sibm, which has a distinctive clinical phenotype, pm-mito is not a clinically well-defined entity; pathologically, however, it shows some sibm-like features (lymphocytic endomysial inflammation, muscle fiber invasion by inflammatory cells, and mitochondrial pathology / cox-deficient fibers) but lacks rimmed vacuoles, chronic myopathic changes, and p62or tdp-43-positive protein aggregates (blume et al., 1997; hiniker et al., 2013). although most patients with pm-mito over time progress to sibm, at least partial response to immunosuppression has been observed in a subset of cases (levine and pestronk, 1998; winkler et al., 2021), raising the possibility that pm-mito and sibm exist on a spectrum, with pm-mito an early – and potentially treatable – stage of sibm. the findings reported by kleefeld et al. support this model: they found an essentially identical inflammatory molecular signature in pm-mito and sibm biopsies, with greater activation of type ii than type i interferon response and expression of cryptic exons in both sets of cases (although frequency of cryptic exon expression was greater in sibm than pm-mito biopsies). in contrast to these similarities, klrg1 and gbp6 (guanylate binding protein family member 6) genes were differentially expressed, with both klrg1 and gbp6 mrna upregulation detectable only in sibm biopsies. in agreement with this finding, gbp6-positive macrophages, gbp6-expressing myofibers, and klrg1-positive t cells were significantly more abundant in sibm than pm-mito samples. overall, most differences between pm-mito and sibm biopsies were quantitative rather than qualitative, with a gradient of severity observed even within the pm-mito group (which could be histologically divided into “mild”, “typical”, and “pre-ibm” subgroups). importantly, this progression of severity was also observed in serial biopsies from two individuals, the first showing pm-mito and the second (5-7 years later) sibm. based on these findings, kleefeld and colleagues recommended that pm-mito should be renamed early sibm and that both disorders together should be classified as “sibm-spectrum disease” (sibm-sd). in addition, they suggested that the difference in klrg1 expression between early and late sibm stages reflected disease duration, with emergence of exhausted t cells a consequence of prolonged inflammation / antigenic stimulation and a harbinger of transition into a therapy-resistant end stage of sibm-sd. in agreement with that hypothesis, another recent study used “deep immunophenotyping” of blood t and nk cells from sibm patients to show that increased cd8+ t cell differentiation correlated with sibm duration (goyal et al., 2022). among many questions that still need to be answered is the “status” of polymyositis without mitochondrial abnormalities, which is histologically identical to pm-mito except for the absence of ragged red and cox-negative fibers (hiniker et al., 2013). it is not currently known whether polymyositis is a separate disease, or an even earlier stage of sibm-sd that can be effectively treated by standard immunosuppressive therapy and is therefore less likely to progress to a chronic inflammation-driven, treatment-resistant end stage. given that inflammation can lead to secondary mitochondrial abnormalities in other inflammatory myopathies (hedberg-oldfors et al., 2022), it is possible that emergence of cox-negative fibers in pm-mito represents the first step on the pathway towards treatment resistance in the setting of persistent chronic inflammation. advances in neuromuscular disease diagnostics 8. improved genetic testing techniques for facioscapulohumeral and myotonic muscular dystrophies despite widespread availability and relatively low cost of advanced genetic testing techniques (such as syndrome-specific multigene panels and whole exome sequencing), 50-75% of presumed genetic neuromuscular disease cases currently remain undiagnosed (cummings et al., 2017). the underlying reasons for these diagnostic challenges are complex and varied, but include the inability of these sequencing techniques to detect structural rearrangements and copy number variants. fortunately, two new genetic testing strategies developed in 2022 will alleviate some of these challenges, simplifying the diagnosis of two common muscular dystrophies (erdmann et al., 2022; stevanovski et al., 2022). facioscapulohumeral muscular dystrophy (fshd) is the third most common muscular dystrophy; it has a fairly unique clinical presentation, with the facial and shoulder girdle weakness that progresses to involve distal legs and the pelvic girdle. however, the age of onset is highly variable (from infancy to late adulthood) and the phenotype can also vary, making the diagnosis challenging. histopathologic features of fshd are relatively unique (fig. 6), but not sufficiently specific to enable definitive diagnosis; hence, directed genetic testing is generally required at some point of diagnostic work-up. molecularly, fshd is caused by aberrant expression of dux4 (double homeobox 4) protein in skeletal muscle, which has myotoxic effects. dux4 is a transcription factor that regulates expression of genes important for preand post-implantation development, but is epigenetically silenced in most adult tissues except in thymus and testis, where it has an unknown function (mocciaro et al., 2021). in fshd, there is a loss of dux4 repression, but that derepression occurs through mechanisms that differ between two disease subtypes, fshd1 and fshd2. in fshd1 (~95% of cases), dux4 derepression is caused by contraction of the d4z4 macrosatellite repeat array in the subtelomeric region of chromosome 4q35 to <12 repeating units; in fshd2 (~5% of cases), there is global hypomethylation of the d4z4 array due to mutations in one of the several genes that encode proteins required for epigenetic suppression. (a permissive haplotype 4qa or 4qal, which provides a polya signal for dux4 mrna, is required for phenotypic disease expression in either disease subtype.) currently, genetic diagnosis of fshd requires (i) confirmation of the permissive haplotype, (ii) determination of the d4z4 repeat length, typically via southern blotting (which requires a large amount of high molecular weight dna), and (iii) sequencing of epigenetic suppressor genes if the d4z4 repeat length is found to be within normal limits. the new, much more streamlined approach for fshd testing developed by erdmann et al. is based on methylation profiling: following confirmation of the permissive haplotype, a high-throughput methylation profile analysis is used to determine the global methylation level of the entire d4z4 repeat array as well as the regional methylation of its most distal repeat unit; individuals with isolated distal hypomethylation have fshd1, while individuals with both global and distal hypomethylation have fshd2 (erdmann et al., 2022). not only does this new diagnostic approach require much smaller quantity of dna and less laboratory effort, it also detects cases that are missed with traditional methods (for example, patients with complex chromosomal rearrangements that cannot be detected by southern blotting) or cases that are in the gray zone between fshd1 and fshd2 (which represent the two ends of the epigenetic disease continuum). in addition, erdmann et al. have shown that the d4z4 repeat methylation profile is a biomarker of fshd severity: in their cohort of 148 patients, the distal methylation level was better correlated with clinical findings (including the age of onset) than the d4z4 repeat length. finally, this study provides definitive evidence that fshd pathogenesis is ultimately driven by epigenetic rather than genetic mechanisms. figure 6. histologic findings in facioscapulohumeral dystrophy (fshd). (a) a representative h&e-stained section shows fiber size variation, endomysial fibrosis, and perimysial inflammation that focally extends into the endomysium. (b) moderately frequent lobulated fibers (arrowheads) are best seen on nadh-tr histochemistry. (c) mhc-i is diffusely upregulated in muscle fibers. (d) cd31 immunohistochemistry highlights enlarged endomysial capillaries. based on the biopsy findings, the patient underwent genetic testing for fshd that showed pathogenic d4z4 repeat contraction (8 repeats) detected on a 4qa permissive haplotype, diagnostic of fshd1. all stains were performed on cryosections; scale bar, 50 µm. myotonic dystrophy (dystrophia myotonica; dm) is the most common muscular dystrophy in adults and the second most common muscular dystrophy overall. as with fshd, there are two distinct dm subtypes, dm1 (which accounts for >95% of cases) and dm2 (which is much rarer). although their clinical features and causative genes differ, both dm1 and dm2 are short tandem repeat (str) expansion diseases: dm1 is caused by expansion of the ctg repeat in the 3’ untranslated region of dmpk gene, while dm2 is caused by expansion of the cctg repeat in intron 1 of cnbp/znf9 gene. interestingly, the mechanism of myotonia is the same in both dm subtypes: mutant rnas sequester rna-binding and processing proteins, leading to incorrect splicing of clc-1 chloride channel (which is required for the maintenance of normal resting membrane potential). histopathology of dm1 and dm2 is strikingly similar, with very high internal nucleation and frequent subsarcolemmal nuclear aggregates in the setting of usual dystrophic changes (fig. 7); therefore, the definitive diagnosis requires genetic testing. although small str expansions (< 300 bp) can be detected by conventional pcr, genetic diagnosis of most str expansion disorders (which in addition to dm1 and dm2 include huntington’s disease, fragile x syndrome, hereditary cerebellar ataxias, c9orf72-related als / frontotemporal degeneration, and other neurologic diseases) currently relies on southern blotting and/or tandem repeat-primed pcr, and therefore faces many challenges already described for fshd in addition to requiring separate probes and/or primers for each str. these challenges are either solved or bypassed by the new genetic strategy developed by stevanovski et al., which relies on programmable targeted long-read nanopore sequencing to genotype all known neuropathogenic strs in parallel, using a single molecular assay (stevanovski et al., 2022). one particularly appealing feature of the platform used for this assay is that it permits flexible inclusion of targets, so that a specific assay used for any given patient can be individually tailored based on the clinician’s input and/or the patient’s preferences; another is that sequencing can be combined with methylation profiling, providing epigenetic as well as genetic information in a single step. as a result, this new genetic testing approach will likely advance basic research in addition to patient care: much remains to be learned about the fundamental biology of the str expansion disorders and the key variables that distinguish normal and disease-causing alleles, and the availability of a relatively inexpensive high-resolution molecular str assay should accelerate research progress in all these areas. figure 7. histologic findings in myotonic dystrophy (dm). (a) a representative h&e-stained formalin-fixed, paraffin embedded section shows fiber size variation, endomysial fibrosis with fatty infiltration, frequent subsarcolemmal nuclear aggregates, and nuclei running in chains along the longitudinal fiber axis (arrowhead). (b) an h&e-stained cryosection shows similar findings, with prominent subsarcolemmal nuclear aggregates and a marked increase in internal nuclei. (c) an acid phosphatase-stained cryosection shows a fiber with prominent sarcoplasmic acid phosphatase-positive granules (asterisk). based on the biopsy findings, the patient underwent genetic testing for myotonic dystrophy that showed >15600 repeats in one allele of cnbp/znf9 gene, diagnostic of dm2. (histologic findings in dm1 are very similar.) scale bars: a and b, 100 µm; c, 50 µm. 9. accumulation of phosphorylated tdp-43 in motor nerves and intramuscular nerve twigs is a diagnostic marker of amyotrophic lateral sclerosis als is a neurodegenerative disease that typically affects both upper and lower motor neurons and is the most common cause of motor neuron disease (mnd) in adults; pathologically, it is characterized by selective motor neuron loss that is accompanied by aggregates of phosphorylated tdp-43 (ptdp-43) in the remaining motor neurons and glia. during life, als diagnosis is based on the clinical criteria; however, there is a significant phenotypic heterogeneity among als patients, and up to 20% of patients with an lmn-predominant form of als are misdiagnosed prior to death. based on the recent work from two separate research groups in italy and japan, immunohistochemical detection of ptdp-43 aggregates in motor nerve biopsies (riva et al., 2022) and/or motor nerve twigs in muscle biopsies (kurashige et al., 2022) may be a useful tool for closure of that diagnostic gap. riva and colleagues performed a retrospective cohort study to assess whether tdp-43 accumulation in motor nerve biopsies can serve as a useful biomarker of als. they retrospectively reviewed the clinical course of 113 patients who underwent a diagnostic biopsy of the anterior motor branch of the obturator nerve, typically for evaluation of a diagnostically confusing lmn syndrome, during the 25-year period between 1994 and 2019. of these 113 patients, 102 had sufficient clinical data to be included in the study; 71 (69.6%) were ultimately diagnosed with als. in the first part of this study, the authors performed blinded histopathology review of original slides for all 102 study participants; 61 biopsies were diagnosed as pathologic mnd, 16 were diagnosed as pathologic motor neuropathy, while 25 were non-diagnostic. excluding the cases with pathologic diagnosis of motor neuropathy (which had 100% sensitivity and 100% specificity for the ultimate diagnosis of motor neuropathy), the specificity of pathologic mnd pattern for the final diagnosis of als in that patient cohort was 66.6%, while its sensitivity was 78.9%. (interestingly, the degree of axon loss in als cases was predictive of the overall and post-biopsy survival length.) in the second part of the study, immunohistochemistry with polyclonal anti-tdp-43 and anti-p(s409/410)tdp-43 antibodies was performed on biopsies from 80 patients for which sufficient archival tissue was available; in this subcohort, 57 patients (71.3%) were ultimately diagnosed with als. accumulation of tdp-43 in myelinated axons and sc cytoplasm was more commonly detected in als patients than in non-als patients, but this difference was statistically significant only when anti-ptdp-43 antibodies were used; overall, the presence of ptdp-43 aggregates had the specificity of 65.2% and sensitivity of 98.2% for the final diagnosis of als. interestingly, some of the patients with a false-positive ptdp-43 signal in their motor nerve biopsies were ultimately diagnosed with inclusion body myositis or idiopathic motor neuropathy. while this finding represents a limitation from the diagnostic perspective, it highlights the mechanistic connections between these disorders: multisystem proteinopathies, which clinically manifest as one or more of these diseases, have been etiologically linked to impaired biology of tdp-43-containing rna stress granules (korb et al., 2021). interestingly, axonal ptdp-43 aggregates were detected in 11 of 11 als cases with normal (and therefore nondiagnostic) motor nerve biopsies, suggesting that ptdp-43 accumulation is an early event in the disease progression that precedes lmn degeneration and axon loss. one limitation of the current study is that the authors did not evaluate whether specificity and sensitivity of the motor nerve biopsy in the als diagnosis could be improved by combining the results of standard histopathologic analysis with ptdp-43 immunohistochemistry; this important question should be assessed in future studies from this or other research groups. kurashige et al. approached the same question in a slightly different way, by focusing on ptdp-43 accumulation in the intramuscular motor nerve twigs (kurashige et al., 2022). in the first part of their investigation, these authors performed an autopsy case-control study that included 10 patients with autopsy-confirmed sporadic als and 12 control, non-als patients; for each case, muscle samples were obtained from the tongue, diaphragm, biceps brachii, and rectus femoris. the second part of the project was a retrospective cohort study, which started by screening 450 patients who underwent a diagnostic muscle biopsy at the authors’ institutions between 2004 and 2019; after all patients with a myopathy and patients with pathogenic variants in the als-associated genes were excluded, the study cohort consisted of 114 patients, 71 of whom had biopsies that included intramuscular nerve twigs. all study samples were stained with both polyclonal and monoclonal anti-p(s409/410)tdp-43 antibodies, which gave identical results. in autopsy cases, axonal ptdp-43-positive aggregates (but no fus-, p62-, or ubiquitin-positive aggregates) were detected in all als patients but not in any of the controls (fig. 8); ~50% of nerve twigs were involved in any given case, with no significant differences among the four sampled muscle groups. in the biopsy portion of the study, 33 of 71 cases with intramuscular nerve twigs (46.5%) showed axonal ptdp-43-positive aggregates; all these patients were ultimately diagnosed with als (9 with the lmn-predominant form), while none of 38 ptdp-43-negative patients received the als diagnosis, and were instead ultimately diagnosed with other mnds or neuropathies. (interestingly, only 4 of the 114 patients in the muscle biopsy cohort were clinically suspected of having als prior to the biopsy.) figure 8. axonal ptdp-43–positive accumulations in intramuscular nerve bundles of skeletal muscle in the postmortem case-control study. (a and d) h&e-staining in axons of intramuscular nerve bundles showed no differences between patients with amyotrophic lateral sclerosis (als) and those without als except for the density of intramuscular nerves (original magnification: a, ×40; d, ×60). (b) immunohistochemical analysis with mouse monoclonal ptdp-43–positive accumulations in intramuscular nerve bundles (arrowheads) of patients with spontaneous als (sals) (original magnification, ×40). (e) immunostaining with mouse monoclonal antibody against ptdp-43 in patients with non-als diseases did not reveal any abnormalities in intramuscular nerve bundles (original magnification, ×40). (c) immunohistochemical analysis with rabbit polyclonal ptdp-43 antibody also revealed ptdp-43–positive accumulations in intramuscular nerve bundles (arrowheads) of patients with sals (original magnification, ×60). (f) immunostaining with rabbit polyclonal antibody against ptdp-43 in patients with non-als diseases did not reveal any abnormalities in intramuscular nerve bundles (original magnification, ×40). [this figure and its legend were adopted from figure 2 in (kurashige et al., 2022); this use is permitted under the creative commons attribution 4.0 cc-by international license.] taken together, these two studies provide compelling evidence that immunohistochemical detection of ptdp-43-positive protein aggregates in motor axons is a useful diagnostic marker of als. while the results of both studies need to be replicated by other researchers, these findings suggest that muscle biopsy evaluation is a particularly promising diagnostic approach: not only are muscle biopsies easier to do and have lower morbidity than motor nerve biopsies, they also showed higher overall specificity and sensitivity than nerve biopsies (perhaps because potentially confounding myopathic biopsies could easily be excluded). of course, the utility of muscle biopsies for als diagnosis is limited by the lack of intramuscular nerve twigs in many specimens (~40% in this study cohort); obtaining larger tissue samples and performing serial level sections will probably help decrease the number of inadequate biopsies if this evaluation becomes a standard component of the als diagnostic work-up. advances in neuromuscular disease treatment 10. sarm1 inhibitors block wallerian degeneration in preclinical models of axonal neuropathy as discussed in advance #4, sarm1 is the central regulator of intrinsic axon destruction program / wallerian degeneration; deletion of sarm1 in mice prevents axonal degeneration in both cns (for example, in mouse models of traumatic brain injury) and pns (in mouse models of axotomy and chemotherapy-induced peripheral neuropathy). the central role of sarm1 in wallerian degeneration raised the possibility that sarm1 blockade would benefit patients with a broad spectrum of neurologic disorders, leading to a major effort by multiple different research groups (both in academia and biotechnology companies) to develop small molecule inhibitors of sarm1. initial proof-of-principle studies with catalytic site sarm1 inhibitors were published in 2021 (bosanac et al., 2021; hughes et al., 2021), with more comprehensive preclinical studies of catalytic inhibitors (bratkowski et al., 2022; shi et al., 2022) and a discovery of a new class of allosteric sarm1 inhibitors (feldman et al., 2022) reported in 2022. sarm1 is a multi-domain enzyme that functions as an oligomer; it contains an auto-inhibitory n-terminal armadillo repeat (arm) domain, repeat sterile alpha motif (sam) domains that are required for dimerization / oligomerization, and a catalytic c-terminal domain that includes the toll/interleukin receptor (tir) motif. the tir domain contains the nad+ hydrolase activity that catabolizes nad+ into nicotinamide, adenosine diphosphate ribose (adpr), and cyclic adpr (which is the second messenger that triggers the wallerian degeneration pathway). under normal conditions, nad+ is bound to the arm domain, resulting in autoinhibition of the sarm1 activity; when the nmn/nad+ ratio increases (typically due to a decreased production of nad+ by nmnat2), nmn binds to the arm domain instead of nad+, resulting in a conformational change that leads to oligomerization of the tir domain and relief of sarm 1 autoinhibition (feldman et al., 2022). several groups have developed non-competitive, adduct-forming small molecule inhibitors of the sarm1 nad+ hydrolase site, with most comprehensive characterization to date reported by bratkowski and colleagues. these non-competitive sarm1 inhibitors function as pseudo-substrates / pro-drugs that intercept nad+ hydrolysis and form inhibitor-adpr adducts, which act as highly potent inhibitors of the catalytic site, with slow on and off rates and the dissociation constant in the low nanomolar range. while all studies have shown that these catalytic site inhibitors prevent axonal degeneration in vitro, bratkowski et al. went beyond cellular axon injury models: they used preclinical mouse models of acute peripheral nerve injury (a sciatic nerve transection model and a model of vincristine-induced peripheral neuropathy) to demonstrate that these drugs are both safe and effective in vivo, with efficacy approaching the efficacy of genetic sarm1 deletion. specifically, treatment with nb-3 (the compound with chemical properties most suitable for in vivo use) suppressed an increase in the plasma level of neurofilament light chain (a biomarker of axonal injury) and led to preservation of distal nerve fiber integrity in both acute nerve injury models; in addition, it attenuated toxin-induced allodynia / neuropathic pain in the vincristine model. (the transection model cannot be used for functional studies because connections between the cns and periphery are completely disrupted.) critically, the nb-3 treatment was effective not only when it was administered prophylactically but also when it was given several hours after injury, a treatment paradigm that is more relevant for potential clinical use. in a separate line of the sarm1 inhibitor research, feldman and colleagues used a chemical proteomic approach to discover an entirely novel class of sarm1 inhibitors: these electrophilic compounds (tryptoline acrylamides) target cysteine c311 in the autoregulatory arm domain, resulting in allosteric inhibition of sarm1 enzymatic activity (feldman et al., 2022). thus far, these new compounds were evaluated only in the cellular models of axon degeneration, where their efficacy was similar to the efficacy of catalytic site inhibitors; it remains to be determined whether they will be similarly safe and effective in vivo. regardless of the in vivo effectiveness of these specific compounds, this study provides initial evidence that sarm1 can be pharmacologically inhibited through allosteric as well as enzymatic mechanisms; because different classes of sarm1 inhibitors are likely to have different side effects and different optimal clinical uses, this finding is both important and exciting. where is this research headed? first, there should be additional preclinical testing of all compounds discovered to date using models of chronic axonal injury (including the nmnat2 deficiency model described in advance #4) that more closely resemble clinical scenarios where sarm1 inhibitors will be used. [in the setting of catastrophic acute axonal injury, degeneration of the distal segment of the injured axon creates a cellular and molecular environment that promotes effective axon regeneration (simon and watkins, 2018; li et al., 2023); therefore, inhibiting sarm1 activation in human patients who experienced traumatic or ischemic nerve injury would probably not be beneficial, and might even be harmful.] second, there should be a continued effort to discover additional sarm1 inhibitors that belong to different chemical classes and/or have different mechanisms of action. finally, the best performing compounds should rapidly proceed to clinical trials. given the incredible pace of research in this field over the last few years, it will probably not take too long before axon-protective medications become available for treatment of human patients with a broad array of peripheral – and possibly central – nervous system disorders. disclosure statement the author receives research support from astellas gene therapies (formerly known as audentes therapeutics, inc) as a member of the muscle biopsy review committee for the aspiro (nct03199469) and fortis (nct04174105) clinical trials, which are evaluating the safety and efficacy of gene transfer therapy for x-linked myotubular myopathy (aspiro) and late onset pompe disease (fortis). acknowledgements i am grateful to drs. nigel g. laing, gina ravenscroft, and benedikt schoser for helpful input during the conceptualization stage of this review. in addition, i would like to thank ms. christine lin for assistance with figure preparation. references abrams, r.m.c., simpson, d.m., navis, a., jette, n., zhou, l., and shin, s.c. 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(2021). diagnosis and clinical development of sporadic inclusion body myositis and polymyositis with mitochondrial pathology: a single-center retrospective analysis. j neuropathol exp neurol 80, 1060-1067. https://doi.org/10.1093/jnen/nlab101 copyright: © 2023 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. 50 years of surgical pathology / 36 years of neuropathology. my way / a personal journey feel free to add comments by clicking these icons on the sidebar free neuropathology 2:28 (2021) reflections 50 years of surgical pathology / 36 years of neuropathology my way / a personal journey sverre j. mørk university of bergen, clinical institute 1, jonas lies vei 91b, 5021 bergen, norway corresponding author: sverre j. mørk · medvitno · skeielia 62 · jonas lies vei 91b · 5239 bergen · norway mork@uib.no submitted: 13 october 2021 accepted: 19 october 2021 copyedited by: henry robbert published: 08 november 2021 https://doi.org/10.17879/freeneuropathology-2021-3639 keywords: neuropathology, reflections, autobiography i: the start 1 drøbak i was born in drøbak (norway) in 1942. drøbak is a village-like small town at the eastern shoreline of the oslofjord (the photo at the top, from the 1960s, shows our dark house dead centre). i grew up here, where everyone ‘knew’ each other. my neighbourhood is depicted in the oil painting from the spring of 1945 (the artist, anton thoresen, lived in the red house). in these safe surroundings, we played cowboys and indians and hide and seek. 1 drøbak 2 giessen 3 oslo 4 kongsberg 5 larsnes 6 ørland air base 7 meråker i always wanted to be a physician. after 12 years of effortless schooling, my final grades lacked a few points for studying medicine in oslo or bergen, the only two medical faculties in norway at that time. in the 1960s and 70s, 50% of norwegian physicians studied medicine abroad. germany, austria, switzerland, great britain, and ireland were among the most popular countries. i applied to and was accepted by universities in scotland, austria, and germany. i did not fancy studying in big cities like berlin, hamburg, munich, or vienna, with all their social diversions (or ‘opportunities’). so, among the actual medical faculties, i chose the one that looked ‘safest’ to me. 2 giessen the medical school of the justus liebig university in (hessen, deutschland) presented a bed-side-teaching concept where the 11 half-year semesters were organised in a school-like schedule (i.e., less freedom of choice for composing your curriculum). the fact that only a few students were accepted every year (‘…kleine zahl seiner medizinstudenten…’) was a perfect fit for me. low numbers of consemesters and transparency (‘who is missing today? almost all on board!’) are two ways to harness young and drifting spirits. steady surveillance by my peers turned out to be a helpful remedy. so, on october 30th 1961, i listened to the first lecture in german. after 3 weeks, i lowered the language barrier enough to understand most of what i heard. although generally restless, i stayed at the justus liebig university from october 1961 until august 1967. i kept the same room (#19) in the new dormitory ‘landgraf ludwig haus’ from autumn 1961 until i left for norway in august 1967. the drawing (45x15cm) by my little brother erling hung above my bed; it has been with me ever since. it depicts the cycle of life and i have been through six of the depicted stages. the right figure fits me best now. sverre, sabine, hans, and peter minutes after the last examination, 1967, and 50 years later (2017). the 12th semester was a tough stretch of examinations, all oral, 19 in all. we were examined in groups of four (the same four students during the whole ‘staatsexamen’). we all did well. here, in the summer of 1967, we were a relieved and happy quartet standing in front of my 11m2 studio after the very last examination. 3 oslo back in oslo, half a year of additional studies (‘tilleggskurs’) were required to pass the last hurdle to be an authorised physician. i was granted preliminary permission to practice medicine for the duration of this course. for the rest of 1967, i worked at the acute psychiatric ward for men of the oslo city hospital, which added to my mental health. i successfully completed ‘national examinations’ in forensic psychiatry, forensic medicine, hygiene, social medicine, and prescription theory, and was awarded my diploma on december 12th 1967. it states that with my staatsexamen from giessen, with the additional course included, i was granted the norsk medisinsk embedseksamen. no crystal ball could tell that four years later i would face many examinations every day executing diagnostic anatomical pathology (histopathology). i learned to see and register what the microscope shows you, internalizing what you see, processing these images into a conclusion, and revealing a correct and crisp diagnosis. 4 kongsberg on january 1st 1968, i started 1.5 years of internship (six months of surgery, six months of internal medicine, lastly half a year of general practice). there was an official raffle for a number that would give you your place in the queue for choosing ‘your’ hospital as well as the country district where you’d spend the last stretch before being certified to practice medicine. i drew a low number, and decided to stay in the south of norway, where i picked the community hospital in kongsberg. there i met and worked together with another intern, tore böhmer halvorsen, who three years later was to be decisive in my way into pathology. i had family and friends in and around drøbak on the east shore of the oslofjord. kongsberg was located inland on the west side of the fjord. during 1968, i’d travel those one-hundred-and-twenty kilometres countless times, through nights and early mornings, probably driving too fast. the interns and other young doctors lived in small townhouses close to the clinic. i had memorable times as a bachelor: socialising, playing bridge, driving on ice-covered lakes, skiing, dancing, sailing, and soccer. 5 larsnes the final six months of my internship took place within the boundaries of the district sande, north of stad, the roughest coastal stretch of norway. my assignment was to assist the county doctor’s general practice. from monday to friday, the local bus unloaded patients at 8:30 in front of the district doctor’s office in larsnes. at the very start most ‘customers’ wanted the attention of the ‘old’ doctor. with some luck in handling, i was quickly accepted by the inhabitants, giving the older doctor and me equal workload. in this large district with several islands and no bridges i had many demanding visits during the winter and spring, going out to islands in bad weather. but in june of 1969, military service in the norwegian airforce called. 6 ørland air base students of medicine, theology, and dentistry could absolve part of the 15 months of mandatory military service during their studies. i finished my recruit training (‘boot camp’) in between semesters. in the summer of 1969, i did a six-week officers course followed by one year as a flight surgeon in a care-of-the-flyer program at ørland air base west of trondheim. to fill this assignment, it was important to get an impression, a real feeling, of how it is to be a fighter pilot. some of the enlisted doctors in the air force did get one single flight with a fighter jet. airtime for the northrop f-5 freedom fighter was precious. all non-fighter pilots within the air base would give ‘anything’ to be taking off in a fighter jet. i was lucky to be granted this experience five times for a total of 6 hours and 25 minutes. i flew tactical missions, night flight, interceptions, and granted control of the jet for some minutes, pretending that the top of the clouds was solid ground! exciting and unforgettable! i met my wife kari during this period and made many life-long friendships. 7 meråker after i completed my obligatory military service, i had not yet decided what kind of a physician i wanted to be. a friend in the military persuaded me to spend the next year in rural meråker (approx. 3,000 inhabitants). the single country doctor was leaving to specialise in psychiatry and had no successor. following my friend’s call, i filled the vacancy for 12 months (1970-1971). it was quite a challenge for a green and inexperienced 28-year-old physician. i still remember my existential thoughts passing the local church that had a huge and grassy graveyard with only a few stones in a corner… now (photo as of july 2021) the trees have multiplied and grown; still there is abundant free space. decades ago, the people in rural areas of norway were not accustomed to unnecessarily bothering the doctor. calls during late evenings and nights occurred, on average, once a month. it was like a fairy-tale for rural doctors today. my medical practice went rather smoothly. when more complicated situations occurred (e.g., marriage counselling), i referred my patients to other competent persons or institutions. generally, childbirths are among the most exciting things in life but i was terrified to being called to a difficult one. thankfully, for me and the meråker community, we were lucky enough to have a brilliant country nurse who acted as a midwife when necessary. her soothing expertise was not called for during my tenure but nurse (söster) inger’s familiarity with generations of inhabitants was indispensable for my practice of medicine there. one story has stayed with me to this day: an old man was found in a river on top of his volvo van. he had fallen asleep on his way to his swedish home, and then the local river flooded after heavy rainfall. the roaring river swept his van away and took it 100 meters downstream, where it snagged on large rocks. a farmer saw the incident and fetched me and other neighbours. we dropped a small boat in the river and secured it with an exceptionally long rope and then made our way to the driver. i was in the back of the boat to catch him. a journalist happened to pass by and shot the telling photo (from the newspaper adresseavisen): my colleague and friend tore b. halvorsen (from internship in kongsberg) had started pathology training in trondheim, the third largest norwegian city. trondheim was located 1.5 hours from meråker. he tried to convince me to start working in the pathology department with him. we had many arguments back and forth, but he won the day he said, ‘the workforce is young, enthusiastic, and easy-going’. i realised that whatever i ended up doing, basic knowledge in general and special pathology would be a benefit. after one year of being the only md for 3,500 persons, it was tempting to enter the safe harbour of a hospital. during my last months in meråker, i had already sent two work applications to hospitals in hamar (internal medicine) and arendal (general surgery). these were specialties miles apart thematically and geographically. the idea was to have some time before choosing the eventual ‘right' specialty. none of my applications were even confirmed received. as the saying goes, ‘one bird in the hand is better than ten on the roof’. i accepted the ‘offer’ from the pathology department in trondheim. late letters of acceptance from both clinics arrived, but too late. i had already promised to join my friend in trondheim. we (kari, baby pia and i) left meråker without having contributed to an increase in the number of tombstones. luckily. ii: the start of my surgical pathology trondheim 1971-1974 sentralsykehuset i sør-trøndela ‘aller anfang ist schwer’. my backbone in anatomical/surgical pathology was formed in the practice of a rather large community hospital. during the years in trondheim, i was influenced by two colleagues, who had entered the pathology field some 8 to 12 months earlier than me. they were experienced fellows compared to the ‘rookie’. my colleagues (tore b. halvorsen and arne ødegaard) managed to keep me ‘downstairs’ doing autopsies for half-a-year. i was told that was the customary entrance to anatomical pathology. starting out with post-mortem examinations should give you ample time (no hurry) to get a grip on the microscopy of tissues and changes therein. thus, my friends (tore and arne, see the next photo) had it easy, cuddled with biopsies, while i, lifting the burdens of autopsies off their shoulders, strived to ‘see’ and to ‘read’ what microscopy offered me. i longed for the real thing to experience the task of being the doctor’s doctor in real time. that is what surgical pathology is about. in goethe’s words: ‘vor die therapie haben die götter die diagnose gestellt’. but i was doing the essential work of diagnostic pathology: establishing borders between normal appearances, physiological changes within the boundaries of normality, and initial changes signalling disease within tissues. after only autopsies for month after month, i longed to address pathology in real time – biopsies. the chief physician of the pathology lab in trondheim was painstakingly meticulous. his goal was not to miss anything in an autopsy or biopsy. he wanted minimal aberrations from the ‘normal’ to be written down on the final ‘list of diagnosis’ on the front page of the autopsy report. i recall 20 or more items on the final list. we’d discuss the individual order of findings with minor, or even no clinical significance to give it its proper position on the list. i still use some of his elegant, descriptive phrases which characterise the microscopic picture in a few, well-chosen words. an awkward situation in the autopsy room: the ‘whole’ lung department came for a demonstration. we had interesting findings and good discussions. in the end, there was something in the air… they complained about the long time it took to get autopsy reports. i protested, knowing that we were pretty prompt. later the same day, a doctor from the lung department informed me that one of the secretaries had a drawer where she puts reports related to ‘dead patients’ without telling anyone. my second ‘boss’ had been on a leave-of-absence as a surgical pathologist in uganda. he was quick to recognise the important players in a tissue section and was effective in his diagnostic service. nor did he dwell on details of no or minimal clinical significance. he was an efficient surgical pathologist. i learned from both, as i did from all the continued education courses in the following years. in 1973 i attended a tumour biology course in uppsala, sweden. the main lecturer was professor jan pontén who headed the institute of pathology at uppsala university. this course became a key experience for me. pontén had a unique presence; he was part of what he presented be it cells, tissues, neoplasia (‘tumours’, new growths, cancers). his research led to insights into growth capacities and proliferation dynamics in normal and neoplastic cells in cultures (in vitro) and in tissues (in vivo). the course involved exciting presentations on glioma tissue culture, multicellular spheroids, contact inhibition, growth capacity and proliferation dynamics. bengt westermark’s lectures also left a lasting impact. he presented many things that were new to me in my second year of diagnosing common surgical biopsies. for me, he added a third and a fourth dimension – he revealed the sheer complexity of the still-life lying in front of your eyes on the glass slide. you would have to imagine what is just a few microns below and above the actual tissue level. you have to think of the 4th dimension: the time aspect, how the present lesion could have evolved and what lies ahead (prognostication). the equation of ‘positive’ (proliferation/cell divisions/mitoses/mitotic figures) and ‘negative’ elements (cell death/apoptosis/apoptotic figures/necroses) defines the biological aggressiveness of a neoplastic tumour. location and eventual spread add to the equation. the tumour biology course contributed to my interpretation ability but it also instilled in me the importance of trying to understand the biology and natural history of human cancers. since there were no neurosurgeons north of oslo, the ‘university hospital’ trondheim planned to start a neurosurgery unit at the hospital where i worked. the plan materialised in the mid-1970s with the appointment as rolf ringkjöb as the first neurosurgeon in trondheim. to be prepared for the challenges this would bring, i applied for a junior position in the neuropathology section, department of pathology, rikshospitalet (the national hospital of norway) in oslo. the main task was to get accustomed to biopsy material from the peripheral, and central nervous system, the eye and orbit, as well as from skeletal muscle. the plan was to return to trondheim to serve the neurosurgery department there. iii: the start of my neuropathology rikshospitalet (the national hospital of norway) 1974-1976 rikshospitalet (rh) was a hospital where special, rare, and difficult cases found their way. the neurosurgical department in oslo served the whole country (about 3.500.000 individuals in the 1970s). the need for a set of criteria for histopathological diagnoses grew in the 1960s. we needed a necessary foundation for relevance of international cooperation in epidemiology and clinicopathological studies on cancer incidence and survival. the first ‘blue book’ was: kreyberg l: histological typing of lung tumors. geneva, world health organization, 1967. the pathology department at the national hospital (rh) was influenced by the prominent pathologist leiv kreyberg (1896-1984). he headed the institute of general and experimental pathology from 1938-1964. professor kreyberg’s scientific contributions were many. he left permanent footprints on carcinogenesis research, in typing and classification of lung cancer as well as diagnostic histopathology with emphasis on tumour biology. olav hilmar iversen (1922-1997) headed the merged pathology department from 1964 to 1990. he recruited ‘aspiring scientists’ to focus on skin cancer (chalones), cell proliferation and growth control. one of them, ole didrik laerum was to become a major influencer in my personal and scientific journey. my mentor to be, ole didrik laerum, had done his phd thesis on metabolic events related to cell population kinetics during early stages of mouse skin carcinogenesis. laerum was inspired to follow a scientific career, and joined manfred rajewsky’s group at the max planck institute of virus research in tübingen, germany, in 1971 as a postdoc. around that time n-nitroso compounds were known to induce brain tumours in many different animal species. the rat turned out to be the animal of choice for experimental tumour production. laerum and rajewsky focused on the induction of brain tumours in the offspring of bdix-rats after administration of ethyl-nitrosourea (enu) at the 18th day of gestation, a standardised system developed by ivancovic, druckrey and others. carcinogenesis was simultaneously followed in cell culture (in vitro) as well as in enu-treated rats (in vivo). aagot christie löken (1911-2007), led the neuropathology section from 1952 until 1978. the official painting of her indicates the gentle intelligent person she was. to get into this new (for me) exciting field, works by percival bailey & harvey cushing, hans joachim scherer, klaus joachim zülch, lucien j rubinstein, for example, were mandatory reading. not least, the works of the neuropathologist scherer deserve a note. he published in german during the late 1930s and 1940s (also in english). scherer performed enlightening histological studies on the development of malignant brain tumours in humans. he described how tumour cells invade the adjacent brain tissue by following existing anatomical structures and taking shape after them (secondary structures of scherer). this was in distinction to the primary arrangement of the cells in the tumour itself. and this was just one of his many pioneering assessments. my neuro-oncological literature studies were educational and helpful, and i’ve returned to these books throughout my career. from the fall of 1974, the section of neuropathology was to be my workplace for the next two years. here i was to meet the spectrum of primary brain tumours, the challenge of cell culture, and the excitement of experimental brain tumour research. this photo of a brain section with grey and white matter and a brain tumour with a bleed (hemorrhage) was taken by norbert wey from a case i examined with a young colleague, jens pahnke, in 2002. (department of pathology, university hospital zurich, switzerland) early on, dr. löken asked me if i would be interested in working up some special brain tumour cases for eventual publication. of course, i said ‘yes’. she suggested to focus on a group of neoplastic brain lesions with a varied connective tissue-like arrangement mixed with a common primary pattern of a malignant brain tumour. this new growth (tumour) is called gliosarcoma, a tumour pattern that needed clarification. as a newcomer to this intricate field (neuropathology/neuro-oncology), the first thing i had to do was to find out what kind of pattern warranted the use of this wording/diagnosis. microscopically, i saw biphasic growth with one clear cut, intrinsic, malignant brain tumour, and another component with aggressive mesenchymal features. evidently, this last tissue component could present in many forms. long story short, my brain and i needed to start out with a tumour that had a more delineated histology. new to brain tumours, ependymoma seemed to be a relatively clearer defined entity. therefore, i proposed to go through the national hospital’s files to collect all tumours with this diagnosis. it turned out to be 148 cases of ependymomas of the brain and spinal cord in the archives from 1953 to 1974.this number reflects occurrence in the whole population of norway during that period. after revisions and exclusion of plexus papillomas and subependymomas, we started a follow-up study of 101 cases. more on this project later (vide infra, part 2). the mds of the pathology department met every morning at 8:00. i was early for my first morning meeting. i placed myself at the long table. rather quickly i realised that my table position was wrong and unwanted. i repositioned, back to the wall (‘junge burschen, zweite reihe’) and fell in line. time and again visiting pathologists and scientists ‘misplaced’ themselves, finding a professor standing tall alongside his/her assigned chair. writing this, i recall another feeling: the anxiety fuming around the more scientifically oriented leading pathologists when, on rare occasions, a frozen section from a tumour-suspect breast lesion arrived under the microscope. my backbone (i felt like having one at the time!) in anatomical/surgical pathology was formed in the practice of a rather large community hospital. thus, i felt confident evaluating the more common surgical biopsies. my self-centered ‘expertise’ was not often called for. our section was run by two technicians and two mds. i was in the starting block and enjoyed the challenges. we had a small frozen section area in front of the operating theatre on the 7th floor of the old block. when we were called upon, we had to leave our new high-rise and go into the main building which had a pater noster (see illustration) for staff only. a fun ride, especially when daring to go over the top. photo credit: l. weber, 2014 my first frozen section: i have a habit of examining the slide before reading clinical info. i thought (and still think) that swiftness is a way of signalling proficiency. i quickly gave my interpretation over the intercom: ‘meningioma’. my name said nothing to the neurosurgeon, nor did my diagnosis impress the operator, who called out that he was in the middle of the pituitary gland. he said that there was no way the lesion was a meningioma. probably followed by a few more words not fit to print. the next few days, i checked through all files and found close to ten cases where ‘meningioma’ was called on frozen and changed to adenoma after viewing the permanent sections. a comfort of sorts. the work with the special brain tumour, ependymoma, stayed with me for a couple of years. at that time, the norwegian population was ethnically consistent. we had one of the oldest cancer registers in the world, kreftregisteret (est. 1951), with complete, mandatory reports of cancer cases from all pathology departments in norway. this registry could be linked up to the mortality register of the ‘statistics norway’ (statistisk sentralbyrå, est. 1876). thus, we could obtain a follow-up rate of 100%. having such a setting, we wanted to establish the frequency of ependymomas in a well-defined, homogeneous population, and determine survival times after brain surgery. the final stages of the manuscript had a special and fortunate course. karin (blikstad) miller, a colleague of mine, knew that i worked on a brain tumour project, and also that i had little experience in such an undertaking. she ‘offered me’ her husband saying that he loved to help aspiring authors with all sides of a manuscript. after retirement in 1968, ashton miller (1908-1992) moved to oslo with his wife, karin. he had been president of the section of urology of the royal society of medicine in england. the result of his help with language, disposition, and corrections led to our shot at a high-ranking journal for eventual publication. i chose cancer, a top cancer journal (of the american cancer society) at that time. miller’s ingenious help with corrections and suggestions proved invaluable. it turned out to be a bull’s-eye (see below). juan rosai asked for and got permission to use one figure (a kaplan-meyer plot showing survival related to location of the tumour) in ackerman’s surgical pathology (juan rosai / sixth edition, 1981). six years later lucien j rubinstein ‘confessed’ to me that he was one of the reviewers. he had advised the editor to ‘accept it as is’. ependymoma paper: manuscript mailed form bergen, november 8th, 1976 to the editorial office of cancer, washington, d.c. acknowledged as received, november 12th, 1976. letter of acceptance, december 3rd, 1976. accepted directly, unchanged after 3 weeks. published, cancer 40:907-915, 1977. ole didrik laerum returned to oslo in 1972, where he continued in vitro and in vitro research on human and experimental brain tumours. he had brought with him a breeding pair of bdix-rats, a gift from druckrey personally. in the summer of 1973, he got a call for an academic position at the gade institute, haukeland university hospital, in bergen. the call was furnished with means to continue and expand his research facilities. he accepted the call and moved to bergen with his family and a pair of rats! from the summer of 1974, he embarked on a unique academic and scientific career in bergen. in 1976, a flow cytometer (an advanced cell sorter based on laser measurements) enforced the research landscape at gade’s. he successfully guided 44 candidates to phds since then. in addition, laerum served as rector of the university of bergen, 1990-1995. i took over parts of laerum’s cell culture duties in oslo. from 1975 on, i was repeatedly asked to join laerum in bergen. going there would probably secure an academic career for me. so… iv: the gade institute, department of pathology, haukeland university hospital, bergen, norway gade’s institute was originally named ‘dr. med. f.g. gades pathological anatomical laboratory’. fredrik georg gade (1855-1933) was the son of a wealthy merchant in bergen. he was a pathologist who defended his thesis ‘about pathological anatomical changes in the tissues of neurotrophic origin’ in 1900. gade donated nok 150.000 (inherited from his father) to build a house exclusively for pathology (which included bacteriology at that time). the gade institute was inaugurated on march 15th 1912. on friday october 1st 1976, i started on another stretch of my way as a consultant in surgical pathology and neuropathology at the gade institute in bergen. that same autumn, i was board certified in the specialty of pathology. going to bergen, i brought three years of anatomical/surgical pathology with two years of neuropathology, added as a subspecialty to complete the fiveyear minimum of specialty training for being a certified pathologist in norway. this duality, surgical and neuropathology, stayed with me from then on. the beginning was demanding, with lots to learn, in many ways and in many fields. ernest glück was the oldest and the most experienced surgical pathologist in bergen at that time. he took me under his wing and enabled me to become a doctor’s doctor at the haukeland university hospital. he had an open door, always willing to help when i felt insecure diagnostically. on a double-headed microscope with him, my suggestions were corrected or affirmed. it took almost half a year before i was joined by my family, and we could start settling in. however, in my scientific life, i settled in immediately with the group laerum had started to build at the gade institute. the long presence of the hanseatic league merchants in the city of bergen (1350-1750) has contributed to the unique bergen dialect. bergeners have a saying: ‘i’m not from norway, i’m from bergen’. the wet climate is another well-known fact. many jokes about the climate in bergen are ‘floating’ around you probably have heard at least one? neurosurgeons, neurologists, ophthalmologists, and ear-nose-throat doctors in bergen were pleased to have a ‘fresh neuropathologist’ aboard. recent biopsies and brain autopsies were presented in the presence of neuroradiologists, neurologists, neurosurgeons, and others, with interest in the actual case. my colleagues interacted for the best of the person in focus (also called patient). in other words, these were clinicopathological conferences in the same form as at the rh in oslo (all over, for that matter). except that i always used gloves. i recall that dr. löken sometimes demonstrated bare-handed. a young and enthusiastic neurologist, harald nyland, contacted me early on. he tried to persuade me to take specimens from fresh autopsy brains. ☹ that was against my neuropathological workbook. to find and eventually confirm a suspected pathology in the central nervous system, you should first secure the brain, eventually with the spinal cord, trough fixation in 10% formalin (4% formaldehyde solution) for at least 2 weeks. harald insisted upon the necessity of using unfixed brain tissue in the search for immune-active cells in the plaques, typically seen by the naked eye (grossly/macroscopically) in this brain disorder. this research resulted in significant findings (to be presented further down the road). however, since immunohistochemistry emerged to be effective on formalin-fixed tissue, the standard fixation method was used on most post-mortem brain examinations. a thorough study of post-mortem brains is still fundamental to understand neurological diseases. gade’s staff around 1980, ‘none mentioned, none forgotten’. exception: professor emeritus erik waaler, in front, who discovered the rheumatoid factor in 1939. he went to bergen and opened the medical faculty here in 1947. an open mind a touch of subjectivity is inherent in histopathological diagnoses. a reality is evidenced through inter-observer as well as intra-observer variance in histopathological assessments. since i am easily influenced, i long ago settled into a routine of ignoring the specimen’s clinical, anamnestic data. my policy is to grab the object glass, glance at the slide on its way to the position between light source and objectives. to look through the microscope before reading at the clinical data (age, gender, actual problem et cetera) is my way to lower bias in reading the histology. that is, before i acknowledge the clinical problem, and the eventually suggested, tentative diagnosis (tx). starting every biopsy assessment with an open mind, trying to decide the source of the material (organ?), then a quick glance for changes, has worked well for me. it is not a time-consuming ordeal, one second, sometimes a few, does it. the trick is to convert the evaluation (reading) of every biopsy specimen into an internal competition. this habit makes the daily workload fascinating to handle. i’ve not had a single day of boredom in the company of my microscope and a carefully produced tissue section. i’d say that this habit (modus operandi) has fed alertness and positivity. in my office i had two computer screens in front of me. one hooked up to the pathology department and the other connected to the servers of the university for surfing the web to keep updated. in this way i could confer with the newest data without having to shut down the one with sensitive patient information. as mentioned before, the bdix rat was an asset that laerum used to the fullest extent for the years to come. the breeding of the bdix rats was in the hand of animal technician tore-jacob raa. he kept breeding 35 generations! the +40-year-old photo shows us injecting tumour cells intraperitoneally: during the 1970s, marc mareel and leo de ridder at the university of ghent, belgium, developed organotypic methods for the study of invasiveness by malignant tumour cells in vitro. chick heart tissue was found to be well suited for invasion studies. in bergen, laerum cooperated with the group in ghent, and i was naturally involved in these activities. this cooperation led to my thesis: ‘characterization of premalignant and malignant cells from the nervous system’, which i defended publicly at the university in bergen, may 1983. v: international activities the idea to start a society of neuropathology in the nordic countries originated chiefly from erna christensen and edith reske-nielsen in denmark, aagot christie löken in norway, and patrick sourander in sweden. ansgar torvik, oslo and yngve olsson, uppsala were involved as well. the scandinavian society of neuropathology was to be a forum for persons interested in clinical and experimental neuropathology. the founding meeting was held at rigshospitalet, copenhagen, denmark on january 23rd 1965. erna christensen and patrick sourander were among those few who re-formed the international committee of neuropathology into the international society of neuropathology in copenhagen in 1967. sourander was the grandfather of modern neuropathology in scandinavia. the scandinavian society had joint meetings with british, german, italian and other nation’s neuropathological societies. these were friendly and inspiring conferences with social events. several nordic neuropathologists were members of other nations’ neuropathological societies. the winter and summer meetings of the british neuropathological society (bns) were especially popular among scandinavians, i would say. in the bns summer meeting 1979 in oxford i presented ‘ploidy of human intracranial neoplasms studied by flow cytometry’. i did not follow up ploidy and brain tumours, since i did not feel that this would have an impact on prognostication of an individual person’s brain tumour. (two roads) the first large and overwhelmingly impressive meeting on my way was that of the viiith international congress of neuropathology, washington, d.c., usa, september 24-29th 1978. i still have the program and the list of participants, as well as the abstracts of that meeting. this event was special for americans too, since it represented the first international congress of neuropathology to be held in the united states. the earlier congresses are listed here: i 1952 rome ii 1955 london iii 1957 brussels iv 1961 munich v 1965 zurich vi 1970 paris vii 1974 budapest viii 1978 washington, d.c. kenneth m earle was the president and henry de forest webster the secretary general of that congress. i have good memories of both these gentlemen. the same applies to officers of the aanp from that meeting: asao hirano, john j kepes, richard l davies, and michael n hart. from the congress, one memorable recollection: a big audience in a dark, huge room with a large picture projected onto the wall. there was a tiny, fuzzy structure barely visible, in the middle. this was stanley prusiner’s first image of a scrapie associated particle. if body language and unclear murmurs were any indication, the audience was not convinced that there was a specific structure to be seen in the image. in the program leaflet, i ticked off a platform presentation: ‘gfa and intermediate filament protein in glioma cells’, given by anders paetau of helsinki university, helsinki, finland. it must have been my first acquaintance with this household molecule to be. the 1980s in 1980, i returned to washington, d.c., to spend three weeks at the armed forces institute of pathology (afip) in bethesda, maryland, just outside the city limits. the afip had made life easier for the world’s surgical pathologist through their soft-covered fascicles (‘a set of books being published in instalments as separate pamphlets’), which presented the tissue patterns of tumours. these inexpensive atlases of tumor pathology standardised nomenclature, classification, and diagnostic, histological criteria of human tumours of all primary sites. i got permission to visit the departments of neuropathology and ophthalmic pathology. after a quick photo session i had an id card, and then the huge concrete building (‘atom bomb secure’, they said) was free to me. my plan for the first week was to see as much eye tumours as possible in the unit of ophthalmic pathology, headed by lorenz e zimmerman. on monday morning, ‘second in command’, ramon l font welcomed us (around 10 us and international eye aficionados) with actual, current cases of diseases of the eye and orbit. he really grilled us for diagnostic suggestions. originally from cuba, he spoke with an accent. i recall him correcting the eye doctor (from mexico) in front of me, who had answered ‘leukemia’; ramon font with a prompt correction: ‘leukemia, leukemia’. the doctor in front of me uttered in his cupped hand, ‘at least i speak two languages’. zimmerman had finished editing the blue book on eye and orbital tumors. the manuscript was being printed. he was so kind to give me access to the background material, most importantly, a box with glass slides. the content was identical to the material sent to leading ophthalmic pathologists worldwide. the rest of the week i was completely occupied by ‘reading’ the different lesions through the microscope. with the box came the diagnoses offered by every one of the cooperating international ophthalmic pathologists (more than 10 of them). i kept my eyes (see ‘an open mind’ above) away from the judgment of the international expertise. i worked the microscope in a relaxed mood (‘no rush’) and wrote down my assessment of every case. only then would i check the diagnoses of the specialists from the international editorial board. the head of department of ophthalmology in bergen, professor torstein berthelsen (1923-2008) did contribute financially to my study tour to the afip. the week ended with a feeling of having the stamina to adequately serve the eye doctors of western norway. the last two weeks of my way of the afip experience turned out to be great as well. i was the only ‘free player’ there. i was fortunate to have the personal attention of the neuropathology staff, especially gary clark and james henry, who helped in picking interesting cases to study during my stay. vernon armbrustmacher supplied me with muscle biopsies. i recall memorable lunches with kenneth earle (chief of neuropathology) and leslie h sobin (editor of innumerable books on histological classification of tumours, and poetry (see example in box)). despite all the studies we try some tumours our efforts defy; we know not their name nor what is their game so we say they are unclassified l h sobin i made life-long friends at the afip. the afip experience added substantially to my brain’s library of diseases. afip is history now. wikipedia: in a blow to the us’s pathology community, the armed forces institute of pathology will close its doors sept. 15 (2011), the victim of government cost-cutting initiatives. the washington, d.c.-based facility, which holds around 95 million tissue samples in its repository and employs platoons of ‘renowned scientific consultants’, has been a ‘global resource for disease diagnosis and analysis’ for nearly 150 years. returning to scandinavia in 1980 on the flight back to scandinavia, i was seated next to a young japanese who told me of his years in palo alto at stanford university which was the highlight of his time in the us. his positive tale about palo alto/stanford planted a seed in me. wasn’t the eminent diagnostician and renowned author of the brain tumour ‘bible’ situated in stanford? he was. the unrivalled textbook on brain tumour pathology used since the 1960s was pathology of tumours of the nervous system (published by edward arnold). this book was co-authored by dorothy s russel and lucien j rubinstein. it was first published in 1959. in total, five editions of this critically acclaimed, highly appraised book have been published. after defending my thesis in may 1983 i felt ready for a change. i planned to join lucien j rubinstein for a one-year sabbatical. i wrote a letter (september 20th 1980) introducing myself, asking for a possible stay under his guidance. professor rubinstein’s response came some weeks thereafter. he was pleased with my ‘interest in coming to work with us’ and he detailed interesting projects they were pursuing. he asked for three letters of reference, ‘possibly one from dr. löken, whom i know of course quite well’. the letter also stated: ‘we are planning to move our neuropathological operation from stanford university to the university of virginia in charlottesville’. this was a surprising statement; i had planned for both: stanford and rubinstein. the latter had to be the preference, of course. so, instead of sunny california, virginia would be my state of residence, if i got the opportunity to team up with ljr. opportunity knocked, and a few years later, i went through the door. (see viii) ix international congress of neuropathology, vienna, austria september 5-10th 1982 wednesday, september 8th: at 16:30 i presented ‘in situ characterization of t lymphocyte subpopulations in multiple sclerosis (ms) brains’. at 16:45 ute traugott presented ‘localization of t and b cells in multiple sclerosis (ms) plaques’. these presentations were awaited with interest, not least due to the take-home message of traugott and raine’s abstract: ‘shown here for the first time in the human brain’. the chairpersons (ingrid allen and ellsworth c alvord) decided that the two last oral presentations of the afternoon were to be discussed together at the end. the discussion i was nervous and cannot recollect much about the discussion. i recall that the meeting room was half dark, with thick curtains hiding the sun outside. one person stood up, asking me if i ‘believed’ those cells i presented really were lymphocytes. i ended up saying, in the affirmative, that ‘i believe they are’. then he pointed at me, looked around in the audience and called out (screamed, in my ears/brain): ‘he’s a believer…, he’s a believer!’. this aggressive comment hit me in the chest, like a heart attack. after the session closed, i was told that this person was known to act rudely/impolitely in discussions. he, a virologist, from the us west coast, was mikkel gammelsten (name changed to old norse by me). i still remember the few neuropathologists who patted my back afterwards. but this unpleasant occurrence is still with me. to cure ‘it’, i reset my focus to the message we presented in 1982. the cedric raine group penned it for us: ‘t cells, shown here for the first time in the human cns’. we could not have written it better ourselves. now it pleases me to look back on that afternoon session in vienna 40 years ago and re-establish the fact that our group from haukeland university hospital (harald nyland, department of neurology), roald matre (immunologist, broegelmann research laboratory, and i) were the ‘first’ to show evidence that t-cells were at the site of ms lesions. we even gave an estimate of the ratio (5:1) between helper and suppressor lymphocytes, as well as presenting cd8 positive cytotoxic t-cells in the parenchyma. our findings were published in a letter to the editor under the heading ‘t lymphocyte subpopulations in multiple sclerosis lesions’ in december 1982 (new england journal of medicine 307:1643-1644, 1982) and those of traugott and raine in january 1983. this fact seems forgotten in most references to this topic. the results presented in vienna 1982 have been repeatedly confirmed during later years. in 1986 the scandinavian neuropathological society hosted the xth international congress of neuropathology. this meeting took place in stockholm, sweden with patrick sourander as president of the congress. it was a huge success, scientifically as well as financially, not least through the economic efficacy of yngve olsson. to my knowledge, the number of participants (1,300) have never been surpassed by later international and european neuropathology congresses. in 2002 our society hosted the euro-cns meeting in helsinki, finland, with matti haltia and hannu kalimo as chief organisers. the 12th european congress of neuropathology was organised by bjarne winther kristensen, denmark from may 31st – june 3rd 2021. our society also organises the northern lights neuroscience symposia. vi: northern lights neuroscience symposia during the preparation for the 1986 event in stockholm, the idea came up to start a high-quality series of neuroscience symposia. i took the challenge of organizing the first such meeting. my cooperation with neurologist harald nyland had focused on multiple sclerosis for some years, and it was convenient to select themes related to this disease. the leader gerd hagen of the norwegian ms society provided financial support so we could invite active researchers from any country to give the newest research in their field of expertise. all the 17 internationally acknowledged scientists complied with our invitation to contribute to the first northern lights neuroscience symposium. the scientific focus was ‘myelin and demyelination’. one of the invited, bruce d trapp, would turn out to be a valuable research partner for the next 30 years! bergen is the hometown of the composer edvard grieg. no rain and extraordinary nice weather, cloudless blue skies, and up to 25oc daily for the meeting (may 20-23rd 1987), which coincided with the opening of the bergen international music festival. this is a yearly main norwegian cultural event existing since 1953. a fine combination of science and culture. kuo-chun ma (shanghai) with yngve olsson (uppsala). most participants came from europe, many from north america and a few from asia and africa. some of us still remember dr. kuo-chun ma from shanghai. he had been taken from the dinner table and imprisoned for eight years by the gang of four. he had learned english well enough that he could serve as an interpreter. that was reason enough for imprisonment. he was one of the first mds allowed to travel out of china. kari skullerud and yngve olsson could tell you more about the astonishing dr. ma, a highly intelligent and flexible personality. probably they will do so here in free neuropathology. sam ludwin (kingston) and hans lassmann (vienna) enjoy luncheon at hotel norge, bergen (first nlns, may 1987). there have been numerous nlns since then. to mention a few: the one in 1995 (the vith nlns on inflammatory muscle disorders) was an adventurous and highly praised event. the location was svalbard/spitsbergen and kari skullerud (neuropathologist, oslo) had the idea and organised the symposium. ellsworth c ‘buster’ alvord (1923-2010) and wife nancy (left) took part in our first nlns. he was an outstanding neuropathologist and scientist from seattle (university of washington). numerous attendants attested to the quality of all aspects of this very first nlns gathering with a focus on myelin and demyelination. see reactions tagged for the facsimile. harald nyland and i organised another symposium on multiple sclerosis in 1996. we used a painting by the artist nicolai astrup (1880-1928) as an eye catcher. in english the title of the oil painting is ‘midsummer eve bonfire’. for us it symbolises the various aspects of demyelinating lesions. multifocal fires of different age and intensity, some burned out, others with flames high! by the way, nicolai astrup was introduced for the first time in the usa just this august 2021. the venue: the clark art institute, williamstown, ma. the boston globe writes: ‘the best artist you’ve never heard of’ and adds: ‘the wall street journal and the new yorker, no less, are cheerleaders’. vii: international activities continue the ixth nlns in reykjavik, 1998, was organised by my good friend gudmundur georgsson. he is standing on the left. in front peter stubbe-teglbjerg (aalborg) with wife, center mara popovic (ljubljana) and marie bojsen-möller (aarhus), then henning laursen (copenhagen), inger mazanti (southampton), and henrik schröder (odense). american association of neuropathologists inc. and other activities i have attended many annual meetings of this society, and met so many people who left an impression on me. the list of all those whom i feel a special bond with would be too long. but i must mention here two persons: one a neuropathologist and the second the spouse of a neuropathologist. i met bernd walter scheithauer in june 1984 at my first annual meeting of the american association of neuropathologists. during a lunchbreak i saw him having a beer at the pool bar in the holiday inn at the embarcadero, san diego, ca. when i think of him, i recall a quote from the tv show mad men: ‘when a man goes into a room, he brings his whole life with him’. i joined him and swapped stories interchanged with information about our similar ‘upbringing’ in europe. in later meetings, i noticed that bernd was augmenting newcomers, disregarded their origin. he trained in the department of pathology at stanford university school of medicine during the last half of the 1970s. there he was influenced by rubinstein who was the director of neuropathology at stanford at that time. from 1979 bernd worked in the department of laboratory medicine and pathology at the mayo clinic in rochester, minnesota. his neuro-oncological expertise was undisputed, and he enjoyed displaying it. he welcomed many norwegian pathologists to stay with him at the mayo clinic in rochester, minnesota. we met many times on both sides of the atlantic, presenting cases at workshops etc. in prague, toronto, zurich, and at other venues. both of us, among others, were searching for histological features that could predict survival of persons with oligodendrogliomas. at the 1984 meeting i presented preliminary data on oligodendroglioma. the full work ‘oligodendroglioma. histologic evaluation and prognosis’ was published in 1986. in a review titled ‘oligodendroglioma: diagnosis and prognosis’ (seminars in diagnostic pathology 4:352-261, 1987) janet m bruner (chief neuropathologist, m. d. anderson hospital and tumor institute, houston, texas) notes: ‘detailed clinical analysis in two large series of cases,2,3 with similar attention to the relation of prognosis and histologic features in two companion studies4,5 have provided concrete and apparently valid criteria for grading’. grading of oligodendrogliomas was an issue of interest not only for neuropathologists. bernd was used to the so-called st. anne-mayo grading system, which was primarily for astrocytomas. nuclear atypia, mitosis, endothelial proliferation, and necrosis as the morphologic criteria to assign a grade, 1-4, according to presence of cero to all four criteria. the norwegian experience was that microcystic change, necrosis, and cell density were the only histologic features of prognostic significance. it was surprising that nuclear atypia, mitosis, and endothelial proliferation did not display effect on survival. it was a fertile soil for discussions and further studies (more on this topic under ‘1990’ later). he opened his home to me, and i am happy to say that he also stayed in our home in norway a couple of times. bernd died in 2011. he is very much missed. since san diego (1984), i have visited arlington, atlanta, boston, cleveland, denver, minneapolis, new orleans, orlando, pittsburgh, portland, st. louis, salt lake city, san francisco, seattle, and washington, d.c., but have not done much tourism, unfortunately. usually, it is airport hotel airport like for most readers of free neuropathology, i’m sure. annual meetings usually occurred in the first half of june, starting on thursday, and ending early afternoon on saturday. when your home base reimburses attendance fees, with paid leave of absence included, you are reluctant to leave a meeting. but i could get in some exploring by having a stayover to sunday (ostensibly to save on airfare). i will never forget one such sunday: june 11th 2000, in atlanta. it was the day of pentecost. i went for a walk around noon. a woman in a glorious white dress, came towards me down the sidewalk. to my surprise, i recognised a lady i had met once or twice in charlottesville 12 years before. she beamed, telling me about her experience in an episcopal church just minutes ago. after entering the packed church, she stood out. she was called by the chaplain to present herself, and then experienced open arms. she ended up singing and swaying with the whole vibrant parish. she had planned this months ago when she realised that the meeting her husband was going to also coincided with the celebration of pentecost in the deep south. later we got acquainted with each other well. working in zurich, i spent many a weekend enjoying the hospitality of elisabeth and elias perentes who both worked at novartis in basel and lived just over the border to france. in 2003, a lorry ran a red traffic light, crashed into two cars, and killed both drivers, one of whom was elisabeth driving home after work. i will never forget the flamboyant elisabeth perentes on that hot sunday in atlanta. viii: lucien julien rubinstein, uva, charlottesville university of virginia, charlottesville the brilliant thomas jefferson (1743-1826) was born in shadwell, just outside charlottesville, virginia. he was one of the ‘founding fathers’ and finalised the wordings of the ‘constitution of the united states of america’. jefferson planned a new university close to his home monticello in albemarle county, virginia. he was the architect and designer of the campus with its special features like the rotunda, colonnade and the serpentine wall, qualities that make the university of virginia stand out even today. after defending my thesis in may 1983, i was ready to join lucien j rubinstein for a one-year sabbatical leave of absence from haukeland university hospital in bergen. late june 1983 lucien met me at the little charlottesville-albemarle airport outside of charlottesville, virginia. leaving the airplane, i was struck by the burning sun and the warm, moist air (40oc). in other words, my first experience with steaming hot virginian weather. the air was ‘dead’, not even a little breeze gave relief. after a few welcoming words, he told me that when you live in america, the first thing you should do is to buy a car. mobility is a must here, he stressed. in the parking lot, lucien’s open mustang was a pleasant surprise. i recall his gray hair in the wind speeding down route 29 south to the small town (approx. 25,000 inhabitants). he took me to my temporary quarters. i was to live in a huge white barn, from another century with barren rooms, high ceilings and worn-out furniture. in most rooms, there were slowly rotating ceiling fans. i did not notice any students. it was hotter inside than out, and ‘summer empty’. as we looked around, i think my facial expression and body movements signalled reluctance to be ‘dumped’ there. after some minutes of silent disbelief, it must have dawned upon lucien that this residence was not appropriate for a 41-year-old cold-blooded norwegian visiting associate professor of the honourable uva. from norway, i had tried to find a flat or townhouse in charlottesville or outskirts (albemarle county) without success. it would be easier when being there in person, i thought. it took some weeks before i found a fitting place for my family (wife kari and our children, pia, 12, ine, 11, and erik, 2 years). in the intervening time, i was offered lodging in ljr’s home, a one-stock redbrick house on the hillside close to the famous resort boar’s head inn. the adjacent, even posher, farmington country club had been their first choice, but ljr’s application for membership was declined for unknown reasons. the interior of lucien’s and wife mary m herman’s home was elegant and even cosy. i was offered a nice bedroom with a queen-size bed, soft and comfortable. the room had a feminine flavour. it took a while before i realised that i occupied mary’s bedroom… i felt welcome and even comfortable in mary and lucien’s house the two weeks of my stay in ednam drive. mostly i was offered a quick breakfast with lucien. he worked on a new edition of pathology of the nervous system, usually from early morning, not seldom in his morning-robe. therefore, he often came in late. taking lucien’s advice, i took my first test drive, which proved to be an unpleasant encounter. raised on cars with manual gearshift, my initial attempt to shift led to an immediate clash with the windshield. without prolonging my essay, i will skip all the details about problems with old and big american cars, only to say that i needed assistance to find where to fill the gas tank. one of my first days in charlottesville, i was introduced to lennart heimer, a neuroanatomist/physiologist, originally from gothenburg. uva was evidently proud to have him in their midst. heimer spoke english with a heavy swedish accent, which didn’t seem to hurt him in academia. i felt relieved to continue with my own scandinavian drawl. neuropathology had offices, labs and special rooms for cell/tissue culture, electron microscopy (ultrastructure), and biochemistry as a special section of the department of pathology. it was situated within the old colonial redbrick building with ‘medical school’ above the entrance. an antique, thin, squeaky, and almost worn-out door can be seen above. exiting it, you entered a small lawn close to a cosy, little street, called an avenue (university avenue). although not broad, wide, or lined by trees, ‘avenue’ makes sense (avenir – access to) since it leads to the original uva campus. university avenue had some nice small restaurants, coffee shops, fast-food spots, and bookstores. i remembered the site of the very first lunch break lucien invited me to. this picture, taken through the front screen of my rental car, shows the red front of the smallest joint in the avenue. we had sloppy joes (without the bun). my assigned office space was shared with two neuropathology fellows, estelle t may and josé m bonnin. we had weekly lab meetings, were we talked projects, progress, assigned workplaces and equipment. my first lab meeting was rather special. a disagreement about glass pipettes used by sozos was awkward to listen to. they were taken from mary’s side of the lab bench! i uttered something about spoiling time in discussing pettiness. my voicing did not fall in good soil, as the saying goes in norwegian. i was registered as a difficult new boy on the block, a notion further enabled a few weeks later. the summer of 1983 was very warm with record highs. on my lunch break each day, it was a shock to leave the cool air-conditioned interior entering a sauna with clothes on (never did that, by the way). now and then we (estelle, josé and i) had our burger king whopper on the grass close to the serpentine wall surrounding the original thomas jefferson’s uva. our office door had a little window, on the inside of which i put a little black and white version of edvard munch’s ‘the scream’. it was facing the hallway and hung there long enough to annoy passers-by. someone tacked up a message telling me to remove the image. it was an intuitive or subconscious cry from me that i see clearly now. amazing grace comes to my mind: ‘was blind, but now i see’. i recently wondered if my fellow fellows felt the same unease at the time. they most probably did. as the time went by, i felt more relaxed, especially during the weekends. this ‘saturday in the office’ posture was more casual than necessary. note the small microscope compared to the ‘monsters’ of today. family life in charlottesville expanded just a few days after kari, pia, ine and erik’s arrival in the us on august 11th 1983. we rented a townhouse in ‘four seasons’ just outside the charlottesville city limits. after a few days, we found out about a lake not too far away. at chris green lake we enjoyed cooling off in the hot and humid august with swimming and making ‘bombs’ (pictured here). on the first day in the water, a passing young swimmer asked our girls if they were from norway. her mother was. the young girl came up to us on the beach with the telephone number to her home. one phone call and we quickly got anchored to people that we still have as friends, close to 40 years later. other social activities: the head of neurological surgery (john a jane) was exceptionally gracious. i was able to attend two of his garden parties. ‘christmas in june’ was a yearly event hosted by ‘dr. and mrs. john a. jane’. it was a fantastic party, which people looked forward to for weeks. in a corner of their huge lawn, a dixieland band entertained with feel-good vibrations. there was plenty of food and beverages. some persons even followed the suggested theme of the party for ‘optional masquerade and/or masks.’ the christmas party in june was an extraordinary successful socialising event. i was introduced to many interesting people. i recall: ‘you have to meet lennart heimer, he is from sweden, and has recently published a neuroanatomy textbook.’ i had not heard heimer’s name before. his ‘the human brain and spinal cord: functional neuroanatomy and dissection guide’ had just been published in 1983. the reviews were excellent and so was the book, which i promptly acquired. lennart’s wife was norwegian, and there were further scandinavian ladies married to american scientist and businessmen. they met once a month, and kari was wholeheartedly taken in. in our four seasons neighbourhood we connected to neighbours with children from abroad, like peru, israel, finland, and canada. after a harsh start, our two girls thrived in the public jack jouett middle school. they were considered just very shy americans. after those first awkward weeks, things turned around. neuropathology continued lucien’s office was relatively modest in size, soft-coloured interior, decorated in beige and brown. a two-headed microscope was the first thing you saw when entering this sacred place. you did not want rush in there. i felt tension in the air from the start. (was it only me?) however, i found the weekly review of the recently arrived consultation cases (‘ccs’) most educational. brain tumour cases came from all over the world. the fellows and i were assigned cases for presentation at the review meeting, and i greatly valued the scholarly discussions related to these rare and mostly difficult cases. the significance of patterns and elements of the microscopic changes were deliberated in extenso. back at our desks, we dictated microscopic descriptions and diagnoses. the front page, for the ease of the submitter to see, gave a clear conclusion/diagnosis. this was followed by a very wordy description to ensure that every possibility was taken into consideration. rubinstein’s wordings in letters to editors (and others) were polite, starting and ending with ‘thank you for’ with interspersed ‘...would you be so kind as to…’, and ‘…please find...’, a custom i have adopted which has suited me well. another matter was his quest for excellence in microphotography with many retakes and increases of magnification (‘go to a higher mag!’). he typically stressed the fact that you are writing for educated and interested readers, so you do not have to explain everything. trust that your audience is ‘level’. i recall a special incidence: at the double microscope, he rubbed his face and moaned over the quality of the tissue section we were looking at. a long silence, and then ‘who ordered the lab to do this (immunostaining, my comment) gfap?’ i did. more moaning, then: ‘technicians must respect the persons who give them orders’, meaning that he himself had to order immunohistochemistry if he wanted the result to be ‘perfect’. in other words, i was not respected. a deep sadness hit me, i felt both bad and sad. ‘they’re like animals’ he said. i thought i didn’t hear right! lucien was unique in his position as a world authority on brain tumours with a more than complex personality, not without ‘musicality’ and a sort of sweetness and vulnerability. in january of 1990, lucien j rubinstein died at 65. the eulogy, written by scott r vandenberg, bernd w scheithauer, and darrell d bigner, has a passage that i would like to share with you. ‘lucien rubinstein had a remarkable dramatic presence. not many people could fail to remember the first encounter with him. all were affected by his charming manner, subtle and, at times mischievous wit, or lancinating wrath’. my wife would easily have stayed in charlottesville for another year or two. i, however, longed for doing neuropathology and diagnostic surgical pathology back home. (two roads) lucien had a special liking for embryonal tumours, so i’ll close the uva experience with three images. ix: the 1980s continued during the year as a visiting associate professor at uva, i continued work on my oligodendroglioma project, which i had started years earlier. through recommendation by kari skullerud (neuropathologist, the national hospital of norway, oslo), we (the norwegian cancer society) hired a young, inexperienced person, arnhild tollefsrud. her intelligent suggestions and hard work included correct registration of hundreds of data points from 208 persons with a oligodendroglioma diagnosis. her insightful work provided sound foundation for three manuscripts. the first, ‘oligodendroglioma. histologic evaluation and prognosis’, was sent to the journal of neuropathology and experimental neurology (jnen). it turned out to be quite an ordeal to get through the review process of the jnen, with many back-and-forth letters between me and the powerful editor-in-chief, john mossy. some sentences from our correspondence might be of interest. i wrote: ‘our contribution is one for (and by) neuropathologists and surgical pathologists, and not for experimental neurologists. we feel that our manuscript reveals the materials, methods, and results in a way that compares favourably with other contributions in this field. we therefore hope you will accept it in its present form. for example, to give a more ‘precise definition’ of oligodendroglioma than used in authoritative textbooks and in the who classification seems beyond our competence and scope’. i asked mossy to return the manuscript to us, and politely added: ‘i do not regret sending it to you, as your comments and criticism have improved the article’. but then it was accepted for publication. janet m bruner (houston) found an error in figure 7 of our jnen oligo-article. we were impressed by her ingenuity and gave a reply in a letter to the editor (journal of neuropathology and experimental neurology 45, 1986). ellsworth c alvord suggested further avenues of studying histological correlation of oligodendroglioma with prediction of survival. (two roads) the two other ‘oligo-manuscripts’ were accepted and published by the journal of neurosurgery after a thorough review. high on life, after getting an extremely nice comment from the editor-in-chief, william f collins, i sent in a suggestion for the cover page of the issue in which our article would appear. i got no feedback. oh well… after publication of the oligodendroglioma papers in jn and jnen, many neuropathologists and neurosurgeons reacted in a positive manner. i gave ‘oligo-talks’ in europe and in the states. following one in in memphis, tennessee, the neuropathologist, f curtis dohan jr, wanted to discuss some interesting cases with me. entering the microscopy room, i was met by a 12-headed microscope loaded with 10 residents. ten (!)and all were residents of the largest neurosurgical department in the southern us. the cases ‘curt’ had sought out for discussion were certainly not straightforward. i remember that we had lengthy discussions before concluding. thinking back, i must say that the quality of questions and comments from those mainly neurosurgical residents surpassed anything i had experienced before. the 1990s two key events in my way occurred in 1990. in january i received an invitation from paul kleihues (zurich/lyon) to participate in a brain tumour meeting in zurich. the invitation had a questionnaire attached, where you answered, ‘by circling the answers which correspond best to your current opinion on classification and grading’. at the end of march 1990 paul called together a who working group to discuss histological typing of tumours of the central nervous system. the main purpose was to get consensus on the nomenclature to be used in the next version of the iacc brain tumour classification. although the meeting took place more than 30 years ago, you can recognise the expertise of the participants by reading some of the last names in the official photo. scott vandenberg sat in for lucien rubinstein who died in january 1990. there were slide-viewing sessions, short presentations on specific problems followed by panel discussions, and occasional decisions by voting. for me, a highly educational and excellent experience. one evening, peter burger, bernd scheithauer, roy weller and i discussed criteria for grading oligodendroglioma among other things. bernd and i agreed to take this further. we wanted to do an ‘oligo study’ (heading of a letter from bernd to me, july 9th, 1990) comparing different grading systems. i had the impression that ‘my’ system would be part of the study, but something went wrong. i am not sure what it was. the study was now ‘oligodendroglioma grading project’. in the summer of 1993 i had fulfilled my contribution to the somewhat changed study/project. caterina gianinni compiled the input from six neuropathologists and six surgical pathologists and was the first author of ‘oligodendrogliomas: reproducibility and prognostic value of histologic diagnosis and grading’ (journal of neuropathology and experimental neurology 60(3):248-62, 2001). the second event came after the xith international congress of neuropathology, kyoto, japan 1990, which was my first and only visit to asia. i had a gap of two or three days between the end of the congress and an ms meeting in the same city. i was completely on my own; no one understood what i said. i did not interact with anyone for days. at least it felt so. (writing this, i reflect that feeling down and being depressed is not the same.) that aside, for the second time in life, i felt so down that i considered it depression. i avoided entering the balcony of my hotel room… on the way back home, i bought a cd at the airport in singapore. it was the misspelling on the cover that hit me: beethoven’s third symphony (‘erotica’)! back in norway, beethoven helped me over my down period/depression. from the first day, i played his ‘eroica’ very loudly (very, very) driving to and from work. it helped very much. i did not miss a day’s work. the ‘third’ is a dynamic source of mental energy. x: department of neurosciences, lerner research institute, cleveland clinic, cleveland, ohio the first time i met bruce trapp was in vienna, 1982. he was in company with my friend henning laursen (neuropathologist at rigshospitalet, copenhagen). they knew each other from the time they spent as postdocs at the national institutes of health, bethesda, maryland (laboratory of neuropathology and neuroanatomical sciences, national institute of neurological and communicative disorders and stroke). after short introductory remarks, henning suggested that we come together in a ‘heuriger’ the same evening. ‘heuriger’ is a word for austrian wine taverns serving this year’s wine (heuer: this year) that they produce themselves. they serve their wines in gardens, and welcome everyone warmly; just what i needed following the infamous afternoon session. outside on bleachers, a couple of americans and scandinavians had a jovial and laid-back time in a pleasant atmosphere. since then, bruce trapp and my way have crossed many times. in one aanp annual meeting, bruce asked me if i knew any ‘bright norwegians’ (!) who could be recruited to join him at the department of neurology, johns hopkins school of medicine, baltimore, maryland. i said yes. back in bergen, a young md, lars bö, took the challenge and met with bruce trapp and the chairman of the neurology department, john ‘jack’ griffin, to check out the ‘premises’ in 1990. lars and his wife, elisabeth, went to baltimore in 1991, supported by the norwegian research council. he developed into an open-minded, innovative, and rational researcher. bruce trapp was, and still is, a well-known myelin researcher. his ultrastructural studies on myelin are of exceptional quality and won him the ‘weil award for best paper on experimental neuropathology’ presented at the aanp annual meeting 1986. so, when harald nyland and i prepared for a meeting on myelin and demyelination, bruce trapp was one of the 17 international experts we invited to bergen. in the 1990s the lerner research institute at the cleveland clinic inc. (cleveland, oh) wanted to add a department of neuroscience to its stock of nine research departments. bruce trapp applied and was offered the position of director of the newly established unit. he started developing neuroscience at ccf in 1994, and lars fit well into the structure that bruce had in mind. we, wife kari and son erik (born 1980) moved to join the ‘trapp lab’ for a one-year-sabbatical in 1997/98. meeting the trapp family was an enjoyable event. after a short while we were treated and felt like family. our families are closely connected still. i could write pages about caring carol, the twins brian & dan the man, sara, david, and bruce. i hope it will be done by others. the collaboration of neuroscience in cleveland and neuropathology in bergen developed in a persistently friendly and inspiring atmosphere. i learned the basics of preparing and immunostaining free-floating 30μm thick sections for confocal microscopy by ansi chang. she was, and still is, an extremely skilled and intelligent person who i am glad i met and with whom i still keep in touch. she instructed me to be an approved user of the leica aristoplan laser scanning microscope. through confocal microscopy and computer-based, three-dimensional reconstructions, we studied pathological changes in multiple sclerosis brains. many hours were spent on the confocal microscope looking for clues. the attraction of the ‘collapsed’ stacks of the laser-scanned immune-stained 30μm thick sections from the frozen specimen of brain tissue was evident in images. the image is stained for myelin and microglia. it was a good feeling to know that you had removed the cns at post-mortem, fixated, selected areas to study, cut, and stored tissue wet or in paraffin blocks. i brought some of the tissue to the us in my hand luggage. the co-operation between cleveland and bergen resulted in many publications (the first results were published in 1994, and the last paper last year in 2020. one, in the new england journal of medicine had a special impact since our findings initiated an editorial (‘demyelinating diseases ― new pathological insights, new therapeutic targets’) in the same journal. our results were presented on national tv and, in february of 1998, reached the front page of the new york times. in other words, the old knowledge that ms lesions affect grey matter of the brain as well as white matter came in ‘new wrappings’ and got some great attention. i had long since noticed that the characteristic lesions in ms brains did not spare the grey matter. so i got the idea to sample grey and white matter from preselected areas disregarding macroscopic changes/lesions. we wanted to see what the eyes did not see; therefore, we searched for a new way to examine ms brains. we did not sample macroscopic lesion. rather we stuck to sampling brain material strictly from preselected areas. we weren’t looking for macroscopically evident ms lesion; instead, we took tissue probes from predetermined areas. (i was experienced in cutting formalin fixed brain in the frontal plane coronal sectioning). a section through the anterior commissure turned out to be a fine landmark for getting similar brain areas from different individuals with ms. from this section we sampled the superior temporal, the cingular gyrus, and parietal tissue. we revealed the extent and pattern of demyelination in the cerebral cortices and underlying white matter of 20 ms brains. tissue from the same preselected brain areas from people without neurological disease served as controls. our findings were published in the jnen in 2003 and undermined the amount and importance of grey matter (cortical) changes in the brains of ms patients. lars bö defended his phd dissertation ‘multiple sclerosis: immunopathological studies of inflammatory central nervous system demyelination’ at the university of bergen, in 1998. the five peer-reviewed articles of his dissertation have, as of may 2021, been cited 5,639 times. space was precious in the energetic and crowded neuroscience floor. so, when the newly ‘added’ neuropathologist/researcher susan staugaitis arrived in cleveland, i had to ‘give up’ my office to her. she was split between diagnostic service for pathology and research in neuroscience. susan was intelligent, sharp, and well informed. she was an outspoken and confident contributor in many aanp and other scientific meetings. we had lot of engaging discussions. susan sadly had a glioblastoma diagnosed in 2014, a fact that she was extraordinarily open about. in 2017, i sent her a christmas greeting with this photo from the library room. drs susan staugaitis (✝) and ansi chang were treasured colleagues of mine in the trapp lab. susan: ‘it goes without saying that i am still alive. i never believed i would be around this long. i am doing my best to stay as strong and safe as possible as i continue to live alone in my home. it helps when we can look at things from multiple perspectives and find the part of the glass that is half full’. she died in august 2018. xi: other activities medical university of lübeck in the year 1360 the hanseatic league (the german guild of merchants) created an overseas office in bergen. for almost 400 years, they dominated commerce and social life here. hanseatic influence is still very much evident in surnames, buildings, and the wharf (‘german wharf’ or ‘tyskebryggen’, after wwii best just called ‘bryggen’) in the centre part of the city. in the 1970s, a ‘hansa-related’ idea was born at an international forensic science meeting where professors otto pribilla from lübeck and johan chr. giertsen from bergen happened to meet. they found it timely to try to establish an official contact between the two hanseatic cities through their respective universities. a general agreement of cooperation was officially acknowledged by both parties in 1976. the aim was and is to foster better contact through student exchange, postgraduate education, and research within the fields of medicine and natural sciences. i had the honour of taking giertsen’s place as coordinator of this cooperation from 1993 to 2011; most of the time together with the surgeon professor jan fredrik halvorsen. we enjoyed interactions with many team players in lübeck. manfred oehmichen, eberhard schwinger, and michael seyfahrt were among the proactive professors, to name a few. the main burden of this successful treaty was carried by the administrators peter mühlhausen (oberamtsrat) in lübeck and johannes (johs) teigland (faculty director), as well as torill knag in bergen. in 1995 the general agreement of cooperation between the universities was extended without limitation. universitäts spital zürich 2002 in 2002, i spent an interesting year at the universitäts spital zürich. i was asked to take care of the diagnostic neuropathology for a year or so. phillip u heitz was the director of the institute of pathology and molecular pathology, with its highly qualified medical and technical staff. i especially remember the productive, pleasant, and friendly atmosphere within the department. social skills were ‘comme il faut’, highlighted by free ski weekends in davos. lifts, lodging, and food were paid for by the department! the institute of neuropathology was completely focused on prion research, enigmatically led by adriano aguzzi. this meant they were continually searching for a clinically oriented neuropathologist to take care of the diagnostic routine for a longer or shorter period. i went for one year (december 2001march 2003). i intended to combine diagnostic ‘neurosurgical’ work with microglia research but that did not materialise. i did, however, learn a lot about prions and psychology, and witnessed the immense effort of postdocs to satisfy their manager during the weekly progress report (and in-between). i connected with many of the optimistic, eager, dedicated, young scientist during my period there. 12 years later, one of them, jens pahnke, became professor of neuropathology at the oslo university hospital. his track record has been and is superb. i appreciated working with the fine technical staff of the institute of neuropathology. this photo shows that we had a good time in between skiing, needless to mention afterwards! miscellaneous major changes have increased the effectivity of doing diagnostic pathology and research over the years. i’ll just mention a few elements that made my way easier. one general problem that gummed up the works all over was the backlog created by secretaries typewriting manuscripts. if you had a few minor changes, pages or the whole report had to be typed anew. from 1973 the ibm ‘golf ball’ typewriter with an internal correction feature reduced the backlog for typing and retyping manuscripts. another problem was that all manuscripts had to be submitted with original photos to be accepted for review. there were strict instructions for authors, especially regarding illustrations, and you had to pay for coloured illustrations. it seemed that every journal had different rules. the invention of the letraset’s dry rub-down instant lettering made it easier to put arrows on figures. they were large, half transparent sheets with letters, stars, arrows and more. letraset was ‘expensive’ and popular; you had to ask around to find the right sheet. it was not uncommon to find the tokens you needed for your illustration ‘scratched out’. it took time to order new sheets. letraset was very popular for years before desktop publishing took over. to find and check out correct references, libraries had meter after meter with index medicus occupying their shelfs. they received new, heavy books annually. you had to carry one to your desk, turn the pages, carry it back to the shelf, get the next one, and so on. during the 1990s, the introduction of the internet and world-wide-web (www) accelerated the shift from printed to online information, especially with the start of pubmed. also in the 1990s, powerpoint transformed the process of preparing and giving presentations. the development of immunohistochemistry (ihc) opened possibilities in characterizing tissue, cells, and cell products. at first, you were lucky if you knew someone who had produced an antibody and was willing to share it. now the production of antibodies is a huge industry providing opportunities for research and industrial applications. (order online and receive your antibody in a couple of weeks!) for me, it was great experience to effectively pinpoint the inborn superintendent of the microenvironment of the normal brain, the microglia (mg)! they are almost invisible in conventional staining (he). the importance of mgs cannot be overrated. they are everywhere in the cns, acting as sensors (housekeepers) of the local milieu. they are constantly working, switching between quiet monitoring physiologic activity and active efforts to keep changes from doing harm. tissue micro arrays (tmas) became popular in the 2000s. small cores of tissue (for example tumour material) could be cut from a ‘donor’ paraffin block and, one by one, placed in an ordered way in columns and rows to the recipient block. as large number of samples could be examined in a single cut from one recipient paraffin block, thus creating an exceptionally cost-effective and timesaving research tool. the digitizing of specimen slides with computer-assisted technology has led to digital pathology as a powerful and expanding line of work for easing many aspects of diagnostic pathology. special scanning machines convert the tissue specimen on the glass slides to a seamless image on your computer screen. this allows for a wealth of possibilities related to histological assessment (diagnosis), research and teaching. i have worked as a digital pathologist for many years now. my first experience was in costa del sol (spain) in 2015. there i performed microscopic evaluation and diagnoses on surgical biopsies in between rounds of golf and trips to the beach. digital pathology allows for quicker measurements, easier comparisons, straightforward sharing of information, and effortless transfer of digitised images for consultation and second opinions. this home-office type of diagnostic, digital pathology is perfect in this sars-cov-2/covid-19 pandemic. polymerase chain reaction (pcr) needs no introduction, but i will tell the following historical anecdote: as a postdoc at the institute for enzyme research, university of wisconsin 1968-1970, the norwegian kjell kleppe (bergen based) found ways to copy larger amounts of dna from a limited quantity (studies on polynucleotides. xcvi. repair replications of short synthetic dna’s as catalyzed by dna polymerases. journal of molecular biology 56(2):341-361, 1971). kary b. mullis used more heat resistant enzymes in the reactions and was given the nobel prize in chemistry for 1993 ‘for his invention of the polymerase chain reaction’. neither the nobel committee nor the laureate mentioned the pioneering work of kjell kleppe. the importance of kleppe’s work has been emphasised recently (see jonathan d kaunitz in digestive diseases and sciences 60(8):2230-2231, 2015; https://en.wikipedia.org/wiki/pcr). haukeland university hospital, bergen, norway the norwegian cancer registry contains complete, mandatory reports of all cancer cases in the country. since the registry can be linked to the mortality register of ‘statistics norway’, a follow-up rate of 100% is obtainable. in cooperation with fröydis langmark (director of the norwegian cancer registry) and arild helseth, we performed population-based epidemiological studies on intracranial gliomas, primary intraspinal neoplasms, and meningioma. we also analysed the occurrence of multiple primary neoplasms in people with meningioma. results were published in 1988 and 1989. in 1989, are helseth defended his thesis ‘a population-based survey of neoplasms of the central nervous system in norway’ in bergen, for the phd degree. another fruitful collaboration was that with christos d katsetos, the neuropathologist of the departments of pathology and laboratory medicine at hahneman university and st. christopher’s hospital for children hospital, philadelphia. we knew each other from mutual uva experience (both, at different times, disciples of lucien j rubinstein). this cooperation led to his successful public defence of ‘neurobiology of class iiib-tubulin isotype: differential cellular expression in human development and neoplasia’ for the phd degree at the university of bergen in 2002. paul kleihues (zurich/lyon/zurich) and roy weller (southampton) were the evaluators of and opponents in the public discussion of the dissertation at the haukeland university hospital, bergen. (sadly, christos passed away in 2017.) in the central block of haukeland university hospital, forensic pathology is located next to labs and offices of neuropathology. we have had much to do with each other. years of oil-related professional diving in the north sea has claimed many lives. likewise, amateur diving is another diving activity that sometimes can be fatal. forensic post-mortem examinations of such cases always gave rise to examinations of brains and often the spinal cord too. surprisingly, there is very little damage to be found even in long-time professional saturation divers. one should think that the pressure and the stress of such activity takes its toll. we were surprised, after examining 8 to 64 (!) tissue blocks from the spinal cord of twenty divers with immunohistochemistry, to conclude that the cords did not reveal tissue changes. while i am happy that the study was published in the journal undersea & hyperbaric medicine, i now think that i perhaps should have published this thorough report somewhere else with wider reach. norwegian ms registry & biobank research related to brain tumours, and ms have been closest to my heart. in cooperation between the norwegian multiple sclerosis competence centre and the norwegian ms registry & biobank, we have organised cns material for research purposes. we have stored clinical data, paraffin blocks, stained, and unstained slides from more than 40 persons with ms. in many cases bioengineers brynhild haugen and laila vårdal embedded whole coronal sections from ms brains into paraffin. they are cut by a large sliding microtome (not many of them around) into 20μ sections, mounted on glass slides in glycerol, and cover slipped for microscopic analysis. the manoeuvring via water bath to glass is technically challenging, as is the staining process. the images (above and below) show how it can be done! regarding ms, there is so much to wonder about. looking through the microscope on ihc stained lesions, i still haven’t been able to grasp who or what is the wrongdoer? angiocentricity of lesions is for me not a general feature. staring at different highlighted antigens from the same lesion gives me an uneasy feeling that the key is there, you just cannot add up the clues to unlock the enigma. i think of the philosopher henri bergson: ‘the eyes only see what the mind is prepared to comprehend’. above, a thin (20μ) coronal brain sections immunostained for myelin (dark brown). it shows bilaterally irregular, and sharply bordered, large, demyelinated areas adjacent to lateral ventricles. look closely and you will notice the amount and variation of the widespread myelin deficits in the cortex. to me it looks as if the players in demyelinated foci have their own agenda! to the left, a single reactive astrocyte dominates the field with scattered myelin rests. (two intact internodes?). to the right you see a tiny myelin debris in a cortical (autofluorescence) field with total loss of myelin and a solitary microglia cell (watch dog) with multiple tiny cell processes, which do not signal activation. what happened to the myelin? one can wonder… then i consider: what about the bioactive substances that are present in the same spot, untagged? this slightly tilted 3d presentation shows a very active cortical ms lesion; the most active picture i have seen. microglia is in red, and myelin is in green. the yellow colour signals co-localisation. the morphology is clearly that of acutely activated microglia engulfing myelin. think about this image as a point in time in a video, with a dynamic remodelling of the cytoplasm, with extensions and retractions in an undulating mode. it goes from slim microglia via this morphology to full-blown macrophages. hopefully methods of the future will start to bring us nearer to the ultimate goal: to unravel the mystery of multiple sclerosis. above a conventionally cut section where demyelination (black=myelin in subcortical white matter) affects the full thickness of the cortex can be seen. the extremely sharp border between affected and normal brain should say something about how the wrongdoer moves in the microenvironment. it is a well-known fact that the movement of cerebrospinal fluid (csf, oedema) differs greatly within the cns. the restriction of fluid movement is much higher in grey matter than in white matter. the complexities of the multi-layered and compact grey matter lead to slower drive of substances here. our (bö et al.) findings presented in jnen in 2003, showed purely cortical subpial demyelination in an extent not demonstrated earlier. this indicates that the csf transports myelin-harming substances which lead to demise of myelin in ms. (fluid transport through the ependyma of the ventricular walls is unrestricted; this being so, white matter oedema drains directly into the csf.) i have a gut feeling considering initial events in ms. recent studies have revealed prominent clonal expansion of cd8 positive t-cells within the cns during ms (journal of immunology 206(1), 2021). this is just one example that has confirmed our findings, first reported in vienna in 1982. the photograph shows my closest co-workers and friends from before 2000 to 2012 and beyond: the bioengineers edith fick, laila vårdal, and brynhild haugen. many are those who have helped me along my way. in bergen, i have had expert assistance on different occasions by anne marie austerheim, karin eliassen, gerd lillian halseth, nina holmelid, randi lavik, karen böhm nilssen, bendik nordanger, tore-jacob raa, laila vårdal, and grethe waaler. i left diagnostic neuropathology after turning 70 in 2012. i felt like i was one of the last surviving members of the he only (+ some ihcs) era of neuropathologists squeezing the most out of basic, classic, cost-efficient, affordable, and fast histopathology. after april 2012, i continued as a consultant in general surgical pathology (at 30% of full time) through 2019. that was the end of my affiliation to the haukeland university hospital. however, i am still engaged in digital diagnostics. i have spent my pathology career believing in short, relevant descriptions, quick responses, and up-to-date diagnoses. i am content as an emeritus professor sharing a 15 m2 office with two other emeriti. one of them is ole didrik laerum, the one who got me to bergen in the first place. each of us have a desk, one microscope, and one computer. rarely are all three of us present at the same time, so the space is adequate. i hope to accomplish a few more things yet. the last years have come and gone like snow in april. in the last decade, my beloved wife, kari, who has been a source of love and order for our close family, started to lose us and herself. she was diagnosed with dementia. she was so full of life, a loving mother, and a talented artist. her care enabled my career and family life, so it was difficult when the tables were turned, and i found myself caregiving for her. my close-knit family has helped me greatly in coping with this challenging time, for which i am endlessly thankful. she has transitioned to a care home, but i visit her frequently, and she frequently comes back to herself. her face often lights up when she sees a frequent visitor, whom she sometimes recognises as ‘sverre’. i have told myself, ‘de-mentia is not a-mentia’, and for kari, that is true. she has retained her use of language. on one recent visit, she exclaimed, ‘there are more people here!’ ‘just me’, i said. ‘well’, kari said. ‘i am here too!’ for the last 50 years, she has been a wonderful companion and remains one now, even as we come to the end. the challenge i was given by the free neuropathology has helped me to concentrate on times past. the time spent has been valuable. i have by now wound up most items that i had put aside. private letters, reports, material related to international co-operation, journals, books, folders with obituaries, reprints, and photographs. i thank werner paulus and tibor hortobágyi for this opportunity to illuminate my existence. brian trapp helped me with language corrections and inputs. a mild acceptance of how things have turned out, the ‘you could have done better’ have been overtaken by the perception of the facts of my way. all the way, robert frost’s poem (the atlantic monthly, 1915) has been with me. the road not taken by robert frost two roads diverged in a yellow wood, and sorry i could not travel both and be one traveler, long i stood and looked down one as far as i could to where it bent in the undergrowth; then took the other, just as fair, and having perhaps the better claim, because it was grassy and wanted wear; though as for that the passing there had worn them really about the same. and both that morning equally lay in leaves no step had trodden black. oh, i kept the first for another day! yet knowing how way leads on to way, i doubted if i should ever come back. i shall be telling this with a sigh somewhere ages and ages hence: two roads diverged in a wood, and i--i took the one less traveled by, and that has made all the difference. with my friend, tore, who led me into the road taken 50 years ago copyright: © 2021 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. my pathway to a career in neuropathology feel free to add comments by clicking these icons on the sidebar free neuropathology 3:10 (2022) reflections my pathway to a career in neuropathology clive harper university of sydney and royal prince alfred hospital corresponding author: clive harper · rpa neuropathology · 94 mallett st · camperdown, nsw 2050 · australia clive.harper@sydney.edu.au submitted: 09 march 2022 accepted: 10 march 2022 copyedited by: henry robbert published: 12 april 2022 https://doi.org/10.17879/freeneuropathology-2022-3809 additional resources and electronic supplementary material: supplementary material keywords: neuropathology, reflections, alcohol brain damage, thiamine deficiency, brain banking and premortem donor program, international collaborations i grew up in balgowlah heights in sydney. my father was a physicist at csiro but was also involved with the national standards commission. in 1970 he was appointed executive member of the metric conversion board that was established to oversee the metric conversion process in australia. it has been said that the reason that this proceeded so smoothly over the next ten years was due to his remarkable management skills. he was awarded an australian honour (ao) for this contribution. i attended the local primary school and, for my secondary education, the manly boys high school. i won a commonwealth scholarship to sydney university and commenced dentistry in 1961. after the successful completion of my first year i applied to transfer from dentistry to medicine and was accepted. i completed my medical degree in 1966 and began working as a resident medical officer at the royal north shore hospital (rnsh) in 1967. after two years i commenced another five years of post-graduate training in pathology. the course included separate years in histopathology, microbiology, haematology and biochemistry. towards the end of this pathology training all i could think of was travelling overseas. i think that my father’s travels and connections with overseas friends had caused my ‘travel bug’. i had no idea what i wanted to do or where i wanted to go. figure 1: clive sits at his microscope in neuropathology at royal perth hospital in 1984. however, at a dinner party one evening i met a pathologist from glasgow who mentioned that one of his colleagues was looking for a locum pathologist. i immediately wrote (no emails or faxes in those days) and waited patiently to hear from professor hume adams, head of the department of neuropathology at the university of glasgow/southern general hospital. i remember how excited i was when i received a letter from him offering me a job as lecturer in neuropathology in glasgow, beginning september 1972. hume even organised accommodation for us and it was all go. i was married with two children aged five and two. at that time australia was actively repatriating mentally disturbed immigrants. the government always sent a doctor along with the patients. i requested such a position and was invited to meet stephan in ryde psychiatric hospital. he was to return to zagreb in yugoslavia (now croatia). he was only about 21 years old and the nurses said that he could not speak english. a government car picked me up in the morning and we drove to the hospital to pick up stephan. then on to mascot to take the qantas flight to rome and then zagreb. i had bought my onward ticket to london. jann and the kids travelled on the same flight, direct to london. stephan and i were seated in a separate zone with no one close by and i had my medical case with drugs and syringes to keep stephan quiet, if necessary. as we took off stephan turned to me and said, “why don’t we have a drink”. he had been foxing about his english the whole time just to get a free trip home. i was able to wander about and keep an eye on the kids. stephan and i had to change planes in rome while my wife and kids went straight on to london. when we arrived in zagreb stephan’s family were there to meet him. he had lost a lot of weight, grown a beard and his hair was very long so they did not recognise him! immigration immediately picked up on this and i was dragged off by security and interrogated as to his true identity. in those days yugoslavia was totally communist and everyone seemed to be carrying guns. i was terrified and the australian embassy official who was meant to meet us had not turned up (surprise, surprise!). finally, stephan’s sister and uncle arrived and they immediately hugged him and everything settled down. i was very pleased to get back on the next flight and get out of my first communist country. figure 2: professor hume adams and clive enjoy a moment in neuropathology at the southern general hospital (university of glasgow) in 1973. arrival in glasgow, scotland hume adams had organised accommodation for us and we were able to move straight into our new home in white inch, about three miles from the southern general hospital. it was a 2-story tenement with three bedrooms and was very comfortable but pretty cold, even for summer. the owner even offered to babysit for us – what a bonus. we organised a local school for our son dal and i went to the hospital to meet my new colleagues. i found hume to be a delightful chap, about 45 years old. the department seemed to be ‘buzzing’. the hospital was a new innovative ‘one-stop shop’ for the neurosciences. it combined neurology, neurosurgery, neuroradiology and neuropathology in the one building and the clinical groups met every week to ensure the smooth running of the institute. getting back to work was a bit of a challenge. i had not done any anatomical pathology for three years so was pretty rusty. i had been training in biochemistry, microbiology and haematology. moreover, neurosciences had always been a bit lost on me so it was a sharp learning curve! fortunately, hume believed in a long training/apprentice period and so i spent a lot of time observing the techniques of brain dissection. my main role was doing the autopsies. we moved about a lot each week working in different hospitals all over glasgow and in some of the major country hospitals. soon after i started work the hospital/institute had an official opening. on the first evening, there was a huge formal dinner at the university refectory. the invited guest for the official opening was wilder penfield, director of the montreal neurosciences institute in canada. his neurosurgical fame related to in-vivo electrical stimulation of the brain to identify specific regions that control motor function. he would buzz the brain with electrodes during his operations and watch to see which muscle contracted! sounds primitive but this work was at the forefront of neuroscience at the time. i found his speech inspiring and felt an almost ‘religious conversion’. this really was the beginning of my major interest in neuropathology. a few weeks after we arrived in glasgow, hume had a dinner party and we met a number of other pathologists. several became famous pathologists in later life – alistair cochrane and roddy mcsween. there was also an ophthalmological pathologist who asked me to be involved in a research project involving the electronmicroscopic study of optic nerves from a group of baboons who had been subjected to ‘toxic amblyopia’. this was my first real taste of neuropathological research and probably set the direction of my future career. hume’s hospitality was extraordinary. in the first four months he had invited us to his home twice, took us to a dinner party and a wine tasting and paid for all of this – so different to the australian work scene. holiday in europe may 1973 in may 1973 the weather was warming and my parents arrived to visit. i organised to take my annual holidays and we all drove in our camper to europe. i had taken the opportunity of organising several job interviews in europe. hume had asked me to stay in glasgow but the weather drove us all mad and we were keen to experience life in a ‘foreign’ country and to master a second language. i had done a lot of school french and, during our time in glasgow, i had done private french lessons with a friend. he said “i don’t need any payment, just give me as much whisky as i want during the lessons”! it cost a fortune but my french did improve. i had interviews for jobs in neuropathology in paris and in geneva and lausanne in switzerland. i was offered all three jobs and chose lausanne. we moved in september 1973. hume had a farewell party for us and i was very sad to leave. i still consider hume to be my mentor. we remained friends for life and visited him in 2012 in glasgow. this was 40 years after we first met. he was then 84 years old. as most of you know hume passed away in 2020 aged 90 years. lausanne, switzerland 1973-74 we drove down through london, crossed the channel and drove on to lausanne. it took about two weeks to find an apartment. it was a six-story block in the hills behind lausanne, overlooking the town and across the lake to the alps. the neuropathology laboratory was beside the hôpital cantonal de lausanne. it was in an office block and we were housed in the basement. there was a nice café on the ground floor and we often went upstairs at morning tea for a coffee and ‘pomme de vie’ liqueur. there were five other medical staff in neuropathology – professor rabinowicz (swiss), dr. alphonse probst (swiss), deraux (german), and two young portuguese trainees (cousins, manuel and juan sanches). it was an excellent setup with lots of technical staff and lots of specialised equipment (em included). the department specialised in developmental neuropathology with lots of quantitative analyses. quantitation later became my speciality in the alcohol cases we studied in perth and sydney. one of my responsibilities was to do most of the autopsies. the other trainees did not have autopsy experience. the department provided a service for the whole canton (state) so i was often sent off with a technician to do autopsies in country towns, usually high up in the mountains. in wintertime i sometimes packed my skis in the van and, after finishing work, would go for a ski. the french language came slowly. i was challenged after six weeks by having to present a case report to an evening conference. i practiced for ages and did a reasonable job until question time. i had no idea what they were asking in their rapid french. future directions should i try to take a clinical job next year? neurology is the obvious choice if i wish to proceed in neuropathology. the idea of a university appointment is still in the back of my mind. teaching would give me a lot of satisfaction but i must remember what a small fraction of teaching occurs in neuropathology – hours per year. the idea of having to do a phd or md is not very appealing. perhaps these things will become clearer in time? despite all of this self-analysis i have not made any decisions. if only i could do clinical medicine without having to work on nights and weekends – that would be hard to take again! i have been reading my old swiss diaries and i constantly refer to my lack of direction and confusion about my career pathway. correspondence with the clinical superintendent in my previous sydney hospital (rnsh peter williamson) led me to believe that i should not return to australia until i had done at least one year in the usa. one week later i wrote in my diary: “here i am with a letter half written to apply for a job as assistant neuropathologist in perth. it is not really like going home so i don’t have the anxieties re arriving home dissatisfied with lack of travel”. i am still waiting for a reply from the cornell medical school in new york but can’t really see it matching up to the perth job with byron kakulas. in fact, as we walked around the lausanne lakefront on sunday, looking at the boats on the lake, we began talking about getting a boat in western australia. the idea was very appealing. anyway, it is just as well that the perth job came up because the new york job did not come through. i guess i should set my sights on my career for a while and try to achieve something really great by the time i am 35-40 years. a professorship would suit me fine but a lot of water under the bridge before then! it’s the idea of publishing papers that really upsets me – they are just not my scene. no one reads them so what’s the point? i wish i could develop some original thought pattern. currently, i am fully occupied trying to learn the basics of neuropathology!” whilst working in lausanne i attended neuropathology conferences in innsbruck, austria and in budapest, hungary (international society of neuropathology). at the latter conference i met byron kakalus from perth, australia. we had never met and he seemed delighted to meet another australian training in neuropathology. he immediately invited me to join his department in perth after i finished working in lausanne. i told him that i was waiting to hear about a position in boston but he said that the perth position was only available if i came immediately. since the family had already planned a three-month trip in italy, yugoslavia and greece after leaving lausanne we compromised and i arranged to commence in perth after this holiday. perth 1975 we flew into perth from switzerland via the uk and were met by my new boss, professor byron kakulas. i started work at the royal perth hospital immediately. byron had set up the biggest and best neuropathology department in australia. it was big even by international standards. he had trained in boston at the massachusetts general hospital with two world famous neurologists/neuropathologists, raymond adams and pearson richardson. there were excellent facilities and large numbers of secretarial and technical staff. there were even two registrars and several supplementary overseas trainees. the registrars, peter blumbergs and tony tannenberg and i worked closely together for several years until they took up senior neuropathology positions in adelaide and brisbane, respectively. they are good friends to this day and we often laugh about that first week and the tough time that they gave me! i really loved my work – a mixture of clinical neuropathology, teaching and small clinical research projects. byron loved having international visitors and we ran an annual neuropathology course for trainee registrars as well as undergraduates. i met henry urich (london university), raymond adams, pearson richardson (mass general) again and many others. there was an excellent clinical program for all of the neuroscience groups in perth. the group met weekly, on a rotation basis, in the four main hospitals (royal perth, fremantle, sir charles gardiner and the children’s hospital). this created a harmonious working relationship between neurology, neurosurgery, neuroradiology and neuropathology. figure 3: staff and visitors to neuropathology at royal perth hospital in about 1981. byron kakulas (front row left) , had a wide group of international colleagues including the famous neurologist/neuropathologist raymond adams, bullard professor of neuropathology at harvard medical school and chief of neurology at massachusetts general hospital. professor adams is sitting next to byron. one of the enjoyable and challenging weekly clinical programs was the clinico-pathological conference. we (the neuropathologists) would prepare a case as an unknown and ask one of the neurologists to discuss the case. some of these cases were so good that i decided to write them up and publish them in the medical journal of australia (mja) following the example in the nejm . this was a great way to start getting publications together. i negotiated directly with the editor of the medical journal of australia and sent him two cases each year (see below): harper cg. progressive difficulty in walking and speech. med j aust 1977, 2:370-3. harper cg. recurrent subarachnoid haemorrhage and obesity. med j aust 1977, 2:865-7. harper cg. multisystem febrile illness. med j aust 1978, 1:205-8. the department had a huge workload of cases, mostly from the department of forensic medicine (coroners). every forensic autopsy brain was sent to us for examination. in addition, we did many of the autopsies for royal perth hospital and did the brain examinations on all of the cases. the total number of brains examined each year was more than 1000. each week we had a ‘brain review’. the most interesting cases were selected and demonstrated to pathology, neurology and neurosurgery trainees. some medical students also attended and it was run as a question and answer session. i replicated these sessions when i moved to sydney. as time went by i became aware that some additional training in neuropathology in the usa would enhance my career. i asked byron what he would recommend and he helped me obtain a fellowship (paid by us grant money) in philadelphia with a greek/american colleague, nicholas gonatas. i applied for leave without pay for 12 months and the hospital supported my application and even paid for my travel. a locum was appointed to my position victor ojeda was from south america but had been working in new zealand. travel to philadelphia via europe – 1977-78 it was blizzarding when we arrived in new york. we stayed in a hotel that night and then took the amtrak train to philadelphia. we were met at penn station by my father’s friend, bill zeiter. he and his wife had offered us accommodation until we found a house. the hospital work was very stimulating. i was responsible for some of the brain biopsy reporting with the head of department, nick gonatas and several other trainees – tomahiko mizutani and stavros balloyannis. they were both neurologists from japan and greece, respectively and were in the usa to get some neuropathology experience. we have kept in touch from time to time. the three of us were responsible for the hospital autopsies and for preparing the clinico-pathological cases each week, as we did in perth. we were working with and meeting many famous neurologists, neurosurgeons and neuropathologists every week. there were regular meetings and conferences and nick gonatas travelled constantly in the usa and to europe. it was very stimulating. nick was the elected president of the american neuropathology association which meant that we had even more interaction with american and international neuropathologists. by chance, there was also a link with my old glasgow colleagues. david graham (i was his locum in glasgow while he worked in philadelphia) had been working with the neurosurgeons in philadelphia on an animal model (monkeys) of head injuries. each of the fellows was allocated a research project and our main laboratory supervisor was nick’s wife, jackie. she had meticulous laboratory habits and trained us well. we were working with animal models of neurotoxicity using toxic ricin. we were studying the effects of toxic ricin on animal nervous tissues using electron microscopy and immunohistochemistry. i was able to publish three papers in the first year. even more importantly, the personal interactions in the department were invaluable to my research education and intellectual development. gonatas nk, harper cg, mizutani t, gonatas j. superior sensitivity of conjugates of horseradish peroxidase with wheat germ agglutinin for studies of retrograde transport. j histochem cytochem 1979, 27:723-34. harper cg, gonatas jo, steiber a, gonatas nk. in vivo uptake of wheat germ agglutinin horseradish peroxidase conjugates into neuronal gerl and lysosomes. brain res 1980, 188:465-72. harper cg, gonatas jo, mizutani t, gonatas nk. retrograde transport and effects of toxic ricin in the autonomic nervous system. lab investigations 1980, 42:396-404. my first significant clinico-pathological manuscript was published while i was in the usa in 1979. the work had been done in perth using forensic autopsy material. this was really the beginning of my lifetime interest in the primary and secondary effects of alcohol on the human brain. wernicke’s encephalopathy is caused by thiamine (vitamin b1) deficiency and is very common in alcoholics. harper cg. wernicke’s encephalopathy a more common disease than realised. j neurol neurosurg psychiatry 1979, 42:226-31. by chance, the editor of this journal was prof. ian simpson from glasgow where i worked in 1973. i do not like to admit it but i think that this helped significantly in getting the paper published. i was not just a name without a face! this was the beginning of an important realisation that interpersonal relationships are critical to one’s career. i would go so far as to say that it was one of the main factors that contributed to the success of my career. during spring and summer our family travelled almost every weekend in our camper van. we had medical friends in washington dc (colin and helen masters), connecticut (michael and heather greenaway) and in boston (chris and stephanie burke). colin was working with dr. carlton gajdusek, who discovered the cause of prion disorders like kuru and creutzfeldt-jacob disease. he was awarded the nobel prize for this work. both hume adams and henry urich visited and stayed with us in philadelphia. figure 4: staff from the departments of neuropathology and neurology at the hospital of the university of pennsylvania. nick gonatas, head of neuropathology, is on the left of the front row next to clive. after finishing my 18 months work in philadelphia, we took a well-earned break of three months and went camping in mexico and guatemala. en route, i attended the annual conference of the american association of neuropathology (aanp) in kansas city. this was where i first met jim powers. he was working at einstein, ny, with bob terry and cedric reine. jim and i have been friends ever since. in 2013 jim and i arranged to meet with bob terry in california. therese and i had invited jim and cedric raine to travel with us through arizona in our hired rv. we were all taking bob terry out to dinner in the rv. we stopped to pick up another person, dik horoupian. when he stepped into the rv bob nearly fell over – they had worked together for many years. jim had coordinated all these arrangements without anyone knowing. at the aanp conference in 1985 jim introduced me to paul garren. paul came to work with me in sydney as assistant neuropathologist in 1986 for one year. jim has now retired as neuropathologist from rochester, new york, and is living in north carolina. in 2013 my wife and i travelled with jim powers and cedric raine in a hired rv (motorhome) through arizona – we had a ball! figure 5: special get together in 2013 in san diego to meet robert (bob) terry. left to right: cedric raine. bob terry, dik horoupian, and jim powers. royal perth hospital 1980 i returned to perth in 1980 full of enthusiasm for research, teaching and clinical work. the weekly program and thursday ‘brain cut’ demonstration was always popular with the hospital clinicians and trainees. i was keen to bring the western australian neuroscientists together and, along with serge bajada, decided to start an ’annual neurosciences colloquium’. i organised a venue (perth railway station – where the indian pacific arrives) and funding from several pharmaceutical companies. we invited an interstate guest and called for papers. the meeting was a great success and we ran it for about five years. it generated a number of collaborations between groups like veterinary science and medicine in perth. i did some work with the vet school at murdock university on ‘staggers’, a nutritional disease of sheep and cattle that damages the brain (rye grass toxicity). many famous visitors came to visit our department. byron had worked in boston during the 1960s and had many international connections. they included raymond adams who i had already met because he held a position at l’hôpital cantonal in lausanne, switzerland where i worked in 1973. he was a very modest man. he wrote the classic text on wernicke-korsakoff disease (caused by vitamin b1 deficiency). i studied the prevalence of this disease in perth and australia in the 1970s and 1980s and our data was used by the federal department of health to mandate the addition of vitamin b1 (thiamine) to bread to help improve everyone’s b1 levels in australia. harper cg. the incidence of wernicke’s encephalopathy in australia a neuropathological study of 131 cases. j neurol neurosurg psychiatry 1983, 46:593-8. raymond adams worked closely with another delightful fellow – pearson richardson. sir john walton, senior neurologist at queens square in london also visited the department. i was delighted when my mentor from glasgow, hume adams decided to visit perth. it was fabulous to catch up with hume and eileen again. my time in the usa had given me a real taste for research and i now had a feel for how to move ahead. i decided to begin an active research program and to apply for research funds to support the work. we had a very close working relationship with the department of forensic medicine in perth (coroners) and studied most of the brains from the coroner’s autopsies (about 3,000 per year). this was a unique source of cases and allowed us to carry out high quality epidemiologic studies. john hilton was the head of department and we became good friends. later, i encouraged him to move to sydney to join the university of sydney and he became head of department of forensic medicine in glebe. my research began with the simple aim of establishing whether or not people who drank alcohol had smaller brains than those who were nonor social drinkers. this work was published in two articles and was the beginning of my life-long thematic research on the effects of alcohol on the brain: harper cg, blumbergs pc. brain weights in alcoholics. j neurol neurosurg psychiatry 1982, 45:383-40. harper cg. neuropathology of brain damage caused by alcohol. med j aust 1982, 2:277-82. the neurosciences in perth was very strong and a new initiative was started in the early 1980s – ‘the australian brain foundation’. this was an australia wide foundation but western australia was one of the leading states and raised most of the start-up funds. i was the medical director of the state foundation for a period. later, through the foundation, i put together a proposal to the wa lotteries commission for funds to purchase an electronic counting machine for brain nerve cells in autopsy material. in june 1982 i did a reconnaissance trip around the world to check out different systems for automated cell counting. next, i flew to new york to meet ralph holloway, an anthropologist at columbia university. he was interested in our work on aboriginal brains. ralph and i worked together for a number of years and published several papers. this was the beginning of a whole new area of research for our group. figure 6: the local media were entranced with our research and came up with some clever headlines. the staff at royal perth hospital by 1984 i was developing an active research program and was looking for honours and phd students. i put out a leaflet to the science and medical students inviting them to apply for an honours year at the hospital and had an application from jillian kril. i think she was 17 years old at the time! jill is now professor of neuropathology at the university of sydney. she completed her honours year on the project ‘brain shrinkage in alcoholics’. we made foam casts of the inside of the skulls of hundreds of autopsy cases so that we could estimate the intracranial volume and compare it with the brain volume (estimated by archimedes principle). this enabled us to calculate how much the brain had shrunk during life. the catchphrase was: “too much drink and your brain will shrink”. the media loved the work and we had lots of publicity. several important manuscripts arose from this research in perth. harper cg, kril jj, backhouse a. measuring brain atrophy. neurology 1986, 36:1147 harper c, kril j, daly j. are we drinking our neurones away? brit med j 1987, 294:534-6. in june 1985 i visited my first ‘brain bank’ in boston – the beginning of another important part of my professional life. our decision to move back to sydney at the end of 1985 and leave our idyllic lifestyle in perth was not an easy one. my principal motivator was professor jim mcleod, head of neurology at royal prince alfred hospital (rpah). neuropathology in sydney was pretty weak compared to the strength of all of the fabulous clinical and experimental neurosciences. it was quite a task to convince my family that this was the right thing to do. personally, i was very anxious that i was moving beyond my level of competence and thought that i might not cope with the challenge. however, jim mcleod convinced me that ‘all of the stars were aligned’ and promised his personal help and advice. one of the main players was the dean of medicine, professor richard guy. he was a senior neurosurgeon and was still working at rpah. in those days the dean of medicine was able to handle both the clinical and administrative jobs. another important player was john allsop, a senior neurologist and chairman of the rpah board. in retrospect, it was the best thing i could ever have done. i was getting into a ‘semi-retirement’ mode in perth at the age of 42 years – far too young. a major challenge was just what i needed. since that time, i have not met anyone who has made a similar major change in direction in their professional career who has regretted his/her decision and i encourage others to do the same – go for it!! i reflected back to a statement i wrote while i was in switzerland in 1974: “i guess i should set my sights on my career for a while and try to achieve something really great by the time i am 35-40 years. a professorship would suit me fine but a lot of water under the bridge before then!” mind you, it was not all that straightforward. the position was advertised and then interviews were arranged in sydney. i think i was the only applicant but, even so, it was a harrowing experience. there were 10 on the interview panel including the head of medicine, professor bickerton blackburn. i remember him asking, “what is all this nerve cell counting about, is that real research? ” i had known prof. blackburn as a medical student and he always seemed totally unapproachable – my interview did not change this opinion. ironically, in 1994, i rented prof. blackburn’s apartment in paris for 6 weeks when i was working with jean-jacques hauw at l’hôpital salpetriere. the blackburn family were a medical force to behold. sir charles blackburn, the father of bickerton blackburn, was a pioneer of clinical research at the university of sydney. in the 1960s he forged strong links between researchers and clinical practitioners at rpah and within the faculty of medicine. he was responsible for establishing key academic and research posts and for appointing highly competent staff like john allsop and jim mcleod to fill these positions. my wife and i flew to sydney in november 1985 to look for houses before we moved from perth. she had already decided that we would live on the northern beaches. after about three days of house hunting we walked into the house in bayview and we both said: this is it! there were lots of farewell parties but the final party was held in the qantas lounge at the airport on the 7th december 1985. my parents were delighted that we were finally returning to sydney after 13 years away. dad wrote me a letter congratulating me – i am still touched when i read it today. dad was not an ‘expressive’ person! i never remember him telling me that he loved me. while still working in perth we (myself and the university design team) had put together plans for the new neuropathology laboratories $250,000 had been donated by the ramaciotti foundation. i based the plans on the neuropathology laboratories in perth that worked very well. i discussed my appointment with roger pamphlett who was about to head off to london for more neuropathology training and he said he would love to come to work with me as senior lecturer in neuropathology once he completed his training and examinations. it all seemed to be falling into place. i also asked jillian kril if she would be interested in coming to the new department in sydney. she was keen to do a phd and i was able to offer her a job as a research assistant. she joined our group in 1986. building works on our labs did not commence until i had been in sydney for many months. i was given tremendous support by my colleagues at the university department of pathology. david cameron was the head of department but sue dorsch, a dynamic immunologist/pathologist also played a key role. by choice, sue later became involved in university administration and finally became the deputy vice chancellor. for many years she actively supported the development of neuropathology. there were many professorial staff who liked the old style of university life. you taught a few students and played about with a bit of ‘hobby’ research. some disappeared for months at a time without explanation and there was little that the administration seemed to be able to do! finally, the laboratories were finished. professor guy officiated at the formal opening of the neuropathology labs on 26th november 1986. the minister for health attended as well as many dignitaries from the university and rpah. the nsw minister for health, the hon. peter anderson, gave his welcoming address and declared the neuropathology laboratories open. when i commenced work at the university, i was invited by the vice chancellor to a luncheon. he held these lunches regularly to meet his new senior staff. it was delightful to meet professor ward. he was very receptive to the needs of our new department and, in no time, i had significant additional funding. today, one is lucky to ever meet the vice chancellor in your entire career! at the luncheon i sat next to the head of telecom. he asked me if i was familiar with faxes. i had to declare my ignorance but, after he described the efficiency of faxes, i returned to my office and called the university telecommunications office. i explained the situation and said that i would like an additional telephone connection in my office for a fax machine. the director said, “who do you think you are – what if every professor calls and asks for an additional phone connection?” i made some enquiries and found out that i could simply buy a ‘line splitter’ and plug it into my existing phone outlet. i could then use the second line for my new fax machine! one of the delightful things about coming “home to sydney” was that there were staff in the university, hospital and state government who were fellow university medical students. when i called to make an appointment with the new south wales (nsw) dept. of health i found that i would be talking with sue morey. she and i had been good friends at uni and, over the next couple of years, she opened doors and provided support that would otherwise have been impossible to achieve. its ‘who you know not what you know’! here, the vice chancellor is opening our new-look pathology museum. my relationships with my pathology colleagues in rpah were far less cordial. the head of the department wanted me to go onto the regular general pathology reporting roster for skin, breasts, bowels etc. i explained that my training was exclusively in neuropathology. after some tense discussions it was agreed that i would take on the responsibility of the hospital autopsies and i did not have to go onto the general roster. frankly, it would have been dangerous and the risk of errors in diagnosis would have been significant. there were several of the hospital pathologists who i knew very well – peter russell and stan mccarthy. they were very supportive. many years later peter became head of the department and things ran much more smoothly. i had studied with stan when we did our pathology training. i also played against him in the inter-hospital rugby – these were ferocious games. we used to train 2-3 times each week and then play a ‘round robin’ against all of the teaching hospitals attached to the university of sydney. i had to stop playing in about 1970 when i broke two lateral spines on my lumbar vertebrae in one of the games. our coach was max elliott, an ex-wallaby representative for australia and respiratory physician. the history of the department of pathology at the university of sydney in october 1850 australia's first institution of tertiary education was established in sydney. the faculty of medicine at sydney university formally came into being on 13 june 1856 and thus is the oldest faculty of medicine in australia and new zealand. provision for teaching of pathology was made in 1883 after funds were made available in 1882 for the establishment of a chair of anatomy and physiology, and for lectureships in subjects including pathology. a significant event in the history of the department of pathology was the opening of the neuropathology unit in 1986. the establishment of this new facility resulted from the combined efforts of the nsw department of health, through the royal prince alfred hospital and the university of sydney. the refurbishment of the laboratories was made possible by generous funding to the university of sydney from the nathan roberts and phyllis henderson bequests and the ramaciotti foundation. the neuropathology unit also has responsibilities for medical undergraduate and postgraduate teaching, together with instruction groups which include nurses, physiotherapists and speech pathologists. international travel and research staff i had told sydney university and rpah before i accepted my appointment that i had already planned a big round-the-world scientific trip in september 1986. they had agreed that this would be ok. the hospitals funded three weeks of conference travel for staff specialists annually. first i flew to london where i had arranged to meet up with roger pamphlett and to meet the staff at queens square. roger had worked with us in neuropathology in perth for a year of his post-graduate neurology training. he enjoyed the neuropathology so much that he decided to make it his career. when i was invited to take the sydney chair in 1985 we made a pact that he would come and join me when he returned from queens square, london. we had a fabulous working relationship for 24 years at usyd. i next visited cambridge and gave a lecture at the maudsley hospital. i met peter lantos and prof. lishmann who were very interested in our research. peter lantos invited me to his “london club” for lunch. we remained friends for many years. international fellows jörg klekamp, a german medical student visited me when i was in perth in 1980. he came as part of an optional clinical term in his german medical curriculum. this was the beginning of a long working collaboration with the hannover medical school. his mentor, prof. kretschmann, had been doing volumetric and morphometric studies for many years and had established normal numbers during brain development. i visited hannover in 1982 and 1984 for this collaboration. one of the high points of the 1984 visit was a night of jazz in an underground cellar – i got so involved that i almost missed my early morning flight! jörg and his partner aggie met me again in london in 1985 to discuss if they could come to sydney to work for a year or so (they stayed three years). they wanted to come to sydney to do more work on brain volumes in aboriginal cases. prof. kretschmann organised a fellowship to help support their visit to sydney. jörg and aggie arrived in sydney in may 1986. they were a lovely couple and worked incredibly hard on their project (comparative neuroanatomy between caucasian and aboriginal brains). we published four manuscripts together. sadly, the work was seen as ‘racist’ by some international colleagues and my abstract submitted to the american neuropathology association (aanp) meeting was rejected. this was my only rejection (of a paper) in my career! klekamp j, riedel a, harper c, kretschmann hj. morphometric study on the postnatal growth of non-cortical brain regions in australian aborigines and caucasians. brain res 1989 riedel a, klekamp j, harper c, kretschmann hj. morphometric study on the postnatal growth of the cerebellum of australian aborigines and caucasians. brain res 1989 klekamp j, riedel a, harper c, kretschmann hj. quantitative changes during the postnatal maturation of the human visual cortex. j neurol sci 1991 riedel a, klekamp j, harper c, kretschmann hj. morphometric study on the postnatal growth of the hippocampus in australian aborigines and caucasians. brain res 1991 the interesting results of this research were picked up in the 1990s and were used in a film entitled ‘the difference’, a bbc documentary, produced and directed by mike smith (1999). this film highlighted the difference between aboriginal and caucasian children in their abilities to use either visual or auditory memory. the aboriginal children did extraordinarily well when they were challenged using visual memory compared with the caucasian kids. the opposite was seen when the children used auditory memory. this supported our research findings that aboriginal brains have larger, and presumably better organised visual cortices than caucasians. the implications of these scientific findings for educating aboriginal children are self-evident. jörg is now professor of neurosurgery in germany and we still keep in touch. it always creates a great sense of satisfaction to see students and colleagues who you have trained and worked with succeed in their chosen professional pathways. media presentations on alcohol our biomedical scientific alcohol story had a great attraction for the media and, over the years, i think i became quite adept at dealing with interviews. below are some of the projects that we were involved with. i was invited back to perth in 1987 by the wa department of health to launch a ‘healthy drinking program.’ in 1992 i ‘starred’ in a documentary made by the abc. i had a dreadful time trying to remember my lines and finally the director said “just ad-lib please clive”. i am now far more respectful of actors! alcohol and brain damage – produced by barbara edwards, dept. of health, wa 1987 choose your poison! documentary by abc 1988 film on addiction produced by robyn davidson – “this time next time”, 1992 research and international friends international interest in our research really began to take off after i visited peter carlen in toronto, canada in 1989. peter worked at the addiction research foundation. i had an introduction through an aussie (jim rankin) who had worked with peter. he was a leader in ‘reversibility of alcohol related brain damage’ and was very interested in our quantitative work on the autopsy cases. partially reversible cerebral atrophy and functional improvement in recently abstinent alcoholics. carlen pl, wilkinson da, wortzman g, holgate r. can j neurol sci. 1984 a fascinating area of research. commenting on a previous editorial by clive g. harper. carlen pl. br j addict. 1988 peter opened the door on international alcohol research for me. peter invited me to the next combined conference for the international society for biomedical research on alcohol (isbra) and the research society of alcoholism (rsa). there are very close ties between the international (isbra) and the us societies (rsa) for alcohol research. this was really the beginning of our research becoming recognised internationally. for the rest of my career the rsa and isbra conferences became the main focus of my international conference travel. previously, i had been attending pathological, neuropathological and neurological conferences but the more specialised meetings were much more relevant. two isbra conferences have been held in australia – 1994 and 2006. i was heavily involved in the organisation of both of these meetings. the first rsa meeting that i attended was on marco island in florida. i was welcomed into the society by a group with whom i just clicked. they included john littleton and susan baron, david lovinger, dennis twombly, rubin and kathy gonzales, fulton crews, and the society ceo, deb sharp. most of this group were basic neuroscientists so they welcomed someone with a clinical background. it was a revelation – so many people working on alcohol related organ damage. deb sharp ran/runs the conferences brilliantly and was always able to help and support our australian research group. deb encouraged therese and i to attend the rsa conference in denver, colorado in 2017 even though we had retired. one of the more senior rsa members was oscar parsons, a clinical psychologist. oscar recognised the importance of the structural brain changes we had described in alcoholic cases. we really clicked at the dinner dance each year because he also loved to dance. oscar appreciated my unorthodox style – not everyone does! i also met most of the more senior members who played an important role in my subsequent research and in helping me obtain grants from niaaa (national institute for alcohol addiction and alcoholism) in the usa (director tk lee). i met matz berglund, a psychiatrist from malmö, sweden. he was keen for us to study the autopsy brains of alcoholics from sweden. he suggested that we could transfer the brains to the new south wales tissue resource centre (trc). every person in sweden has a centralised medical record that includes data on general health, eating and drinking habits. this information is invaluable when correlated with the harvested brains. i visited matz in malmö in 1989 to discuss this possibility. i recall being in the hotel room when cnn announced the huge earthquake that had struck san francisco. i watched in horror as they showed the motorways, bridges and buildings that had collapsed. unfortunately, the swedish government would not let the brains leave the country so that our collaboration was cut short. there was a lot of serious science at these conferences and a lot of wonderful collaborations were developed. for many years we worked closely with edie sullivan and dolf pfefferbaum at the sri in palo alto, california. they invited us to their labs at sri in palo alto for six weeks to complete a study using mri on formalin fixed brains. we had intended to camp in a friend’s vw camper but there were no camping areas close by. edie booked us into a downtown motel called ‘the mermaid inn’. it became obvious on the first night with all the movement about the corridors and banging of doors that this was a brothel. edie was horrified at her mistake and she invited us to stay in their beautiful home. most recently we stayed at edie and kevin’s home in palo alto when we were in the usa in 2017. in 1992 i attended the isbra conference in bristol, in the uk. i met another australian who was big in the alcohol field john saunders. he and i ran two subsequent isbra conferences in australia (gold coast in 1994 and sydney in 2006). i also met many of the key european alcohol research workers like karl mann from heidelberg. figure 7: professors edie sullivan (left) and dolf pfefferbaum (right) from stanford university joined my wife (therese garrick) and me at the isbra conference dinner in sydney in 2006. neuropathology training and courses i saw training of staff in neuropathology as one of our critical roles. this included not just pathologists but also neurologists, neurosurgeons and radiologists. as a result, i began negotiations soon after i arrived in sydney with these medical groups. senior neurology and neurosurgery staff realised the enormous benefits of adding neuropathology to their trainee programs. we soon had regular rotating appointments with trainees from neurology, neurosurgery and pathology. two of the rotating neurosurgical registrars are still good friends – david mcdowell, a senior neurosurgeon in canberra and charlie teo, a well-known neurosurgeon in sydney. charlie was awarded the prestigious australian of the year award in 2016. one of my pathology rotating registrars was michael buckland. he now holds my position as head of neuropathology at royal prince alfred hospital. one of his main interest is the molecular biology of brain tumours. michael has also been instrumental in starting a brain bank for sports head injuries. the australian sports brain bank is located at the brain and mind centre and is partnered with the concussion legacy foundation in the usa. in addition, i decided to start an annual postgraduate neuropathology training course. this was a huge undertaking. i was able to convince my australian neuropathology colleagues (and friends) to contribute to this enterprise. initially, the main support group were peter blumbergs (adelaide), tony tannenberg (brisbane) and colin masters, catriona mclean and michael gonzales from melbourne. my own staff were also involved – roger pamphlett, michael rodriguez, roger stankovic, therese garrick, steve kumjew, donna sheedy and jillian kril. we limited the number of students to about 20 initially because each student needed a microscope and set of histological slides. there was a big emphasis on practical aspects of neuropathology and each student dissected a brain. the first year was a huge amount of work for our technicians but, after that, it became easier. the program was a huge success and, over the years, there were others who visited and contributed including our mate jim powers from rochester, new york. the students paid to attend so travel, accommodation and social event costs were covered for our teaching staff. each january they would fly into sydney for the four-day course over a weekend. we had a lot of fun together and i still receive thanks from many of those clinicians who attended. we even had international participants from new zealand and jordan. figure 8: michael buckland, then registrar in neuropathology, attended and presented at the 2001 american association of neuropathologists. it was about this time (1993) that i met therese garrick. she had been appointed to a research position with the biological schizophrenia research team (bsrt). her role was to attend the department of forensic medicine each morning to review the cases of the day to see if any were suitable for inclusion in our new brain bank (nsw tissue resource centre). on her first day she was told by stan catts (director of the bsrt) “meet professor harper at the forensic institute and he will show you around”. the meeting went something like: “hello professor harper, my name is therese garrick”. i responded with “hello therese (incorrect pronunciation) please call me clive”. she responded with the correct pronunciation of her first name and i repeated it incorrectly again. i am not sure why i did not get it but, to this day, i still pronounce it incorrectly! this was the beginning of a wonderful friendship/relationship and we have been married for 20 years. jim powers flew out from rochester to join us for the celebrations in 2000. figure 9: official dinner at the 2001 american association of neuropathologists. from the left, gavin dixon, michael buckland, maria sarris, therese garrick and clive harper. we were all working in neuropathology at the university of sydney. fern yang was another of my favourite staff members. she and her husband migrated to australia in 1990 and both had medical degrees from china. these degrees could not be registered in australia so they decided to do master’s/phd degrees at the university of sydney. fern came to work with me in neuropathology and her husband found a position in physiology. she had a delightful buoyant personality and was a pleasure to work with. after completing her master’s degree she became our muscle/nerve biopsy technician. sadly, in about 2000 she developed a cancer of the bile duct and died after a 4-year battle. jordan and syria – april 1992 bashar anabtawi, a jordanian/palestinian pathologist had come to our annual postgraduate neuropathology course in sydney in 1990. he wanted to develop his skills in muscle and nerve pathology. we got on very well and, as he left sydney he asked me if i would be prepared to come to jordan to help him develop a nerve and muscle laboratory for the middle east. at the time, most of the biopsies from jordan were sent to london. i jumped at the opportunity. bashar was good friends with the australian ambassador in jordan and the next thing we knew we were invited to stay at the consulate in jordan – what an opportunity. this adventure was amazing. apart from the work we visited damascus, and the carved city and temples in petra. in addition, we visited pella, with its ancient greek ruins. we climbed mount sinai to see the sun rise in the morning and then, in the afternoon, went for a swim in the ‘dead sea’. the ambassador was bob bowker – we are still friends with bob and his wife jenny. we share our love of camping around australia and own identical camping rigs (active campers). working in paris 1993 in 1993 i was invited to work in paris for six weeks with jean-jacques hauw at the hôpital universitaire pitié salpêtrière. my aim was to review all of their cases of the wernicke korsakoff syndrome and determine the prevalence of that disease in their paris forensic population. we published the data in 1995. harper c, fornes p, duyckaerts c, lecomte d, hauw j-j. an international perspective on the prevalence of the wernicke-korsakoff syndrome. metabolic brain disease, 1995. nisad in 1994 i negotiated to work with the nisad (neuroscience institute for schizophrenia and allied disorders) group of researchers and to take on some new staff to help with the brain bank. i went to forensic medicine each morning looking for donor cases for the brain bank. if there were any cases that had a history of schizophrenia or alcoholism that might be suitable for the brain bank the nisad staff called the relatives of the deceased to ask if they would consider donating their loved ones brain to the brain bank – a tough job! figure 10: students at our annual postgraduate neuropathology course at the university of sydney studying the histopathology slides for the course in 2004. research grants the neuropathology lab was going gangbusters with good financial support from the nh&mrc and nisad and various other smaller funding bodies. in 1990 i had given a lecture in washington, dc, to the niaaa. in discussions with the director, enoch gordis, he asked if i had ever considered submitting a grant application to the niaaa to support the australian brain bank. when dr. glenda halliday joined our research group we put together a research grant application to niaaa in the usa. the niaaa is one subsection within the huge us national institutes of health (nih). we were successful with this application in 1993 and were awarded $750,000. this allowed us to appoint several new staff members for the brain bank and therese garrick was reappointed as clinical manager. as part of the grant i was expected to travel to the usa each year to attend the research society of alcoholism. i formed many wonderful working relationships and friendships with members of the rsa over the next 20 years. the grant was a great honour – only one or two other australian research groups received grants from the nih. the funding has continued to this day (2020). figure 11: lecturers at the annual postgraduate neuropathology course at the university of sydney in 2004. from the left: jillian kril (sydney), clive harper (sydney), judith fryer (sydney), renata kalnins (melbourne), roger pamphlett (sydney), tony tannenberg (brisbane) and peter blumbergs (adelaide). ‘using our brains’ – a new initiative at the university of sydney in the early 2000s we realised that we needed to obtain more ‘normal brains’ to allow our brain bank research to prosper. we came up with the idea of asking the public to consider donating their brains while they were alive. here is the documentation of the huge public program that we launched in 2002. i was blessed by having a young friend, james pegum, assist with the six-month lead-up and launch in the nsw parliament house. he ably assisted with all of the subsequent media activity. i think that i was on every sydney television and radio station during the launch week. one of the main reasons for the success of this initiative was that gough whitlam agreed to become a donor and help launch the program. james went on to a career in it and is now a part-owner of privia. he is married to ria and they are living in orange. they have two boys, william and charlie and we see each other regularly. figure 12: official launch of the ‘using our brains’ donor program in the new south wales state parliament house in 2002. clive harper is talking with retired prime minister gough whitlam (centre) who volunteered as our first donor. sri lanka – 2002, 2007 and 2008 throughout the 2000s forensic medicine at glebe had been inviting postgraduate pathology students from sri lanka to sydney for an additional one year’s training. this was supported strongly by the university of colombo. in fact, it was a requirement that each student spend one year in another country to complete their training in pathology. i spoke to john hilton (director of forensic medicine) and he put me in touch with the coordinator of the colombo program. subsequently, a number of students came and worked with us in pathology at rpah. the first student was geethika jayaweera we are still good friends. geethika is now one of the most senior pathologists in the sri lankan army. there were many other trainees who visited sydney for 12 months and this helped to build strong links between our countries. as a result of this program i was invited to visit colombo as their external examiner in postgraduate anatomical pathology in 2002, 2006 and 2007. on each occasion, after we had completed the examinations with the postgraduate students, we spent two weeks travelling around their beautiful country. back in colombo we met up with geethika and her family and went to the local cricket club. we met arjuna ranatunga, the captain of the sri lanka team when they won the 1996 cricket world cup. he was nicknamed captain cool and is regarded as the pioneer who helped to lift sri lankan cricket from underdog status. after retirement, he worked in many posts of sri lankan cricket administration and then entered politics in 2005. ranatunga became the cabinet minister of transport and civil aviation. he was very charming. the sri lankan cricketers have a soft spot for australia as shane warne raised funds to rebuild the galle cricket ground after the 2004 tsunami. muscat, oman – 2008 and 2009 in 2008 i was invited to run a neuropathology course in muscat, oman. it ran very smoothly and was well accepted. following this, the dean of medicine at the sultan qaboos university approached me to ask if i would be interested in coming to work at the university/hospital in the future. i was very attracted to the idea and thought that it might fit in well with our plans for retirement. i negotiated with my staff and the university and rpah and changed my role to part-time. i was responsible for doing the neuropathology reports for the brain bank cases a 20 hour per week appointment. the pathology sides of the cases could be sent to me anywhere in the world – including oman. our neuropathology team was now 26 people. our five months term in 2009 in oman was fantastic. we enjoyed the enthusiasm of our students and the atmosphere of both the hospital and university. we lived on campus but took the opportunity of exploring oman at every opportunity in our borrowed tent and sleeping bags. on many occasions, as we camped on the beaches and in the deserts, we were offered food and drinks by passing locals. the omani’s hospitality was amazing. this was the perfect ‘exit plan’ and we moved into retirement smoothly and easily. i have no regrets about retiring and have enjoyed every moment. one of the main activities has been travel and catching up with our world-wide network of friends from both our work and social lives. we are also doing volunteer work with a local toy recycle group, catering to the nsw rural fire services and on our extended travels, working with blazeaid. they are another volunteer group who help aussie farmers after fires and floods to refence their properties. awards royal prince alfred research foundation medal for excellence in research i was encouraged to put my name ‘in the ring’ for the prestigious royal prince alfred (rpa) research foundation medal for excellence in research. i was one of two doctors selected by the committee and we were invited to present our research in lecture format so that the committee could select the winner. my younger son, rye, was able to come along –this was the first time he has heard me speak in public. i won the rpa foundation medal for excellence in research in 2006. this was the press release: “rpa medal winner identifies approaches to repair alcohol related brain damage. professor clive harper has won the prestigious rpa foundation medal for excellence in research for his ground-breaking research into alcohol-induced brain damage.” figure 13: farewell luncheon with all of the examiners at the university of colombo after completion of the examinations for postgraduate anatomical pathology in 2002. australia day honours award in the following year (2007) i was delighted to be included in the australia day honours list and was awarded an am (member of the order of australia). this is the citation: professor clive gordon harper department of pathology, university of sydney nsw 2006 for service to medicine in the field of neuroscience, particularly research into the neuropathological consequences of alcohol-related brain damage, and through contributions to public health policy. the presentation of awards was at nsw parliament house. professor marie bashir was the governor general and officiated. i had met her professionally when she was the clinical director of mental health services for the central sydney area health service. she was very charming and told me that she had been following my career and the development of the brain bank. the henri begleiter excellence in research award in 2009 i received another award that i am very proud of: the henri begleiter excellence in research award. nominations for this award are accepted from research society of alcoholism members. recipients of this award receive a plaque and cash award at the annual meeting. this award is given to an individual demonstrating innovation or creativity and excellence in their research and/or someone whose work has a major impact on the field. recipients of this award: ting-kai li (2007), adolf pfefferbaum (2008), clive harper (2009), jan hoek (2010), marlene oscar berman (2011), linda spear (2012), kathleen grant (2013), george fein (2014), john krystal (2015), bernice porjesz (2016), raymond anton (2017), sarah jo nixon (2018). the award was made at the san diego rsa conference by the president, peter monti. my personal philosophy has always been to find the best staff to work with regardless of whether or not they are smarter than you … most have been smarter! always be available to staff and to look for opportunities to help staff in their personal development, such as encouraging attendance at conferences, even for junior staff. always be on the lookout for new opportunities and do not be afraid of the extra work. go the extra yard and don’t be afraid to ask the hard questions. be totally honest and up front with staff and colleagues. i have rarely been disappointed by my staff and consider that i have been blessed in my career. i was awarded emeritus status by the university of sydney and give occasional consultative advice to my colleagues. in our retirement my wife and i have joined a number of volunteer groups including blazeaid. the peninsular senior citizens toy recycle group and the local rural fire service (catering division). blazeaid is particularly challenging. it is a volunteer-based organisation that works with families and individuals in rural australia after natural disasters such as fires and floods. working alongside the rural families, we help to rebuild fences and other structures that have been damaged or destroyed (https://blazeaid.com.au). in our spare time we swim, cycle, sail and travel in our 4x4 camper rig. last year we were on the road, around australia, for seven months! after covid we hope to travel overseas again to meet up with our international colleagues and friends. figure 14: camping in 2021 on magnetic island in queensland with the local koala. copyright: © 2022 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. adrenomyeloneuropathy (amn): myelinopathy or axonopathy? feel free to add comments by clicking these icons on the sidebar free neuropathology 4:24 (2023) letter adrenomyeloneuropathy (amn): myelinopathy or axonopathy? comment on: considering the myelin-centric hypothesis: insights from budka's historical adrenomyeloneuropathy case report by e. salsano and c. benzoni herbert budka division of neuropathology and neurochemistry (obersteiner institute, formerly institute of neurology / neurological institute), department of neurology, medical university of vienna, austria corresponding author: division of neuropathology and neurochemistry (obersteiner institute, formerly institute of neurology/neurological institute) · department of neurology · medical university of vienna · vienna · austria herbert.budka@meduniwien.ac.at submitted: 13 december 2023 accepted: 17 december 2023 copyedited by: georg haase published: 21 december 2023 https://doi.org/10.17879/freeneuropathology-2023-5227 keywords: adrenomyeloneuropathy (amn), axonopathy, axonopathy, myelinopathy, demyelination i am grateful to drs. salsano and benzoni [1] for their appreciation of our original adrenomyeloneuropathy (amn) report [2] and of my recent account on its history [3], but still more for raising an interesting scientific issue that, in my opinion, has not been completely resolved: whether the characteristic affection of long cerebrospinal tracts in amn develops as either original myelin or axonal pathology, or even as both. they think i overlooked the issue. in fact, i have been well aware of it but decided – maybe wrongly – to omit such a discussion from my recent contribution that i considered as a primarily historic account in the sense of a "nice story", as it does not provide new scientific data [3]. however, i am now glad that drs. salsano and benzoni’s letter offers the opportunity to comment on myelinopathy versus axonopathy in amn. i agree with them that this pathogenetic issue is not purely academic: with future progress in molecular and personalized medicine, it becomes relevant to tailor therapy to the right target. in a potentially confusing way, the terminology of "demyelination" has been in twofold use. first, more generally and somehow imprecisely, it has been applied to plain myelin loss, including descriptions of histological stainings for myelin, regardless of the underlying pathogenesis. second and more specifically, it has signified dismantling of the myelin sheath from the (at least initially) intact axon, resulting in myelino-axonal dissociation, as may be seen in oligodendrogliopathies (best example: the lytic infection by the john cunningham virus/jcv in progressive multifocal leukoencephalopathy/pml) and schwannopathies; cellular or humoral immune attacks against myelin, ideally to be visualized by myelin "stripping" by macrophages or binding of antibodies; or electrolytic or metabolic myelin impairment e.g. in central pontine myelinolysis/cpm or leukodystrophies. indeed, x-linked or classic adrenoleukodystrophy (ald) has been categorized with other leukodystrophies among demyelinating diseases. however, specific demyelination and axon injury are often concurrent and can have common pathogenic mechanisms and physiological effects [4]. thus the situation is complicated by the mutual dependency between myelin sheath and axon, leading to secondary axonopathies in primary myelinopathies, and vice versa. so it might resemble the classical chicken-or-egg dilemma. in addition, the dissection of such pathogenetic events requires specific methodologies that can be met best in experimental models, but only to a limited degree in human autopsy tissue that provides a glimpse at one specific point in time, not over a period, and may be prone to autolytic and preparatory constraints as well as subjective interpretation. in our original amn report [2], we described the lesioning of long tracts as "incomplete demyelination", meant in a more general sense and not considered to imply a specific pathogenesis that was then well beyond our original intent. we mainly emphasized perivascular histiocytic cuffs with pathognomonic ultrastructural inclusions that guided us towards ald. however, we mentioned "well preserved axons" and "no loss of oligodendrocytes", based on then available basic histological stains without immunohistochemistry. now, with hindsight by my 52 years of neuropathological experience and the stunning progress of biomedical sciences including neuropathology, i must criticize these imprecise descriptions of my early career. indeed, when i made preparations for my recent historical account [3], original paraffin blocks were retrieved and immunostained with up-to-date methodology and antibodies, including axonal markers (phosphorylated and non-phosphorylated neurofilament proteins/nfp), ubiquitin and the autophagosome cargo protein p62, tau, and phosphorylated and non-phosphorylated tdp-43. while it is not easy to recognize relevant features in the original stainings of histological sections from our case that are now available to the public by virtual microscopy [3], re-examination with modern molecular markers gives a more clear-cut feeling of what happened in the affected tissue. first, affected cerebrospinal tracts show not only diffuse loss of myelin, resulting from moderate loss of myelinated fibers, but also moderate diffuse loss of pnfp-immunoreactive axons (fig. 1) that seems to be commensurate with myelin loss. second, there are some disseminated dystrophic axonal swellings (fig. 2) that are occasionally found to be surrounded by a still intact myelin sheath (fig. 3), and show immunoreactivity for p62 (fig. 4) and ubiquitin (fig. 5). such axonal swellings are, to a lesser degree, disseminated also in the tegmentum. third, rare tegmental neurons express pnfp in their cytoplasm (fig. 6), suggesting a proximal "axonal reaction" to distal degeneration, whereas other neurons do not have pnfp in their cell bodies, the normal finding. inclusion bodies or deposits of other proteins as examined were absent. in sum, these neuropathological features document an ongoing axonopathy with dystrophic features in long nerve fiber tracts, whereas evidence for a primary myelin affection is lacking, albeit difficult to appreciate in such tissue. in the literature on amn, the pathology observed in the affected tracts has been usually described to reflect a distal axonopathy [5,6]. my re-examination of the original amn case is in accordance with this interpretation. on the other hand, griffin et al. [7] described, in two sural nerve biopsies of their five first serial amn cases, onion bulbs, a feature diagnostic of primary demyelination in the peripheral nerve. in their letter, drs. salsano and benzoni suggest a "hypothesis of a myelin-centric nature of early-stage amn", and provide arguments for this viewpoint, mainly based on in vivo quantitative mri techniques, and rescue from disease by human abcd1 expression in oligodendrocytes of an ald model in zebrafish [1]. in addition, they interpret neuropathological findings in our original case with a relatively short clinical duration to support their myelin-centric hypothesis. i apologize to drs. salsano and benzoni that our imperfect description in the original report might have been misleading. however, i agree with them that more evidence on the fate of the myelin-axon axis in amn is needed, including their proposal to study a mouse model with conditional knock-out of abcd1 in distinct cns cell types. acknowledgement i am greatly indebted to prof. romana höftberger, pd ellen gelpi, and the histo lab members of the obersteiner institute vienna, for their support in re-examining the original amn case. all figures are taken from the pyramid or tegmentum of the lower medulla oblongata in new immunohistochemical sections (ihc, performed in a dako autostainer with dako envision kit and diaminobenzidine/dab as chromogen) or luxol fast blue (lfb) stained sections of the original amn case. note also the characteristic perivascular histiocytic sleeves. fig. 1. moderate diffuse loss of axonal profiles. ihc with anti-pnfps (smi31) x 40. fig. 2. disseminated axonal swellings. ihc with anti-pnfps (smi31) x 200. fig. 3. in center, a large axonal swelling surrounded by normally appearing myelin sheath. lfb and nuclear fast red x 500. fig. 4. axonal swellings contain p62. ihc with anti-p62 (clone 3/p62 lck ligand, bd transduction laboratories, franklin lakes, nj, usa) x 100. fig. 5. axonal swellings contain ubiquitin. ihc with anti-ubiquitin (clone ubi-1, aka 042691gs, millipore, burlington, ma, usa) x 100. fig. 6. a tegmental neuron with pnfps in its cell body in the upper right corner; other neurons are negative. ihc with anti-pnfps (smi31) x 200. references 1. salsano e, benzoni c: considering the myelin-centric hypothesis: insights from budka's historical adrenomyeloneuropathy case report. free neuropathol. 4: 23 (2023). https://doi.org/10.17879/freeneuropathology-2023-5218 2. budka h, sluga e, heiss wd: spastic paraplegia associated with addison’s disease: adult variant of adreno-leukodystrophy. j neurol. 213: 237–250 (1976). https://doi.org/10.1007/bf00312873 3. budka h: a historical look using virtual microscopy: the first case report of adrenomyeloneuropathy (amn). free neuropathol. 4: 18 (2023). https://doi.org/10.17879/freeneuropathology-2023-5115 4. moore grw, stadelmann-nessler c: demyelinating diseases. in: love s, budka, h, ironside jw, perry a (eds.) greenfield’s neuropathology, 9th ed., vol. 2, pp. 1297-1412. crc press, boca raton fl 2015. 5. faust pl, powers jm: peroxisomal disorders. in: love s, budka, h, ironside jw, perry a (eds.) greenfield’s neuropathology, 9th ed., vol. 1, pp. 562-588. crc press, boca raton fl 2015. 6. schaumburg hh, powers jm, raine cs, spencer ps, griffin jw, prineas jw, boehme dm: adrenomyeloneuropathy: a probable variant of adrenoleukodystrophy. ii. general pathologic, neuropathologic, and biochemical aspects. neurology 27 (12): 1114-1119 (1977). https://doi.org/10.1212/wnl.27.12.1114 7. griffin jw, goren e, schaumburg h, engel wk, loriaux l: adrenomyeloneuropathy: a probable variant of adrenoleukodystrophy. i. clinical and endocrinologic aspects. neurology 27 (12): 1107–1113 (1977). https://doi.org/10.1212/wnl.27.12.1107 copyright: © 2023 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. survey of neuroanatomic sampling and staining procedures in alzheimer disease research center brain banks feel free to add comments by clicking these icons on the sidebar free neuropathology 4:6 (2023) original paper survey of neuroanatomic sampling and staining procedures in alzheimer disease research center brain banks juan c. vizcarra1, andrew f. teich2, brittany n. dugger3, david a. gutman4, and the alzheimer’s disease research center digital pathology working group5 department of biomedical engineering, emory university & georgia institute of technology, atlanta, usa department of pathology and cell biology, department of neurology, the taub institute for research on alzheimer’s disease and the aging brain, columbia university, new york, new york, usa department of pathology and laboratory medicine, university of california-davis, sacramento, california, usa department of neuropathology, emory university, atlanta, georgia, usa members of the group and their affiliations can be found in supplementary material 2. corresponding authors: brittany n. dugger · department of pathology and laboratory medicine · university of california, davis · research iii, third floor, suite 3400a · 4645 2nd ave. · sacramento, ca, 95817 · usa, bndugger@ucdavis.edu david a. gutman · department of pathology and laboratory medicine · emory university · 1364 clifton rd · atlanta, ga, 30322 · usa, dgutman@emory.edu additional resources and electronic supplementary material: supplementary material 1 supplementary material 2 supplementary document github repository: https://github.com/gutman-lab/adrc-np-survey-2023 submitted: 25 february 2023 accepted: 28 march 2023 copyedited by: georg haase and cinthya aguero published: 13 april 2023 https://doi.org/10.17879/freeneuropathology-2023-4696 keywords: neuropathology, adrcs, brain banks, tissue repository, biobank abstract the collection of post-mortem brain tissue has been a core function of the alzheimer disease research center’s (adrcs) network located within the united states since its inception. individual brain banks and centers follow detailed protocols to record, store, and manage complex datasets that include clinical data, demographics, and when post-mortem tissue is available, a detailed neuropathological assessment. since each institution often has specific research foci, there can be variability in tissue collection and processing workflows. while published guidelines exist for select diseases, such as those put forth by the national institute on aging and alzheimer association (nia-aa), it is of importance to denote the current practices across institutions. to this end a survey was developed and sent to united states based brain bank leaders, collecting data on brain region sampling, including anatomic landmarks used, staining (including antibodies used), as well as whole-slide-image scanning hardware. we distributed this survey to 40 brain banks and obtained a response rate of 95% (38 / 40). most brain banks followed guidelines defined by the nia-aa, having h&e staining in all recommended regions and targeted region-based amyloid beta, tau, and alpha-synuclein immunohistochemical staining. however, sampling consistency varied related to key anatomic landmarks/locations in select regions, such as the striatum, periventricular white matter, and parietal cortex. this study highlights the diversity and similarities amongst brain banks and discusses considerations when amalgamating data/samples across multiple centers. this survey aids in establishing benchmarks to enhance dialogues on divergent workflows in a feasible way. introduction brain banks are a fundamental resource to facilitate scientific research to better understand the complex biology of the human brain. human tissues are a critical component in understanding a wide range of disorders that affect human health, including alzheimer disease, parkinson’s disease, and chronic traumatic encephalopathy (cte), as well as understanding the effects of normal aging.1,2 within the alzheimer disease and related disorders (adrd) community, the national institute of health (nih) has supported many brain-banking efforts, including alzheimer disease research centers (adrcs).3–5 many brain banks have been collecting tissue for decades and have developed specific protocols on sectioning, staining, and preserving samples typically driven by a specific research area or focus. for example, in a research repository focused on amyotrophic lateral sclerosis (als), the spinal cord may be routinely included in the sampling schematic, while this may not be routine in one focused on alzheimer disease (ad).6,7 a key mandate of adrcs is to aggregate and share the data from research participants, including clinical data, neuropsychological assessments, patient demographics, and when available, neuropathology (np) assessments as well as tissue samples.8 the np data provides the ground truth diagnosis, based on a review of a specific set of stained brain region slides, using standardized semi-quantitative rating scales developed over the past several decades. for ad these include the consortium to establish a registry for alzheimer's disease (cerad) score, braak neurofibrillary tangle stage, thal amyloid phase, and others.9–13 guidelines regarding adrd diagnosis have been previously published and updated as research progresses.14–17 following initial autopsies, a typical protocol will involve the dissection of brain tissue and placement into small cassettes (∼30x20x5 mm, but this can vary) to be paraffinized, sectioned, and then stained either histochemically or immunohistochemically for select markers. however, numerous variations exist, such as large format free floating sections that may be 40-80 µm thick. the specific staining protocols, sample preparations including slice thickness, antibodies or other reagents used for processing, and the anatomic regions surveyed are just a few parameters that can vary from center to center, bank to bank, or even case to case.18,14 furthermore, with the advent and more widespread availability of whole slide image (wsi) technology, it is now feasible to digitize neuropathology datasets at high resolution. these datasets are more easily shared than physical slides and there is a need to understand how best to harmonize workflows. data harmonization efforts will require the creation of a standardized data dictionary in order to facilitate data sharing in the spirit of the fair guidelines (findability, accessibility, interoperability, and reusability).19 given potential variability in staining, sectioning, blocking parameters, and naming conventions, we created, distributed, and evaluated a survey that assessed the current landscape across research institutes to identify common practices in adrd brain banks (supplementary document). materials and methods the authors, along with input from the adrc digital pathology working group, produced and disseminated a survey to brain bank leaders in the spring / summer of 2022, see supplementary table s1 for list of submitted surveys and supplementary material 2 for names of members of the adrc group and their affiliations. survey questions focused on obtaining data for the immunohistochemistry (ihc) procedures used, sampling of brain hemispheres, wsi scanners available, antibody usage, and brain region staining and sampling procedures. for anatomic areas, select landmarks were surveyed to identify heterogeneity between sampling procedures among adrcs (for the complete survey, see supplementary material 1). the survey questions were developed using the jotform.com platform (jotform inc, san francisco, ca). a url link to the survey was emailed to 39 past and current adrc neuropathology core leaders and one non-adrc brain bank leader. participation was voluntary. responses contained no personally identifiable information and results were anonymized. survey responses were compiled in the fall of 2022. survey entries were exported into an excel spreadsheet file for analysis. all analyses were done using open-source python libraries through custom code found in the github repository, including the resulting excel file: https://github.com/gutman-lab/adrc-np-survey-2023. categorical data were presented as frequencies and percentages. most questions in the survey had multi-select answers that were not mutually exclusive (multiple answers possible for each question) or were free text-answers. questions regarding the counter staining-process for ihc were yes/no questions. results the survey requests were sent out to 39 past and present adrc np core leaders, plus one additional brain bank leader not affiliated with an adrc. the survey obtained a response rate of 95% (38/40). most surveys were completed by the center’s np leader or co-leader (33/38). the survey collected center information regarding the ihc procedure used, sampling of brain hemispheres, wsi scanners available, antibody usage, and brain region staining and sampling procedures. ihc processing and counterstaining were similar across centers, with most respondents stating they use hematoxylin counterstain with diaminobenzidine (dab) as the chromogen (brown) without enhancement (30/38). other responses included using neutral red with dab as chromogen with nickel enhancement (3/38) and only dab with nickel enhancement (1/38). four participants specified only using hematoxylin counterstain but neither dab as chromogen with or without nickel enhancement, presumably meaning no implementation of ihc, an alternative approach, or a misinterpretation of the survey question. four of the 38 respondents did not specify the brain region they sampled. of the remaining 34, most denoted sampling the brain's left hemisphere (32/34), 13 sampled both hemispheres, and two sampled only the right hemisphere. answers to this question were not mutually exclusive and included sampling the brain's left, right, or both hemispheres. average section thickness varied considerably across centers, with the most common section size being 5 µm (12/38), followed by 8 µm (7/38), 4 µm (6/38), and 6 µm (5/38). some centers (3/38) sample within a range of sizes, i.e., 5 7 µm. the thinnest section size sampled was 2 µm and the thickest was 80 µm. most centers have a single expert or neuropathologist do all the blocking (23/37), with the remaining utilizing a group of individuals that routinely perform the blocking (14/37); responses were free text answers. the type of wsi scanner available was also surveyed for all institutions. the most common type of scanner denoted was the aperio / leica (25/38). other scanners included olympus, zeiss, huron, philips, hamamatsu nanozoomer, keyence, and 3d histotech. most respondents had access to only a single type of scanner (29/38), while four had two scanner types available. five respondents stated they had no access to a wsi scanner. these data are similar to previously published results.20 with respect to ihc, most respondents utilize the at8 antibody when staining for tau (23/37, antibody information missing for one center) or the phf1 antibody (14/37). some respondents stated using more than one antibody variant for tau staining, with two centers using three, and two centers using two antibodies. other tau antibodies used include cp13 and rd3/rd4. amyloid beta (aβ) staining showed more antibodies used by respondents, with 12 different antibodies in current use. 4g8 (13/37) and the 6e10 (9/37) antibodies are the most common. only one respondent stated using multiple antibodies for aβ staining (10d5, 4g8, and 6e10). for alpha-synuclein (ɑsyn) and tdp-43, respondents specified phospho-specific or non-phospho-specific antibody use. most respondents use a phospho-specific antibody for tdp-43 (29/37). for ɑsyn, about half of the respondents use each type of antibody (phospho-specific: 18, non-phospho specific: 17), and two respondents specified alternative approaches (lb509 emd millipore & millipore #ab5038p). additionally, one respondent used both phospho and non-phospho-specific antibodies. nineteen brain regions were surveyed to assess the stains applied, anatomical landmarks included, and sampling methodology. the survey identified a set of highly consistent combinations of stains and regions across centers/banks. for example, the h&e stain was denoted to be used by all respondents. however, there was a set of six regions (central gyri, periventricular white matter, anterior hippocampus, olfactory bulb, posterior cingulate gyrus, and temporal pole) that were not universally stained with h&e. other stains, like ɑsyn, were more targeted, with frequent use in the midbrain, amygdala, and anterior cingulate gyrus (n≥30) and moderate use for the frontal gyri, posterior hippocampus, temporal lobe, medulla, olfactory bulb, parietal gyri, and the pons (n≥13) (figure 1). figure 1. heatmap showing the number of centers using specific stains for different sampled brain regions. the vertical axis displays the 19 brain regions surveyed and the horizontal axis displays the major stains surveyed. the number in the heatmap signifies the number of centers that use the stain for that region. ɑ-syn: alpha-synuclein, aβ: amyloid-beta, h&e: hematoxylin & eosin. as anatomic regions can be vast and vary in mediolateral, superior/inferior, or rostrocaudal aspects, we also surveyed specific anatomic landmarks within select sampled brain regions. for all landmark questions, the responses were not mutually exclusive. the inclusion of specific anatomic landmarks varied considerably across respondents. for example, for the frontal gyri region, most respondents consistently sample the middle frontal gyrus (32/37 centers that collect the region), but only a few respondents stated that they also sample the inferior frontal gyrus (8/37). for the cerebellum, nearly all respondents stated they sampled the dentate nucleus (36/37), but less than one-third of respondents sampled the vermis (11/37). similarly, for the thalamus and subthalamic nuclei region, not all respondents included the subthalamic nuclei (30/37). other landmarks in this region were sampled by a subset of brain banks (figure 2 shows responses for select regions, additional results are included in supplementary figure s1). figure 2. bar plots showing the anatomical landmarks sampled in four brain regions. the dashed line represents the total number of submitted surveys (38). the dotted line represents the number of respondents that answered the question (varies per region). the n value in the vertical label corresponds to the value of the dotted line. all answers were non-mutually exclusive. mt: mammilla-thalamic, ac: anterior commissure, additional region landmark responses can be found in supplementary figure s1. for most regions, the majority of respondents stated sampling after coronal slicing (>80%) as opposed to before. for the cerebellum region, the survey asked if the sampling was done with longitudinal, transverse, or coronal slicing. most centers use longitudinal sectioning when sampling the cerebellum (21/38), with a subset using transverse (n=6) or coronal (n=5) sectioning, or some other approach (n=5) (figure 3). additional questions not reported are those regarding the antibody vendor, the select set of regions and the number of gyri and sulci targeted during sampling (survey answers can be found at https://github.com/gutman-lab/adrc-np-survey-2023). these questions were free text answers, and while we attempted to glean data from these, there was too high a level of variability to draw conclusions. figure 3. for 18 out of the 19 regions (olfactory bulb excluded), the number of centers that sample the region via different sectioning approaches is shown using stacked bar plots. for 17 regions, the survey asked if the centers sampled the region after or before coronal slicing. for the cerebellum region, the options were: longitudinal, transversal, and coronal slicing. the numbers show the value of the stacked bar. the label in each bar is the brain region. discussion overall, we received survey responses from 95% of sites, providing detailed information regarding neuropathology (np) protocols in select brain banks within the united states. across respondents, the most consistently sampled regions include the frontal gyri, visual cortex, midbrain, posterior hippocampus, and striatum (37/38 centers). the least sampled regions include the posterior cingulate gyrus and the temporal pole (18/38). sampled regions were denoted to be stained with h&e most often, concerning ihc, ɑsyn staining was most frequently conducted on the midbrain & amygdala (>32 centers), and aβ staining was most frequently used in striatum, frontal gyri, cerebellum, and posterior hippocampus (>29 centers). there was variability for tissue thickness, and the specific antibodies/epitopes for tau, ɑsyn, and aβ. the use of specific landmarks to aid in localizing sample regions showed a much higher degree of heterogeneity (figure 2). brain regions can be relatively large, and assigning generic terms or talairach/mni space or brodmann area may not be sufficient for optimal consistency in sampling.21,22 for denoting the presence/absence of a particular neuropathologic feature, a level of precise anatomic location within a nucleus may or may not be necessary, especially when assessing overall diagnoses. however, depending on what nuclear subregions are examined, the distribution of aggregate proteins can vary, for example in the amygdala, and these may alter specific correlations.23–25 there is also variability in the performance of staining procedures, and how effectively they may reveal pathologies, as some have reported for ɑsyn ihc.26 additional studies, including those from the brainnet europe consortium and from persons within the adrc network, have aided in understanding intra-rater reliability, accuracy to clinical diagnoses, and validation of methods in multi-institutional cohorts.10–13, 27–30 depending on the specific scientific question, understanding these additional details may be important.14,15 given the recent advancements in digital pathology, to examining other communities that have converted to a digital format, such as the radiology community, can be advantageous. within the radiology imaging community, standardization of imaging protocols has been achieved across centers, driven in part by the alzheimer’s disease neuroimaging initiative (adni), now starting its fourth iteration.31,32 despite significant differences in mri scanners across individual institutions, these efforts have facilitated the development of comparable imaging acquisition protocols across centers. the inherent digital nature of mri datasets has facilitated not only the sharing of the original imaging sequences but also allows the sharing of standardized quantitative measures of brain structure and pathology. for example, mri volumes are often run through a freesurfer pipeline that produces volumetric information on individual brain structures, cortical thickness, and white matter/gray matter volume, among other detailed statistics.33–36 while data sharing is also a key focus of the adrc network, sharing the physical autopsy slide sets between research centers may often be impractical. there is a great expense in properly packing and shipping large sets of glass slides between centers, although individual sections or blocks can reasonably be shared at a limited scale. the increased availability of whole slide imaging (wsi) platforms is enhancing the ability to share high-resolution digital pathology images.37,38 these, in turn, can be utilized alongside machine learning/artificial intelligence (ml/al) workflows to tackle questions that are otherwise difficult to solve by conventional or traditional approaches. for example, early work in this domain has shown that computational workflows can identify and quantify neuropathological hallmarks of the disease, such as aβ plaques, in a scalable manner.39–43 however, before similar studies can be replicated at scale across a large, diverse collection of images, it is imperative to begin developing a standardized data model to capture pre-analytic variables, as these have been denoted to alter machine learning algorithm outputs.42,44 ml models are notorious for making mistakes in unpredictable ways when being subject to novel images (i.e., different from images used during model training).45–48 thus, it is to be expected that models trained on images from 5 µm sections would not have similar results when assessing images from 80 µm sections. similarly, the different staining or sampling approaches between adrcs might produce images that vary slightly or significantly when viewed through an ml workflow. understanding the data is equally important as developing the computational approach, and the results of this survey will help in understanding the depth of heterogeneity in the adrc’s collective database. conflicts of interest dr. gutman has done consulting work with histowiz inc llc. acknowledgements we immensely thank the adrc digital pathology working group, dr. jonathan d. glass, dr. marla gearing, dr. katherine l. lucot, and kevin nzenkue for their helpful suggestions and feedback on this project. funding this work was supported in part by grants from the national institute on aging (nia) of the national institutes of health (nih) under award numbers p30-ag066511-03s2, nih u01 ag061357-04s1, p30ag072972, r01ag052132, r01ag056519, r01ag062517 and p30ag066462. references 1. carlos, a. f. et al. from brain collections to modern brain banks: a historical perspective. alzheimers. dement. 5, 52–60 (2019). 2. beach, t. g. alzheimer’s disease and the ‘valley of death’: not enough guidance from human brain tissue? j. alzheimers. dis. 33 suppl 1, s219–33 (2013). 3. yong, w. h., dry, s. m. & shabihkhani, m. a practical approach to clinical and research biobanking. methods mol. biol. 1180, 137–162 (2014). 4. alzheimer’s disease research centers. national institute on aging. https://www.nia.nih.gov/health/alzheimers-disease-research-centers. 5. beekly, d. l. et al. the national alzheimer’s coordinating center (nacc) database: an alzheimer disease database. alzheimer dis. assoc. disord. 18, 270–277 (2004). 6. pantoni, l. et al. postmortem examination of vascular lesions in cognitive impairment: a survey among neuropathological services. stroke 37, 1005–1009 (2006). 7. 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neuropathol. 141, 159–172 (2021). 13. newell, k. l., hyman, b. t., growdon, j. h. & hedley-whyte, e. t. application of the national institute on aging (nia)-reagan institute criteria for the neuropathological diagnosis of alzheimer disease. j. neuropathol. exp. neurol. 58, 1147–1155 (1999). 14. montine, t. j. et al. national institute on aging-alzheimer’s association guidelines for the neuropathologic assessment of alzheimer's disease: a practical approach. acta neuropathol. 123, 1–11 (2012). 15. hyman, b. t. et al. national institute on aging-alzheimer’s association guidelines for the neuropathologic assessment of alzheimer's disease. alzheimers. dement. 8, 1–13 (2012). 16. lucot, k. l. et al. assessment of current practices across alzheimer’s disease research centers biorepositories. biopreserv. biobank. 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(2023) doi:10.1093/jnen/nlac132. 43. signaevsky, m. et al. artificial intelligence in neuropathology: deep learning-based assessment of tauopathy. lab. invest. (2019) doi:10.1038/s41374-019-0202-4. 44. jones, a. d. et al. impact of pre-analytical variables on deep learning accuracy in histopathology. histopathology 75, 39–53 (2019). 45. finlayson, s. g. et al. adversarial attacks on medical machine learning. science 363, 1287–1289 (2019). 46. eykholt, k. et al. robust physical-world attacks on deep learning models. arxiv [cs.cr] (2017). 47. cisse, m., adi, y., neverova, n. & keshet, j. houdini: fooling deep structured prediction models. arxiv [stat.ml] (2017). 48. szegedy, c. et al. intriguing properties of neural networks. arxiv [cs.cv] (2013). copyright: © 2023 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. yap1 fusion-positive ependymoma presenting in an adult with a pigmented phenotype and association with superficial siderosis feel free to add comments by clicking these icons on the sidebar free neuropathology 5:23 (2024) letter yap1 fusion-positive ependymoma presenting in an adult with a pigmented phenotype and association with superficial siderosis osorio lopes abath neto1, leonardo furtado freitas2, martha quezado3, zied abdullaev3, kenneth aldape3 department of pathology, university of iowa hospitals and clinics, 200 hawkins dr, iowa city, ia 52242, usa department of radiology, university of iowa hospitals and clinics, 200 hawkins dr, iowa city, ia 52242, usa laboratory of pathology, center for cancer research, national cancer institute, national institutes of health, 10 center dr., room 2s235, bethesda, md, 20892, usa corresponding author: osorio lopes abath neto · department of pathology · university of iowa hospitals and clinics · 200 hawkins dr · iowa city · ia 52242 · usa osorio-lopesabathneto@uiowa.edu submitted: 19 august 2024 accepted: 23 september 2024 copyedited by: vanessa s. goodwill published: 07 october 2024 https://doi.org/10.17879/freeneuropathology-2024-5817 keywords: pigmented, ependymoma, yap1, siderosis, dna methylation ependymomas containing pigmented tumor cells are rare, with only 17 reported cases in the literature1,2,3,4, most of which arrising from the fourth ventricle or adjacent locations in the infratentorial compartment. the pigment frequently consists of neuromelanin5, lipofuscin, or a combination of both1. the cases reported so far have not been characterized with molecular testing, except for the index case of himstead et al.1, which was shown to represent a posterior fossa type b ependymoma by methylation profiling, as expected from the demographics (46-year-old female) and tumor location (floor of the fourth ventricle). here we describe a case of a pigmented ependymoma with multiple unusual features: adult age, location in the supratentorial compartment, presence of a yap1 fusion, and association with superficial siderosis. this case expands the spectrum of demographic and morphologic features associated with yap1 fusion-positive ependymomas and represents to our knowledge the first supratentorial pigmented ependymoma with complete molecular characterization. a 66-year-old female with a history of smoking and localized squamous cell carcinoma of the right nare diagnosed 4 years prior presented to the emergency room with one week of confusion and falls, which had worsened in the last day. brain imaging revealed a heterogeneously enhancing right anterior temporal lobe mass with solid and cystic components. there was evidence of hemorrhage within the tumor and in the subarachnoid space, as well as supraand infratentorial superficial siderosis (figure 1). the patient underwent gross total tumor resection and recovered well after the surgery. figure 1. axial brain mri (a–h) and ct (i) images. large right temporal solid and cystic lesion with blood level (red arrows) and heterogeneous contrast enhancement in the medial margin (blue arrows). there was no restricted diffusion (c and d). t2* magnetic susceptibility sequence (g and h) showed extensive supraand infratentorial superficial siderosis (white asterisks). multiple punctate calcifications along the solid tumoral medial margin are seen on ct imaging (i, black arrows). the neoplasm was composed of relatively monomorphic cells with round to oval nuclei with a dispersed chromatin distribution and variable amounts of fibrillary cytoplasm. scattered areas showed perivascular pseudorosettes (figure 2). there was a sharp interface between the tumor and surrounding reactive brain parenchyma, which exhibited astrogliosis and rosenthal fibers. multifocal areas of hemorrhage and hemosiderin-laden macrophages were identified in subependymal and superficial cortical areas. many tumoral cells contained a coarse dark cytoplasmic pigment, which was not highlighted by fontana masson or iron stains, showed autofluorescence and pas positivity after diastase treatment, and was electron-dense and associated with vacuoles on ultrastructural studies, most consistent with lipofuscin. there were no areas of necrosis and mitotic figures were not apparent. neoplastic cells were positive for gfap, negative for synaptophysin, and showed a prominent paranuclear dot-like staining pattern for ema, but only rare cells showed olig2 and sox10 expression. figure 2. morphologic and molecular features of the pigmented ependymoma. the neoplasm is composed of monomorphic cells with oval nuclei arranged in an ependymoma-like architecture with vascular pseudorosettes (a; h⁍&⁍e, 100x). neoplastic cells are positive for gfap (b; 400x), negative for olig2 (c; 200x), and show a prominent paranuclear dot-like staining pattern for ema (d; 200x). synaptophysin (e; 100x) is negative in the tumor but positive in the adjacent neuropil at the right. in multiple foci of the tumor, neoplastic cells contain cytoplasmic accumulation of a dark pigment (f; h⁍&⁍e, 400x), which is negative for melanin (g; fontana mason, 400x), positive for pas with diastase (h; 400x) and shows autofluorescence on fluorescence microscopy (i; 100x). electron microscopy (j; 25000x) shows electron-dense structures associated with vacuoles within the cytoplasm of the tumor cells. superficial areas of the cortex uninvolved by the tumor contain dispersed deposits of hemosiderin (k; h⁍&⁍e, 200x), which are positive with an iron stain (l; iron, 200x). molecular testing revealed copy number loss of chromosome 22q (m; copy number variation profile from methylation analysis) and detection of a fusion involving yap1 and mamld1 (n). dna methylation profiling resulted in concordant matches with high confidence scores to the yap1 fusion-altered supratentorial ependymoma class in multiple versions of dna methylation classifiers (dfkz version 12b6 score 1.0, bethesda version 2 score 0.96). an rna exome fusion panel detected a yap1::mamld1 fusion, consistent with the methylation-based classification. ependymomas with yap1 fusion in adults are exceedingly rare, with only a few reported cases outside the pediatric population. in a series of 13 confirmed yap1 fusion-positive ependymomas at the national cancer institute (nci), only two were identified in adults: a 45-year-old female and an 86-year-old female. both cases occurred in the cerebral hemispheres (unknown specific location for the first case and right occipital lobe on the second), but none had pigmented cells or evidence of prior hemorrhage. this seems to suggest that the presence of pigmented neoplastic cells in ependymomas is unrelated to the underlying molecular abnormalities, with now two confirmed cases having differing molecular classification: supratentorial yap1 fusion-positive and posterior fossa b ependymoma1. interestingly, all three of the yap1 fusion-positive tumors in the adult demographic (this case and the two cases at nci) were females, in keeping with the predominance of this tumor in females in pediatric series6. this case also showed imaging evidence of superficial siderosis involving the supraand infratentorial compartments, confirmed in the surgical specimen with the presence of hemosiderin deposition in neocortical sections adjacent to and within a small subependymal area overlying the tumor. superficial siderosis has been rarely reported in spinal ependymoma cases7,8. considering the combination of siderosis indicating chronic bleeding and the presence of prominent rosenthal fibers and calcifications, all morphologic markers of a longstanding reactive process, the tumor we report most likely had a slow growth. furthermore, the presence of lipofuscin argues in favor of postmitotic tumor cells that have aged long enough to allow for the accumulation of non-degradable proteins within the cytoplasm. lipofuscin aggregates in neurons are a hallmark of senescence9, and yap1 fusion-positive ependymomas are likely to arise from neural progenitor cells10. the understanding of the behavior of yap1 fusion-positive ependymomas is limited given its rarity but in pediatric series they appear to show a favorable prognosis compared to other ependymal neoplasms11. the patient of this case underwent successful gross total resection with uneventful recovery, but long-term follow up data and accumulation of more cases are needed to reliably establish expected outcomes for these tumors. conflicts of interest statement the authors have no conflicts of interest to report. funding statement the authors acknowledge that they received no funding in support for this work. references 1. himstead as, perez-rosendahl m, fote gm, zhang a, kim mg, floriolli d, et al. pigmented ependymoma, a tumor with predilection for the middle-aged adult: case report with methylation classification and review of 16 literature cases. free neuropathol 2022;8;3:3-16. https://www.doi.org/10.17879/freeneuropathology-2022-4076 2. malhotra a, rao s, santhoshkumar r, muralidharan n, mitra s, shetty s. pigmented ependymoma of the fourth ventricle-a curious entity: report of a rare case with review of literature. int j surg pathol 2021;29(1):80-84. https://www.doi.org/10.1177/1066896920926700 3. chan ac, ho lc, yip ww, cheung fc. pigmented ependymoma with lipofuscin and neuromelanin production. arch pathol lab med 2003;127(7):872-5. https://www.doi.org/10.5858/2003-127-872-pewlan 4. rosenblum mk, erlandson ra, aleksic sn, budzilovich gn. melanotic ependymoma and subependymoma. am j surg pathol 1990;14(8):729-36. https://www.doi.org/10.1097/00000478-199008000-00005 5. mccloskey jj, parker jc jr, brooks wh, blacker hm. melanin as a component of cerebral gliomas: the melanotic cerebral ependymoma. cancer 1976;37(5):2373-9. https://www.doi.org/10.1002/1097-0142(197605)37:53.0.co;2-2 6. pajtler kw, witt h, sill m, jones dt, hovestadt v, kratochwil f, et al. molecular classification of ependymal tumors across all cns compartments, histopathological grades, and age groups. cancer cell 2015;11;27(5):728-43. https://www.doi.org/10.1016/j.ccell.2015.04.002 7. pikis s, cohen je, vargas aa, gomori jm, harnof s, itshayek e. superficial siderosis of the central nervous system secondary to spinal ependymoma. j clin neurosci 2014;21(11):2017-9. https://www.doi.org/10.1016/j.jocn.2014.05.020 8. zhuang j, duan q, liang c, chang y, yin d. superficial siderosis of the central nervous system caused by myxopapillary ependymoma of cauda equine: a case report. neurol sci 2024;45(3):1303-1306. https://www.doi.org/10.1007/s10072-023-07099-8 9. moreno-garcía a, kun a, calero o, medina m, calero m. an overview of the role of lipofuscin in age-related neurodegeneration. front neurosci 2018;5:12:464. https://www.doi.org/10.3389/fnins.2018.00464 10. eder n, roncaroli f, domart mc, horswell s, andreiuolo f, flynn hr, et al. yap1/taz drives ependymoma-like tumour formation in mice. nat commm 2020;11(1):2380. https://www.doi.org/10.1038/s41467-020-16167-y 11. andreiuolo f, varlet p, tauziède-espariat a, jünger st, dörner e, dreschmann v, et al. childhood supratentorial ependymomas with yap1-mamld1 fusion: an entity with characteristic clinical, radiological, cytogenetic and histopathological features. brain pathol 2019;29(2):205-216. https://www.doi.org/10.1111/bpa.12659 copyright: © 2024 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. cell-in-cell phenomena of intracellular neutrophils in a recurrent pleomorphic xanthoastrocytoma feel free to add comments by clicking these icons on the sidebar free neuropathology 5:17 (2024) letter cell-in-cell phenomena of intracellular neutrophils in a recurrent pleomorphic xanthoastrocytoma gianna m. fote1,2, kamran urgun2, jordan davies1, alexander s. himstead1, kevin gramajo-aponte3, alexander lopez1, frank p.k. hsu1, william h. yong2 uc irvine school of medicine, department of neurosurgery, university of california, 200 south manchester ave, orange, ca 92868, usa uc irvine school of medicine, department of pathology and laboratory medicine, university of california, 101 the city drive south, orange, ca 92868, usa uc irvine school of medicine, university of california, 101 the city drive south, orange, ca 92868, usa corresponding authors: gianna m. fote · uc irvine school of medicine · department of neurosurgery · 200 south manchester ave · orange, ca 92868 · usa gfote@hs.uci.edu: william yong · uc irvine school of medicine · department of pathology and laboratory medicine · 101 the city drive south · orange, ca 92868 · usa yongwh@hs.uci.edu submitted: 29 january 2024 accepted: 11 june 2024 copyedited by: michel mittelbronn published: 08 august 2024 https://doi.org/10.17879/freeneuropathology-2024-5341 keywords: glioma, brain tumor, pleomorphic xanthoastrocytoma, phagocytosis, case report, emperipolesis, cell-in-cell abstract we describe a case of a young patient with a recurrent pleomorphic xanthoastrocytoma (pxa) showing unusual cell-in-cell (cic) phenomena. we observed mostly viable but also necrotic neutrophils engulfed within tumor cells. the recurrent tumor was immunopositive for brafv600e mutant protein and showed cdkn2 homozygous deletions typical of pxa. both genetic alterations were also reported in the original primary tumor. unlike the original tumor that was gfap and olig-2 immunopositive, the recurrent neoplasm was largely negative for gfap and olig-2 suggesting dedifferentiation. the large malignant cells that contained the neutrophils were negative for histiocytic and lymphohematopoietic markers. whereas cdkn2 homozygous deletion is common in pxa, its presence is rare in histiocytic neoplasms. both reactive astrocytes and glial neoplasms very rarely may engulf neutrophils in a process resembling emperipolesis or cellular cannibalism. future work may clarify which type of cic pathway is involved. introduction pleomorphic xanthoastrocytomas (pxas) are astrocytic tumors presenting in children or young adults, and generally have a good prognosis, although cns who grade 3 pxas with elevated mitotic activity and necrosis have a 5 year survival rate of 65 % 1. histologically, they are characterized by pleomorphic cells including spindle cells, mononucleated and multinucleated giant cells, eosinophilic granules, reticulin deposition, and large amounts of cytoplasmic lipids 2, and are typically positive for gfap and s100 3. common molecular alterations include homozygous cdkn2a/b deletion in 60–90 % and braf v600e mutation in 60–80 % of cases 4,5. standard treatment includes maximal resection for all grades with postoperative radiation for grade 3 pxas, and braf v600e mutant tumors can be treated with a combination of dabrafenib and trametinib to inhibit braf and mapk pathways 6. cell-in-cell phenomena (cic), or the internalization of one cell within another (figure 1), has only been reported in one previous case of malignant glioma 7. here, we present a case of an aggressive, recurrent cns who grade 3 pxa with cdkn2 deletion and brafv600e mutation with a rare histologic finding of cic in tumor cells with engulfed neutrophils, many of which appeared viable. figure 1. general characteristics of cell-in-cell processes in endocytic processes host cells internalize a target cell. invasive processes are driven by activity of the internalized cells. “cell types” refers to the host cells. homotypic refers to the engulfing cell and the internalized cell being of the same cell type. heterotypic means that the engulfing and internalized cell types are different. emperipolesis historically involves survival of the internalized cell, but some authors extend its usage to cases wherein there may be death of the internalized cells. cell-in-cell processes have been previously reviewed 13,17. previous literature is available on cannibalism 18–20, phagoptosis 21, phagocytosis 12 and emperipolesis 22. clinical history a 10-year-old male presented to outside hospital with a right parietal tumor involving the petrous and mastoid bones with extension into the middle and posterior fossa. the patient had a near total resection followed by radiotherapy and temozolomide. the patient underwent a second resection five months later and was started on daily dabrafenib and trametinib. available records state that pathology on first resection was right parietal anaplastic pleomorphic xanthoastrocytoma who grade iii, braf mutant. ten months later, the patient presented with bleeding and swelling of the right ear, then developed right face weakness, difficulty swallowing, dyspnea, and aphonia. fifteen months after the initial diagnosis, he presented with culture negative febrile leukocytosis (white blood count (wbc) 39 preoperatively). he was started on vancomycin and ceftriaxone empirically. ct and mri revealed increased size of pxa, extending into temporal bone, right cerebellum, right temporal lobe, and the cerebellopontine angle, involving cranial nerves vii and viii (figure 2a). angiogram revealed that the sigmoid sinus and the internal carotid artery were occluded, although collateral circulation was intact. the patient was taken to the operating room for mass resection. the patient’s prior craniotomy was extended in a translabyrinthine approach, and epidural and intradural tumor was debulked. a mastoidectomy and petrosectomy were performed, and the facial nerve was transected. the semicircular canals, cochlea, and vestibular aqueduct were opened and obliterated. tumor within the sigmoid sinus was removed en bloc. a frozen specimen collected during the case was consistent with patient’s known pleomorphic xanthoastrocytoma. tissue was sent for cultures (aerobic, anaerobic, fungal) but these ultimately did not grow out any organisms. following the procedure, the patient was taken to the postoperative recovery unit in stable condition. post-operatively the patient failed a swallow test and required percutaneous endoscopic gastrostomy placement. repeat blood cultures were negative, and wbc diminished to 30. the patient was transferred nine days after admission to a children’s hospital. at discharge, he had unilateral facial swelling, decreased facial sensation, face asymmetry, and absent hearing. an mri subsequently demonstrated residual tumor regrowth and he underwent further resection the following month. his post-operative course was complicated by pseudo-meningocele for which a subdural drain was placed. the drain subsequently clogged and began to leak, and neuroimaging demonstrated further tumor progression. the patient deteriorated clinically, became unresponsive on exam and required supplemental oxygen. the patient’s family elected for comfort-focused care and the patient passed away 16 months after his initial diagnosis. pathology the final diagnosis was recurrent pleomorphic xanthoastrocytoma (pxa) cns who grade 3 with brafv600e mutation and homozygous cdkn2 deletion. the recurrent tumor showed large, markedly pleomorphic epithelioid cells with frequent mitotic activity, necrosis and intracellular neutrophils (figure 2b). associated tumor necrosis was bland (figure 2bii) or contained abundant neutrophils (figure 2biii). intracellular, often viable though sometimes degenerating or necrotic, neutrophils in vacuolar spaces were present within the tumor cytoplasm (figure 2biv–vi). the tumor cells were often intact with well-preserved nuclei but some cells showed degenerated nuclei (figure 2bvi). in many areas, extracellular neutrophils were sparse or inconspicuous despite the presence of abundant intracellular neutrophils (figure 2biv–v). unlike the original tumor that expressed both gfap and olig2, the recurrent tumor was largely immunonegative for gfap and olig2. the large tumor cells were brafv600e immunopositive (figure 2bvii) and fish analysis demonstrated a cdkn2a homozygous deletion identical to what was reported in the original tumor. testing for microorganisms including gram, gms, pas, and afb stains as well as spirochete and tb/mycobacterium immunostains was negative. histiocytic and lymphohematopoietic markers were negative or largely negative in the large tumor cells, including s100, cd163, cd68, cd1a, langerin, factor xiiia, cd123, and cd35. there was also a population of small histiocytes that were immunopositive for s100, cd68 and cd163 but immunonegative for brafv600e, cd1a and langerin compatible with a reactive non-neoplastic infiltrate. the contrasting brafv600e, s100 and cd68 immunoprofiles of the large tumor cells (brafv600e+, s100-, cd68-) and the small histiocytes (brafv600e-, s100+, cd68+) are seen in figures 2bviii–ix. the ki-67 proliferation index of the tumor was high (figure 2bx). there was negative immunostaining of the large tumor cells for map2, sox10, ema, keratin ae1/3, melan a, sma, cd21, cd15, cd45, cd23, cd33, desmin, mpo, lysozyme, hmb-45, cd20, cd3, cd43, and cd42b. ini-1 nuclear staining was retained. neutrophils within tumor cells were immunopositive for mpo (figure 2bxi). figure 2. (a) mri of a heterogeneously enhancing mass with perilesional edema and progression over time including t1 post-contrast and t2 flair. (i) 12 months post-diagnosis. (ii) 14 months post-diagnosis. (iii) 15 months post-diagnosis, pre-operatively. (iv) 15 months post-diagnosis, post-operatively. (b) histology. (i) epithelioid tumor cells sometimes have a “ball of neutrophils” appearance 200x. (ii) bland necrosis 200x. (iii) necrosis with abundant neutrophils 200x. (iv) mostly viable neutrophils within intracytoplasmic vacuolar spaces of cell (arrow) 600x. (v) varying degrees of engulfed neutrophil degeneration 600x. (vi) degenerate basophilic nucleus in tumor cell (arrow) 400x. (vii) brafv600e immunopositive tumor cells 200x. (viii) s100 immunonegative tumor cells; s100 immunopositive small histiocytes 200x. (ix) cd68 immunonegative tumor cells; cd68 immunopositive small histiocytes 200x. (x) ki-67 immunopositivity in many tumor nuclei 200x. (xi) myeloperoxidase immunostaining highlights the predominantly intracytoplasmic localization of neutrophils 400x. (c) timeline of the patient’s clinical course. histology in (b) is from the patient’s resection at 15 months post-diagnosis. discussion we present a case of a pxa with several unusual features, including invasion through bone and soft tissue, loss of gfap and olig2 immunoreactivity, severe acute inflammatory infiltrate, and unusual cic with internalized neutrophils. alternate or concurrent diagnoses of infection or histiocytosis were considered, but cultures and special stains for microorganisms and histiocytic and lymphohematopoietic markers were negative. rosai dorfman disease (rdd) can occur in the cns and is characterized by abundant histiocytes with emperipolesis. however, host tumor cells were immunonegative for histiocyte marker s100, had loss of cdkn2a inconsistent with rdd, and internalized neutrophils rather than lymphocytes, a rare finding for rdd 8. ultimately the diagnosis of recurrent pxa with brafv600e mutation and cdkn2a deletion was favored due to the patient’s history of tumor with an identical molecular phenotype. the loss of gfap and olig2 staining, tissue necrosis with invasion of surrounding tissue, and history of multiple recurrences suggests that this patient’s pxa dedifferentiated. within the central nervous system, cic has been observed only in highly anaplastic tumors: large cell medulloblastoma 9–11, and one prior case of malignant glioma. in this 1986 glioma case, pleomorphic, sparsely gfap positive “monster cells” with cytoplasmic lipids, reticulin fibers, and multiple nuclei had internalized viable polymorphonuclear neutrophils as well as mononuclear and small tumor cells 7, similar to the present case. these observations pose the question for future study of which molecular mechanisms facilitate cic in these aggressive gliomas. healthy astrocytes conduct phagocytosis physiologically 12, but would typically result in destruction of the internalized material 13. neutrophils participate in a unique type of emperipolesis in which they invasively enter megakaryocytes and then donate their membranes for the production of platelets; it is unknown whether rapidly dividing cancer cells could use neutrophils for membrane production in a similar manner 14,15. cdkn2a mutation is associated with homotypic cic (entosis) 16, but it is unknown whether cdkn2a mutation may contribute to heterotypic cic as well. as the field evolves, cases of cic in new contexts may supplement rigorous molecular analyses to characterize the pathways involved and better understand the biology of aggressive gliomas. conflicts of interest statement the authors have no conflicts of interest to report. funding statement the authors acknowledge that they received no funding in support for this work. references 1. lee c, byeon y, kim gj, et al. exploring prognostic factors and treatment strategies for long-term survival in pleomorphic xanthoastrocytoma patients. sci rep. 2024;14(1):1-10. doi: https://doi.org/10.1038/s41598-024-55202-6 2. kepes j, 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https://doi.org/10.1002/ajh.23892 12. konishi h, koizumi s, kiyama h. phagocytic astrocytes: emerging from the shadows of microglia. glia. 2022;70(6):1009-1026. doi: https://doi.org/10.1002/glia.24145 13. brown gc. cell death by phagocytosis. nat rev immunol 2023 242. 2023;24(2):91-102. doi: https://doi.org/10.1038/s41577-023-00921-6 14. cunin p, bouslama r, machlus kr, et al. megakaryocyte emperipolesis mediates membrane transfer from intracytoplasmic neutrophils to platelets. elife. 2019;8. doi: https://doi.org/10.7554/elife.44031 15. yener y, dikmenli m. the effects of acrylamide on the frequency of megakaryocytic emperipolesis and the mitotic activity of rat bone marrow cells. j sci food agric. 2011;91(10):1810-1813. doi: https://doi.org/10.1002/jsfa.4388 16. mackay hl, muller paj. biological relevance of cell-in-cell in cancers. biochem soc trans. 2019;47(2):725-732. doi: https://doi.org/10.1042/bst20180618 17. borensztejn k, tyrna p, gaweł am, et al. classification of 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within glial cells (“emperipolesis”) in a case of a granular cell tumor. acta neuropathol. 1978;44(2):163-165. doi: https://doi.org/10.1007/bf00691486 copyright: © 2024 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. fast-track neuropathological screening for neurodegenerative diseases feel free to add comments by clicking these icons on the sidebar free neuropathology 5:16 (2024) original paper fast-track neuropathological screening for neurodegenerative diseases benjamin englert1,2,*, sigrun roeber1, thomas arzberger1,3, viktoria ruf1, otto windl1, jochen herms1,2,4 center for neuropathology and prion research, faculty of medicine, lmu munich, munich, germany german center for neurodegenerative diseases (dzne), munich, germany department of psychiatry and psychotherapy, lmu university hospital, lmu munich, munich, germany munich cluster of systems neurology (synergy), munich, germany * present address: department of pathology, university of helsinki and hus diagnostic center, helsinki university hospital, helsinki, finland corresponding author: jochen herms · center for neuropathology and prion research · faculty of medicine · lmu munich · feodor-lynen-strasse 23 · 81377 munich · germany jochen.herms@med.uni-muenchen.de submitted: 07 june 2024 accepted: 21 july 2024 copyedited by: georg haase published: 06 august 2024 https://doi.org/10.17879/freeneuropathology-2024-5643 keywords: neuropathology, dementia, movement disorders, prion disease, alzheimer disease, lewy body disease abstract background: the postmortem diagnostic of individuals having suffered presumptive neurodegenerative disease comprises exclusion of a prion disease, extensive brain sampling and histopathological evaluation, which are resource-intensive and time consuming. to exclude prion disease and to achieve prompt accurate preliminary diagnosis, we developed a fast-track procedure for the histopathological assessment of brains from patients with suspected neurodegenerative disease. methods: based on the screening of two brain regions (frontal cortex and cerebellum) with h&e and six immunohistochemical stainings in 133 brain donors, a main histopathological diagnosis was established and compared to the final diagnosis made after a full histopathological work-up according to our brain bank standard procedure. results: in over 96 % of cases there was a concordance between the fast-track and the final main neuropathological diagnosis. a prion disease was identified in four cases without prior clinical suspicion of a prion infection. conclusion: the fast-track screening approach relying on two defined, easily accessible brain regions is sufficient to obtain a reliable tentative main diagnosis in individuals with neurodegenerative disease and thus allows for a prompt feedback to the physicians. however, a more thorough histological work-up taking into account the clinical history and the working diagnosis from fast-track screening is necessary for accurate staging and for assessment of co-pathologies. introduction dementia or cognitive impairment are common clinical diagnoses; however, they often lack neuropathological verification by autopsy. compared to other underlying causes of death, dementia lowers the chances of an autopsy to be performed [1]. the low autopsy rates, in turn, lead to a loss of expertise for diagnosing neurodegenerative diseases with high precision and according to well-established standards. a study by selvackadunko et al., comparing the antemortem clinical diagnosis and postmortem neuropathological diagnosis revealed that in one third of cases the in-life and the neuropathological diagnosis differed [2]. a swedish study found a full accordance of clinical and neuropathological dementia diagnosis in 49 % of cases, in a further 14 % the clinical diagnosis corresponded with some but not all the diagnoses after postmortem evaluation [3]. in alzheimer’s disease (ad), the commonest neurodegenerative disease (ndd), a study showed that 119 out of 533 clinically diagnosed ad cases did not fulfil the neuropathological criteria for definite ad at autopsy, with dementia with lewy bodies (dlb), vascular dementia, frontotemporal lobar degeneration (ftld) and hippocampal sclerosis being the most frequently encountered ad mimics [4]. in progressive supranuclear palsy (psp), only 78 % of clinically diagnosed cases were histologically confirmed as indeed being a psp, clinically incorrect classified cases histologically were classified as parkinson’s disease (pd), ad, multiple system atrophy (msa), pick’s disease (pid), motor neuron disease (mnd) and corticobasal degeneration (cbd) [5]. furthermore, prion disease can be mistaken for various other clinical entities, especially in the elderly due to a less consistent clinical phenotype and a high incidence of dementia [6]. even though new developments in tau positron emission tomography imaging and cerebrospinal fluid analysis have very much improved the clinical diagnosis of ndds in the last three years, these expensive methods are often not available to clinicians [7]. the conventional neuropathological workup for brains of patients with suspected neurodegenerative diseases is extensive, since it typically includes a comprehensive and detailed characterization of all pathologies and co-pathologies present, particularly in a brain bank setting. for this, the analysis of at least seven or eight tissue blocks from different brain regions is required. a report by kovacs and budka from the vienna brain bank describes five immunostainings on seven brain regions as minimal number to assess protein deposition in ndd [8]. the national institute of ageing guidelines recommend the sampling of at least eight brain regions for ad, six for lewy body disease (lbd) [9]. since up to five immunohistological stainings per region are necessary, these approaches lead to about 50 stained sections to be analysed under the microscope, which is costly and time-consuming, taking a considerable time until a final report can be provided. in contrast, the priority for clinicians and relatives is to receive information about the definite (main) diagnosis in a timely manner. to reduce costs and effort, condensed protocols for common neurodegenerative alterations have been deployed, essentially based on the protocol by flanagan et al. [10]. this protocol requires sampling of 20 brain regions (sometimes bilateral sampling of the same region is needed), with four regions embedded in the same tissue cassette, giving a total of five paraffin blocks [10]. following a nontiered approach, all blocks are stained with he and luxol fast blue, and each block with an additional specific stain (bielschowsky silver impregnation or immunohistochemical stains for α-synuclein, β-amyloid or phospho-tau) [10]. clement et al. further simplified the protocol by unilateral sampling of 12 brain regions embedded in six tissue cassettes, with better preservation of neuroanatomical relationships [1]. amended protocols have been published for the diagnosis of frontotemporal lobar degeneration (ftld) and limbic-predominant age-related tdp-43 encephalopathy neuropathological changes (late-nc) [12,13]. condensed protocols have been reliably applied in clinical and medicolegal autopsy settings [14,15,16]. previous studies have already addressed the question of the utility of small-sized tissue samples of few selected brain regions for ndd diagnostics. venetti et al. performed simulated brain biopsies on 73 autopsy cases of various ndd and compared the outcome between assessments of the mid-frontal cortex alone or four brain regions (frontal, temporal, parietal cortex and basal ganglia) to the final autoptic diagnoses [17]. considering solely the frontal sample, the authors indicate an average sensitivity of 64 % and a specificity of 43 %, and 92 % sensitivity and 71 % specificity for all four brain regions, with the highest sensitivities found for ftld with tdp-43 inclusions (ftld-tdp, 88 %) and ad (80 %) and the lowest for psp (0 %) [17]. a study by king et al. evaluated 62 ndd cases using 1 cm3 of prefrontal and middle temporal cortex and compared the results with gold standard autopsy diagnoses [18]. the sensitivity of assessing the frontal lobe alone was 81 % and the specificity 79 % [18]. these figures increased to a sensitivity of 85 % and a specificity of 83 % when taking both regions into account [18]. the diagnostic accuracy of evaluating frontal lobe specimens was high e.g. in ad, cbd and msa (100 % of correct diagnoses) and low in psp (0 %), ascribed by the authors to sparse expression of tau-positive psp lesions in the neocortex [18]. we use the below-described protocol for screening not only to obtain a rather quick working diagnosis that allows to diagnose more than 96 % of ndds but also to protect laboratory personnel from extensive contact with potentially prion-infected tissue. material and methods in our study we included 133 cases who underwent neuropathological assessment as brain donors to the neurobiobank munich (nbm), located at the centre for neuropathology and prion research, ludwig-maximilians-university (lmu), munich, germany. the collection of tissue for the nbm was approved by the ethics committee of the medical faculty of the lmu (no. 345‑13). after the brain had been removed, one hemisphere was cryopreserved while the other hemisphere was fixed for at least two weeks in 4 % buffered formalin. after sufficient fixation, small tissue specimens (1 x 1 x 0.5 cm) from the superior frontal gyrus (approximately ba 8) and the cerebellum (lateral cerebellar cortex) were sampled, treated with 100 % formic acid for one hour [19], rinsed with water, fixed in formalin for half a day and embedded into the same paraffin block. tissue sections were cut and stained with h&e and subjected to six immunohistochemical stainings for prion protein (clone l42, targeting an epitope on the first alpha helix [20], 1 : 50 - 1 : 100, pre-treatment with proteinase k, own), α-synuclein (clone 42, 1 : 2000, bd transduction laboratories); β-amyloid (clone 4g8, 1 : 2000, covance); phospho-tau (clone at8, 1 : 200, thermofisher scientific); phospho-tdp-43 (clone 1d3, 1 : 50, own); p62 (clone 3/p62 lck ligand, 1 : 100, bd transduction laboratories)). stainings were performed using an automatic staining system (roche, ventana benchmark ultra). if required and in accordance with the respective clinical diagnosis, various other immunostainings (e.g. fus, 3-repeat tau) or silver impregnation techniques (bielschowsky, gallyas) were occasionally performed. a schematic overview of the work-up is presented in figure 1. firstly, we assessed the h&e stain for the presence of general neurodegenerative features (atrophy, neuronal loss, spongiosis, gliosis) and various other pathological alterations such as tissue ischemia/hypoxia, haemorrhage, inflammation or neoplasms as well as vascular pathologies. furthermore, disease specific alterations e.g. senile plaques in ad or ballooned neurons in cbd were examined on an h&e stain [21,22]. figure 1: process flow of fast-track screening histology for further neuropathological diagnosis, we evaluated the presence of the following morphological features by immunohistochemistry. prion protein immunoreactive structures in a synaptic, vacuolar, laminar cortical or coarse pattern, plaque-like focal deposits or amyloid-kuru plaques were searched to diagnose prion disease [23]. 4g8-positive amyloid β (aβ) plaques, tau-immunoreactive neurofibrillary tangles (nft), neuropil threads (nt) and neuritic plaques (np) were used for the fast-track diagnosis of higher stages of ad associated pathology [9,24]. the diagnosis of psp was made when nt, nft, neuronal cytoplasmic inclusions (nci), tufted astrocytes (ta) and oligodendroglial coiled bodies (cb) were visible [25,26,27]. nci, nt, cb and most notably astrocytic plaques (ap) were highly suggestive for cbd [27,28]. pick bodies (pib) were the characteristic hallmark of pid [29]. intraneuronal α-synuclein positive lewy bodies (lb), extracellular lewy body-like inclusions, lewy neurites (ln) and grain-like cytoplasmic inclusions are present in lbd [30]. given the overlapping and, if at all, not readily distinguishable neuropathological features of pd, dlb and parkinson’s disease dementia [31,32], we summarized these three entities under the umbrella term lbd. msa is characterised by α-synuclein immunoreactive glial cytoplasmic inclusions (gci) [3]. phospho-tdp-43 positive neuronal and glial intracytoplasmic inclusions as well as dystrophic neurites can be found in ftld-tdp [34]. for ftld cases without tauor tdp-43-positive inclusions, immunostaining for the ubiquitin binding protein p62 detecting cytoplasmic glial/neuronal inclusions, neuronal nuclear inclusions, threads provides a valuable tool prompting further immunohistochemical characterisation [34,35]. representative micrographs of characteristic histomorphological findings and protein deposits are shown in figure 2. a decision diagram showing a representative guide to make a screening diagnosis according to characteristic histological findings is shown in figure 3. figure 2: representative micrographs of characteristic histological findings. a1: eosinophilic plaque in ad. a2: strongly stained neuropil (at8) with numerous intraneuronal inclusions ad. a3: at8-positive neuronal inclusion (star), neuropil thread (arrowhead) and a neuritic plaque (arrow) in ad. a4: cored (arrows) and diffuse aβ-plaques in ad. a5: parenchymal and leptomeningeal amyloid deposits in vessel walls (cerebral amyloid angiopathy, arrows) in an ad patient. a6: signs of aging-related tau astrogliopathy with perivascular accentuation (inset with higher magnification) as common co-pathology in ad. b1, b2, b3: psp with less pronounced positivity of the neuropil, frequent ta (arrows), nft (arrowhead) and oligodendroglial cb (stars). c1: ballooned neuron in cbd. c2, c3: overview of a cbd case showing moderate nt, neuronal inclusions and ap (c3). hallmarks of pid include a ballooned neuron (d1), at8-positive pick bodies (d2, arrowheads) as well as a ramified astrocyte (d3). e1, e2: lewy bodies in ldb (arrowhead, stars; rarely visible in he-stain). f: numerous cerebellar gci in msa. g1, g2, g3: various ptdp-43-positive dystrophic neurites (g1), compact neuronal (g2) and neuronal cat-eye shaped (g3) inclusions in ftld-tdp. g4: tdp-43-negative, p62-positive globular inclusion in ftld-fus (arrowhead). g5: typical star-shaped p62-positive inclusion in ftld-tdp with c9orf72 mutation. h: deposits of prion protein in creutzfeldt-jakob disease. images show stainings with he and with antibodies against β-amyloid (aβ), phospho-tau (at8), α-synuclein (αsyn), phospho-tdp43 (ptdp), p62 and prion protein (prp). scale bars: c1, d1, d3, g5: 5 μm; a1, e1, g1, g4: 10 μm; a3, a4 a6 (inset), b2, b3, c3, d2, e2, g2, g3: 20 μm; a2, a5, b1, c2, f, h: 50 μm; a6: 200 μm. figure 3: decision diagram guiding the investigator to establish a screening diagnosis based on characteristic histological findings. prp: prion protein, at8: phospho-tau, nft: neurofibrillary tangles, nci: neuronal cytoplasmic inclusions, ta: tufted astrocytes, cb: coiled bodies, ap: astrocytic plaques, pib: pick bodies, np: neuritic plaques, nt: neuropil threads, aβ: β-amyloid, αsyn: α-synuclein, lb: lewy bodies, ln: lewy neurites, gci: glial cytoplasmic inclusions, psp: progressive supranuclear palsy, cbd: corticobasal degeneration, pid: pick’s disease, ad: alzheimer’s disease, lbd: lewy body disease, msa: multiple system atrophy, ftld: frontotemporal lobar degeneration. a limited neuropathological disease staging was also performed. a thal-score for aβ deposits was assigned to a stage of either (at least) 1 (frontal beta amyloid plaques) or 5 (additional aβ plaques detected in the cerebellum) [36]. the presence of frontal tau-immunoreactive neurofibrillary pathology allowed for assigning a braak and braak stage of at least iv [37]. recognition and staging of additional caa according to thal et al. [38] is limited to stages 1 (if frontal pathology is present) or (at least) 2 (with cerebellar amyloid angiopathy). frontal α-synuclein inclusions lead to designation of a braak stage of 6 [39]. a full histological work-up according to a standardized, well-established nbm protocol was performed on formalin-fixed hemispheres after complete fixation, taking both the working diagnosis obtained by screening histology and the deceased's medical history into account for a more targeted evaluation. the final neuropathological diagnoses were made in accordance with established guidelines for the histological diagnosis of ndd [9,24,25,30,33,40,41,42]. point estimates and 95 % confidence intervals (ci) for sensitivity and specificity comparing fast-track to final diagnoses were calculated for each disease group using graphpad prism version 9.4.1, graphpad software, san diego, california, usa. statistical significance was set at the 0.05 level (fisher’s exact test). kappa coefficients for inter-rater variability between fast-track and final diagnoses were calculated with a graphpad online tool (https://www.graphpad.com/quickcalcs/kappa1/). according to the kappa values the agreement was considered as poor (kappa = 0), slight (0 - 0.2), fair (0.21 - 0.4), moderate (0.41 - 0.6) substantial (0.61 - 0.8) or almost perfect (0.81 - 1.0) [43]. results fast-track histology was analysed in 133 cases (58 females, 75 males, median age at death: 71.1 years; age range: 38.7 - 95.1 years) which were enrolled in a brain donation program for brain banking and thus underwent brain autopsy at nbm between march 2015 and december 2020. from 156 cases initially selected, we excluded 15 cases because of suspected motor neuron disease (mnd)/amyotrophic lateral sclerosis (since spinal samples were not assessed on screening histology), three cases with clinical restless legs syndrome, one case with suspected fahr’s disease and four cases with huntington’s disease already genetically confirmed during lifetime. the main histological ndd diagnoses without consideration of additional pathology besides lbd pathology in ad shown in table 1 were: psp (n = 26, 19.5 %), ad without lbd pathology (n = 24, 18.05 %), lbd (n = 22, 16.5 %), ad with lbd (n = 13, 9.77 %), cbd (n = 14, 10.5 %), msa (n = 14, 10.5 %) non-tau ftld (ftld-tdp, ftld-fus; n = 9, 6.7 %), prion disease (n = 4, 3 %), pid (n = 3, 2.3 %). in four cases (3 %), no diagnosis could be made using fast-track histology and additional histological work-up was hence required for neuropathological diagnosis. those cases were later classified as psp (two cases), ftld non-tau and lbd (one case each). in 80 cases (60.2 %), the suspected clinical diagnosis could be confirmed by fast-track histology, whereas in 49 (39.8 %) the clinical diagnosis was either ambiguous or different from the histopathological diagnosis. when comparing the initial fast-track diagnosis to the diagnosis made after subsequent conventional pathological work-up of the brain, it became evident that in 128 cases (96.2 %) the ndd entity determined by fast-track diagnosis was correct and only co-pathologies or correct disease staging needed to be determined. the fast-track diagnosis needed revision in only one case of an unusual tauopathy initially classified as psp (3 %). in four cases, the analysis of additional brain regions after fast-track histology was required to reach a conclusive histopathological diagnosis, as mentioned above. there was a high sensitivity for the diagnosis, considering only the main pathology without additional pathology, for ad, braak and braak stage 5 or 6 (1.0, ci = 0.9059 - 1.00, p < 0.0001), cbd (1.0, ci = 0.7847 - 1.00, p < 0.0001), lbd (1.0, ci = 0.8513 - 1.00, p < 0.0001), pid (1.0, ci = 0.4385 - 1.00, p < 0.0001), msa (1.0, ci = 0.7847 - 1.00, p < 0.0001) and ftld non-tau (1.0, ci = 0.7009 - 1.00, p < 0.0001). the sensitivity for the diagnosis of psp was 0.96 (ci = 0.8046 - 0.9979, p < 0.0001). the respective specificity was for ad 1.0 (ci = 0.9615 - 0, p < 0.0001), for cbd 1.0 (ci = 0.9687 - 1.0, p < 0.0001), for lbd 0.9910 (ci = 0.9507 - 0.9995, p < 0.0001), for pid 1.0 (ci = 0.9713 - 1.0, p < 0.0001), for msa 1.0 (ci = 0.9687 - 1.0, p < 0.0001), for ftld non-tau 0.9919 (ci = 0.9557 - 0.9996, p < 0.0001) and for psp 0.9815 (ci = 0.9350 - 0.9967, p < 0.0001). leading diagnosis on screening histology confirmation by full histology ad without lbd (n = 24) 91.67 %* ad with lbd (n = 13) 100 % psp (n = 26) 96.15 % cbd (n = 14) 100 % pid (n = 3) 100 % non-tau ftld (n = 9) 100 % lbd (n = 22) 100 % msa (n = 14) 100 % prion disease (n = 4) 100 % no diagnosis possible (n = 4) table 1: histopathological diagnoses obtained by fast-track screening histology and their confirmation by final histology in percentage of cases. the diagnoses are arranged according to the predominant protein deposit: ad without (green) and with (light brown) lbd, primary tauopathies (blue), tdp-43 or other proteins in non-tau ftld (purple), synucleinopathies (red), prion disease (black). * ad was confirmed in all 24 cases, however, two showed additional lbd pathology (one olfactory bulb only, one lbd braak stage 5) and were thus classified as ad with lbd cases. the kappa coefficient for inter-rater variability comparing fast-track and final histology was as follows: ad 1.0 (ci = 1.0 - 1.0), cbd 1.0 (ci = 1.0 - 1.0), pid 1.0 (ci = 1.0 - 1.0), msa 1.0 (ci = 1.0 - 1.0), ftld non-tau 0.943 (ci = 0.833 - 1.0), lbd 0.973 (ci = 0.921 - 1.0), psp 0.927 (ci = 0.846 - 1.0). among the cases referred to our brain bank with various clinical diagnoses such as atypical parkinsonian disorder, movement disorder of unknown aetiology or behavioural variant frontotemporal dementia (bv-ftd), four cases (3 %) showed positive staining for prion protein. among neuropathologically classified non-tau ftld cases (nine on fast-track, ten in total), one had the clinical diagnosis of cbd, one of lbd, one of pid and of unclassified dementia with oral facial dystonia. the remaining cases were clinically classified as frontotemporal dementia (four cases), suspected bv-ftd or psp (one case) and primary progressive aphasia (one case). the case with clinically suspected lbd showed a peculiar immunostaining with tdp-43 negative sequestome-1 (p62) positive inclusions typical for c9orf72 hexanucleotide expansion-related ftld cases [4]), which prompted us to further assessment and final classification as ftld-tdp. additional cerebral amyloid angiopathy (caa) occurred in 50 cases (37.6 %). we detected caa on screening histology already in 46 cases (92 %). we found lbd co-pathology in 15 out of 37 ad cases (40.5 %) among which 13 cases where lbd co-pathology had already been seen on fast-track histology. another frequent finding (12 / 37 cases, 32.4 % in our study) in ad is tdp-43 pathology, which was only identified after complete histopathological work-up in seven cases. final histology revealed the presence of ad-associated alterations (aβ plaques and / or ad-type tau deposits) in 15 / 23 lbd cases (65.2 %), 3/14 cbd cases (21.4 %), 4 / 10 non-tau ftld cases (40 %), 9 / 14 msa cases (64.3 %), 7 / 27 psp cases (25.9 %). we detected signs of aging-related tau astrogliopathy (artag) in seven lbd, three ad, three psp, two ftld non-tau and one cbd case. we reported artag in five cases (3.8 %) on screening histology. two ad, two cbd, eight lbd, one non-tau ftld and one msa case showed signs of argyrophilic grain disease (agd) on final histology. additional lbd pathology was present in one psp and 15 ad cases upon examination of one hemisphere. except for two cases (one with lbd braak stage 5 and one with sole olfactory bulb pathology), all cases showed a braak lbd stage 6 and could therefore be identified already on screening histology. discussion in our study we demonstrated that a reliable, preliminary main diagnosis in patients with a suspected neurodegenerative disease can be achieved in over 96 % of the cases within a few days using histological and immunohistochemical screening of two selected, easily accessible brain regions (superior frontal gyrus and cerebellum) embedded into one paraffin block. considering the rapid workflow, a prompt response from the neuropathologist to the clinician might improve the accuracy of clinical diagnosis in other patients. furthermore, performing a screening approach might also be feasible in places with limited resources and could also be performed by general or forensic pathologists, perhaps with the aid of online consultation using digitalized micrographs. the fast-track diagnosis may also have consequences for relatives and lead to genetic counselling. however, our approach cannot supersede a thorough macroand microscopic examination with sampling of various other brain regions, given the multitude of possible proteinopathic and vascular co-pathologies, potential extraordinarily rare ndd and alterations relevant for disease staging. by way of example, our approach neither allows for classification into brainstem-predominant, limbic or diffuse neocortical lb pathology according to the consensus report nor for determination of olfactory bulb only or amygdala predominant pathology [40,45,46]. due to the heterogeneous spatial distribution pattern of subpial, subependymal, perivascular, white matter and gray matter artag [47], it is possible that artag pathology will be missed or not fully assessed by screening histology. given the anatomical distribution of argyrophilic grain disease (agd), often limited to the limbic system [48], assessment of this disease occurring frequently with concomitant ndd is limited with our method. this also holds true for the diagnosis of primary age-related tauopathy (part) and late-nc which usually affects the middle frontal gyrus in later stages [49,50]. furthermore, staging tdp-43 co-pathology in ad according to josephs et al. [51] is not possible in our rapid screening approach. independent from its diagnostic benefit, our procedure enhances laboratory safety by detecting unexpected prion protein aggregates before a complete work-up of the brain, as seen in four cases analysed. since up-to-date cerebrospinal fluid based real-time quaking-induced conversion (rt-quic) has high sensitivity (> 90 %) and specificity (up to 100 %) for the detection of prion disease [52,53,54,55], an intravitam diagnosis in patients with suspected prion disease is possible. yet, rt-quic might give false negative results as documented for instance in sporadic cjd cases at younger age or cases with vv1 or mm2 molecular subtype [52,56]. patients with negative rt-quic are also more likely to present with motor symptoms or gait difficulties at initial presentation [56]. additionally, implementing rt-quic assays is difficult and therefore often only a limited number of laboratories offer it [57]. of note, absence of prion protein immunoreactivity in the fontal cortex and cerebellum does not entirely rule out the possibility of a prion disease since thalamic forms of creutzfeldt-jakob disease or fatal familial insomnia might not exhibit pathological prion protein aggregates in the frontal cortex or cerebellum [23,58]. finally, in a different brain bank cohort, the use of two brain regions (frontal cortex and cerebellum) has been considered sufficient for the surveillance of prion disease [59]. the diagnostic accuracy of fast-track histology might even be higher by including temporomesial structures (hippocampus, amygdala) or thalamus (e.g. for the detection of rare prion disease variants). diagnosing part, as defined by tau nft pathology with a braak and braak stage < iv and thal plaque phases 0 - 2 [49], would require sampling of hippocampal structures including entorhinal and transentorhinal cortices as well as adjacent temporal neocortex. in particular, fast-track histology might enhance the recognition of common co-pathologies, such as agd, artag or late-nc which typically or often initiate in the limbic structures of the medial temporal lobe [47,48,50]. in our study however, handling and cutting of the brain was limited to a minimum for the sake of safety in respect to prion transmission and hence focused only on two easily accessible brain regions. furthermore, our study was performed in a brain bank setting and all cases hence underwent a thorough neuropathological evaluation after screening histology, covering the aforementioned disease entities as well. compared to the study by venneti et al. [17] which assessed mid-frontal cortex (and additional cortical regions as well as basal ganglia) we achieved a higher diagnostic sensitivity. remarkably that study [17] showed 0 % sensitivity for the diagnosis of psp by applying only a frontal lobe specimen, whereas we reached a sensitivity of 96 %. in the study by king et al. [18], the sensitivity for the diagnosis of ad, cbd and msa was 100 % for frontal specimens and frontal/temporal specimens combined, which is as high as in our study. however, we reached a higher sensitivity for the diagnosis of psp (96 % vs. 0 %), lbd (100 % vs. 60 %) and ftld non-tau (100 % vs. 83 % (combined) respectively 67 % (fontal sample alone). the authors of both previous studies [17,18] mention the sparse occurrence of frontal tau pathology in psp and thus diagnostic difficulties. on the contrary, we found frontal tufted astrocytes in all cases and frontal coiled bodies in all but one case classified as psp on screening histology. since neuronal, astrocytic and oligodendroglial tau accumulation in the frontal cortex precedes pathology in other neocortical areas [60], we consider assessment of the superior frontal gyrus as useful for an orientating diagnosis of psp. in a proposed stepwise distribution model of psp, frontal tau can already be detected at stage 3, temporally just after anatomically less easily accessible regions of the subthalamic nucleus and basal ganglia [60]. of note, we assessed brain tissue of participants in the frame of a brain bank program, essentially recruiting patients with a known history of a ndd and therefore often presenting with advanced disease and widespread pathology. this might for instance account for the high number of lbd cases showing frontal pathology. conclusion we here developed a diagnostic algorithm based on the histomorphological assessment of two easily accessible brain regions to obtain a working diagnosis for the commonest ndd. our diagnostic algorithm is highly reliable with an over 96 % concordance between the screening and final neuropathological diagnoses. in addition, it enhances the safety of laboratory personnel by excluding prion diseases before exhaustive manipulation of the brain. furthermore, our diagnostic algorithm allows for a prompt feedback to the treating physicians. in conjunction with the clinical history, our screening diagnosis might foster a targeted evaluation of the fixed brain tissue. finally, the approach presented here might be feasible in future biopsy diagnostics of ndd and thereby enhance the diagnostic rapidity of postmortem ndd diagnostics. some caution is however warranted since our approach does not appreciate the manifold co-pathologies that may be present in ndd and since it has limited value for disease staging. therefore, subsequent thorough analysis of various brain specimens is still required, especially in a brain bank setting. acknowledgements we thank all brain donors and their relatives as well as the (neuro)pathologists involved in tissue asservation all over germany for enabling this study. we further thank mr. michael schmidt for excellent technical assistance. conflicts of interest statement all authors do not disclose any conflict of interest. funding statement no funding was received to help prepare this manuscript. references 1. tamsen, 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https://doi.org/10.1007/s00401-020-02158-2 copyright: © 2024 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. 62nd annual meeting of the canadian association of neuropathologists association canadienne des neuropathologistes (canp-acnp) feel free to add comments by clicking these icons on the sidebar free neuropathology 3:25 (2022) meeting abstracts 62nd annual meeting of the canadian association of neuropathologists association canadienne des neuropathologistes (canp-acnp) meeting abstracts october 13–15, 2022 saskatoon, sk submitted: 31 october 2022 accepted: 01 november 2022 published: 16 november 2022   the canadian association of neuropathologist – association canadienne des neuropathologistes (canp-acnp) held their 62nd annual meeting at the delta bessborough in saskatoon, sk from october 13th to 15th, 2022, under the leadership of dr. robert hammond, president of the canp-acnp, dr. peter schutz, secretary treasurer of the canp-acnp, and with technical support from canp administrator, colleen fifield. the academic program comprised 15 scientific abstracts, 9 unknown cases, a mini-symposium on competence based medical education in neuropathology, and the presidential symposium on multiple sclerosis and immune mediated demyelinating disease. digital pathology images from the 9 unknown cases are available for viewing online (www.canp.ca). the unknown case sessions were moderated by dr. andrew gao. the presidential symposium 2022 on multiple sclerosis and immune mediated demyelinating disease featured the gordon mathieson lecture given by dr. g. r. wayne moore entitled demyelination, multiple sclerosis, and mri and the david robertson lecture given by dr. michael levin entitled multiple sclerosis and future therapies. the program was completed by three invited presentations with dr. e. ann yeh presenting on pediatric multiple sclerosis and immune mediated demyelination, dr. tanja kuhlmann presenting on neuropathology of ms and stem cells and dr. pamela kanellis presenting on outlook of patients and public on ms research and treatment in canada. the mary tom award for best clinical science presentation by a trainee went to dr. christopher newell (supervisor dr. j. joseph), and the morrison h. finlayson award for best basic science presentation by a trainee was won by dr. erin stephenson (supervisor dr. v.w. yong). the following abstracts were presented at the 62nd annual meeting of the canadian association of neuropathologists – association candienne des neuropathologistes (canp-acnp) in october 2022.   https://doi.org/10.17879/freeneuropathology-2022-4496 keywords: canadian association of neuropathologists, canp, meeting abstracts, 62nd meeting oct. 2022 contents abstract 1 effects of prenatal cocaine exposure on the human developing brain. a neuropathological study. abstract 2: utilizing neurodevelopmental time windows of hypoxic-ischemic pathology to infer brain maturity in patients with congenital cardiothoracic defects abstract 3: spinal nerve root blood in pediatric autopsy cases is not necessarily a marker of trauma at that site abstract 4: multifocal necrotizing leukoencephalopathy post-covid-19 infection: expanding the “neuro-covid” spectrum abstract 5: the international disseminated pediatric low grade glioma consortium: project goals and preliminary results abstract 6: glioblastoma, idh-wildtype with fgfr3-tacc3 fusion has unusual histologic, molecular and clinicoradiologic characteristics abstract 7: infiltrative pattern in pediatric ganglioglioma abstract 8: clinical implementation of methylation profiling: the sickkids experience abstract 9: cerebral amyloidoma: pathological findings from diagnostic biopsy to autopsy abstract 10: identification and validation of immunohistochemical antibodies against transcriptomically distinct human hippocampal astrocyte subtypes abstract 11: cadherin-23 is essential for the normal organisation of cerebellar mossy fibre synapses abstract 12: the extracellular matrix transcriptome following demyelination abstract 13: nodding syndrome: characterizing the newest tau proteinopathy in africa abstract 14: mri-pathological correlations show differential patterns of ex vivo texture in als abstract 15: lymphoid and plasma-cell infiltrates with amorphous eosinophilic material deposition in the leptomeninges: a challenging case     abstract 1 free neuropathol 3:25:4 effects of prenatal cocaine exposure on the human developing brain. a neuropathological study. catherine fallet-bianco1, mubina jovanovic1, dorothée bouron-dal soglio1 1department of pathology, hôpital sainte-justine-université de montreal, montreal, qc, canada recent studies report that cocaine use among pregnant women continues to be a public health concern. experimental studies have shown that prenatal cocaine exposure is responsible for disturbances affecting neurogenesis, neuronal migration and neurotransmitter systems. however, human-based studies are impacted by confounding factors: multi-drug use, lack of neuropathological studies. we describe the neuropathological phenotype observed in two fetuses after prenatal exposure to cocaine. in a 34-year-old woman using cocaine for several years, ultrasonography at 24 gestational weeks revealed a severe microcephaly with a severe global disorganization of brain development. in the second case, a 19-year-old mother using cocaine, ultrasonography at 20 weeks showed a severe microcephaly with bilateral schizencephaly resulting from multiple hemorrhages. a vasoconstriction of maternal-fetal arteries is, classically, the mechanism considered responsible for the disturbed brain development. a vascular disruption is probably the cause of the lesions in the second case. the changes in the first case are similar to those generated in experimental models and confirm that cocaine has direct toxic effects on the neurogenesis, gliogenesis and neuronal migration in the developing brain. the wide spectrum of brain lesions depends on multiple factors; the most important is probably the moment of exposure in gestation, as well as dose, frequency of cocaine use, and genetic susceptibility.   abstract 2 free neuropathol 3:25:5 utilizing neurodevelopmental time windows of hypoxic-ischemic pathology to infer brain maturity in patients with congenital cardiothoracic defects karina chornenka1, christopher dunham2 1 department of pathology and laboratory medicine, university of british columbia, vancouver, bc, canada 2 division of anatomical pathology, children’s and women’s health centre of bc, vancouver, bc, canada neuroradiologic investigations have demonstrated that cerebral development is delayed by 2-4 weeks in infants suffering from congenital heart defects and congenital diaphragmatic hernia (chd/cdh). these estimates are based upon application of the “total maturation score” (tms) system that evaluates brain development using magnetic resonance imaging (mri) by assessing myelination, cortical gyration, insular development, t1 white matter signal intensity and the involution of the germinal matrix (n engl j med 2007;357: 1928-1938, j pediatr surg 2012;47:453-461). these infants often require surgical correction of their malformations shortly after birth, but unfortunately some do not survive. of those coming to autopsy, it is not uncommon to encounter acute hypoxic ischemic injury (hii). we examined three individuals born at term with chd/cdh, ranging in age from 3 to 5 months, who died shortly after surgery and displayed evidence of neuropathology that included acute hii characterized by periventricular leukomalacia (pvl) and pontosubicular necrosis (psn). pvl and psn are typically encountered in the context of prematurity and occur during specific developmental time windows (i.e., pvl: 24-32 weeks gestational age; psn: 20 weeks gestational age – 2 months postnatal) (acta neuropathol 1995;90:7-10, acta neuropathol 2005;110:563-578). given the ages of the affected individuals herein, we suggest that the presence of premature-type neuropathology in the form of acute hii could be used to support the hat infants with chd/cdh incur delayed brain development. moreover, based on our observations we propose that the delay in cerebral development could be longer than previous estimates using mri.   abstract 3 free neuropathol 3:25:6 spinal nerve root blood in pediatric autopsy cases is not necessarily a marker of trauma at that site marc r. del bigio1 1 department of pathology, university of manitoba and diagnostic services manitoba, winnipeg, mb, canada increasingly, the forensic examination of suspected child abuse cases includes dissection of the entire cervical spine; hemorrhage along nerve roots is postulated to indicate forces exerted by shaking. seventy pediatric spinal cords encased in dura mater were examined (including 12 en bloc cervical spines); age 0-58 months. the mechanism of deaths were: suspected abusive trauma 35; accident 13; undetermined or natural without head/neck trauma 12; forceps injury at birth 3; birth related intracranial hemorrhage 7. prominent spinal subarachnoid hemorrhage was present in 30 cases; hemorrhage was detected along spinal nerve roots at the cervical level in 14/30 and at the lumbosacral level in 8/30 in the absence of definite injury to the spinal column. two cases with definite evidence of bone/ligamentous injury to the spine and the 2 forceps injury cases had extensive epidural hemorrhage ± subarachnoid hemorrhage extending the entire length of the cord. anatomical studies in humans, tracer studies, and experimental hemorrhage show direct communication of the cranial and spinal subarachnoid and subdural spaces with extension to the dorsal root ganglia. i conclude that the majority of spinal nerve root hemorrhages in pediatric trauma cases are simply markers of subarachnoid or subdural hemorrhage elsewhere. additional work is needed to determine if en bloc spine dissection, which is invasive and time consuming, adds significant information to the autopsy.   abstract 4 free neuropathol 3:25:7 multifocal necrotizing leukoencephalopathy post-covid-19 infection: expanding the “neuro-covid” spectrum jacob a. houpt1, lee cyn ang1,2, qi zhang1,2 1 department of pathology and laboratory medicine, london health sciences centre, london, ontario, canada 2 department of clinical neurological sciences, london health sciences centre, london, ontario, canada multifocal necrotizing leukoencephalopathy (mnl), also known as disseminated necrotizing leukoencephalopathy (dnl), is a rare pathological phenomenon defined by numerous discrete foci of necrosis with predilection for ventral pontine white matter. while exact etiology and pathogenic mechanisms have yet to be elucidated, mnl is most often reported on post-mortem examination in the context of immunocompromised (ex: receiving chemotherapy or with untreated hiv-aids) or critically ill patients (ex: metastatic cancer, sepsis). coronavirus disease 2019 (covid-19) is a viral respiratory illness with variable severity depending on host immune response and comorbid conditions. in its most serious cases, covid-19 can elicit both harmfully insufficient and excessive immune responses. we report on a case of covid-19 pneumonia which progressed to a clinical impression of sepsis, declining respiratory status, and death in an unvaccinated, previously well patient in which there were findings consistent with mnl throughout the midbrain and basis pontis. there was no encephalitis, microthrombi, infarcts, or other previously reported neuropathological findings. this index case was again compared and contrasted against four additional mnl cases examined at the london health sciences centre. the neuropathological findings are examined and described for these 5 cases, all with clinical scenarios involving systemic critical illness and/or immune system compromise and with similar ventral pontine focal necrosis, myelin loss, and limited reactive glial response. though numerous radiological studies have suggested a disseminated necrotizing leukoencephalopathic process underlying covid-19-associated encephalopathy, this case represents the first to offer pathological correlation supported by autopsy confirmation of mnl in a patient with covid-19 infection.   abstract 5 free neuropathol 3:25:8 the international disseminated pediatric low grade glioma consortium: project goals and preliminary results adrian levine1,4, julie bennett2, joseline haizel-cobbina3, liana nobre2, michael dewan3, uri tabori2, cynthia hawkins1 1 department of pediatric laboratory medicine, hospital for sick children, toronto, on, canada 2 division of neuro-oncology, hospital for sick children, toronto, on, canada 3 department of neurosurgery, vanderbilt university medical center, nashville, tn, usa 4 clinician investigator program, university of british columbia, bc, canada although most pediatric lgg (plgg) have excellent long-term survival, there is a subset of cases that disseminate (dplgg) and have very poor outcomes. it is unknown why these tumors behave in such an aggressive way. we believe that biological mechanisms, distinct from those present in non-disseminated lgg, underlie this metastatic ability, but so far there has been no in-depth investigation of this. to improve our understanding of this rare patient population, we have created a new consortium to compile dlgg cases across from across the world. we are collecting comprehensive clinical information, including the tumor presentation and pattern of dissemination, surgical outcomes and subsequent treatment courses, and quality of life outcomes. cases with available tissue will be tested with our glioma ngs panel for the most common mutations and fusions, and methylation array for copy number and classification. our data thus far on the first 53 cases demonstrates the dplgg has much worse prognosis than the overall plgg population. virtually all dplgg progress at 5 years, compared to a quarter of other plgg, and the disseminated cases are approximately 5-times more likely to die at 10 years. this can affect children of all ages and does not carry a gender predilection. we observe three patterns of dissemination – 40% of patients present with a localized mass and have secondary dissemination, 35% with disseminated tumor and a clear dominant mass, and 25% with disseminated disease without a dominant mass. the most common molecular alteration is braf fusions.   abstract 6 free neuropathol 3:25:9 glioblastoma, idh-wildtype with fgfr3-tacc3 fusion has unusual histologic, molecular and clinicoradiologic characteristics namita sinha1, sherry krawitz1, jai j.s. shankar2, saranya kakumanu3, ken aldape4, marc r.del bigio1 1 department of pathology, university of manitoba, winnipeg, mb, canada 2 department of radiology, university of manitoba, winnipeg, mb, canada 3 cancer care manitoba, university of manitoba, winnipeg, mb, canada 4 laboratory of pathology, national cancer institute, nih, bethesda, maryland, usa according to 2021 world health organization (who) classification, term ‘glioblastoma’ is reserved for cns who grade 4 astrocytic neoplasm that is idh-wildtype. a small subset of glioblastomas may have unusual clinical, histological and molecular profiles that may need appropriate molecular work-up for definitive diagnosis, and some of them may be reported as high-grade glioma, not otherwise specified, if complete molecular work up is lacking. we present two cases: a 48-year-old female with imaging revealing right occipital solid, cystic, mass demonstrating nodular calcification; and a 53-year-old male with imaging revealing multifocal enhancing mass with extensive calcification in the right parietal, occipital and temporal regions. the cerebral tumors in both patients were relatively circumscribed high-grade glioma composed of gfap immunoreactive monomorphic ovoid cells, with endothelial proliferation, predominantly infarct-type necrosis, and abundant calcospherites. both tumors showed occasional mitosis with ki-67 of ~ 5-10%. mass array analysis showed no idh, h3f3a, braf v600e mutation in these two cases and revealed tert promoter mutation in the second case. because of unusual histology, methylation profiling was pursued in these two cases that matched to glioblastoma. interestingly, ngs study showed fgfr3-tacc3 fusion in both the cases. follow-up imaging showed tumor progression in these two patients, with recurrence at 9 months in the second patient. both the patients are clinically well at 7 months and 15 months of follow-up. a few case reports are published with similar histologic and molecular findings. our cases contribute to expanding histologic, molecular and clinical spectrum of fgfr3-tacc3 fused glioblastoma.   abstract 7 free neuropathol 3:25:10 infiltrative pattern in pediatric ganglioglioma murad alturkustani1 1 department of pathology, faculty of medicine, king abdulaziz university, jeddah, saudi arabia ganglioglioma is a well-circumscribed low-grade glioneuronal tumor with a broad morphological spectrum. diffuse glioneuronal tumors are used to describe cases with infiltrative growth. molecular studies of some of these cases were consistent with ganglioglioma. this work aims to clarify the growth patterns in ganglioglioma. the available slides and clinical and molecular information for ganglioglioma cases under the open pediatric brain tumor atlas from the children’s brain tumor network database were reviewed to confirm the integrated diagnosis and to evaluate the growth patterns in these cases. infiltration is defined as the presence of neoplastic cells among the nonneoplastic parenchyma. the diagnosis of ganglioglioma was confirmed in 16 of 46 cases (nine females and seven males; age ranges from eight months – 19 years with a mean of 9.9 years). the infiltrative pattern was identified in 5 cases as the predominant pattern and in another 5 cases combined with a circumscribed nodule, while only 6 cases had predominant circumscribed growth. this work confirms the presence of an infiltrative/diffuse variant of ganglioglioma. awareness of this variant should help with infiltrative tumors, as the differential includes diffuse glioma, which is usually idh wild type in this population.   abstract 8 free neuropathol 3:25:11 clinical implementation of methylation profiling: the sickkids experience adrian levine1,2, michelle ku1, scott ryall1, cynthia hawkins1 1 department of pediatric laboratory medicine, hospital for sick children, toronto, on, canada 2 clinician investigator program, university of british columbia, bc, canada over the past decade, methylation profiling and machine learning based classification has had a major impact on the diagnostic framework for cns tumors, however there remains inconsistent and limited access to clinical testing for this analysis. at sickkids, we have implemented a clinically validated methylation assay using the illumina epic platform for the heidelberg cns tumor classifier, copy number profiling, and mgmt promoter methylation status. we have now run 136 samples for clinical use (plus additional samples for validation), which have been classified through both v11 and v12.5 of the heidelberg classifier. many external samples were not received with a histologic diagnosis, those that do have a diagnosis include 34 low grade gliomas (lgg), 19 high grade gliomas (hgg), 10 ependymomas, 4 pineal tumors, and 4 medulloblastoma/embryonal tumor. overall, only 34 samples (25%) classified with confidence score >0.85 on the v11 classifier and 77 samples (57%) on v12.5. the medulloblastomas all classified with high confidence scores, as did 7/10 ependymomas, and 2/3 papillary tumors of the pineal region. 9/19 hggs classified with high confidence – most of which were methylation-defined subtypes of idh-mutant and idh-wildtype astrocytomas or glioblastomas, with unclear clinical significance. notably, though 2 of the cases that were referred as hgg were in fact other tumor types, one being an atrt, and the other an ependymoma with zfta-fusion. the classifier had particular challenge with lggs, of which only 17 (50%) classified with high confidence, including one that was erroneously called “control tissue, reactive tumor microenvironment”.   abstract 9 free neuropathol 3:25:12 cerebral amyloidoma: pathological findings from diagnostic biopsy to autopsy shervin pejhan1, adrian budhram2, joseph megyesi2, lee cyn ang1,2, robert hammond1,2, qi zhang1,2 1 department of pathology & lab medicine, university of western ontario, on, canada 2 department of clinical neurological sciences, university of western ontario, on, canada amyloidoma is a rare condition defined by the aggregation of amyloid proteins forming a mass-like lesion. while brain-restricted amyloidomas have been reported, their course and pathogenesis are poorly understood. we are reporting a case of multifocal brain amyloidoma in a 71-year-old man, which is significant for its protracted clinical course and progression of neurological symptoms. a pair of stereotactic biopsies revealed the underlying pathology. the pathological findings were unusual with a dual picture of perivascular lymphoplasmacytic infiltration in the frontal area, accompanied by amyloid deposits in occipital white matter. proteomic analysis of the amyloid protein by mass spectrometry was also curious as it characterised both lambda light chain and amyloid beta-protein without evidence of monoclonality. further assessments found no involvement of other organ systems. with the progression of symptoms and lack of definitive treatment, the patient underwent palliative whole-brain irradiation, with no improvement, and passed away shortly thereafter. assessment of post-mortem brain tissue showed massive amyloid deposition in the periventricular white matter with sparse perivascular lymphocytes and fewer plasma cells than the biopsy samples. special staining, immunohistochemistry, electron microscopy, and mass spectrometry confirmed multifocal cerebral amyloidoma, lambda light chain type. the collection of the biopsy and autopsy findings may be the result of lesions observed at different stages of progression, treatment effects, or a not-previously described pattern of brain amyloidoma. studying this case is beneficial to gaining insight into the pathobiology of this rare condition and to inform future studies and therapeutic interventions.   abstract 10 free neuropathol 3:25:13 identification and validation of immunohistochemical antibodies against transcriptomically distinct human hippocampal astrocyte subtypes brenden joseph1,2, kaitlin sullivan3, mark s. cembrowski3, veronica hirsch-reinshagen1,2 1 department of pathology & laboratory medicine, university of british columbia, vancouver, bc, canada 2 division of neuropathology, vancouver general hospital, vancouver, bc, canada 3 department of cellular and physiological sciences, djavad mowafaghian centre for brain health, university of british columbia, vancouver, bc, canada our understanding of astrocyte function has steadily evolved from an early conceptualization as simplistic support cells to a current appreciation for their diverse and critical physiological roles. there is emerging evidence for the existence of distinct astrocyte subtypes with differential genomic expression, spatial localization, morphology, and physiological function. however, little is known about their specialized functions or the role of specific subtypes in human disease. a lack of established subtype-specific immunohistochemical markers represents a significant barrier to further investigation. to address this limitation, we aim to identify transcriptomically distinct human astrocyte subtypes and validate immunohistochemical markers for these. to identify distinct transcriptomic subtypes of human astrocytes, we used single cell nuclear rna sequencing of 11,204 astrocytes in human hippocampal surgical samples from 5 patients with intractable mesiotemporal epilepsy. canonical correlation and seurat cluster analysis identified 6 distinct clusters of astrocytes with unique transcriptomes and revealed several candidate marker genes unique to each cluster. we are currently validating c8orf34 and nrg4 as subtype specific markers for cluster 5, which was the most transcriptomically unique cluster. the study’s current and future directions include the selection of commercially available antibodies against candidate marker gene products and validation of these subtype-specific antibodies using doubleand triple-label immunofluorescence microscopy with other established markers for various cns cell types. establishing validated antibodies will lead to the discovery of subtype-specific involvement of astrocytes in human neurological and psychiatric conditions and advancements in our understanding of human astrocyte physiology.   abstract 11 free neuropathol 3:25:14 cadherin-23 is essential for the normal organisation of cerebellar mossy fibre synapses madison t. gray1,2,3, julie l. lefebvre2,3 1 department of pathology and laboratory medicine, western university, london, on, canada 2 neuroscience and mental health, hospital for sick children, toronto, on, canada 3 department of molecular genetics, faculty of medicine, university of toronto, toronto, on, canada to understand the physiological basis of neurological disease, one must first identify which neurons are connected and how they form those connections. here, we identify a novel cell adhesion molecule signature in golgi cells, an inhibitory interneuron in the granular layer of the cerebellar cortex. golgi cells receive excitatory input from both precerebellar mossy fibres and cerebellar granule cells, while providing inhibition to those same granule cells in a feedback loop. we show that both golgi cells and their mossy fibre partners express cadherin 23 (cdh23) – a pattern conserved in evolution. using explant cultures in vitro and ectopic viral expression in vivo, we demonstrate that cdh23 is sufficient to induce the formation of synapses by mossy fibres. the known in vivo roles of cdh23 depend on binding protocadherin 15 (pcdh15) in trans. however, here we show that pcdh15 is not expressed in mossy fibre neurons, golgi cells, or their cerebellar targets. a cdh23 mutant incapable of binding pcdh15 nonetheless induces mossy fibre synapses in vitro and in vivo, confirming that the synaptic organiser function of cdh23 is independent of pcdh15 binding. finally, analysis of a cdh23-null mouse reveals that cdh23 is not necessary for anatomical synapse formation but mossy fibres. however, loss of cdh23 results in enlarged, sparse mossy fibre terminals suggesting a role for cdh23 in mossy fibre bouton development.   abstract 12 free neuropathol 3:25:15 the extracellular matrix transcriptome following demyelination erin l. stephenson1, samira ghorbanigazar2, charlotte d’mello2, rajiv william jain2, v. wee yong2 1 department of pathology and laboratory medicine, university of calgary, calgary, ab, canada 2 hotchkiss brain institute, university of calgary, calgary, ab, canada the extracellular matrix (ecm) of the central nervous system (cns) is an interconnected network of proteins and sugars. the ecm has critical roles not only in homeostasis, but ecm remodeling in neurological diseases impacts both injury and repair. multiple sclerosis (ms) is a chronic inflammatory and degenerative disease of the cns. here, we evaluated ecm changes in ms lesions compared to controls using databases generated in-house through spatial rna-sequencing, and through a public resource of single-nucleus rna sequencing. our results found widespread changes in ecm molecules and their interacting proteins, including alterations to proteoglycans and glycoproteins within inactive and active ms lesions. some highly upregulated members, including serglycin and sparc-related proteins, have not previously been investigated and their role on ms lesion evolution and disease course remains unknown. our results emphasize that there are profound changes to the ecm following demyelination.   abstract 13 free neuropathol 3:25:16 nodding syndrome: characterizing the newest tau proteinopathy in africa kenneth g. kodja1,2,3, sylvester onzivua4, david clutterbuck2, david l. kitara5, amanda fong2, michael s. pollanen1,2,3 1 department of pathobiology and laboratory medicine, university of toronto, toronto, on, canada 2 ontario forensic pathology service, toronto, on, canada 3 tanz centre for research in neurodegenerative diseases, university of toronto, toronto, on, canada 4 department of pathology, college of health sciences, makerere university, kampala, uganda 5 department of surgery, gulu university, gulu, uganda nodding syndrome (ns) is an acquired tau proteinopathy plaguing the remote rural communities in eastern sub-saharan africa. it has been proposed that infection with the nematode parasite onchocerca volvulus triggers an autoimmune attack against the human protein leiomodin-1. this theory is dependent on the constitutive neuronal expression of leiomodin-1. we tested this hypothesis by exploring the histologic distribution of this protein in the normal human brain. subsequent immunostaining of cerebellar tissue and c. elegans (a proxy for onchocerca volvulus) using an antibody recognizing the n-terminus of leiomodin-1 was conducted. our study failed to identify the presence of leiomodin-1 immunoreactivity in neurons or glia. our results also suggest the possibility of leiomodin-1 antibody cross-reactivity between human purkinje cell membranes and the body wall of c. elegans; a finding explained by the homology between leiomodin-1 and tropomodulin. this outcome does not support the hypothesis of autoimmunity involving onchocerca volvulus and leiomodin-1. we also investigate the signature laminar distribution of cortical tau pathology in ns. through morphometric analysis, our current study, in agreement with the literature, suggests that primary tauopathies (the likes of progressive supranuclear palsy, corticobasal degeneration, pick’s disease and globular glial tauopathy) are associated with severe lower layer (iv-vi) pathologic burden, independent of clinical symptomology. interestingly, we found predominant involvement of the upper layers (i-iii) in ns; a laminar profile similar to the amyotrophic lateral sclerosis and parkinsonism-dementia complex of guam. the present study lays the foundation for future work investigating potentially unique mechanisms of propagation and neurodegeneration in ns.   abstract 14 free neuropathol 3:25:17 mri-pathological correlations show differential patterns of ex vivo texture in als avyarthana dey1,2, collin luk2, abdullah ishaque1,2, peter seres3, dennell krebs2, yee-hong yang4, julia keith5,6, sumit das1,7, ekaterina olkhov-mitsel5, sanjay kalra1,2 1 neuroscience and mental health institute, university of alberta, edmonton, ab, canada 2 division of neurology, faculty of medicine and dentistry, university of alberta, edmonton, ab, canada 3 peter s. allen mri center, university of alberta, edmonton, ab, canada 4 faculty of science – computing science, university of alberta, edmonton, ab, canada 5 laboratory medicine and molecular diagnostics, sunnybrook health sciences centre, toronto, on, canada 6 department of laboratory medicine & pathobiology, university of toronto, toronto, on, canada 7 department of laboratory medicine & pathology, faculty of medicine and dentistry, university of alberta, edmonton, ab, canada in vivo magnetic resonance imaging (mri) has provided a reliable biomarker for amyotrophic lateral sclerosis (als). however, lack of pathological specificity of these biomarkers impedes their integration in clinical investigations. to address this lack of specificity and establish their relationship with disease pathology, the aim of the current study was to examine the associations between ex vivo neuroimaging and histopathological features. we hypothesized that histopathology of the cervical spinal cord (csc) and precentral gyrus (pcg) shows associations with texture of the posterior limb of internal capsule (plic). formalin-fixed brains of 13 als patients underwent excision of the pcg, plic, and csc. t1-weighted mri was performed for pcg and plic from which texture features – namely autocorrelation (autoc), energy (energ), inverse difference normalized (indnc) – were extracted. quantitative histological assessments were performed using qupath to assess the density of axons in the corticospinal tract on nf immunolabeling, and extent of macrophage infiltration of the corticospinal tract on cd68 immunolabeling in the csc. qualitative assessments were performed to assess superficial spongiosis and presence of tdp-43 inclusions in the pcg. pearson’s-r correlational analysis was performed between texture and histological features. statistical significance was set at p < 0.05. a negative correlation was observed between plic autoc and presence of tdp-43 inclusions (r = -0.652, p = 0.022) as well as superficial spongiosis (r = -0.585, p = 0.046) in the pcg. a positive correlation was observed between plic energ and cd68 density (r = 0.714, p = 0.009). the pcg and csc demonstrate patterns of histopathological changes associated with plic texture features in als.   abstract 15 free neuropathol 3:25:18 lymphoid and plasma-cell infiltrates with amorphous eosinophilic material deposition in the leptomeninges: a challenging case gianluca lopez1,2, karam han1, shino d. magaki1, sophie x. song3, noriko salamon4, kanwarpal s. kahlon5, inna keselman6, ausaf a. bari7, harry v. vinters1 1 section of neuropathology, department of pathology and laboratory medicine, ronald reagan ucla medical center and david geffen school of medicine, los angeles, ca, usa 2 university of milan, milan, italy 3 section of hematopathology, department of pathology and laboratory medicine, ronald reagan ucla medical center and david geffen school of medicine, los angeles, ca, usa 4 department of radiology, david geffen school of medicine, university of california, los angeles, los angeles, ca, usa 5 division of hematology-oncology, david geffen school of medicine, university of california, los angeles, los angeles, ca, usa 6 department of neurology, david geffen school of medicine, university of california, los angeles, los angeles, ca, usa 7 department of neurosurgery, david geffen school of medicine, los angeles, ca, usa a 65-year-old female presented with recurrent seizures. she had a history of intractable epilepsy after a motor vehicle accident, which was treated several decades ago with left anteromedial temporal lobectomy and hippocampectomy; hippocampal sclerosis was noted at the time. at the time of her last hospital presentation, she underwent magnetic resonance imaging, which showed a leptomeningeal enhancement in the left parieto-occipital lobe. she underwent a biopsy for diagnostic purposes, which showed a diffuse lymphoid infiltrate predominantly involving the leptomeninges, with extension into the virchow-robin spaces and with deposits of amorphous eosinophilic material. the lymphoid population comprised small atypical lymphocytes, scattered plasma cells, and plasmacytoid lymphocytes. with immunohistochemistry, the lymphoid infiltrate was positive for cd20, cd19, and bcl2; a subset of cells were positive for cd138 and mum1; lambda light chain restriction was noted on rna in situ hybridization. negative stains included cd3, cd5, cd10, cd21, cd23, and bcl6. the ki-67 proliferative index was estimated at 5%. on congo red stain, the amorphous material showed green birefringence under polarized light. myd88 mutation was not detected. the patient had no evidence of systemic amyloidosis, monoclonal igm gammopathy, urinary bence jones protein, or bone marrow or other organ sites involvement. copyright: © 2022 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. neurotrauma: 2023 update feel free to add comments by clicking these icons on the sidebar free neuropathology 4:14 (2023) review neurotrauma: 2023 update david s. priemer1-3, daniel p. perl1,2 the department of defense/uniformed services university brain tissue repository, bethesda, md, usa department of pathology, f. edward hébert school of medicine, uniformed services university, bethesda, md, usa henry m. jackson foundation for the advancement of military medicine, inc., bethesda, md, usa corresponding author: david s. priemer · department of pathology · f. edward hébert school of medicine · uniformed services university (usu) · 4301 jones bridge rd · bethesda, md 20814 · usa david.priemer.ctr@usuhs.edu submitted: 18 august 2023 accepted: 01 september 2023 copyedited by: s. radhika published: 20 september 2023 https://doi.org/10.17879/freeneuropathology-2023-5076 keywords: traumatic brain injury, chronic traumatic encephalopathy, tau, tdp-43, apoe abstract 2022 was a productive year for research in traumatic brain injury (tbi) and resultant neuropathology. after an extensive review, we present related studies and publications which we felt were of particular importance to the neuropathology community. first, 2022 was highlighted by important advancements in the diagnosis and, moreover, our understanding of chronic traumatic encephalopathy (cte). important publications include a pair concluding that cte primarily concerns neuronal accumulation of phosphorylated tau (ptau), but that glial ptau accumulation often helps to facilitate diagnosis. in addition, a new large community study from australia continues the indication that cte is relatively uncommon in the community, and the first large-cohort study on brains of military personnel similarly demonstrates that cte appears to be uncommon among service members and does not appear to explain high rates of neuropsychiatric sequelae suffered by the warfighter. the causation of cte by impact-type tbi was supported by the application of the bradford hill criteria, within the brains of headbutting bovids, and interestingly within an artificial head model exposed to linear impact. finally, a large-scale analysis of apoe genotypes contends that gene status may influence cte pathology and outcomes. in experimental animal work, a study using mouse models provided important evidence that tdp-43 facilitates neurodegenerative pathology and is implicated in cognitive dysfunction following tbi, and another study using a swine model for concussion demonstrated that evidence that axonal sodium channel disruption may be a driver of neurologic dysfunction after concussion. finally, we end with memoriam to dr. john q. trojanowski, a giant of neurodegenerative research and an important contributor to the neurotrauma literature, who we lost in 2022. introduction the year 2022 was an exciting and highly productive year for research in traumatic brain injury (tbi). accordingly, a large number of papers with notable impact to the neuropathology community were published, and the task of highlighting only a small handful of them for this review proved extraordinarily difficult. nonetheless, and after a thorough review of the literature, herein we recognize the papers that what we felt were of particular interest to the neuropathology community, with a summary of their contribution to our field and thus why we recognized them. as you will see, many of our selections this year involve important work within the field of chronic traumatic encephalopathy (cte). we readily acknowledge that inclusions and exclusions from this list reflect our own bias, and that some of the papers we selected involve our own research efforts. we suspect that at least several of our considerations would have also been chosen by others if given the same task. finally, we conclude our review with memoriam to the life and exemplary career of dr. john q. trojanowski, whose unexpected death in 2022 left a legacy that will not soon be forgotten. chronic traumatic encephalopathy is caused by repetitive impact traumatic brain injury in 1965, sir austin bradford hill proposed what became known as the bradford hill criteria. the bradford hill criteria provide a system to examine the potential role of environmental exposures in causation of disease.1 bradford hill proposed the use of nine different criteria or “viewpoints”, and these were primarily aimed at establishing a causal relationship between smoking and lung cancer. in 2022, authors nowinski et al. attempted to apply all nine viewpoints of the bradford hill criteria through a review of literature in order to investigate the relationship between repetitive tbi and subsequent cte pathology.2 what they established in their review was that: cte pathology had considerable strength of association with repetitive tbi (bradford hill criterion 1); the relationship between repetitive tbi and cte has been consistently demonstrated across multiple studies conducted by independent groups in different patient populations (criterion 2); there is evidence of a dose-response relationship between amount of repetitive tbi over time and cte (e.g., association between years played in contact sports and increasing likelihood to have cte at autopsy; criterion 5); there is evidence suggesting mechanistic plausibility that tbi can result in the unique distribution of phosphorylated tau (ptau) pathology in cte (criterion 6); an association between repetitive tbi and cte is not in conflict with what we know about both tbi and cte (i.e., coherence, criterion 7); and the relationship between repetitive tbi and cte is analogous to similar causal associations (criterion 9). criticism of the application of the bradford hill criteria to repetitive tbi and cte may emerge particularly on the topics of specificity (criterion 3) and temporality (criterion 4), as the literature indicates that cte is not entirely specific to repetitive tbi3-5, and it is difficult to definitively establish a temporal relationship with repetitive tbi and cte in a situation where the disease can only be diagnosed post mortem. finally, the 8th criterion examining experimental evidence demonstrating causality from an environmental exposure, or demonstrating prevention of disease from cessation of an environmental exposure, has not yet been fulfilled in regard to cte and repetitive tbi, and may take many years to definitively address. however, regardless of whether all nine criteria are met, the application of this method to examine the relationship between repetitive tbi and cte is commendable and has made an important contribution to the literature. chronic traumatic encephalopathy-like pathology in the brains of headbutting animals in support of the relationship between repetitive tbi and cte, an interesting study also emerged in 2022 from a unique and unusual source: animals who engage in headbutting. authors ackermans et al. performed postmortem neuropathologic examination of brains obtained from muskoxen and bighorn sheep, two animal species that are well known to engage in headbutting displays at high speeds and with large applied forces.6 brains from seven animals were included, (four bighorn sheep, three muskoxen), involving both sexes and a distribution of ages from young-to-middle age in bighorn sheep and middle-to-old age in the muskoxen. all three muskoxen were from the wild, while three of the four bighorn sheep were from captivity. examination was of the prefrontal cortex +/the parietal cortex and used three different antibodies for ptau (cp13, at8, and phf-1) among other markers. the authors found high degrees ptau immunoreactivity in neurons/neuronal structures in all muskoxen samples, often preferentially in distributions reminiscent of cte (with preference at the bottom of sulci, occasionally in a perivascular distribution, and in superficial cortical layers), and to lesser degrees in the brain of the single male bighorn sheep examined. the pathology was observed in the absence of aβ plaques. the authors speculate that the reduced frequency of ptau pathology in bighorn sheep may be due to the relatively young age and/or captive status of the animals. interestingly, and even though male muskoxen headbutt more frequently and with greater force than female, the female muskoxen tended to demonstrate more severe ptau pathology than the male. the authors suggest that this may be due to individual differences (small sample size), or anatomical differences in the male such as increased skull thickness and more pronounced forehead fat pads (figure 1). regardless of these details, this study provides the first direct evidence of ptau accumulation in patterns similar to cte within the brains gyrencephalic animals naturally exposed to repetitive tbi. further investigation of brains from additional animals whose nature exposes them to repetitive tbi is warranted, and encouraged. figure 1. comparison of male and female muskoxen. anatomical differences in male versus female oxen may play a role in protecting male muskoxen from traumatic brain injury relative to females. mechanistic evidence for sulcal depth-predominant injury from impact brain trauma mechanistic plausibility for the distinct pattern of sulcal depth pathology in cte as due to impact tbi was further supported in 2022, with an important work from researchers kerwin et al. titled “sulcal cavitation in linear head acceleration: possible correlation with chronic traumatic encephalopathy”.7 the researchers applied linear impact, via a drop from height, to an artificial, polymer-based head model (“biofidelic brain phantom”) complete with a 3d-printed skull, deionized water representing cerebrospinal fluid, and a simplified cerebrum constructed with grey and white matter components at appropriately different weight concentrations (i.e., densities), and with gyri and sulci. the model was designed to create a force of impact similar to an average boxing punch, and the physical distortion of the artificial brain to the linear impact was recorded at 25,000 frames/second. researchers qualitatively observed a pattern of cavitation within the brain, via the formation of vapor bubbles (“inertial cavitation bubbles”) due to the change in intracranial pressure induced by the impact, most notably in the contrecoup region. fascinatingly, their subsequent computational measurements showed that the strain from the fluid cavitation was particularly focused at depths of sulci, providing evidence that physical disruption to the brain from impact head injury is most pronounced at depths of sulci and thus consistent with the pattern of pathology observed in cte. importantly, in the year prior the same laboratory published the results from a different experiment in which the artificial brain model was exposed to blast wave; this resulted in an entirely different pattern of tissue distortion with physical strain maximized at interface regions, particularly the subpial region and gray-white matter junction, closely corresponding to the pattern of pathology observed in interface astroglial scarring.8,9 chronic traumatic encephalopathy is primarily a neuronal disease debate over how to identify and classify cte pathology continued in 2022, with a focus being on the relevance of astrocytic ptau pathology in cases of cte and largely in response to the de-emphasis of astrocytic ptau as necessary for a cte diagnosis in the most recent consensus diagnostic statement.10 authors butler et al., with the resources of the boston university cte brain bank, examined dorsolateral frontal cortex samples from the brains of 150 decedents with known cte for the precise cellular distribution of ptau pathology in the cerebral cortex.11 individuals whose brains were examined in the study ranged from 21 to 80 years of age. analysis of which cell types were involved by ptau pathology was accomplished via co-labeling with markers for astrocytes (gfap) and neurons (map2). the authors identified that not only was neuronal ptau predominant in cases of cte, but extent of neuronal ptau pathology was significantly correlated with age, extent/years of repetitive tbi exposure, and assessed severity of cte, while astrocytic ptau pathology only had a significant association with age. indeed, many brains from younger decedents (age of death <40) with diagnostic cte pathology had sparse, if any, astrocytic ptau pathology (figure 2a). the authors also observed that at sulcal depths glial ptau pathology was largely superficial/subpial in orientation, whereas neurons were predominant in the distinctive perivascular localization of ptau pathology of cte (figure 2b). while they could not refute that astrocytic ptau pathology may be exacerbated by cte, particularly at sulcal depths, or that astrocytic ptau pathology may play some role in cte pathobiology, the authors ultimately conclude that astrocytic ptau pathology in cte is an age-driven finding akin to age-related tau astrogliopathy (artag), which itself is also not considered to be related to tbi exposure. as such, astrocytic ptau in cte cases may simply represent a co-existent pathology in brains from aged individuals. figure 2. chronic traumatic encephalopathy (cte) pathognomonic lesions without (a) and with (b) superficial glial phosphorylated ptau accumulation. evidence from 2022 supports that cte is primarily a neuronal disease, in that neuronal ptau pathology in cte cases is significantly correlated with age, extent/years of repetitive tbi exposure, and severity of cte, while astrocytic ptau pathology is only correlated with age. however, other research also suggests that astrocytic accumulation of ptau may nonetheless facilitate better recognition of a pathognomonic cte lesion in practice. (scale bars: a, 500µm; b, 700µm) astrocytic ptau pathology aids the recognition of chronic traumatic encephalopathy pathology although astrocytic ptau in the context of cte seems to be age-driven and possibly independent of tbi exposure, the presence of astrocytic ptau pathology, or at least ensuring that the antibodies used to identify ptau include astrocytic ptau, may nonetheless be critical to enabling the reliable diagnosis of cte in clinical practice. authors ameen-ali et al., including the investigators of the connect-tbi research group, published fascinating data in this regard within their manuscript entitled “detection of astrocytic tau pathology facilitates recognition of [cte] neuropathologic change”.12 twelve cases from contact sports athletes with known cte were identified, along with four cases of alzheimer disease neuropathologic change (adnc). one cortical tissue block known to contain representative pathology was selected from each case, and four adjacent/consecutive sections from each block were stained with four different ptau antibodies, including ptau antibodies that would exclusively identify neuronal ptau in cte (3r and gt-38) and antibodies that would label both glial and neuronal accumulations (4r, phf-1). a total of 9 neuropathologists assessed digital slide sets that collectively included all stained sections from all sixteen cases, and did so while completely blind to case information (e.g. decedent demographics, prior neuropathologic diagnoses, etc.) and also to the precise antibody that they were evaluating on any given slide. reviewing pathologists were then simply asked if they would classify a given slide as containing cte pathology according to current consensus criteria, and to provide a degree of confidence in their opinion from 0-100%. the resulting data showed that the type of antibody used dramatically impacted the detection, and confidence of detection, of cte pathology. the authors calculated significantly reduced sensitivities for the detection of cte using antibodies that exclusively labeled neuronal ptau (gt-38, 51.9%; 3r, 15.7%) in comparison to the antibodies that were inclusive of both neuronal and astrocytic ptau (phf-1, 80.6%; 4r, 73.0%); cte was detected by the majority of pathologists in 75% of cases using phf-1, 70% of cases using 4r, 57% of cases using gt-38, and in only 5% of cases using 3r. using this novel approach, the authors conclude that identification of cte pathology is optimized when the presence of both neuronal and astrocytic ptau accumulations can be evaluated, and the visualization of neuronal ptau pathology alone leads to inconsistent diagnoses. the juxtaposition of the data from this study and the study recognized in the prior section clearly demonstrates the ongoing need for continued refinement, validation, and investigation into the practical application of current diagnostic criteria for cte. it is our hope that manuscripts like these will spur further critical investigations of the current cte diagnostic criteria, and eventually the convening of additional consensus efforts. more evidence that chronic traumatic encephalopathy is uncommon in the community continuing from important work that has suggested that cte in the general community is rather uncommon13-15, particularly outside the context of contact sports or other forms of impact-type tbi, the year 2022 also saw a new large-cohort community-based study, this time in australia. researchers suter et al. published data on prospective cte screening of 180 consecutive brains from a routine neuropathology service, including 162 cases comprising an “unselected clinical cohort” and 18 cases which were donated due to known dementia.16 the cohort demographics were broad: 58% of the cases were from males and 42% from females, the age of death ranged from 18-101 years (average age of death: 55 years), and 14% of the cohort had known tbi exposure including from contact sports, physical assaults, neurosurgery, falls, and other sources. in addition, 28% had a history of alcohol or other substance abuse. all cases were screened for cte as part of the initial diagnostic evaluation. for the 162 routine brain referrals the screening process included performance of ptau immunohistochemistry (at8 antibody) on sections from three blocks containing superior and middle frontal gyri, peri-rolandic cortex, and superior and middle temporal gyri. the known dementia donations were more extensively examined for ptau pathology in accordance with institutional dementia protocols. the researchers identified a myriad of neurodegenerative pathology in the series, including four cases (2.5%) with cte pathology. all four cte cases were identified following the three-block screening protocol instituted for unselected clinical cases (not donated for known dementia). all four cte cases involved death in the sixth decade of life. two of the four cases had known tbi history, in the form of a history of tbi from boxing and history of prior brain surgery. interestingly, the other two cases which the authors did not classify as having a definite tbi history had a known history of epilepsy, which has been described to be associated with tbi sufficient to produce cte pathology.17,18 in order to investigate sensitivity of the three-block cte screening protocol, the authors also applied the protocol to materials from twelve additional cases of known cte from the australian sports brain bank; they found that the protocol was able to detect cte definitively in nine cases (75%) and in one other case the authors felt there were features that would have been suggestive enough to spur more comprehensive evaluation after initial screening. ultimately, and while acknowledging that the three-block screening was imperfect and may have resulted in a small number of missed cases, the authors demonstrate further evidence that cte is uncommon in the general community and particularly in the absence of known or implied tbi history. they also provide evidence against the notion that alcohol or substance abuse is a factor in the pathogenesis of cte pathology. chronic traumatic encephalopathy is not the “invisible wound” of war addressing cte in another critical community, in 2022 we reported the first large-scale analysis for cte in the brains of military personnel.19 our laboratory is the department of defense/uniformed services university brain tissue repository, which is a brain bank exclusively dedicated to the collection and study of brain specimens derived from service members. we accept and comprehensively evaluate brain donations from anyone who has served in the military, irrespective of tbi history, neuropsychiatric disease, or other factors. among our primary missions is identifying the underlying pathology for the so-called “invisible wound” of war: the frequent development of persistent cognitive, behavioral, and/or motor symptomatology following exposure to combat-related, and particularly blast-related, tbi which by routine head imaging shows no consistent anatomic defects. for our report, published in the new england journal of medicine, we evaluated 225 consecutive military brain donations involving a cohort with an age range of 18-87 years (average age of death: 48 years) and representing active duty and retired military personnel from all service branches. represented were high frequencies of known military tbi exposure (20% with blast exposure, 9.3% with military impact-type tbi unassociated with blast), contact sports history (26.7%), and history of significant impact-type tbis in civilian life unrelated to sports play (e.g. from motor vehicle accidents, assaults, falls leading to concussion, loss of consciousness/coma, skull injury, intracranial bleeding, and/or significant post-traumatic sequelae). further, the cohort had high rates of psychiatric disease (39.1%, most commonly post-traumatic stress disorder), alcohol and/or substance abuse (43.1%), and death by suicide (22.7%); these rates are unfortunately comparable to reported rates in active duty and/or retired military populations.20-24 we blindly assessed all cerebral-cortex containing, at8-immunostained slides that were available for each case, which amounted to a degree of evaluation that met or far exceeded current diagnostic guidelines for cte10 (average: sections from 13 blocks per case, range: 5-23). we identified cte pathology in ten of the 225 cases (4.4%). as can be inferred, cte pathology did not correspond to the large majority of cases involving psychiatric disease, alcohol and/or substance abuse, and/or suicide. further, when cte was identified, it tended to be mild, or only barely diagnostic: five of the ten cte cases only involved a single cte pathognomonic lesion. importantly, all ten of the cte cases identified in our series involved a past history of participation in contact sports with or without an additional history of significant civilian impact-type tbi unrelated to sports (e.g. motor vehicle accident, physical assault, etc.). the relative risks for civilian tbi exposures, especially contact sports participation (relative risk not computable owing to 100% frequency of contact sport history in cte cases; 95% ci: 6.16 to infinity) were substantially higher than those for military exposures, especially blast exposure. though our study was underpowered precisely because there were few cases of cte, in comparison to impact-type tbi exposures evaluated in our study, blast exposure history was the only tbi measure that had no significant association with cte pathology (relative risk: 1.71; 95% ci: 0.46-6.37). ultimately, our study suggests that cte is relatively uncommon in the brains of military personnel, and when identified is overwhelmingly in the context of civilian tbi exposures, namely contact sports participation, and therefore is potentially unrelated to a military occupation at large. further, cte does not seem to account for the high rates of alcohol and/or substance abuse, psychiatric diagnoses suffered by service members. finally, our series also suggests that blast exposure is not an independent risk factor for cte development, though further research is needed. relevance of apoe gene status and chronic traumatic encephalopathy? because not all of those who are exposed to impact-type tbi develop cte pathology, even when repetitive and even in the context of contact sports participation, it certainly stands to reason that other risk factors or predispositions may be involved in pathogenesis. to date, pursuits toward the identification of potential genetic risk factors for cte have been relatively lacking. however, in 2022 researchers atherton et al. published findings on the “association of apoe genotypes and chronic traumatic encephalopathy” in the journal jama neurology.25 a total of 364 consecutive brain donations sourced from the veterans affairs-boston university-concussion legacy foundation brain bank were utilized in the study, including from 294 donors whose brains had known cte pathology and 70 controls with histories of repetitive tbi exposure but no cte pathology, all aged 47-77 years at death. the overall allele frequency for apoeε4 was 0.20. while the researchers found no significant association between cte status and apoe genotype, they did find statistically significant associations between apoeε4 status and cte stage (mckee stages i-iv) when considering donors aged >65 years, and even when adjusted for adnc. the authors also suggest through from their statistical assessment that apoeε4 status is associated with a similar risk of a given cte stage as that from playing more than 7 years of american football. further, the researchers report an association between apoeε4 status and dementia in repetitive tbi-exposed individuals >65 years of age, however this association is lost after adjusting for adnc. regardless, the authors ultimately conclude that they have provided evidence that apoeε4 status may be a risk factor for cte pathological and clinical outcomes, and imply that knowledge of apoeε4 status may be relevant towards precision medicine and with regard to individual decision-making on contact sport participation. obviously, more research using larger sample sizes, prospectively gathered materials for this purpose, and performed in other independent laboratories will be necessary to validate and support these suggestions. there are a number of important concerns that may emerge from this impactful paper with regard to interpreting the findings. first and foremost, there were no statistically significant associations between apoeε4 status and outcomes of cte status (i.e. yes or no), cte stage, or clinical dementia in repetitive tbi-exposed individuals ≤65 years-old. while the authors excluded cases with adnc to support their conclusions for individuals aged >65 years, this does not account for age-related ptau accumulation that may be incidental nor does it account for the 22 total cases with diagnoses of frontotemporal lobar dementia-tau that are reported to be within the study; certainly, these factors may impact cte staging efforts. further, in addition to analyzing associations with cte stage, the researchers also performed a digital, quantitative assessment of ptau burden in dorsolateral prefrontal samples. while the authors calculated statistically significant p-values for the association of apoeε4 status and this quantitative measure of ptau burden, the relatively weak 1.04 or after excluding adnc cases is coupled with a 95% ci of 0.48-1.60. interestingly, and unexpectedly, the results from weighing apoeε4 status with the quantitative assessment of ptau burden also did not correlate with the results analyzing cte stage with respect to apoeε4 status; granted, while the assessment of ptau burden was performed only in the dorsolateral prefrontal cortex, this lack of correlation may nonetheless have implications regarding the mckee cte staging scheme. finally, what we found most interesting was the authors’ assessment of only former american football players within the study, which included a total of 323 cases. in this particular cohort, the authors found no significant association between duration of play and outcomes of clinical dementia or quantitative ptau burden in the dorsolateral prefrontal lobe. the authors did report statistical associations between duration of play and cte status and stage, however these were surprisingly quite weak. the or for cte status and years of play was 1.25 (95% ci: 1.13-1.38) for individuals aged ≤65 and 1.32 (95% ci: 1.13-1.55) for individuals aged >65 after exclusion of adnc cases. this data appears to suggest that our widely held concepts regarding the clinicopathologic correlation of this disease continue to need further investigation. evidence of sodium channelopathy and altered node of ranvier structure following concussive traumatic brain injury little is understood regarding the physiology of the immediate axonal response to tbi, including pathologic alterations such as the formation of axonal swellings and other processes that may be related to or co-occur with diffuse axonal injury (dai). further, a comprehensive understanding of pathophysiologic mechanisms underlying concussion and post-concussive symptoms and recovery continues to elude us. in 2022, authors song et al. reported their use of a miniature swine model, relevant due to their gyrencephalic brain structure, for concussion/dai to study alterations in axonal voltage-gated sodium channels and the structure of the nodes of ranvier (nors) after acute tbi.26 nine animals were subjected to rotational tbi and sacrificed at different time intervals following injury (6 hours, 72 hours, and 2 weeks) and four served as controls. in parallel, researchers also studied human brain tissues derived from five individuals who died after acute, moderate-to-severe tbi in time intervals that were ≤14 days after injury (survival time ranged from 23 hours to 14 days, median survival time was 3 days), along with five sexand age-matched controls. in both the pig and human materials, the authors confirmed evidence of dai via demonstration of increased in app immunolabeling in all injured tissues, and not in control tissues. from there, the authors used immunofluorescent antibodies targeting sodium channels within the nors, using antibody to nach isoform 1.6 (nav1.6), as well as antibodies targeting other axonal proteins including caspr, which is an axonal membrane protein accentuated in the paranodal region, and axonal cytoskeletal proteins βiv-spectrin, ankg, and nfasc186, which are more highly expressed within the nor itself. in the pigs, the authors demonstrated marked and statistically significant loss of nav1.6 labeling, indicating axonal sodium channel loss, at 6and 72-hours following injury, with a recovery to the level of control animals at 2 weeks following injury. in association with sodium channel loss, the authors noted significant increase in nor length 72-hours following injury, with partial recovery at 2 weeks, along with formation of so-called “void nodes” (defined by the complete loss of nav1.6 with normal paranodal caspr labeling) and “heminodes” (defined by loss of caspr on a single side of the nor, associated with normal or reduced nav1.6). finally, the authors noted loses of βiv-spectrin, ankyrin g, and neurofascin 186 within the nors following acute tbi, with evidence diffusion of the proteins into the paranodal space. the above observations were also noted in the human tissues, which demonstrated marked loses of nav1.6 and similar changes to the structure of the nors and analyzed proteins. considering the vital role of voltage-gated sodium channels and nor structure for the generation of action potentials, these findings provide critical evidence that neurologic dysfunction following concussion, or other tbi, may in part be due to sodium channelopathy and altered nerve conduction by these mechanisms. further, researchers in the study also found that many of the nor changes were observed adjacent to app-positive axonal swellings, including at the 6-hour mark of survival in the swine model. this observation potentially indicates new and potentially more sensitive biomarkers by which we can detect dai. the role of tdp-43 in exacerbating neuropathology and outcomes following traumatic brain injury beyond tau there is increasing interest in the role of other known neurodegenerative proteins, whether directly or otherwise, involved in neuropathologic and ultimately clinical outcomes following tbi(s). moreover, mechanisms by which tbi may be related to exacerbated neurodegenerative pathologies and/or multiple neurodegenerative pathologies are a highly active area of investigation. in 2022, authors gao et al. reported their findings from a study focused on the influence of tdp-43 in driving adnc and cognitive impairment in mice following tbi.27 as part of their impressive study, the authors utilized 5xfad app transgenic mice exposed to a model of mild closed head injury using a well-published, commercially available impact device. trauma was induced at 2 months of age. via novel object recognition assessment and a maze test, the researchers found that a single mild tbi exposure was associated with the accelerated development of memory and cognitive deficits in the app transgenic mice, compared to non-exposed app transgenic mice and app wildtype mice; the deficits in the app transgenic mice were recognized at 3 months of age, whereas typically these mice do not show such deficits until beyond 5 months of age. further, the authors demonstrate significantly increased deposits of aβ plaques and significantly increased expressions of ptau, acetylated tau, β-site amyloid precursor protein cleaving enzyme 1 (bace1, an enzyme implicated in aβ plaque formation), and tdp-43 in comparison to non-exposed app transgenic mice and app wildtype mice. it is interesting to note that app transgenic mice, as they age, do not develop tauopathy. further, via reverse transcription and real-time polymerase chain reaction analysis performed on harvested hippocampal tissue 24 hours after tbi exposure, the authors report significantly elevated inflammatory markers and pro-inflammatory cytokines (vimentin, tnfα, il-1β, and il-6) in app transgenic mice following tbi. as such, the authors demonstrate that a single impact-type tbi exacerbates neuropathological and clinical deficits in mice predisposed to aβ pathology, and notably among the neuropathological observations were increases in tdp-43, tau, and indices of neuroinflammation. the researchers then sought to address the question of how tdp-43 may be involved in the exacerbation of the neuropathological and clinical deficits noted in the app transgenic mice. this was accomplished via a series of experiments inducing single and/or multiple tbis following the injection of rna products via viral vectors, which included those designed to silence endogenous tdp-43 expression, into the hippocampus of the app transgenic mice. the authors report that cognitive and memory deficits, as well as neurodegenerative indices (including immunohistochemical assessment of aβ plaques and western blot analyses including for aβ, bace1, ptau, and acetylated tau) and neuroinflammatory indices (immunohistochemical analysis for astrocytes [gfap] and microglia [iba1]) after single and repeated tbi in the app transgenic mice were all prevented/alleviated following induction of tdp-43 knockdown in comparison to the control group, and were also reverted in a group which was administered a tdp-43 knockdown rescue protocol. furthermore, app transgenic mice injected with a viral vector to induce tdp-43 overexpression alone, without tbi, also demonstrated accelerations in memory and cognitive impairments, mimicking those who had sustained tbi. these cases also showed dramatic increases in indices including bace1, acetylated tau, ptau, phosphorylated glycogen synthase kinase-3β (pgsk3β, an enzyme known to play an important role in tau phosphorylation and adnc) and phosphorylated nf-κb, and also increased labeling of astrocytes and microglia. other components of the study demonstrate evidence that tdp-43 overexpression in app transgenic mice, whether induced by tbi or by viral vector injection, downregulates the expression of synaptic proteins including various glutamate receptor subunits and psd95, and thus interferes with synaptic integrity. finally, the researchers report findings investigating the role of tdp-43 in tau phosphorylation and aβ deposition through work with cultured hippocampal neurons from tau transgenic mice and app transgenic mice. they report evidence that tdp-43 knockdown in tau transgenic neuronal cells is associated with reduced expression of ptau and pgsk3β, and also that tdp-43 is upstream from gsk3β in driving tau phosphorylation. in neuronal cells from app transgenic mice, the researchers report that tdp-43 knockdown was associated with reduced expression of bace1 which provides further evidence that tdp-43 has a driving role in aβ formation. ultimately, the extensive and thorough research highlighted by this study provides fascinating evidence that not only does tdp-43 play an instrumental role in driving both tau phosphorylation and aβ formation in the context of ad, but also may be a principle link that connects tbi with the subsequent development of neurodegenerative disease. in memoriam: john q. trojanowski, m.d., ph.d. we conclude this year’s listing on a solemn note, but also in awe of the legacy left by a titan in the field of neurodegeneration, neuropathology in general, and in the context of this review, neurotrauma in particular. on february 8th, 2022, we lost dr. john q. trojanowski due to fatal complications from a fall. dr. trojanowski received his m.d. and ph.d. degrees from tufts university in 1976. following this, he completed residency training in pathology and fellowship training in neuropathology at the massachusetts general hospital and the university of pennsylvania, respectively. he stayed in pennsylvania and followed his training with an extraordinary career that included the establishment and direction of the university of pennsylvania center of neurodegenerative disease research center with his wife and scientific partner, dr. virginia lee (figure 3), co-authorship in over 1,400 peer-reviewed papers, an abundance of accolades, and an immeasurable degree of influence that he impressed upon a lifetime’s worth of trainees, other mentees, and professional colleagues. pertaining to work in neurotrauma, john was one of the foundational members of the collaborative neuropathology network characterizing outcomes of tbi (connect-tbi).28 he was a co-author on several important and highly cited studies concerning pathologic protein accumulation due to tbi in both animal and human studies from the late 1990s and early 2000s.29-34 more recently he had been involved in cte research which included but certainly was not limited to a major 2020 study published in brain which established that the immunophenotypes of astroglial and neuronal ptau in cases of cte are distinct and individually resemble age-related tau astrogliopathy (4r-tau immunoreactive, thorn-shaped astrocytes) and adnc (3r and 4r tau), respectively.35 john’s life and legacy are beautifully memorialized in writings by several of our peers and in the media36-42, and we strongly encourage readers to visit these remembrances. > figure 3. dr. john q. trojanowski (right) and his wife and scientific partner, dr. virginia m. lee (left). together, john and virginia co-founded and directed the center for neurodegenerative disease research at the university of pennsylvania acknowledgements we would like to thank ms. sofia echelmeyer in the office of external affairs and university media services at the uniformed services university for her magnificent illustrative work (figure 1). we also thank dr. edward b. lee from the department of pathology and laboratory medicine at the university of pennsylvania for providing a beautiful photograph of drs. john trojanowski and virginia lee (figure 3). disclaimer the information/content, conclusions, and/or opinions expressed herein do not necessarily represent the official position or policy of, nor should any official endorsement be inferred on the part of, uniformed services university, the department of defense, the us veterans administration, the u.s. government or the henry m. jackson foundation for the advancement of military medicine, inc. conflicts of interest the authors do not have any conflict of interest to declare. references 1. hill ab. the environment and disease: association or causation? proc r soc med. 1965 may;58(5):295-300. pmcid: pmc1898525 2. nowinski cj, bureau sc, buckland me, et al. applying the bradford hill criteria for causation to repetitive head impacts and chronic traumatic encephalopathy. front neurol. 2022 jul 22;13:938163. pmcid: pmc9355594 doi: 10.3389/fneur.2022.938163 3. johnson ve, stewart w, smith dh. widespread tau and amyloid-beta pathology many years after a single traumatic brain injury in humans. brain pathol 2012;22:142–9. pmcid: pmc3979351 doi: 10.1111/j.1750-3639.2011.00513.x 4. shively sb, edgerton sl, iacono d, et al. localized cortical chronic traumatic encephalopathy pathology after single, severe axonal injury in human brain. acta neuropathol 2017;133(3):353-366. pmcid: pmc5325841 doi: 10.1007/s00401-016-1649-7 5. iverson gl, gardner aj, shultz sr, et al. chronic traumatic encephalopathy neuropathology might not be inexorably progressive or unique to repetitive neurotrauma. brain. 2019;142(12):3672-3693. pmcid: pmc6906593 doi: 10.1093/brain/awz286 6. ackermans nl, varghese m, williams tm, et al. 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14. forrest sl, kril jj, wagner s, et al. chronic traumatic encephalopathy (cte) is absent from a european community-based aging cohort while cortical aging-related tau astrogliopathy (artag) is highly prevalent. j neuropathol exp neurol 2019;78(5):398-405. doi: 10.1093/jnen/nlz017 15. postupna n, rose se, gibbons le, et al. the delayed neuropathological consequences of traumatic brain injury in a community-based sample. front neurol 2021;12:624696. pmcid: pmc8008107 doi: 10.3389/fneur.2021.624696 16. suter cm, affleck aj, lee m, et al. chronic traumatic encephalopathy in a routine neuropathology service in australia. j neuropathol exp neurol. 2022;81(10):790-795. doi: 10.1093/jnen/nlac071 17. geddes jf, vowles gh, nicoll ja, et al. neuronal cytoskeletal changes are an early consequence of repetitive head injury. acta neuropathol 1999;98:171-8. doi: 10.1007/s004010051066 18. thom m, liu jy, thompson p, et al. neurofibrillary tangle pathology and braak staging in chronic epilepsy in relation to traumatic brain injury and hippocampal sclerosis: a post-mortem study. brain. 2011;134(pt 10):2969-2981. pmcid: pmc3187539 doi: 10.1093/brain/awr209 19. priemer ds, iacono d, rhodes ch, olsen ch, perl dp. chronic traumatic encephalopathy in the brains of military personnel. n engl j med. 2022;386(23):2169-2177. doi: 10.1056/nejmoa2203199 20. howard jt, stewart ij, amuan m, janak jc, pugh mj. association of traumatic brain injury with mortality among military veterans serving after september 11, 2001. jama netw open 2022;1;5(2): e2148150. pmcid: pmc8837911 doi: 10.1001/jamanetworkopen.2021.48150 21. richardson lk, frueh bc, acierno r. prevalence estimates of combat-related post-traumatic stress disorder: critical review. aust n z j psychiatry 2010;44(1):4-19. pmcid: pmc2891773 doi: 10.3109/00048670903393597 22. tanielian t, jaycox l. (eds.). invisible wounds of war: psychological and cognitive injuries, their consequences, and services to assist recovery. santa monica, ca: rand corporation, 2008. (https://www.rand.org/pubs/monographs/mg720.html). 23. meadows so, engel cc, collins rl, et al. 2015 health related behaviors survey: substance use among u.s. active-duty service members. santa monica, ca: rand corporation, 2018. (https://www.rand.org/pubs/research_briefs/rb9955z7.html) 24. teeters jb, lancaster cl, brown dg, back se. substance use disorders in military veterans: prevalence and treatment challenges. subst abuse rehabil 2017;8:69-77. pmcid: pmc5587184 doi: 10.2147/sar.s116720 25. atherton k, han x, chung j, et al. association of apoe genotypes and chronic traumatic encephalopathy. jama neurol. 2022;79(8):787-796. pmcid: pmc9237800 doi: 10.1001/jamaneurol.2022.1634 [published correction appears in jama neurol. 2023 feb 1;80(2):215. pmcid: pmc9577875 doi: 10.1001/jamaneurol.2022.3673] 26. song h, mcewan pp, ameen-ali ke, et al. concussion leads to widespread axonal sodium channel loss and disruption of the node of ranvier. acta neuropathol. 2022;144(5):967-985. doi: 10.1007/s00401-022-02498-1 27. gao f, hu m, zhang j, hashem j, and chen c. tdp-43 drives synaptic and cognitive deterioration following traumatic brain injury. acta neuropathol. 2022 aug;144(2):187-210. pmcid: pmc9945325 doi: 10.1007/s00401-022-02449-w 28. smith dh, dolle jp, ameen-ali ke, et al. the collaborative neuropathology network characterizing outcomes of tbi (connect-tbi). acta neuropathol commun. 2021;9(1):32. pmcid: pmc7919306 doi: 10.1186/s40478-021-01122-9 29. smith dh, uryu k, saatman ke, trojanowski jq, mcintosh tk. protein accumulation in traumatic brain injury. neuromolecular med. 2003;4(1-2):59-72. doi: 10.1385/nmm:4:1-2:59 30. chen xh, johnson ve, uryu k, trojanowski jq, smith dh. a lack of amyloid beta plaques despite persistent accumulation of amyloid beta in axons of long-term survivors of traumatic brain injury. brain pathol. 2009;19(2):214-223. pmcid: pmc3014260 doi: 10.1111/j.1750-3639.2008.00176.x 31. ikonomovic md, uryu k, abrahamson ee, et al. alzheimer's pathology in human temporal cortex surgically excised after severe brain injury. exp neurol. 2004;190(1):192-203. doi: 10.1016/j.expneurol.2004.06.011 32. uryu k, laurer h, mcintosh t, et al. repetitive mild brain trauma accelerates abeta deposition, lipid peroxidation, and cognitive impairment in a transgenic mouse model of alzheimer amyloidosis. j neurosci. 2002;22(2):446-454. pmcid: pmc6758680 doi: 10.1523/jneurosci.22-02-00446.2002 33. uryu k, chen xh, martinez d, et al. multiple proteins implicated in neurodegenerative diseases accumulate in axons after brain trauma in humans. exp neurol. 2007;208(2):185-192. pmcid: pmc3979356 doi: 10.1016/j.expneurol.2007.06.018 34. smith dh, chen xh, nonaka m, et al. accumulation of amyloid beta and tau and the formation of neurofilament inclusions following diffuse brain injury in the pig. j neuropathol exp neurol. 1999;58(9):982-992. doi: 10.1097/00005072-199909000-00008 35. arena jd, smith dh, lee eb, gibbons gs, irwin dj, robinson jl, lee vm, trojanowski jq, stewart w, johnson ve. tau immunophenotypes in chronic traumatic encephalopathy recapitulate those of ageing and alzheimer’s disease. brain. 2020 may 1;143(5):1572-87. pmcid: pmc7241956 doi: 10.1093/brain/awaa071 36. lee eb. john q. trojanowski: neuropathology icon. acta neuropathol. 2022 apr;143(4):419-425. pmcid: pmc10259176 doi: 10.1007/s00401-022-02413-8 37. lee eb. the continuing legacy of john. acta neuropathol. 2022;144(6):1063-1064. doi: 10.1007/s00401-022-02514-4 38. nelson pt, lee eb, judkins ar. john q. trojanowski, md, phddecember 17, 1946–february 8, 2022. j neuropathol exp neurol. 2022 jun 1;81(6):434-6. doi: 10.1093/jnen/nlac029 39. chen-plotkin as. john q. trojanowski, 'tour de force' in neurodegeneration (1946-2022). nat neurosci. 2022;25(6):675-676. doi: 10.1038/s41593-022-01087-5 40. irwin dj, lee eb. a tribute to john q. trojanowski (1946-2022). j clin invest. 2022 may 16;132(10):e161019. pmcid: pmc9106356 doi: 10.1172/jci161019 41. lashuel ha. remembering john q trojanowski, in his own words: a life dedicated to discovering building blocks and using them to build bridges of knowledge, collaboration, and discovery. npj parkinsons dis. 2022;8(1):43. pmcid: pmc9005626 doi: 10.1038/s41531-022-00310-1 42. kolata g. john q. trojanowski dies at 75; changed understanding of brain diseases. the new york times, 2022 mar 1 (https://www.nytimes.com/2022/03/01/health/john-q-trojanowski-dead.html). copyright: © 2023 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. selective cellular and regional vulnerability in frontotemporal lobar degeneration: a scoping review feel free to add comments by clicking these icons on the sidebar free neuropathology 6:11 (2025) review selective cellular and regional vulnerability in frontotemporal lobar degeneration: a scoping review kashif ravasia1 and veronica hirsch-reinshagen2 school of medicine, university of british columbia, vancouver, canada division of neuropathology, vancouver general hospital and university of british columbia, vancouver, canada corresponding author: veronica hirsch-reinshagen · department of anatomical pathology · vancouver general hospital and university of british columbia · rm 1207899 w12th ave · vancouver · canada veronica.hirsch@vch.ca submitted: 15 august 2024 accepted: 13 march 2025 copyedited by: georg haase published: 09 april 2025 https://doi.org/10.17879/freeneuropathology-2025-5812 keywords: ftld, selective vulnerability, human post mortem, histology, tau, tdp-43, fus abstract the three main types of frontotemporal lobar degeneration (ftld) are characterized by the accumulation of abnormal proteins, namely tau, tdp-43 and fus. the distribution of these proteins within different human brain regions is well known, as is the range of morphological variability of the cellular inclusions they form. compared to the extensive knowledge that exists about distinct protein aggregates in ftld, surprisingly little is known about the specific cell (sub)types that these inclusions affect. even less is known about disease-specific abnormalities other than protein inclusions in affected and unaffected areas. these are non-trivial knowledge gaps. first, knowing which cell subtypes are vulnerable or resilient to the development of pathological protein inclusions is crucial to understand the cellular disease mechanisms. second, mounting evidence suggests that non-cell autonomous mechanisms may play important roles in neurodegenerative conditions. for example, astrocytic tau pathology is associated with synaptic loss in corticobasal degeneration but not in progressive supranuclear palsy. furthermore, changes that are more difficult and time-consuming to quantify, for example loss of a specific neuronal subtype that does not develop pathological inclusions, remain virtually unexplored and their relevance for disease progression are unknown. this scoping review is an attempt to collate all histological evidence from human studies that address the question of cell-specific vulnerability in the most common ftld subtypes. by taking a systematic approach including various brain cell types such as neurons and their subtypes as well as astrocytes, microglia and oligodendrocytes and the entire central nervous system with its affected and unaffected regions, this review summarizes the current status in the field and highlights important knowledge gaps. introduction frontotemporal dementia (ftd) is a clinical neurodegenerative syndrome characterized by prominent deficits in behaviour, language, and/or personality, with relative sparing of memory early in the disease course. ftd is the third most common form of neurodegenerative dementia after alzheimer’s disease (ad) and dementia with lewy bodies, and affects about 20,000 to 30,000 people in the united states according to estimates by knopman and roberts1 alone. this prevalence is similar to that identified in the united kingdom2. ftd is phenotypically heterogeneous, including cases of behavioural variant ftd (bvftd) and primary progressive aphasia (ppa), the latter of which can be additionally subclassified into nonfluent variant ppa (nfvppa), semantic variant ppa (svppa), or logopenic variant ppa (lvppa)3. these subtypes can often be difficult to disentangle clinically, as additional symptoms may develop as the disease progresses, and symptoms can mimic or overlap with other neurodegenerative conditions such as ad, parkinson’s disease (pd), or amyotrophic lateral sclerosis (als)3. the neuropathological condition underlying most cases of ftd, termed frontotemporal lobar degeneration (ftld), is also highly heterogeneous. the clinical diagnosis of ftd and the neuropathological diagnosis of ftld are not always concordant. patients with pathological ftld and significant loss of memory may be diagnosed in life with ad, and those with significant motor symptoms may be diagnosed with als or pd4,5. conversely, some cases of clinically diagnosed bvftd and lvppa meet neuropathological criteria for ad6,7. as the name implies, ftld is characterized by preferential atrophy of the frontal and temporal lobes and abnormal protein inclusions in neurons and glial cells. historically, the first inclusions to be recognized were argyrophilic pick bodies in pick’s disease (pid)8. until the 1980s, cases with clinical ftd and a ftld pattern of brain atrophy but without pick bodies were described as atypical pid cases8,9. with the advent and widespread adoption of immunohistochemistry (ihc), it became possible to identify novel protein inclusions in ftld cases. the first one to be identified was hyperphosphorylated tau10,11, which ultimately led to a revised and expanded categorization of tau-positive ftld, including pid. in 2006, another protein, transactive response dna binding protein 43 (tdp-43), was identified in about 90 % of cases of tau-negative ftld12,13. most of the remaining cases were later found to contain a protein known as rna-binding protein fused in sarcoma (fus)14,15. while there remains a small group of cases that stain positive for ubiquitin but negative for tau, tdp-43, and fus, over 99 % of ftld cases can now be classified into tauopathies (ftld-tau), ftld with tdp-43 pathology (ftld-tdp), and ftld with fus pathology (ftld-fus)15. each of these neuropathological entities exhibits known patterns of neuronal and glial inclusions16–18 in the neocortex, deep grey nuclei and infratentorial structures. in the different ftld pathologies, the selective vulnerability of different brain cell types such as neuronal subtypes, astrocytes, microglia and oligodendrocytes is incompletely understood. selective neuronal vulnerability has been a focus of recent research on neurodegenerative diseases including ad and pd. in each of these syndromes, specific sets of neurons degenerate more quickly and more consistently than other neuronal populations19,20. furthermore, in each disease affected neuronal populations display similar alterations in organelle distribution, neurotransmitter receptors, electrophysiology, and/or morphology19,20. this patterned neurodegeneration has been less studied in ftld. in addition, ftld pathology prominently affects glial cells whereas in ad or pd the pathological inclusions are mainly neuronal. the pathophysiological effects of the glial involvement in ftld are largely unknown. pathological glial involvement has been shown in amyotrophic lateral sclerosis (als), where progressive motor neuron degeneration has been shown to be modulated by non-neuronal cells through a process known as non-cell autonomous neurodegeneration21,22. therefore, studies that seek to understand the processes responsible for patterned neurodegeneration should consider and include the evaluation of non-neuronal dysregulation as one possible pathological mechanism of disease progression. here, we review the available literature on selective neuronal vulnerability in ftld and include data on glial pathology and its relationship to neuronal pathology wherever possible. the review is organized primarily according to the type of ftld proteinopathy and the ftd disease subtype. this review places special emphasis on human post mortem studies involving colocalization of pathology using ihc or immunofluorescence, as these studies allow identification of specific cell populations and definitive neuropathological diagnosis of each case. in the ihc studies described below, the most commonly used subtype-specific markers include parvalbumin and calbindin for inhibitory neurons and subtype-specific neurotransmitters or enzymes involved in neurotransmitter metabolism such as choline acetyltransferase (chat) or tyrosine hydroxylase (th). glial cell-specific markers include glial fibrillary acidic protein (gfap) and aquaporin 4 (aqp4) as pan-astrocytic markers, and vimentin and cd44 as markers of activated or reactive astrocytes. microglial markers include cluster of differentiation 68 (cd68), human leukocyte antigen – dr isotype (hla-dr) and ionized calcium-binding adapter molecule 1 (iba1). 1. frontotemporal lobar degeneration with tau pathology tauopathies, named for the accumulation of microtubule-associated protein tau, account for approximately 40 percent of ftld cases23. while numerous tauopathies have been described and characterized, this review focuses on the three ftld tauopathies for which the existing literature is most robust: pick’s disease (pid), corticobasal degeneration (cbd), and progressive supranuclear palsy (psp). 1.1 pick’s disease 1.1.1 general features pid typically presents with bvftd or lvppa, and the gross appearance involves frontotemporal atrophy with relative sparing of the rolandic cortex and the posterior superior temporal gyrus24 (figure 1). neuropathologically, pid demonstrates astrocytosis, loss of neurons, ballooned neurons with eosinophilic cytoplasm, and extensive spongiosis25 (figure 1). most strikingly andof pathognomic value, pid demonstrates pick bodies, i.e. round, solitary, argyrophilic neuronal cytoplasmic inclusions (ncis) primarily located in pyramidal neurons and in the dentate granular neurons of the hippocampus26 (figure 1). glial pathology has also been observed in pid cases , including ramified astrocytes and small oligodendroglial inclusions24. pick bodies consistently display 3-repeat (3r) tau pathology, while astrocytes display a variable combination of 4r and 3rtau16,25,27,28, depending on the cases studied. many of the studies evaluating the selective cellular vulnerability in pid were conducted before the modern classification scheme for ftld making it difficult to confirm whether these cases actually correspond to pid. figure 1. pathological features of pick’s disease. a. low power image reveals relative preservation of the posterior aspect of the superior temporal gyrus (asterisk, 50x). b. hematoxylin and eosin stain reveals neocortical ballooned neurons (600x). c. bielschowsky silver stain reveal numerous argyrophilic round inclusions (pick bodies) in the granular neuronal cells of the hippocampal dentate fascia (600x). d. tau immunohistochemistry reveals numerous tau-immunoreactive pick bodies and granular neuropil staining (600x). 1.1.2 neuronal pathology table 1 provides a summary of the known neuronal subtypes that bear tau pathology and/or undergo selective neurodegeneration in pid. many regions of the frontal and temporal cortex display marked neuronal loss which begins in the outer cortical layers and later progresses to involve the deeper layers of the cortex and extracortical regions25. pyramidal cell density is most markedly reduced in lamina ii of the cortex, with lesser involvement of laminae iii and v, and occurs in the early stages of the disease29. this finding is consistent with studies of laminar distribution of tau pathology in pid, which have demonstrated that pick bodies are prominent in laminae ii and iii, with variable involvement of deeper cortical layers25,30. table 1: summary of specific neuronal subtypes susceptible to tau accumulation and/or neurodegeneration by brain region. region pick’s disease cbd psp neocortex superficial pyramidal cells; von economo neurons deep and small superficial neurons – unknown subtype; loss of excitatory synapses corticocortical projection neurons (pre-supplementary motor area); inhibitory interneurons (primary motor cortex); upper motor neurons; loss of excitatory and inhibitory synapses hippocampus dentate granule cells dentate granule cells and pyramidal ca2 neurons parahippocampal gyrus – unknown subtypes; pyramidal ca1 neurons basal ganglia caudate > putamen – unknown subtype caudate and putamen > gp – unknown subtype gabaergic neurons thalamus mediodorsal nucleus – unknown subtype ventrolateral portion – unknown subtype intralaminar nuclei (centromedian and parafascicular); ventrolateral nucleus brainstem raphe nuclei (5-ht) and lc (na) > sn; pontine nuclei; dm x sn – both da and gaba snr (parvalbumin); snc; ventral tegmental area and the parabrachial pigmented nucleus (th); lc; mesencephalic motor nuclei; mesopontine nuclei (cholinergic and non-cholinergic cells) cerebellum mossy fibers, monodendritic brush cells, dentate projection neurons purkinje cells, cerebellar dentate neurons purkinje cells, cerebellar dentate neurons abbreviations: cbd: corticobasal degeneration; da: dopamine; dm x: dorsal motor nucleus of the vagus nerve; lc: locus coeruleus; na: noradrenaline; sn: substantia nigra; snr: substantia nigra pars reticulata; snc: substantia nigra pars compacta; psp: progressive supranuclear palsy; th: tyrosine hydroxylase; 5-ht: 5-hydroxytryptamine or serotonin. von economo neurons (vens) are found in lamina v of the anterior cingulate and frontoinsular regions of the cortex and are morphologically distinct from neighboring pyramidal neurons31. vens have been shown to be significantly affected in confirmed pid cases and degenerated more rapidly than neighboring cells32. moreover, vens demonstrate tau aggregation and cytoplasmic swelling in all stages of pid31, which is consistent with the observation that early neuron loss and astrocytosis are most significant in the orbitofrontal and mediofrontal cortices in this disease25,33. in the hippocampus, the dentate gyrus is most significantly involved in pid34, with greater tau pathology in granule than in hilar cells35. one study found a trend toward decreased calbindin immunoreactivity in dentate granule cells in pid, as compared with other pathological subtypes of ftld. this trend did not reach statistical significance, and loss of immunoreactivity may also be secondary to generalized neuronal atrophy rather than to selective vulnerability of dentate cells36. as early as 1998, it was observed that the striatum can exhibit atrophy in severe or longstanding cases of pid, with greater involvement of the caudate than the putamen26. these findings have been confirmed in a more recent study on the progression of pid, in which the researchers also detected late developing tau depositions in the globus pallidus25. these tau inclusions are primarily intra-neuronal, but the phenotypic features of the affected neurons have not been studied in detail25. limited evidence suggests the mediodorsal nucleus of the thalamus is preferentially affected in pid37. furthermore, asymmetric thalamic atrophy has been observed by volume-based magnetic resonance imaging (mri)38, but the affected neuronal populations remain unknown. the brainstem can also be neuropathologically affected in pid. in one study, pontine involvement was found to develop after the onset of limbic and neocortical disease, and the serotonergic and noradrenergic nuclei of the raphe nuclei and locus coeruleus exhibited more severe pathology relative to the remaining brainstem25. involvement of the substantia nigra, the dorsal motor nucleus of the vagus nerve and other brainstem nuclei was observed to coincide with pontine pathology, while involvement of the inferior olivary nuclei and medullary pyramids occurred only in advanced disease stages25. there is rare literature on the involvement of the cerebellum in pid. in 1999, braak et al. determined that mossy fibers, monodendritic brush cells, and dentate projection neurons were involved in a set of cases classified as pid, but these findings have not been replicated yet39. in summary, the distribution of tau pathology and neuronal loss in the cortex of patients with pid seem to be correlated, particularly with regard to vens. yet, the relationship between tau pathology and rate of loss of different neuronal populations is still unknown for the remaining brain areas such as hippocampus, brainstem and cerebellum, where the distribution of the pid tau pathology has been described in detail. 1.1.3 glial involvement pid cases display significant astrogliosis and astroglial tau pathology in cortical regions. astroglial tau pathology is most notable in the orbitofrontal cortex and mediofrontal cortices, which show significant neuronal loss and tau-immunopositive ramified astrocytes even in early stages of disease25. in early stages of pid, ihc for gfap, a marker that is increased in reactive astrocytes, demonstrates widespread astrocytic reaction in laminae i, iii, and iv33. some of these astrocytes also stain positive for tau, with one double-labelling ihc experiment showing that 23 % of cortical gfap-positive astrocytes were also positive for tau and that tau-positive filamentous inclusions can sometimes displace gfap-positive fibrils29. interestingly, markers of astrocytic apoptosis and dysregulated ceramide metabolism, thought to be neuroinflammatory and pro-apoptotic, have been found in regions of neuronal loss in pid33,40. oligodendroglial coiled bodies are restricted to areas affected by neuronal tau pathology and degeneration in pid41. interestingly, a pid-specific oligodendroglial inclusion has been described24,41, but its relationship to oligodendrocyte degeneration or axonal loss is unknown. similar to astrogliosis, microgliosis has been consistently described in the frontal and temporal cortex of pid for decades. this finding has been confirmed in cases meeting revised criteria following the development of the modern ftld classification system13,18,42. cortical microglial cells demonstrate activation by enhanced staining for hla-dr29 and this increased microglial activation is especially notable in areas displaying a high burden of pick bodies43. the extent of grey matter microglial involvement in pid seems to be quite substantial. indeed, comparative studies have shown a significative increase in grey matter microgliosis not only relative to controls but also relative to cases of cbd, psp, and rare tauopathies42. these differences are observed regardless of the ihc protocol used to identify microglia, including cd68, iba1, and cr3/43, a novel antibody that reacts with the human leukocyte antigen isotypes dr, dp, and dq42. additional studies consistently show microgliosis in the white matter underlying the frontal and temporal cortex29,42,43. when activated microglia are measured specifically, whether by using cd68 or hla-dr, more gliosis is seen in the white matter than in the grey matter29,42. by contrast when total microglial burden is measured using iba1, the difference between these regions is less evident42. microglial dystrophy is also more severe in subcortical white matter than in the cortical grey matter, with many microglia demonstrating a loss of fine branches and unusual cytoplasmic morphology42. together, these data suggest that neuronal loss, astroglial reactivity and tau pathology significantly yet not perfectly overlap in the cortex in pid. how these three alterations relate to each other has not been described in detail. furthermore, microglial activation and dystrophy seem greater in the white matter than in the overlying cortex. the relationship between microglial alterations, oligodendroglial changes, axonal and neuronal loss as well as tau pathology remains unclear. finally, despite the early and consistent involvement of the hippocampus, and the known involvement of subcortical structures later in pid, there are no additional details on glial involvement and their relationship to neuronal tau pathology in these areas. 1.2 corticobasal degeneration 1.2.1 general features corticobasal degeneration (cbd) is a 4r tauopathy initially described as the underlying pathological condition of a clinical syndrome now termed corticobasal syndrome (cbs). cbs is characterized by asymmetric rigidity and apraxia, dystonia, myoclonus, and cortical symptoms44. however, it is now understood that cbd can also present clinically as nfvppa, progressive supranuclear palsy syndrome (psps), frontal behavioural-spatial syndrome (fbs), or the classic cbs motor syndrome45–47. moreover, cases of clinically diagnosed cbs have been shown on post-mortem pathological examination to meet diagnostic criteria for psp, pid, ad, creutzfeldt-jacob disease, and ftld-tdp48,49. neuropathologically, cbd is characterized by cortical atrophy that tends to involve the perirolandic cortex but can also involve temporal regions associated with language, and anterior frontal regions associated with behaviour and personality50. cbs cases exhibit cortical spongiosis and astrogliosis of the superficial laminae and ballooned neurons in laminae iii, v, and vi. diagnostic tau-immunoreactive pathology includes neurofibrillary tangles, numerous thread-like lesions in white and grey matter50 and characteristic glial lesions such as astrocytic plaques and oligodendroglial coiled bodies51 (figure 2). figure 2. pathological features of corticobasal degeneration and progressive supranuclear palsy. a, b. gallyas staining reveals different argyrophilic astrocytic inclusions. in cbd (a) only the distal processes of astrocytes are stained, whereas in psp (b) tufted astrocytes display increased proximal cytoplasmic staining (600x). c, d. tau immunohistochemistry (ihc) reveals differences in astrocytic morphology between cbd and psp with astrocytic plaques in the former (c) and tufted astrocytes in the latter (d) (600x). e, f. tau ihc highlights neurofibrillary tangle pathology in both conditions, with globose tangles (f) being more frequent in psp. note increased background thread pathology in cbd (c, e) (600x). g, h. oligodendroglial coiled bodies are seen on tau ihc in both conditions (600x). 1.2.2 neuronal pathology table 1 provides a summary of the known neuronal subtypes that bear tau pathology and/or undergo selective neurodegeneration in cbd. cbd is characterized by atrophy of the frontal, parietal and temporal cortex, with consistent involvement of the premotor cortex52. in keeping with the diverse range of clinical presentations, 4r-tau can accumulate in a variety of neuroanatomical regions. in classical cbs cases, 4r-tau shows peri-rolandic distribution46, but there are also cases with predominant temporal involvement46,53 and some with an unusual degree of frontal tau pathology53. cortical neurons demonstrate ballooning and achromasia in deep cortical layers and a variety of tau immunoreactive pathology ranging from granular pre-tangles to more filamentous neurofibrillary tangles51,54. small neurons in upper cortical layers are most vulnerable to cbd51. in some cases of cbd, hippocampal neurons in the ca2 region and the dentate gyrus can show tau pathology 50. the extent of tau pathology varies by clinical presentation, being more pronounced in psps than in cbs despite pathological confirmation of cbd in both sets of cases46. cbd also exhibits extensive pathology in the basal ganglia. filamentous neuronal inclusions are often visible in the caudate and the putamen, but less consistently seen in the globus pallidus51. neuronal pathology tends to become more severe with disease progression49,52. confirmed cases of cbd often exhibit mild-to-moderate neuronal loss and tau-positive neuronal inclusions and threads in the thalamus55, which is consistently affected in its ventrolateral portion56. in cbd, the brainstem is not classically considered as a region of interest, but the substantia nigra can be affected. in these cases, the substantia nigra appears markedly depigmented with pale intracytoplasmic inclusions in surviving neurons57, numerous pre-tangles, and severe loss of dopaminergic and gabaergic neurons but without significant astrocytic plaque pathology51,58,59. some cbd cases also show tau deposition in the tegmentum and inferior olivary nucleus. this phenomenon has not been studied in detail, but current evidence suggests that medullary tau deposition is more common in cases that clinically present as psps46. the cerebellum has not been extensively studied in cbd, although variable neuronal loss and gliosis were found in the cerebellar dentate nucleus together with scattered cortical purkinje cell axonal torpedoes and mild bergmann gliosis51. one study found the cerebellum to be involved in approximately half of the cases studied60. cerebellar involvement mainly consisted in diffuse granular accumulation of cytoplasmic tau in the cell bodies of purkinje cells, and of doughnut-shaped structures in the cerebellar molecular layer in a smaller set of cases60. while the latter alterations were not studied directly in any cbd case, ancillary studies in psp cases revealed their location in the gfap-positive radial processes of bergman’s glia60. a relatively detailed map of neuronal tau-positive pathology has been described in cbd, but the degree of correlation between tau-positive pathology and stereotactically measured neuronal loss remains uncertain, especially in subcortical and infratentorial regions. it also remains unclear whether tau-positive pathology predominates in specific subtypes of affected neurons. it would also be of particular interest to understand these relationships in early and later disease stages. 1.2.3 glial involvement glial pathology in cbd is of diagnostic significance51. indeed, the characteristic thread pathology of cbd is likely predominantly glial rather than neuronal, as only a small fraction of thread-like structures are double labeled with neurofilament antibodies41. moreover, studies of astrocytic tau pathology provide evidence of glial cell involvement in regions that are preferentially affected by cbd. astrocytic plaques have been shown to co-localize with cd44, possibly suggesting a reactive change61, but not with gfap62. furthermore, the presence of astrocytic plaques in specific areas correlates with neuronal loss and reduced local density of homer1+ excitatory post-synaptic puncta, providing evidence for a relationship between glial and neuronal pathology63. one study has described tau-positive astrocytic plaques in the superior frontal gyrus prior to the development of symptomatic neurodegeneration64, and another study demonstrated that astrocytes and neurons in the grey matter of the anterior frontal lobe demonstrate tau pathology even in preclinical cbd65. a semiquantitative score for astrocytic plaque density was shown to remain moderate throughout disease progression, while the density of neuronal inclusions in the anterior frontal grey matter increased with disease progression65. taken together, these findings have led some scholars to speculate that cbd is a primary astrogliopathy65,66. astrogliosis and astrocytic plaques have also been observed in the hippocampus67, and are consistently found in the basal ganglia of confirmed cbd cases. astrocytic plaques can be found throughout the striatum, although they are more numerous in the caudate than the putamen65. indeed, the caudate is even more severely affected than the anterior frontal gyrus in preclinical cbd65. it is also worth noting that these astrocytic plaques develop early in the disease process, and one study has found that basal ganglia astrocytic pathology is most severe in the preclinical stage of cbd, with diminished density of plaques in end-stage disease65. these findings suggest an early involvement of basal ganglia astrocytes, in keeping with the frequently observed clinical picture49. astrocytic plaques have also been described in the thalamus, although they are less frequent than in the neocortex or the caudate58. finally, gfap-positive radial processes of bergman’s glia60 may show doughnut-shaped tau-positive structures in the cerebellar molecular layer in a small set of cbd cases60. oligodendroglial coiled bodies are distributed extensively throughout affected areas in cbd, but they are less frequent than in psp41. furthermore, little is known about the relationship between these oligodendroglial coiled bodies and other histological aspects such as myelin density or axonal density in the surrounding area68. microglial activation is observed in confirmed cbd cases when assessed by cd68 immunoreactivity42. in one large comparative study of frontotemporal microglial burden, cd68-positive microglia were significantly more numerous in the frontal grey matter than in the temporal grey matter of cbd cases. there was however no significant increase in the density of cr3/43or iba-1-immunoreactive microglia42. additionally, the parietal somatosensory and the superior temporal cortex demonstrate more widespread microgliosis in cbd than in psp56. white matter microgliosis is a consistent finding in cbd56. significant differences between controls and cbd cases have been observed in subcortical white matter in the frontal, temporal, and parietal lobes42,56. the frontal and temporal subcortical regions display moderate-to-severe microglial dystrophy that is more noticeable in the white matter than in the associated cortical grey matter42. activated microglia are also widely distributed throughout the basal ganglia, with hla-dr immunostaining demonstrating their significant proliferation in the striatum, the lentiform nucleus, the subthalamic nucleus, and the substantia nigra, as compared to controls56. microglial activation is also observed in the ventrolateral portion of the thalamus56. the early and region-specific presence of astrocytic pathology as well as the link between astrocytic and synaptic pathology raises the possibility that astrocytic tau pathology may be pathogenic in cbd. it is therefore somewhat surprising that no more detailed studies exist correlating subtype-specific neuronal loss with astroglial tau pathology. notably, neuronal tau pathology has been described in areas without significant astrocytic pathology such as the brainstem, suggesting that different pathomechanisms may be at play in these regions. microglial reactivity seems to mirror neuronal pathology in cbd. it endeavors to further dissect the relationship between astrocytic and microglial pathology to understand whether microglial activation reflects a primary neuroinflammatory mechanism or a specific response to neuronal injury. 1.3 progressive supranuclear palsy 1.3.1 general features psp is also a 4r tauopathy and presents most often as a movement disorder, yet cognitive decline is quite common and can be the presenting feature45. while the classic psps involves vertical gaze palsy, unprovoked falls, akinesia, and cognitive dysfunction, each of these symptoms can present along a spectrum of severity, and some cases with predominant akinesia or cognitive involvement are clinically diagnosed as cbs or ppa69,70. neuropathologically, psp is characterized by globose neurofibrillary tangles, thin, branching astrocytic tau inclusions (“tufted astrocytes”), and oligodendroglial coiled bodies (figure 2). the basal ganglia and brainstem tend to be especially involved, though cases with features suggestive of frontotemporal dementia often demonstrate substantial cortical involvement8. 1.3.2 neuronal pathology table 1 provides a summary of the known neuronal subtypes that bear tau pathology and/or undergo selective neurodegeneration in psp. historically, psp was thought to be primarily a disease of the basal ganglia and the midbrain with cortical involvement being limited and largely confined to the pre-central gyrus71. more recent studies have however challenged this view. indeed, many psp cases demonstrate widespread frontal and temporal atrophy, and these cases often manifest clinically with cognitive, behavioural, and linguistic symptoms similar to those observed in other forms of ftld72,73. immunohistochemical studies have demonstrated tau-positive neuronal tangles and neuropil threads in the superior frontal gyrus, middle frontal gyrus, and inferior temporal gyrus, with greater cortical tau pathology in cases that clinically manifest with frontotemporal dementia as compared to classical psps74. analyses using confocal microscopy have shown that both excitatory and inhibitory cortical synapses are reduced in pathologically confirmed cases of psp with frontal tau pathology63. in contrast to cbd, astrocytic pathology in psp does not appear to correlate locally with loss of synapses63, suggesting a possible divergence in the mechanisms of synaptic vulnerability between the two diseases. while many of the classical motor deficits in psp are related to subcortical pathology, the primary motor cortex and supplementary motor areas are often affected in psp as well. corticocortical projection neurons in the pre-supplementary motor area and inhibitory interneurons in the primary motor cortex have been identified as particularly vulnerable populations in psp75, but pyramidal neurons also display variable degrees of pathology76. compared to cases presenting clinically with cbs or ppa, cases presenting with classical psps have been found to exhibit greater pyramidal motor neuron involvement76. hippocampal involvement in psp remains poorly characterized in the literature. preliminary studies have demonstrated enlarged neurons and neurofibrillary tangles in the parahippocampal gyrus (phg) and in the ca1 sector of the hippocampus77. neuronal tau pathology appears to precede astroglial or oligodendroglial involvement in the hippocampus, and the burden of neuronal pathology can be quite severe78. by contrast, basal ganglia have been studied extensively in psp with consistent findings of early neuronal and glial tau pathology throughout the striatum, globus pallidus, and subthalamic nucleus8. gaba is the primary neurotransmitter involved in basal ganglia circuitry and decreased expression of gad-67, a marker of gabaergic interneurons, has been confirmed in case-control studies79. however, the relationship between neurofibrillary tau pathology and affected neuronal subtypes has not been evaluated. additionally, the nucleus basalis of meynert exhibits mild-to-moderate neuronal loss and a reduction in chat positivity has been shown in at least some cases. altogether however, the basal forebrain is only modestly affected in psp in comparison to other neurodegenerative conditions80. psp cases demonstrate both neuronal loss and microglial activation in many regions of the thalamus. in particular, the intralaminar nuclei appear to be profoundly affected, with one case-control study reporting a loss of 45 % of neuronal density across the centromedian and parafascicular nuclei in psp cases81. the ventral lateral nucleus also exhibits atrophy and neuron loss, particularly in cases with greater involvement of the primary motor cortex75. the brainstem exhibits striking changes in psp. typically, both divisions of the substantia nigra are affected. in the pars reticularis (snr), there is a loss of overall neuron density and decreased parvalbumin reactivity among surviving neurons, suggesting particularly pronounced vulnerability among the parvalbumin-positive cells82. this selective involvement of circuits involving parvalbumin-positive neurons has also been observed in parkinson’s disease (pd). yet, psp cases appear to exhibit more severe disruptions to the parvalbumin-positive interneurons and more frank atrophy than pd cases82. in the pars compacta (snc), there is a duration-dependant, selective dropout of neuromelanin-positive cells82. indeed, for reasons that are poorly understood, dopaminergic cells appear profoundly vulnerable to the changes induced by psp. tyrosine hydroxylase-immunoreactive cells (th-ir) in the nearby a10 region including the midline ventral tegmental area and the parabrachial pigmented nucleus are also affected with the loss of approximately 50 % of th-ir neurons compared with controls83. disruptions to dopaminergic signalling have been experimentally linked to downregulation of parvalbumin circuitry in mouse models, providing a potential explanation for the selective vulnerability of these two distinct neuronal populations84. psp cases also often demonstrate marked but selective neuronal loss in the locus coeruleus and the mesencephalic motor nuclei85,86. the locus coeruleus displays marked loss of noradrenergic neuromelanin-positive neurons explaining the relative pallor visible on gross inspection. quantification using ihc has revealed a loss of 49 % of neuromelanin-positive neurons relative to controls86. cholinergic neurons in the mesopontine nuclei, including the lateral dorsal tegmental nucleus and the pedunculopontine nucleus (ppn) are also affected80. more recently, these results have been replicated in a case-control study conducted by sébille et. al. (2019), showing that psp cases exhibit greater neuronal loss than controls in both the ppn and the cuneiform nucleus85. both cholinergic neurons, identified using ihc for chat, and non-cholinergic neurons are affected, and the ppn is more severely affected in psp than in pd85. notably, the study found minimal neuronal loss in the surrounding regions85, supporting the hypothesis that disease propagation is not driven by anatomical proximity alone. there is evidence to suggest moderate involvement of the cerebellum in most clinical phenotypes of psp78. there is also increasing awareness of a rare clinical presentation of psp with predominant cerebellar ataxia, which tends to exhibit more pronounced cerebellar neuron loss, tau-positive granular profiles in purkinje cells, and grumose degeneration in the dentate nucleus87. neuronal involvement has been evaluated in greater detail in psp than in cbd and pid. it is interesting to note that inhibitory neurons seem to be affected at least in the cortex, basal ganglia, and substantia nigra in psp. in addition, other neuronal subtypes such as cholinergic and dopaminergic also seem affected, suggesting that neurotransmitter subtype is not the defining feature of vulnerable neurons to psp pathology. based on this, transcriptomic studies may provide additional insights into the similarities of vulnerable neuronal subpopulations in psp. 1.3.3 glial involvement astrocytic pathology in psp is complex and intriguing, as there is not always a direct relationship between astrogliosis and the presence of tufted astrocytes. for example, one study showed that despite a very substantial burden of tau-immunoreactive tufted astrocytes in the motor cortex, many cases exhibit only minimal gliosis when evaluated with gfap88. this finding cannot be attributed to variation between cases, as the cases demonstrated remarkable homogeneity in the pattern of gliosis. tufted astrocytes, similar to astrocytic plaques, have been shown to co-localize with cd4462, possibly suggesting a reactive change61, but not with gfap. some research has been conducted into the involvement of astrocytes and oligodendrocytes in the subcortical white matter, and no significant difference has been demonstrated in the burden of gfap or myelin basic protein (mbp) between psp cases and controls89. curiously, one biochemical study found that insoluble tau was detectable in white matter regions by western blotting despite the absence of tau immunostaining in contiguous sections90. finally, evaluation of oligodendrocyte-specific pathology suggests that psp is not a primary oligodendrogliopathy, in contrast to multiple system atrophy and globular glial tauopathy91. astrocytic pathology in the basal ganglia appears to be an early event, with particularly severe early astrocytic involvement in the striatum78,92. the discrepancy between gfap distribution and astroglial tau pathology has also been described in the basal ganglia. in one study, the caudate and putamen exhibited the highest burden of tufted astrocytes, while the globus pallidus and substantia nigra exhibited most astrogliosis88. no relationship has been found between astrogliosis or neuronal tau pathology and tufted astrocyte density. yet, the severity of astrogliosis has been shown to correlate with the density of neurofibrillary tangles88,93. characteristic astroglial and oligodendroglial inclusions, i.e. tufted astrocytes and coiled bodies, respectively, are consistently found in the thalamus of moderate-to-severe psp cases. conditional probability analyses suggest that in most cases thalamic glial inclusions occur later than striatal inclusions but earlier than neocortical inclusions78. analysis of astrocytic pathology has revealed both astrogliosis and tufted astrocytes in midbrain regions, including the tectum and the red nucleus88. astroglial pathology appears more limited in the pons and medulla, with mild astrogliosis and very few tufted astrocytes78,88. microglial activation has also been demonstrated in psp cases relative to controls. in psp, the frontal cortex exhibits statistically significant microgliosis that can be detected using immunostaining for hla-dr or iba-142,56. the microgliosis is often most severe in the motor cortex56, and microglial pathology in this region correlates with neuronal pathology in the same region56. interestingly, the somatosensory cortex also exhibits statistically significant microgliosis56. while involvement of the neocortex in psp is not as pronounced as in cbd42,56, the presence of microglial activation suggests that it may be worthwhile to more thoroughly investigate cortical pathology in psp. psp cases often demonstrate white matter microgliosis, but the extent and distribution of the latter vary widely across studies. evidence of increased overall microglial density is conflicting, with one study finding an increase in iba-1 positive microglia in the frontal white matter and another study finding no increase in iba-1 positive cells despite an increase in cd68 positivity42,89. evidence of activation is also conflicting, as some studies but not other ones have demonstrated significantly increased microglial burden in the frontal and temporal white matter when assessed with immunostaining for cd68 and hla-dr and compared with controls42,43,56. in the basal ganglia, microgliosis can be extensive, with significant elevations in hla-dr-positive microglial burden throughout the globus pallidus and subthalamic nucleus56. microglial activation in the thalamus also appears to be widespread when assessed using hla-dr immunostaining, with increased burden in the ventrolateral nucleus and anterior nucleus when compared to controls or cbd cases. adjacent structures, such as the mammillothalamic tract and the thalamic fasciculus, also demonstrate increased microglial burden56. finally, the brainstem shows robust microglial activation in the superior colliculus, the medial longitudinal fasciculus, the substantia nigra, the red nucleus, and the pontine base as measured by hla-dr expression56. overall, there is robust knowledge on the distribution of astroglial and microglial activation in psp. less understood is the relationship between astrocytic tau pathology and astrocyte reactivity and how these latter relate to microgliosis. there seems to be at least some correlation between neuronal tau pathology and microgliosis. the relative independence of neuronal tau pathology and microgliosis from astrocytic pathology, as exemplified in the brainstem, is intriguing and requires further exploration. in addition, detailed glial transcriptomic phenotyping may help to identify the astrocytic populations responsible for regional astrogliosis and those most vulnerable to accumulation of 4r-tau. yet, it is also possible that astrocytes undergo proteomic changes and loss of gfap positivity as tau accumulates. this highlights the need for additional studies evaluating glial involvement in psp and its relationship to neurodegeneration. 2. frontotemporal lobar degeneration with tdp-43 pathology tdp-43 was identified in 2006 as the pathological protein present in most cases of ubiquitin-positive, tau-negative ftld12,13. as a result of this discovery, cases that had previously been described as ftld-u (for ubiquitin) were reclassified as ftld with tdp-43-immunoreactive pathology (ftld-tdp). a harmonized histologic classification system for ftld-tdp now exists with four well-defined subtypes lettered a-d and a more recently discovered, rapidly progressive phenotype provisionally labelled “type e”94,95. ftld-tdp type d is very rare and only found in familial cases with a mutation in the valosin-containing protein (vcp) gene94. ftld-tdp type e is also rare and considered by some authors as a variant of type b96. the present review will focus on types a, b, and c, as they collectively account for the significant majority of ftld-tdp cases8,94 (figure 3). figure 3. tdp-43 immunohistochemical features of ftld-tdp subtypes. a,d. ftld-tdp type a shows compact neuronal cytoplasmic inclusions (ncis) and short neurites that predominate in superficial over deep neocortical layers. neuronal intranuclear inclusions (inset in a) are a distinguishing feature (600x). b, e. type b pathology shows frequent granular ncis that affect all neocortical layers (600x). c, f. type c pathology is identified by long, frequently corkscrew-like, neuritic inclusions that preferentially affect superficial cortical layers, but can also affect the deep cortex (600x). sup = superficial. 2.1 ftld-tdp type a 2.1.1 general features clinically, ftld-tdp type a often presents as bvftd or nvppa, though there can also be motor neuron involvement8. an heritable form of ftld-tdp type a caused by a mutation in the progranulin gene (grn) on chromosome 17, and the familial variant often demonstrates more widespread pathology and a younger age at death97,98. moreover, another subset of ftld-tdp cases is associated with a pathological c9orf72 hexanucleotide repeat expansion on chromosome 9. most of these cases fulfill histological criteria of type b, or an overlap between type a and type b (type ab). only a minority present histologically as pure type a99. ftld-tdp type a is characterized by moderate or numerous compact neuronal cytoplasmic inclusions (ncis) and dystrophic neurites in layer ii of the neocortex94 (figure 3). lentiform neuronal intranuclear inclusions (niis) are also commonly identified, but they are usually much less numerous than ncis100. late-stage disease often results in neuronal death, loss of positivity for neuronal ihc markers such as neun, and, importantly, clearance of inclusions101. this can complicate research findings, as tdp-43 positivity can be lost in end-stage disease, necessitating qualitative assessment or adjunctive immunostaining in addition to quantification of pathological inclusions. 2.1.2 neuronal pathology ftld-tdp type a demonstrates extensive cortical atrophy and proteinopathy with prominent involvement of the superficial cortical laminae. there is also evidence of deeper involvement, with many cases exhibiting short dystrophic neurites and compact ncis in the deeper cortical laminae99. cases with low levels of overall tdp pathology demonstrate inclusions in projection neurons and in oligodendrocytes of the orbital gyrus and gyrus rectus102. when the overall burden of tdp pathology increases during the disease progression, inclusions are found in the middle frontal gyrus, anterior cingulate gyrus, and insular cortex, as well as the superior and middle temporal gyri102. these findings provide support for an early involvement of the frontal and temporal lobes. this study however only included bvftd cases and did not distinguish between type a and type b. to identify the type of neuron affected by neurodegeneration in ftld-tdp, a recent study compared the postmortem tissue rna-seq transcriptomes from the frontal cortex, temporal cortex, and cerebellum of 28 control and 30 ftld-tdp cases. the analysis showed that neuronal loss in the cortex mainly concerns excitatory neurons103. unfortunately, this study did not include pathological subclassification of ftld-tdp cases in its analysis. as in pid, vens appear to exhibit selective vulnerability in ftld-tdp104. additionally, a population of pyramidal cells neighboring vens, which likewise express the gaba receptor subunit theta (gabrq) seem vulnerable in ftld-tdp. the loss of gabrq-expressing neurons appears to be correlated with the development of behavioural symptoms at least in ftld-tdp and ftld-fus104. among cases with a c9orf72 repeat expansion, type a cases appear to demonstrate particularly severe loss of vens and neighboring gabrq-expressing pyramidal cells104. ftld-tdp cases with grn mutations have also been shown to exhibit decreased densities of gabrq-expressing cells104. whether vens are involved in sporadic cases of ftld-tdp type a remains poorly understood. while some studies have examined sporadic bvftd105,106, few of the cases examined were clearly type a. ftld-tdp type a often affects the hippocampus, where the burden of disease can be quite severe. the pathological findings frequently meet criteria for hippocampal sclerosis, defined as “severe hippocampal neuronal loss and gliosis”99,107. in the hippocampal dentate gyrus, ftld-tdp type a cases exhibit a moderate burden of compact ncis, though they demonstrate a significantly lower burden of compact ncis than type b or c cases99. unlike other types of ftld-tdp, type a cases also often exhibit niis in the dentate gyrus. additionally, these cases consistently have delicate wispy threads in ca1, a change that is frequently associated with hippocampal sclerosis. this feature was found to be 100 % sensitive and specific for type a cases in one study99. additional studies are needed to address whether these pathological features show selective predilection for specific hippocampal neuronal subpopulations. the basal ganglia are often involved in cases of ftld-tdp type a. long et al. have recently suggested that the degree of atrophy in the bilateral caudate and right putamen can distinguish between types a and b with high sensitivity and specificity108. these findings are consistent with previous research on the extracortical distribution of dystrophic neurites and neuronal inclusions in ftld-tdp subtypes. here, a significant difference was found between dystrophic neurite density in the putamen of type a cases when compared with type b109. furthermore, both ftld-grn and ftld-tdp type a without mutations in progranulin have been diagnosed on autopsy in cases of clinical cbs110, suggesting that involvement of the striatum can be widespread and clinically significant. analysis of striatal neuron populations using anti-calcineurin antibodies has revealed marked loss of substance-p positive efferents to the substantia nigra and the globus pallidus pars interna111. enkephalin-positive efferents to the globus pallidus pars externa were also affected, though not as severely, and chat-positive striatal interneurons were mostly unaffected111. the affected areas also exhibited proliferation of gfap-positive astrocytes, and the severity of neuron loss was correlated with both the accumulation of phosphorylated tdp-43 inclusions and clinical cognitive symptoms111. while these findings have yet to be replicated in a large study powered to distinguish between subtypes of ftld-tdp, they suggest a selective vulnerability of substance-p-positive striatal efferents. thalamic atrophy is a relatively common finding in ftld. mri findings have suggested that ftld-tdp cases exhibit greater thalamic involvement than ftld-tau or ftld-fus112. in particular, ftld-tdp type a cases demonstrate significant atrophy in most nuclei of the thalamus when compared to controls, with the possible exception of the ventral posterolateral, ventral medial, pulvinar, and medial geniculate nuclei, where only a trend has been demonstrated37. however, more research is necessary to replicate these findings at the histological level and to explore selectively vulnerable populations since only a minority of studies on ftld-tdp include thalamic histopathological data113. research into brainstem involvement in ftld-tdp has been limited, and most studies do not explicitly distinguish between subtypes of ftld-tdp. one study found evidence for dystrophic neurites and multiple types of nci in the substantia nigra, without involvement of the hypoglossal nucleus99. another small study found that the superior colliculus was involved in all subtypes of ftld-tdp, and that the substantia nigra, red nucleus, and raphe nuclei also consistently exhibited tdp-43-positive inclusions114. more studies are needed to dissect the corresponding cellular details. the extent of cerebellar involvement in ftld-tdp type a is currently uncertain. although imaging studies of patients with ftld-grn fail to demonstrate significant cerebellar atrophy115, it is unclear whether this is also the case for sporadic ftld-tdp type a cases. this may be partly explained by the lack of cerebellar tdp-43 accumulation apparent on histology even in very late stages of disease progression116. together, the current information on vulnerable neuronal populations in ftld-tdp type a points towards selective involvement of excitatory and gabrq-expressing cortical neurons, substance p-secreting neurons, and to a lesser extent also enkephalin-positive striatal neurons. in the rest of the brain, the distribution of neuronal tdp-43 pathology has been well described, but the degree of subtype-specific neuronal loss relative to this pathology remains unexplored. 2.1.3 glial involvement astrocytic pathology has not been well-studied in ftld-tdp type a. some ftld-tdp type a cases exhibit glial cytoplasmic inclusions, though they do not appear to be as abundant as in ftld-tdp type b99,117. these glial cytoplasmic inclusions appear to localize primarily to oligodendrocytes in the white matter based on morphology and ihc evidence117. other than the above, immunohistochemical evaluation of astroglial and oligodendroglial involvement in the brain has been largely neglected with the exception of the identification of increased gfap reactivity in the basal ganglia in general ftld-tdp111 and thalamus in ftld-grn118. of promise, conventional ihc and mass spectrometry analysis of the insoluble proteome in ftld-tdp cases has recently identified a unique pattern of astrocytic f-box protein 2 (fbxo2) expression specifically in type a cases. further studies will however be required to clarify the proteomic alterations and distribution of these fbxo2-positive astrocytes119. overall, the extent of macroglial involvement in ftld-tdp type a and its significance for pathogenesis remains largely unexplored. microglial activation, assessed using cd68 immunostaining, has been shown to be increased in the superficial cortical laminae i-iii in ftld-tdp type a43. while the genetic and histological ftld-tdp classifications do not perfectly match, cases of ftld with a grn mutation have been shown to exhibit more superficial cortical microglial activation than cases with a c9orf72 expansion. this suggests that inherited forms of fltd-tdp type a due to grn mutations may involve greater microglial activation in affected grey matter regions than inherited type b or ab forms due to c9orf72 expansion120. the rna-seq analysis described above also showed that increases in microglial and endothelial cell expression were highly correlated with neuronal loss103. the frontal and temporal white matter both display consistent microglial activation when assessed morphologically or by positivity for cd6842,43. historically, it was hypothesized that cases with a grn mutation may exhibit more microglial activation based on the inflammatory functions of progranulin. yet, post-mortem studies have found no difference between ftld-tdp cases with or without grn mutations43. one study which included mostly type a cases but did not separately analyze results by subtype, found that microglial activation in the hippocampal white matter is significantly greater in ftld-tdp cases than in controls121. in the ca1 region, however, no significant difference was found between ftld-tdp cases and controls. in the dentate gyrus, the same study found that ftld-tdp cases actually exhibited less microglial activation than controls121. in the thalamus of ftld-grn cases, iba-1-positive microglia and gfap-positive astrocytes were increased in number while myelination measured by myelin basic protein ihc was decreased118. to conclude, in ftld-tdp type a microglial activation appears to largely mirror neuronal pathology and loss. there is however not enough available data to draw significant conclusions on the distribution and severity of macroglial involvement in ftld-tdp type a and its potential pathophysiological relevance. 2.2 ftld-tdp type b 2.2.1 general features ftld-tdp type b is characterized by at least moderate numbers of ncis throughout all layers of the cortex94. these inclusions usually have a diffuse granular morphology, in contrast to the compact elliptical or crescentic inclusions found in type a100 (figure 3). clinically, ftld-tdp type b usually presents as bvftd or as motor neuron disease plus ftd, with a significant clinical and genetic overlap between ftd and als94. there are both sporadic and genetic forms of ftld-tdp type b, and the genetic causes usually overlap with genes implicated in the pathogenesis of als. ftld-tdp types b or ab are most frequently associated with pathological c9orf72 hexanucleotide repeat expansions, and particularly prone to manifest with psychotic symptoms and motor neuron disease (mnd)100. however, it is important to recognize that the association between type b pathology and psychotic symptoms is not entirely explained by the effect of c9orf72 expansions, as sporadic cases with type b pathology also display an increased frequency of psychotic symptoms96. patients with c9orf72 mutations produce aggregation-prone dipeptide repeat (dpr) proteins including glycine-alanine (poly-ga), glycine-arginine (poly-gr), proline-alanine (poly-pa), proline-arginine (poly-pr), and glycine-proline (poly-gp), due to unconventional translation of the abnormal hexanucleotide repeat expansion in c9orf72122. in these patients, the dprs are found throughout the brain, with the cerebellum exhibiting the greatest concentration of total and soluble dprs123. this review will not explore region-specific expression of dipeptide repeat proteins as a definite positive association between dpr burden and disease-associated clinical symptoms has not been established. 2.2.2 neuronal pathology the predominance of early behavioral symptoms in ftld-tdp type b suggests that the orbitofrontal cortex and/or limbic structures are affected early during disease. evidence to support this hypothesis comes from data-driven machine staging models116 and retrospective chart reviews after definitive diagnosis124. however, familial forms of ftld-tdp can have different patterns of clinical and pathological progression, and these differences can be even more pronounced in comparison to sporadic cases23. as described for type a, a transcriptomic study102 showed evidence of selective loss of excitatory cortical neurons in ftld-tdp, but this study did not include ftld-tdp subtyping103. an earlier study from 1993 on cases with ftld-tdp associated with als showed significant decreases in calbindin positivity without changes in expression of parvalbumin, suggesting that neurons expressing calbindin may be affected to a greater degree than neighboring neurons125. yet, this study was performed before the discovery of tdp-43 and thus predated the modern classification of ftld-tdp. like ftld-tdp type a cases, type b cases often involve vens of the anterior cingulate cortex. as described by nana et al., even early-stage cases appear to exhibit disproportionate cytoplasmic tdp-43 immunopositivity in vens, and the accumulation of tdp-43 pathology in this neuronal population correlates with clinical severity126. additionally, fork cells, which are found in the frontal insula alongside vens, have been found to display a similar pattern of early neurodegeneration126. in both vens and fork cells, cells displaying inclusions or nuclear tdp-43 depletion develop somatodendritic atrophy, suggesting direct neurotoxicity126. while some of the ftld-tdp type b cases studied had c9orf72 repeat expansions, most were sporadic. further evidence for the involvement of vens in sporadic ftld-tdp type b comes from earlier studies on sporadic bvftd, in which many cases were either unclassified or type b105,106. in the hippocampus, ftld-tdp type b cases demonstrate a greater burden of both compact and diffuse ncis in the dentate gyrus and in the ca1 region of the hippocampus compared to ftld-tdp type a99. however, ftld-tdp type b cases do not demonstrate hippocampal niis, dystrophic neurites, or threads, and as a result, the total tdp-43 burden seen by ihc is lower than in ftld-tdp type a cases in the ca1 region99. ftld-tdp type b cases also demonstrate diffuse ncis and glial cell inclusions in the basal ganglia and substantia nigra99,109. as discussed above, ftld-tdp type b cases also appear to involve loss of substance-p positive striatal efferents, with milder involvement of enkephalin-positive efferents. furthermore, striatal involvement may be less severe in ftld-tdp type b cases than in type a cases108,111. of note, all ftld-tdp type b cases demonstrating this apparent selective neuronal vulnerability presented clinically as ftd-mnd111. the thalamus appears to undergo minimal changes in most cases of ftld-tdp type b. only small numbers of thalamic ncis tend to develop in type b cases109. imaging studies demonstrate relatively minor focal atrophy in the lateral geniculate nucleus, ventral lateral, and mediodorsal nuclei of the thalamus37. c9orf72 repeat expansions however may predispose to more widespread thalamic atrophy, with greater involvement of the pulvinar37,127, but the exact cellular underpinnings of this change remain elusive. brainstem pathology is a core feature of als, and limited evidence suggests that involvement of the brainstem may be more widespread in ftld-tdp type b than in type a or c114,128,129. in addition, involvement of cranial nerve motor nuclei is a well documented feature of ftld-tdp type b8. ftld-tdp type b often features glial cytoplasmic inclusions in the medulla, in addition to widespread neuronal alterations in the hypoglossal nucleus23,99. the latter are significantly more pronounced in ftld-tdp type b than in other ftld-tdp types. cerebellar tdp-43 pathology in ftld-tdp is poorly documented, and the existing literature often does not stratify by histopathological subtype. nonetheless, some clinically diagnosed cases of bvftd or ftd-mnd, with or without a c9orf72 repeat expansions, demonstrate mild but statistically significant atrophy in various regions of the cerebellum115,130. it is tempting to speculate that some of these cases may represent ftld-tdp type b, but it is impossible to draw firm conclusions without autopsy confirmation. histologically, no significant neuronal tdp-43 pathology or neurodegeneration has been seen in ftld-tdp type b cases99,131, hence the molecular correlate substrate of cerebellar atrophy in ftld-tdp type b remains unknown. overall, current evidence suggests a selective vulnerability of cortical vens, fork cells and motor neurons in ftld-tdp type b. preliminary data suggest that neocortical excitatory and calbindin-expressing neurons as well as substance-p positive striatal efferents are also affected. the selective neuronal vulnerability of other brain regions to tdp-43 pathology remains however largely unexplored. the relationship between tdp-43 pathology and loss of neuronal subtypes has only been established for vens, fork cells and motor neurons. 2.2.3 glial involvement astrocytic pathology has not been well characterized in ftld-tdp type b. white matter pathology is less pronounced in ftld-tdp type b than in type a, and most tdp-43 localizes to oligodendrocytes117. in ftld-tdp type b cases, activated microglia are distributed throughout all layers of the neocortex43. no significant differences have been found between sporadic ftld-tdp and cases associated with a c9orf72 mutation43. in contrast to ftld-tdp types a and c, ftld-tdp type b cases and controls show similar microglial burden as assessed by cd68, iba1, or cr3/43, according to pairwise comparisons between cases and controls42,43. however, a trend towards increased cd68 reactivity was identified in the frontal white matter compared to controls43, suggesting that differences in microglial activation may have been overlooked in published yet underpowered studies. in conclusion, additional human studies are warranted to evaluate the involvement of macroglia and microglia in ftld-tdp type b and their potential pathophysiological roles. 2.3 ftld-tdp type c 2.3.1 general features ftld-tdp type c accounts for 25 % of ftld-tdp cases8. it is characterized by the presence of long dystrophic neurites, which tend to be concentrated in the superficial cortical laminae but are also present in deeper cortical layers94,99 (figure 3). in ftld-tdp type c there are few neuronal cytoplasmic inclusions and very few neuronal intranuclear inclusions94,99. clinically, this form of ftld usually manifests as svppa, and sometimes also as nfvppa or bvftd94. 2.3.2 neuronal pathology type c cases typically demonstrate asymmetric cortical atrophy predominating in the temporal lobe38. in affected regions, superficial dystrophic neurites are visible on ihc, which are usually long and thick, in contrast to the shorter, comma-shaped dystrophic neurites of ftld-tdp type a cases94. it is unclear which type of neuron is most vulnerable to the development of these inclusions. for instance, vens have not been studied in a cohort enriched for ftld-tdp type c cases. in type ftld-tdp type c cases, the dentate gyrus of the hippocampus exhibits compact ncis that have been described as “pick body-like” due to their uniformity99. the ca1 region does not generally contain ncis, but does exhibit dystrophic neurites, which are usually not seen in ftld-tdp type a or type b cases99. notably, only a minority of cases demonstrate frank hippocampal sclerosis109. like the hippocampus, the striatum displays dystrophic neurites99 and compact ncis with round contours in ftld-tdp type c99. the nucleus accumbens appears to accumulate a greater pathological burden than the dorsal striatum, though the mechanism for this apparent regional selectivity is unknown132. riku et al. found that two out of five ftld-tdp type c cases displayed a selective degeneration of striatal efferents as described above for ftld-tdp type a. caution is however warranted given the low number of ftld type c cases studied111. in contrast to ftld-tdp type a and type b cases, type c cases typically do not exhibit thalamic ncis109. while one imaging study has found mild atrophy in the mediodorsal nucleus relative to controls, there was less thalamic atrophy than in any other pathological subtype of ftld37. ftld-tdp type c cases also exhibit only mild dystrophic neurites in the substantia nigra. as in type a, the hypoglossal nucleus is largely spared99. one study involving nine ftld-tdp type c cases found no evidence of brainstem pathology in the midbrain, hypoglossal nucleus, or inferior olivary nucleus109, while another study involving five type c cases found tdp-43 deposits consistently in the superior colliculus, with occasional involvement of the inferior olivary nucleus and the red nucleus114. the cerebellum has not been systematically studied in ftld-tdp type c. as discussed above for ftld-tdp type a, some indirect evidence exists for cerebellar involvement ftld-tdp in genera, but there have been no large studies examining differences between pathological subtypes, and the clinical significance of these findings remains unclear. unlike ftld-tdp type a and b cases, ftld-tdp type c cases appear to show concentrated tdp-43 pathology in the neocortex, hippocampus, and anterior striatum. it is surprising that the identity of vulnerable neurons that develop the characteristic long ‘corkscrew’ neurite is still unknown more than a decade after the formal recognition of this pathological ftld subtype. 2.3.3 glial involvement it appears that glial inclusions, particularly subcortical white matter ones, are uncommon in ftld-tdp type c117. it remains however unclear why tdp-43 does not tend to accumulate in cells of oligodendroglial morphology in ftld-tdp type c cases, in contrast to the situation in types a and b. microglial activation has been studied to a significant degree in ftld-tdp type c. here, it appears to follow a similar pattern to that observed in type a cases, with more activation in the superficial cortical laminae and predominant white matter microgliosis in the frontal lobe42,43. caution is however warranted, since the corresponding studies included only five42 and seven43 cases with ftld-tdp type c pathology. these limited data suggest that macroglia are involved in ftld-tdp type c in a less prominent manner than in other ftld-tdp subtypes. further studies evaluating astrocytic and microglial reactivity as well as oligodendroglial loss throughout the brain of ftld-tdp type c cases could provide powerful insights into this question. 3. frontotemporal lobar degeneration with fused in sarcoma (fus) pathology 3.1 general features about five percent of ftld cases exhibit neither tau nor tdp-43 pathology, and many of these cases are positive for fus on ihc8. three phenotypes of fus-positive, tdp-43-negative, tau-negative ftld have been described: atypical frontotemporal lobar degeneration with ubiquitin inclusions (aftld-u), basophilic inclusion body disease (bibd), and neuronal intermediate filament inclusion disease (nifid)133. the most common subtype of ftld-fus, aftld-u, is characterized histologically by round, oval, or bean-shaped fus-immunoreactive ncis with a wide pattern of distribution134. these inclusions are also positive for transportin 1, which mediates the nuclear import of fus135. additionally, aftld-u cases demonstrate long, thin, curved niis that have been described as “vermiform”136. bibd cases exhibit severe striatal and nigral atrophy, with varying degrees of cortical atrophy corresponding to behavioural symptoms137,138. the disease gets its name from basophilic cytoplasmic inclusions that are negative for neurofilament and tau but positive for fus and occasionally positive for ubiquitin or p62138. colocalization immunofluorescence has shown that fus accumulates in inclusions identified by hematoxylin and eosin staining and inclusions identified by p62 ihc. in addition, fus also accumulates in many more inclusions that could not be detected using other methods than ihc138. for this reason, bibd is now generally classified as a fus proteinopathy, though there are still many open questions about its pathogenesis and presentation. nifid cases also demonstrate involvement of various cortical and subcortical regions, and the observed ncis and niis are heterogenous in their morphology and immunoreactivity139. the ncis can be round, crescentic, annular, or tangle-like. in addition there are so-called “hyaline conglomerate inclusions” with a filamentous appearance and an eosinophilic core140. both vermiform and round niis have been reported, and these appear to be more common in neurons that also exhibit round cytoplasmic inclusions139,140. these inclusions are immunoreactive for intermediate filament proteins and fus, and colocalization of the latter has been confirmed using double-label immunofluorescence139. nonetheless, some ftld-fus cases are immunoreactive for the intermediate filament alpha-internexin but not for fus. interestingly, one such case has been reported to exhibit both tdp-43 and alpha-internexin positivity141. clinically, aftld with ubiquitin inclusions (aftld-u) typically manifests early in life as an unusual form of bvftd that can involve obsessions, pica, ritualistic behavior, and hypersexuality133. due to their rarity, other forms of ftld-fus have not been characterized as systematically, but case reports suggest that they are also likely to present with early-onset bvftd or ftd-mnd133,137,138,141,142. while stereotyped and repetitive behaviours are reported in some cases, these symptoms are not as consistent as in aftld-u. conversely, prominent mnd and parkinsonism seem to be more common in both bibd and nifid133,137,141, and bibd more commonly involves memory impairment and apraxia133,137. 3.2 neuronal pathology cases of aftld-u demonstrate widespread ncis throughout the frontal and temporal cortices143 and occasional niis in pyramidal neocortical neurons14. bibd and nifid cases also sometimes demonstrate neuronal inclusions in the frontal and temporal cortices137, but there are case reports of late-onset bibd that preferentially affects the motor system, with minimal limbic or prefrontal involvement143. ftld-fus cases may also demonstrate selective involvement of vens. while the sample size was small (n=8), one study found consistent and severe degeneration of gabrq-positive neurons104. moderate to severe hippocampal involvement is a common feature in aftld-u, with fus-positive ncis and vermiform niis in the granule cells of the dentate gyrus and, to a lesser extent, in the subiculum and ca1 regions14,133. in contrast, bibd demonstrates less consistent involvement of the dentate granule cells and more consistent involvement of the pyramidal neurons134,136,138. hippocampal inclusions can also be found in nifid140,144, but in at least a subset of cases, inclusions in the hippocampus are not nearly as numerous as in the frontal lobe144. severe striatal atrophy has been reported in all three major subtypes of ftld-fus136–138,140. in aftld-u, there are often crescentic ncis and small numbers of niis136, and the striatum can exhibit varying degrees of involvement14,143,145. in bibd, severe striatal atrophy is a consistent finding, and in some cases, striatal pathology is considerably more severe than cortical or hippocampal pathology138. both caudate and putamen demonstrate severe neuronal loss, gliosis, and basophilic inclusions138, and early involvement of the striatum and the pyramidal motor system are more common in bibd than in aftld-u143. as in bibd, nifid cases are thought to show severe, consistent striatal atrophy136,137, though a recent case series has presented numerous cases without evidence of atrophy141. the globus pallidus136–138,144 is also involved in ftld-fus, particularly in bibd and nifid but also in aftld-u14,136. however, some cases appear to exhibit only mild pallidal atrophy despite significant involvement of the striatum133,137, and there are some cases of aftld-u that involve extreme caudate atrophy with relative preservation of both putamen and globus pallidus133. in aftld-u cases, the thalamus is not a consistent site of pathology, but many cases show small numbers of ncis and niis136,140. in contrast, bibd appears to consistently demonstrate moderate to severe pathological burden in the thalamus, including non-compact collections of coarse granules and even occasional niis140. finally, nifid also shows relatively consistent involvement of the thalamus, though the level of pathology can vary considerably136,140,144. the brainstem also seems to be involved in aftld-u, with greater involvement of rostral regions143. however, many cases demonstrate only mild fus-immunoreactive pathology in the pons and midbrain, including the substantia nigra but sparing the red nucleus140. in bibd, there are basophilic, fus-immunoreactive inclusions in the brainstem136–138, particularly in the pontine nuclei and the inferior olivary nucleus136. indeed, along with the basal ganglia, the brainstem appears to be among the regions with the highest density of fus-positive inclusions in bibd137. caudal regions of the brainstem display more pronounced pathology than rostral regions in bibd143. furthermore, lower motor neurons in the spinal cord are affected136,137. nifid also shows more consistent and widespread brainstem pathology than aftld-u, including involvement of the locus coeruleus, the red nucleus, and the basis pontis140, though gross atrophy is not always apparent144. much of the brainstem pathology presents as coarse granules rather than as compact inclusions139. cerebellar involvement appears to be an uncommon finding in aftld-u, though some cases exhibit a small number of ncis in the dentate nucleus136. cerebellar involvement seems to be somewhat more common in bibd, with ncis and gcis often present in the dentate nucleus137,138,143. nonetheless, the cerebellar cortex is not generally affected143. nifid cases also sometimes demonstrate neuronal loss and fus-immunoreactive pathology in the cerebellum136,139. however, this does not appear to be a consistent finding144, and there are fewer fus-immunoreactive inclusions in the cerebellum than in virtually any other grey matter region that has been subjected to study134. in summary, the distribution of fus-immunoreactive pathology has been described in all three types of ftld-fus. however, only vens and gabrq+ neuronal subtypes have been proven to be affected by the pathological inclusions. further subtype-specific studies of neuronal loss and correlative studies with fus inclusions in each ftld-fus subtype are needed to understand the pathogenic mechanisms of disease in these entities. 3.3 glial involvement all three major forms of ftld-fus are associated with glial cytoplasmic inclusions in the white matter14,134,139,140. these inclusions are typically oval or flame-shaped134. similar inclusions have been identified in als with a fus mutation145–147. double labelling has demonstrated that the inclusions localize to oligodendrocytes145. based on these findings and the morphological characteristics of the involved cells in ftld-fus, it seems reasonable to conclude that the gcis represent pathological involvement of oligodendrocytes. the distribution and protein expression of affected cells have however not been systematically studied. in one study comparing microglial activation across subtypes of ftld, ftld-fus cases did not appear to demonstrate greater grey matter microglial activation than controls. yet, only four cases were included, and all cases were aftld-u42. in the white matter, there appears to be a possible trend toward increased microglial activation compared to controls in frontal and temporal white matter, but a statistically significant difference has not been demonstrated42. conclusion there is clear evidence for selective regionand cell-type specific vulnerability in all forms of ftld, which helps explain the distinct clinical and pathological features associated with each subtype. where there is evidence of selective neuronal vulnerability, such as vens in pid and ftld-tdp or striatal substance-p-positive efferents in ftld-tdp, further characterization of the changes to those populations and comparisons to unaffected neuronal types would be of value. where there is little evidence on selectively vulnerable populations, such as in ftld-fus, systematic attempts to identify and characterize vulnerable cell types could help focus further research. as the scientific community has learned from attempts to understand other neurodegenerative conditions, abnormal protein inclusions are excellent markers for disease classification and diagnosis but are unlikely to be the sole pathophysiological factor at play. this highlights the importance of systematic studies to understand selective vulnerability of both neurons and glia and the precise distribution of these cell types in ftld. when a selectively vulnerable population is identified, exploration of associated glia, synaptic inputs, and projections can provide mechanistic information beyond that afforded by characterization of isolated affected neurons. this review suggests a number of areas for further research, including the evaluation of neuronal subtype-specific vulnerability; the significance and distribution of oligodendroglial and astrocytic pathology; the relationship between activated microglia and vulnerable neuronal populations; and the relative importance of cell-autonomous versus non-cell-autonomous mechanisms of neurodegeneration. this latter aspect has not been extensively evaluated in humans. closing the knowledge gap on some of these areas may identify 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(berl) . 2011;122(1):87-98. https://doi.org/10.1007/s00401-011-0838-7 146. kobayashi z, tsuchiya k, arai t, et al. occurrence of basophilic inclusions and fus-immunoreactive neuronal and glial inclusions in a case of familial amyotrophic lateral sclerosis. j neurol sci . 2010;293(1-2):6-11. https://doi.org/10.1016/j.jns.2010.03.029 147. hewitt c, kirby j, highley jr, et al. novel fus/tls mutations and pathology in familial and sporadic amyotrophic lateral sclerosis. arch neurol . 2010;67(4):455-461. https://doi.org/10.1001/archneurol.2010.52 copyright: © 2025 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. my recollections of my 50 years as a neuropathologist from a neurologist’s perspective feel free to add comments by clicking these icons on the sidebar free neuropathology 4:16 (2023) reflections my recollections of my 50 years as a neuropathologist from a neurologist’s perspective yoshio hashizume institute of neuropathology, fukushimura hospital, noyori-cho, toyohashi, aichi, japan corresponding author: yoshio hashizume · institute of neuropathology · fukushimura hospital · noyori-cho · toyohashi · aichi · japan yhashi@chojuken.net additional resources and electronic supplementary material: supplementary material submitted: 04 september 2023 accepted: 02 october 2023 copyedited by: georg haase published: 18 october 2023 https://doi.org/10.17879/freeneuropathology-2023-5108 keywords: neurodegenerative disease, pathology of dementia, spinal cord pathology figure 1: a recent photo of me taken in the outpatient department of fukushimura hospital. nurse korin kondo is on the left. introduction i am very honored to be able to write about my neuropathology background in the reflections section. fifty years have passed since i started researching neuropathology, and i am grateful for the good fortune that i have enjoyed to be able to continue working in this field. in this section, i would like to describe the history of my neuropathological work in the tokai region, which is located in the central part of japan. figure 1 is a recent photo of me taken in the outpatient department of fukushimura hospital. my background i was born on august 25, 1944 at the end of world war ii in the mountain village of haze in mie prefecture, which is located in central japan. the village of haze was two hours by car from matsuzaka, a city in the center of mie prefecture, and it was located in a rural area where buses came only three times a day on unpaved roads. both my parents were from the village of haze. the village was my hometown. it was where the kushida river flowed, and it had clean water and beautiful mountains. my father worked as a police officer in the city of tsu, the prefectural capital of mie prefecture. i still vividly remember the city in ruins from when it was destroyed by air raids during the war. i grew up as the second son of four siblings. i was brought up with the immeasurable affection of my parents in a chaotic post-war japan with scarce supplies. figure 2 shows a picture of me when i was four years old. figure 2: a picture of me when i was four years old. after attending elementary and junior high school in the city of tsu, i began attending tsu high school, one of the leading preparatory schools in the prefecture in 1960. even though my parents weren’t wealthy, they created a warm home and let me study to my heart‘s content. during summer vacation back then, i was studying for 16 hours a day, except for eating and sleeping. when i was in high school, my mother got sick and had to go to the hospital. that‘s why i chose to become a doctor: because i wanted to help people. in 1963, i was admitted to the nagoya university school of medicine, which is where i wanted to go. having grown up in the countryside, nagoya was a big city for me. i lived in a small boarding house in a room with only 3 tatami mats and no windows. while studying medicine, i was able to lead a fulfilling life with extracurricular activities, student movements, and a part-time job. figure 3 shows a lecture on neurology by professor itsuro sobue, who was an associate professor at the time. the arrow is to professor sobue, and the arrowhead is to me. figure 3: a lecture on neurology by professor itsuro sobue, who was an associate professor at the time. the arrow is professor sobue, and the arrowhead is me. after school, i was active in the chorus, and this is where i got to know my wife, who was in the chorus at another university. i was actively involved in political and social issues and participated in student movements. at the time, japan was in the midst of the okinawa reversion movement. i wanted to see okinawa before it was returned to japan, so i traveled there with a tent. i also worked hard at various part-time jobs, such as selling ice cream and oranges on the platforms of nagoya station. figure 4 is a picture of me when i graduated from nagoya university school of medicine. figure 4: a picture of me when i graduated from nagoya university school of medicine. before i started studying neuropathology after graduating from nagoya university school of medicine in 1969, i worked as a physician at anjo kosei hospital in the mikawa area for two years. this hospital is a leading hospital for acute care in the mikawa district of aichi prefecture, and i was able to diagnose and treat various medical conditions. during these two years, i was involved in pathology autopsies on many patients for whom i was the attending physician. this was a major impetus for me to major in pathology. around that time, dr. izumi kano, my advisor, told me to “be the master of an art.” when i later majored in neuropathology, i always ruminated on the phrase “be the master of an art” during the procedure to remove the spinal cord at autopsy. i got married while working at anjo kosei hospital and later had a son and two daughters. after practicing general internal medicine for two years, i was clinically involved in neurological diseases in the neurology laboratory at the first department of internal medicine, nagoya university school of medicine. in the laboratory, i was advised by professor itsuro sobue. figure 5 is a group photo taken during a trip to kashikojima with my colleagues from the department of neurology, nagoya university school of medicine. dr. sobue was one of the founders of neurology in japan, a specialist in peripheral nerve diseases, and he advanced japanese research in gerontology as a leader in research on geriatric medicine. dr. sobue served as president of aichi medical university for many years and trained many neurologists in the nagoya area. the starting point for me in neuropathology was the department of neurology, nagoya university. clinical experience in neurology was extremely vital to my subsequent interest in neuropathology. at the time, however, there were not enough brains to autopsy in the nagoya area to study neuropathology. i wanted to search for the brains of patients for whom i was the attending physician and on whom pathological autopsies were performed. brains for autopsy belonged to the department of pathology, and in order to do neuropathology there was no other way than to join the department of pathology. therefore, i joined the department of pathology at nagoya city university in 1974 and began studying systemic pathology under professor hidemasa kishimoto. dr. kishimoto specialized in clinical pathology and was of a good character, and many students and clinicians came to study at his laboratory. at the time, my goal was to acquire the skills to perform pathology autopsies by myself even if i was assigned to a local hospital later on. during this period, i trained in basic pathology, including organ excision after fixation, embedding, sectioning, staining, microscopy, summarizing findings, and giving presentations at clinical pathology conference. figure 5: a group photo taken during a trip to kashikojima with my colleagues from the department of neurology at nagoya university school of medicine. in the back row, 1st from is dr shigemitsu nishigaki, 2nd is me, 5th is professor itsuro sobue, 1st from the left in the front row is dr. akira takahashi, and 3rd is dr tsutomu yanagi. studies abroad in munich in 1974, dr. shigemitsu nishigaki from nagoya university school of medicine introduced me to the max plank institute for psychiatry in munich, germany, and i decided to study there. i studied german language for two months at the goethe institute in grafing in the suburbs of munich before starting my life at the institute. learning german with many students from all over the world was a very valuable experience. the max plank institute for psychiatry is one of europe‘s leading neuropathology institutes. a professor at the time was professor gerd peters, editor of acta neuropathologica and author of klinische neuropathologie [clinical neuropathology]. this book is an excellent work on neuropathology. on the left in figure 6 is professor peters when he gave a special lecture at the japanes society of neuropathology, and on the right is the textbook klinische neuropathologie. figure 6: on the left is professor gerd peters when he gave a special lecture at the japanese society of neuropathology. the right is the textbook klinische neuropathologie. the institute received a large number of brains for autopsy from southern germany, mainly from psychiatric hospitals. i observed professor peters cutting the brain and i was able to observe the gross findings from many cases, including multiple sclerosis and wernicke‘s encephalopathy. i was able to live a blessed life, reading the book klinische neuropathologie every day, looking at the pictures in the book, and looking through a microscope every day. dr. parviz mehraein, who was an associate professor in the laboratory at the time, was a boon not only to my research but also to my life in germany, which i was not accustomed to. the upper of figure 7 is the max plank institute for psychiatry in munich, the middle is dr. mehraein, his wife and me, and the lower left is me in the laboratory. because of this study abroad, i decided to make neuropathology my life‘s work. i was able to publish a paper summarizing the clinicopathological findings of cases of cerebral aqueduct obstruction that were compiled at the institute (1). in munich, i was able to study closely with dr. motohiro suetsugu of kyushu university and dr. tatsuji tanabe of nihon university, who came from japan to study abroad. figure 7: upper: the max plank institute for psychiatry in munich. middle: dr. parviz mehraein, his wife and me. lower: me in the lab. obtaining a doctorate in medicine after studying abroad in munich, i returned to japan in 1976 and continued my research on neuropathology while continuing to study general pathology under professor kishimoto in the department of pathology at nagoya city university. advised by dr. kishimoto and dr. tsutomu yanagi, head of the department of neurology, nagoya daini red cross hospital, i received a doctorate in medicine for my pathological research on ossification of the posterior longitudinal ligament (2,3). as a neurologist, dr. yanagi was an excellent doctor who was familiar with spinal cord diseases such as cervical spondylosis, ligamentous ossification, and spinal cord infarction. after i was advised by dr. yanagi, i decided to make spinal cord pathology my life’s work. the research on ossification of the posterior longitudinal ligament was later taken over by dr. takashi kameyama (4) who published a wonderful paper in the journal brain. life as a researcher in the department of pathology, nagoya university in 1980, i was hired as an assistant in the department of pathology at my alma mater, nagoya university. i was able to continue my research in neuropathology under professor soichi iijima, who was an instructor in the japanese society of pathology. dr. iijima specialized in pathology of the spleen, but he allowed me to continue with neuropathology. he told me that i should devote myself wholeheartedly to neuropathology. he had a wonderful attitude toward pathology autopsies, and he wrote in his book that “problems that need to be addressed can always be found in the body of a deceased patient, and how to deal with those problems depends on the ability of the clinician and the pathologist.” taking these words to heart, i have devoted myself to pathology autopsies (5). later, dr. junpei asai became a professor. dr. asai specialized in pathology of the spleen. during his tenure as professor, he was stricken with malignant lymphoma, but he fought the disease with fortitude and completed his professorship. i was indebted to this laboratory for 13 years until i was transferred to aichi medical university. figure 8 shows a photograph of the teachers who taught me pathology. professor kishimoto is on the left, professor iijima is in the middle, and professor asai is on the right. figure 8: the teachers who taught me pathology. from left, professor hidemasa kishimoto, professor soichi iijima, and professor junpei asai. study abroad at montefiore hospital in 1982, i was a researcher of the ministry of education and i studied at montefiore hospital in new york city, where i was able to train in neuropathology under professor asao hirano. this was around the time that aids broke out in new york, and cases for autopsy began to appear. being able to study with dr. imaharu nakano and many other doctors from japan in professor hirano’s laboratory was very helpful in continuing my study of neuropathology in japan. with professor hirano’s guidance, i was able to write a paper on spinal cord intramedullary metastasis and spinal pencil-shaped softening (6,7). dr. hirano’s lab was visited by many doctors from japan who wanted to study neuropathology, such as neurologists, neurosurgeons, psychiatrists, and pathologists. dr. hirano made a massive contribution to the advancement of neuropathology in japan. dr. hirano’s famous book “for those who study neuropathology” became a bestseller. in figure 9, the left is a moment at the laboratory with professors zimmermann and hirano and research students from japan. the center is professor hirano’s famous book “for those who study neuropathology.” the right is professor hirano and me in my professor’s office. figure 9: the left is a moment at the laboratory with professors zimmermann and hirano and research students from japan. the center is professor hirano’s famous book “for those who study neuropathology.” the right is professor hirano and me in my professor’s office. conducting research in the pathology department, nagoya university hospital in 1990, i was appointed as an associate professor in the department of pathology, nagoya university hospital. with the help of pathologists, neurologists, and neurosurgeons from major hospitals in the tokai region of central japan, we focused on collecting brains for autopsy. the brain can be searched for many crucial neurological diseases, and clinical neuropathological conferences are conducted at many hospitals. at the time, the director of nagoya university hospital was professor akira takahashi, a neurologist whom i respect, and i was able to conduct research along with many young neurologists in his department. figure 10 is a group photo of me and my colleagues when i was in the department of pathology, nagoya university hospital. here are the papers with many doctors who were conducting joint research at the time (8-19). figure 10: a group photo of me and my colleagues when i was in the department of pathology, nagoya university hospital. front row from left: toshiaki inagaki, noriko hirunagi, me, and shigeo riku. middle row: mari yoshida and motoko sakai. back row from left: tetsuo ando, tetsuya mizuno, satoshi okuda, takashi kameyama, shinichi miyao, and akito kume. in western japan, there is an academic conference called the clinical neuropathology meeting. every year, we have the opportunity to bring actual specimens and discuss cases while looking at specimens under a microscope in the pathology laboratory of the medical school. in 1992, i chaired this meeting in nagoya. every year, we always receive a lecture from dr. hirotsugu shiraki, a leading neuropathologist in japan, at this meeting, and we heard about als in the kii peninsula. professor shiraki is the former president of the japanese society of neuropathology and the most preeminent neuropathologist i have ever met. he has made vast numbers of achievements in neurodegenerative diseases, minamata disease, carbon monoxide poisoning, toxic diseases such as smon, and the pathology of demyelinating diseases. on the right of figure 11 is the venue for the clinical neuropathology conference, and on the right is professor shiraki giving a special lecture. figure 11: on the left is the venue for the clinical neuropathology conference, and on the right is professor shiraki giving a special lecture. conducting research at aichi medical university in 1993, i became a professor at the institute for medical science of aging, aichi medical university. this research institute was founded by profes-sor hisashi tauchi, professor emeritus at nagoya university and an authority on the pathology of aging. dr. tauchi is a pathologist who pioneered pathological research in geriatric medicine. even after he turned 80, dr. tauchi continued to publish pathological research on japanese centenarians as a book, and he also published a book in english (20). i also wrote an article on the neuropathology of centenarians in that book. dr. tauchi was the first in the world to propose that the number of cells decreases with age, causing organ atrophy. even after being appointed as a professor at the institute for medical institute of aging, aichi medical university, i continued to strive to make the institute one of the leading neuropathological institutes in japan. on the left in figure 12 is the building of the institute, and the right is professor tauchi (marked with an asterisk) with my colleagues at the institute when i was appointed as a professor. figure 12: on the left is the building of institute for medical institute of aging, and on the right is professor tauchi (marked with an asterisk) with my colleagues at the time of my appointment. in my third year at the institute for medical science of aging, dr. mari yoshida, with whom i had previously conducted joint research, joined the institute. dr. yoshida specializes in neurology and has grown to become a leading expert in the neuropathology of neurodegenerative diseases in japan 21,22,23). afterwards, she succeeded me as a professor at the institute for medical science of aging. figure 13 shows a pathology autopsy in the laboratory on the upper left, cutting of a brain by dr. yoshida on the upper right, data on autopsied brains on the lower left, and organized specimens on the lower right. figure 13: a pathology autopsy in the laboratory on the upper left, cutting of a brain by dr. yoshida on the upper right, data on autopsied brains on the lower left, and organized specimens on the lower right. a professor in nagoya university’s department of neurology at that time was dr. gen sobue, who sent many researchers to my laboratory, and we were able to do a lot of joint research. dr. sobue is an extremely talented neurologist, active, and he played a major role in elucidating the molecular pathogenesis of als and bsma (24). he is still active as the president of aichi medical university. figure 14 is professor itsuro sobue, professor akira takahashi, and professor gen sobue from the left. figure 14: from left, professor itsuro sobue, professor akira takahashi, and professor gen sobue. in 1996, i visited oxford university, where i was able to study microscopic specimens directly from professor margaret esiri for two months and study at a prestigious neuropathology laboratory. at the time, i was also able to meet professor j trevor hughes, the author of “pathology of the spinal cord” and someone i have long admired. figure 15 shows professor esiri on the left and professor hughes and me on the right. figure 15: professor esiri on the left and professor j trevor hughes and me on the right. i conducted joint research with many young doctors studying neuropathology such as dr yasushi iwasaki, dr. maya mimuro, dr. nobuko ujihira, dr. motoko sakai, dr. tamaki iwase, dr. keizo yasui, dr. nozomi hishikawa, dr. yoji goto, dr. junichi mizuno, dr. masumi ito, dr. satoshi kuru, and dr. jun sone. we also welcomed many researchers from shanghai. the first person to come to the institute was dr. yin wang. after coming to japan, he started studying neuropathology. he worked incredibly hard and made such amazing progress that he obtained his doctorate in medicine in japan (25). dr. wang’s wife is also an excellent physician and has written a wonderful paper. figure 16 is a photo taken at the meeting of the japanese society of neuropathology. from the left, dr. iwasaki, dr. yoshida, me, dr. yang, and dr. wang. figure 16: a photo taken at the meeting of the japanese society of neuropathology. from the left, iwasaki, yoshida, me, yang, and wang. every year, dr. wang referred a neurologist from shanghai fudan university. exchanges with shanghai continued even after dr. wang returned to china. every year, i visited shanghai for a research conference, and i was invited as a visiting professor at fudan university. in 2006, i visited shanghai, beijing, and changchun with many japanese researchers and became friends with many chinese professors in each place. i would like to thank professor chuanzhen lu and professor zhurong ye in shanghai, professor luning wang, professor dehong lu, and dr. yueshan piao in beijing, and professor yu zhang in changchun. dr. y wang is currently a professor in shanghai. the photographs in figure 17 show a case conference involving group microscopy and cutting of a brain at fudan university in shanghai. figure 17: the photograph shows a case conference involving group microscopy and cutting of a brain at fudan university in shanghai. in 2003, i hosted the japanese society of neuropathology in nagoya, where i invited professor markus tolney from switzerland to give a special lecture on argyrophilic grain disease. at the conference, we were able to hold a symposium on the pathology of spinal cord diseases for the first time as the japanese society of neuropathology. at the conference, we also exhibited the following neuropathological specimens that we had encountered: 1. the brain of gin-san, a famous twin centenarian from nagoya and 2. the brain of shoichi yokoi, who returned to japan after living in the jungles of guam for 28 years after the war and who exhibited parkinsonism. on the upper left in figure 18 is me chairing the 44th annual meeting of the japanese society of neuropathology. the upper right is the photo at the social gathering of the academic society, the lower left is an exhibit of mr. shoichi yokoi’s pathological specimens, and the lower right is the photo at an exhibit of gin-san’s pathological specimens. figure 18: on the upper left is me chairing the 44th annual meeting of the japanese society of neuropathology. on the upper right is the photo at the social gathering of the academic society. from the left are doctors hiroko yamamoto, mari yoshida, kenji kosaka, me, imaharu nakano, seung kim, kazuo nagashima, and mitunori yamada. on the lower left is an exhibit of mr. shoichi yokoi’s pathological specimens. the third from the left is mr. shoichi yokoi’s wife and the fourth is dr. yoko konagaya, an attending physician of shoichi yokoi. on the lower right is a photo at an exhibit of gin-san’s pathological specimens, the center is gin-san’s daughter, and on the right is dr. chisato tanahashi, a doctor at minami seikyo hospital who autopsied gin-san. in addition to researching neuropathology, i also worked to educate medical students at nagoya university, nagoya city university, and aichi medical university, conducting lectures on neuropathology and providing specimens for pathology training. i continued giving lectures on neuropathology to medical students for about 30 years. in 2007, i happened to be appointed as the 6th president of the japanese society of neuropathology. based on exchanges with china, i endeavored to hold the first congress of the asian society of neuropathology. the president was professor kazuo nagashima of hokkaido university, and the vice presidents were dr. luning wang from china and dr. seung kim from south korea. we succeeded thanks to the cooperation of doctors from various asian countries. on the left in figure 19 is a group photo of the organizers of this meeting. the program is on the right. figure 19: on the left is a group photo of the organizers of the first congress of the asian society of neuropathology. from the left in the front row is dr. tumtip sangruchi from thailand, dr. seung kim from korea, dr. luning wang from china, dr. kazuo nagashima, dr. chitra sarkar from india, dr. thong wong from malasia, and dr. chinchen lee from taiwan. from the left in the back row is dr. masaki takao, dr. atsuo koto, me, dr. imaharu nakano, and dr. yoichi nakazato. the program of the congress is on the right. we sought to further advance neuropathology, the bulletin of the japanese society of neuropathology. its content has been enhanced through the efforts of successive editors-in-chief. as chairman, i helped to enhance a brain bank, which was an effort spearheaded by dr. shigeo murayama of the university of tokyo. i also strove to establish a regional branch of the society of neuropathology. this means that doctors who are interested in neu-ropathology will have the opportunity to study neuropathology wherever they are in japan. in 2009, the 50th anniversary ceremony of the japanese society of neuropathology was held in takamatsu under the leadership of president kiyomitsu oyanagi. i described the history of the japanese society of neuropathology. the first meeting of the japanese society of neuropathology was held in 1960 in tokyo, chaired by professor tadashi inose of yokohama city university. initially, the principal members of the society were psychiatrists. since then, the society has developed, and we were able to hold two international congresses of neuropathology in 1992 and 2018. in 2010, i retired from aichi medical university. afterwards, professor yoshida and professor iwasaki took over my research. professor iwasaki specializes in prion diseases and has produced a number of excellent papers (26). i hope that he will continue to strive to advance the institute in the future. thus far, the institute has assembled over 6,000 autopsied brains with neurological diseases, and our laboratory has grown into one of the leading neuropathological research institutes in japan. conducting research at the institute of neuropathology at fukushimura hospital since retiring from the institute of neuropathology, aichi medical university in 2010, i have been working full-time as a doctor at fukushimura hospital in toyohashi. fukushimura hospital is a large hospital with 480 beds that specializes in dementia in the mikawa area. founded by president takayuki yamamoto, the hospital works with many nursing homes and elderly care facilities and is operated by sawarabikai. on the left side of figure 20 is president yamamoto, and on the right side is the full view of fukushimura hospital. figure 20: on the left is president takayuki yamamoto, and on the right side is the full view of fukushimura hospital. president yamamoto created the institute of neuropathology for me in 2010. i am grateful for an environment that allows me to continue working in neuropathology even though i have retired from a university. fukushimura is a facility that can perform pathology autopsies on nearly 30 dementia patients a year. we hold clinical pathology meetings on three autopsies each month, and we invite many doctors to participate a discussion via zoom. i am indebted to many people at fukushimura hospital, including director osamu kohashi, vice director hiroyuki ikari, vice director hidechika okada, vice director yoshiko yamamoto, dr. akira hori, dr. hiroyasu akatsu, dr. keita sakurai, and technicians takeshi kanesaka, norihiro ogawa, naoko sonoda, and chieko taniguchi. the institute can prepare large specimens of the cerebral hemispheres, allowing us to clearly identify findings that are difficult to gauge in small specimens, such as the spread of lesions and the degree of atrophy. as a facility registered with a brain bank in japan, we have preserved a large number of frozen tissues from cases confirmed by a neuropathological examination, and we have created a system that allows joint research with researchers here in japan and abroad. recently, dr. daita kaneda has joined us as a full-time doctor and he has been very active. on the left in figure 21 is cutting of a brain at fukushimura hospital, on the upper right is me observing a specimen under a microscope, and on the lower right is a large tissue specimen of the cerebral hemisphere. figure 21: on the left is cutting of a brain at fukushimuara hospital. from the left is dr. daita kaneda, me, and technician takeshi kanesaka. on the upper right is me observing a specimen under a microscope, and on lower right is a large tissue specimen of the cerebral hemisphere. in 2019, i was able to publish “spinal cord pathology,” co-authored with professor yoshida from miwa shoten, as the culmination of my research (27). this book describes the pathology findings observed during an autopsy of various spinal cord diseases that occur during the process from human conception to aging. the purpose of this book is to provide useful information for those involved in clinical, pathological, and basic research on spinal cord diseases. this is a collection of 900 valuable histopathology photographs of spinal cord diseases that i have compiled over a long period of time, and i am proud of its unique focus on spinal cord pathology. i was very pleased that dr. akira takahashi, professor emeritus of nagoya university and someone whom i respect, praised my work as “a historical masterpiece.” on the left in figure 22 is the cover of “spinal cord pathology” and on the right is the cover of “pathology of the spinal cord” by j trevor hughes, published in 1978. this is the book that has long been my goal. figure 22: on the left is the cover of “spinal cord pathology,” and on right is the cover of “pathology of the spinal cord” by j trevor hughes, published in 1978. this is the book that has long been my goal. in 2018, i was able to give a special lecture entitled the neuropathology of neurodegenerative diseases at the international congress of neuropathology held in tokyo. in this lecture, i was able to present a summary of the neuropathological findings of neurodegenerative diseases that i have been working on for a long time (28). in addition, i was able to make a presentation on observations from a large number of autopsied brains with dementia that i encountered at the institute of neuropathology, fukushimura hospital at the symposium of the japanese society of neurology in 2019. i was able to publish the content of that presentation as “macroscopic findings of brain with dementia” in the journal neuropathology (29). i will be 80 years old in 2024, so i plan to retire from the institute of neuropathology at fukushimura hospital. finally, i will list some of the major papers by joint researchers that could not be included here (30-44). in conclusion i am grateful for the good fortune that i have enjoyed to be able to continuously conduct research specialized in neuropathology for 50 years. i am grateful to the many teachers who have supported my research, my colleagues who have conducted joint research with me, the clinicians and pathologists who have been involved in many pathological autopsies, and the technicians who have prepared so many specimens. i would like to end this article by thanking my family. my wife is a volunteer at a cancer palliative care facility, my eldest daughter is a pediatrician, my eldest son is a researcher at a major electronics company, and my second daughter is a physician. i am glad that each of them is actively living a meaningful life. figure 23 shows my family, from left to right: my eldest daughter yukiko, my second daughter mariko, my eldest son kenichi, and my wife kiyoko. figure 23: my family, from left to right: my eldest daughter yukiko, my second daughter mariko, my eldest son kenichi, and my wife kiyoko. as a hobby, i have been boat fishing for more than 40 years, and i go out with my fishing friends to wakasa bay and ise bay for red sea bream, flounder, and yellowtail. i am happy that i can still drive my own car and attempt to fish. i am grateful for my health. on the left in figure 24 is me on a fishing boat in wakasa bay, and on the right is a print of a red sea bream. finally, i would like to thank professor werner paulus and dr. osamu yokota, okayama university for giving me the opportunity to write this article. figure 24: on the left is me on a fishing boat in wakasa bay, and on the right is a print of a red sea bream. references 1. hashizume y. fünf 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sakai m, kuru s, konagaya m, kameyama t, yoshida m. hereditary spastic paraplegia (in japanese with english abstract). spine and spinal cord 2022; 35:781-786. 44. sone j, ueno s, akagi a et al. notch2nlc ggc repeat expansion causes retinal pathology with intranuclear inclusions throughout the retina and causes visual impairment. acta neuropathol commun. 2023; 11:71. https://doi.org/10.1186/s40478-023-01564-3 copyright: © 2023 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. serum neurofilament light chains in progressive multiple sclerosis patients treated with repeated cycles of high-dose intravenous steroids feel free to add comments by clicking these icons on the sidebar free neuropathology 4:15 (2023) original paper serum neurofilament light chains in progressive multiple sclerosis patients treated with repeated cycles of high-dose intravenous steroids lidia stork1, michael haupts2, niels kruse1, petra spill-askeridis3, adriane kutllovci1, martin s. weber1,4,5, wolfgang brück1, imke metz1 institute of neuropathology, university medical center göttingen, germany department of neurology, university hospital düsseldorf, germany augusta hospital, anholt, germany department of neurology, university medical center göttingen, germany department of translational neuroinflammation and automated microscopy, fraunhofer institute for translational medicine and pharmacology, göttingen, germany corresponding author: imke metz · institute of neuropathology · university medical center göttingen · robert-koch-str. 40 · 37075 göttingen · germany imetz@gwdg.de submitted: 01 august 2023 accepted: 24 september 2023 copyedited by: cinthya aguero published: 05 october 2023 https://doi.org/10.17879/freeneuropathology-2023-5049 keywords: progressive multiple sclerosis, serum neurofilament light chains, high-dose intravenous steroids abstract background and objectives: in progressive multiple sclerosis (ms) patients, cns inflammation trapped behind a closed blood brain barrier drives continuous neuroaxonal degeneration, thus leading to deterioration of neurological function. therapeutics in progressive ms are limited. high-dose intravenous glucocorticosteroids (hdcs) can cross the blood-brain barrier and may reduce inflammation within the cns. however, the treatment efficacy of hdcs in progressive ms remains controversial. serum neurofilament light chains (snfl) are an established biomarker of neuroaxonal degeneration and are used to monitor treatment responses. we aimed to investigate whether repeated cycles of intravenous hdcs reduce the level of snfl in progressive ms patients. methods: we performed a monocentric observational study of 25 patients recruited during ongoing clinical routine care who were treated with repeated cycles of intravenous hdcs as long-term therapy for their progressive ms. snfl were measured in 103 repeated blood samples (median time interval from baseline 28 weeks, range 2-55 weeks) with the single molecular array (simoa) technology. the expanded disability status score (edss) was documented at baseline and follow-up. results: the median age of patients was 55 years (range 46-77 years) with a median disease duration of 26 years (range 11-42 years). snfl baseline levels at study inclusion were significantly higher in progressive ms patients compared to age-matched healthy controls (median 16.7 pg/ml vs 11.5 pg/ml, p=0.002). snfl levels showed a positive correlation with patient age (r=0.2, p=0.003). the majority of patients (72%, 16/23) showed reduced snfl levels ≥20 weeks after hdcs compared to baseline (median 13.3 pg/ml, p=0.03). snfl levels correlated negatively with the time interval from baseline hdcs therapy (r=-0.2, p=0.03). this association was also evident after correction for treatment with disease-modifying drugs (adjusted r2=0.10, p=0.001). the edss remained stable (median 6.5) within a median treatment duration of 26 weeks (range 13-51 weeks). conclusion: although larger studies are needed to confirm our findings, we were able to demonstrate that hdcs treatment reduces snfl levels and therefore may slow down neuroaxonal damage in a subgroup of patients with progressive ms. moreover, a stable edss was observed during therapy. findings suggest that hdcs may be beneficial for the treatment of progressive ms. introduction neurodegeneration and worsening of clinical disability in progressive multiple sclerosis (ms) occur independently from newly formed, acute inflammatory lesions characterized by mri contrast enhancement [34]. pathological findings in progressive ms include so-called slowly expanding lesions, meaning white matter demyelinated lesions with slow expansion at their lesion edge, as well as extensive cortical demyelination and diffuse white matter injury (figure 1 a-c) [21, 39]. in contrast to relapsing remitting ms, the inflammation in progressive ms becomes trapped or compartmentalized behind a closed blood-brain barrier, and typically no mri lesion enhancement is observed [11, 34]. a close association between inflammation and neurodegeneration is also evident in progressive ms [20]. a profound microglial activation and meningeal lymphoid structures are associated with tissue injury and demyelination (figure 1 b-d) [28, 40]. the inflammation causes a widespread neuroaxonal loss not only in white matter lesions, but also in normal-appearing white matter as well as in cortical and deep gray matter [11, 41]. this neuroaxonal loss is thought to be the major cause of progressive, irreversible neurological disability in patients with progressive ms. therefore, treatment of progressive ms needs to target the compartmentalized cns inflammation in order to slow down neurodegeneration and disease progression. figure 1: histological findings in progressive multiple sclerosis typical histological findings in progressive ms include slowly progressing (smouldering) lesions (a, b) as well as meningeal inflammation and cortical demyelination (c, d). a: a sharply demarcated and demyelinated white matter lesion with an increased cellularity at the border of the lesion is shown (lfb/pas); b: the lesion edge is characterized by a microglia/macrophage rim (kim1p); c: a meningeal lymphoid infiltration (arrows) and underlying subpial cortical demyelination is found (anti-plp); d: the same lesion as in panel c reveals numerous b cells in the lymphoid meningeal infiltrate adjacent to the demyelinated cortex (anti-cd20). scale bars: a and b: 500µm; c: 200µm; d: 100µm. lfp/pas = luxol fast blue combined with periodic acid schiff; plp = proteolipid protein most approved disease-modifying therapies (dmts) target the peripheral immune system and reduce the number of new acute inflammatory lesions, but they do not significantly slow disability progression in progressive ms. siponimod and ocrelizumab were shown to reduce confirmed disability progression in progressive ms patients compared to placebo. however, their treatment effects were mostly observed in patients with so-called active disease, meaning in patients with relapses or imaging features of acute inflammatory activity [30]. interferon-β-1b was also approved in europe for the treatment of progressive ms with active inflammation [17]. high dose glucocorticosteroids (hdcs) as a treatment option for progressive ms, administered either alone or in combination with other immunomodulatory/immunosuppressive drugs, have been discussed for decades [5, 10, 25, 44, 46]. steroids cross the blood brain barrier and may thus also target the compartmentalized cns inflammation in progressive ms patients. a previous study suggested that the oral administration of hdcs is beneficial for the treatment of progressive ms and showed a reduction in neurofilament light chain (nfl) levels in csf [45]. nfl are structural proteins of axons that are released into the csf and serum upon neuroaxonal damage. numerous prior studies showed that serum neurofilament light chains (snfl) are a plausible biomarker for neuroaxonal degeneration. this biomarker is widely used for monitoring disease activity and treatment responses in ms [32, 52, 55]. in progressive ms, snfl correlate with brain atrophy and a higher likelihood of disability worsening [31]. treatment of progressive ms with fingolimod, natalizumab, siponimod, ocrelizumab, and autologous hematopoietic stem cell transplantation reduces snfl levels [3, 29, 36, 53]. although snfl levels are well suited for comparing patient groups, natural fluctuations should be taken into account when evaluating individual patients. fluctuations in snfl levels of up to 20% were observed in the majority of a cohort of progressive ms patients [8]. in this study we present a monocentric observational study that evaluates changes in snfl levels in progressive ms patients with repeated cycles of parenterally given hdcs. results indicate that long-term treatment with hdcs reduces the neuroaxonal damage in progressive ms patients. methods standard protocol approval and patient consents the ethics committee of the university medical center göttingen approved this study (#1/2/21). patients gave written informed consent for their study inclusion. the study has been performed in accordance with the ethical standards laid down in the 1964 declaration of helsinki and its later amendments. study cohort the study cohort was recruited in the tertiary neurological clinic augusta hospital in anholt, germany. patients were recruited during ongoing clinical routine care from september 2018 to december 2019. inclusion criteria of the study were: 1) clinically definite ms with a primary or secondary progressive disease course [54], 2) absence of relapse activity at least for 3 months prior to baseline blood sampling and 3) long-term hdcs therapy. initially, 27 patients were included in this longitudinal study. however, two patients had to be excluded due to a marked increase in snfl unrelated to ms disease activity, namely due to a traumatic brain injury in one patient and an intrathecal pump implantation in the other. all patients showed no signs of clinical activity and were thus classified as having inactive progressive ms. clinical assessment clinical information was obtained by the treating physician via medical record review as well as personal interview and examination. baseline was defined as time point of the first blood sampling of the first course of hdcs within the frame of the present study. twenty-one out of our 25 included study patients had previous cycles of hdcs before baseline blood sampling was done, with a median treatment duration of 26 weeks (range 8-120 weeks). the median daily dose of previous hdcs cycles was 1500 mg (range 300-2250 mg). according to the individual clinical situation and patient preferences, the treating physician determined the treatment regimen – the dosage of steroids, duration of cycles and interval between cycles. patients also received symptomatic treatments including physiotherapy. the clinical course was determined according to published criteria [54]. the expanded disability status scale (edss) score was assessed at two time points: at baseline and before the next hdcs cycle. relapse activity during the study period as well as the therapy with dmts were also registered. due to absence of clinical disease activity, mris were not indicated and not available for the study. in addition, comorbidities, the presence of fatigue symptoms as well as a clinical history of smoking and prior infections were noted. patients with diabetes, coronary heart disease and stroke in their clinical history were grouped as having cardiovascular risk factors. the presence of cognitive deficits and psychiatric symptoms were also recorded. blood sampling blood sampling at baseline and within the next 3 weeks (median 1.9, range 0.9 to 2.6 weeks) after baseline hdcs cycle was done for all patients. blood samples taken directly before the next hdcs cycle (median 26, range 13 51.3 weeks) were available for analysis in 24 patients. for a subset of patients, further blood samplings were performed at later time points to assess the change in snfl levels over time (median 4 samples per patient), with a median time to last follow-up of 28 weeks (range 2-55 weeks). blood was also taken from 22 healthy age-matched controls and snfl levels measured to obtain a range for normal values. snfl measurement with simoa technology serum samples were stored at −80 °c until analysis. quantification of nfl was done using the simoa nf-light advantage kit (quanterix, bellerica, ma, usa; cat. no. 103186), as described in detail previously [14]. assays were performed on a simoa hd-1 analyzer (quanterix) according to a quanterix-developed protocol. after completion of the assay, data were analyzed using quanterix software. statistical analyses demographics and clinical characteristics were described by summary statistics appropriate for their scales. differences between two groups in clinical characteristics and snfl levels were tested using the mann–whitney u test. to compare snfl levels in repeated blood samples, the wilcoxon test was used. spearman correlation was performed to address the question of potential associations between clinical parameters and snfl levels. to adjust for relevant covariates, the association of snfl with the time interval that passed from baseline was analyzed using logistic regression models. for multiple linear regression analyses, a logarithmic transformation of snfl levels was carried out. statistical analyses were done with graphpad prism 6 and ssps statistics 28 for windows. p-values smaller than or equal to 0.05 were regarded as statistically significant. data availability the first and the last authors take full responsibility for the data analyses, interpretation, and conduction of the research. they have full access to all of the data and the right to publish these data apart from any sponsorship. results patient baseline characteristics a total of 103 blood samples taken from 25 patients were included in our analyses. the patients’ demographic and clinical data at baseline are shown in table 1. clinical relapses were not observed for 3 months prior to baseline blood sampling (see inclusion criteria). no relapses occurred during the duration of the study. five patients were treated with disease-modifying therapies (n=1 interferon-ß-1a; n=2 dimethyl fumarate and n=2 glatiramer acetate). despite no signs of clinical activity, treatment with dmts was continued due to patients’ personal preferences and the absence of clear recommendations for therapy withdrawal. table 1: demographic and clinical characteristics of the study cohort baseline characteristics number of patients 25 age in years, median (min-max) 55 (46-77) gender m:f (%) 6:19 (24:76) disease duration in years, median (min-max) 26 (11-42) diagnosis at baseline spms: ppms (%) 22:3 (88:12) edss at baseline, median (min-max) 6.5 (5.5-8.5) hdcs equivalent dose per day in mg, median (min-max) 1500 (300-3000) edss before second hdcs cycle in years, median (min-max) 6.5 (6.0-8.5) mean number of blood samples per patient (min-max) 4.1 (3-7) time interval from baseline to last follow-up in weeks, median (min-max) 28 (2-55) snfl levels decrease after hdcs therapy snfl baseline levels were significantly higher in progressive ms patients compared to age-matched healthy controls (median 16.7 pg/ml vs 11.5 pg/ml, p=0.002). within the first two weeks after baseline hdcs therapy, the median snfl level was not significantly reduced (median 15.6 pg/ml, p=0.7). however, ≥20 weeks after baseline hdcs therapy the majority of patients (72%, 16/23) showed reduced snfl levels compared to baseline (median 13.3 pg/ml, p=0.03) (figure 2a). the median snfl level was still higher compared to the healthy control cohort, but these differences no longer reached statistical significance. pairwise comparison of snfl levels at baseline, subsequent hdcs cycle (2nd cycle during the study) as well as at last follow-up revealed that even more patients (76%, 19/25) had reduced snfl levels. snfl levels showed a median reduction of 22% at last follow-up compared to baseline snfl values (p=0.01) (figure 2b). this effect was also observed when excluding the 5 patients treated with dmts (p=0.001; data not shown), indicating that the snfl reduction is not due to dmt treatment. although in individual patients a 20% reduction of snfl levels may be regarded as natural fluctuation [8], on a group level this reduction seems relevant. individual changes in snfl levels are shown in figure 2c. the percentage change of snfl levels comparing values from baseline and last follow-up for all patients included in the study are given. fourteen patients (56%) had a reduction in snfl levels >20% (figure 2c), with some patients showing a decrease of snfl levels up to 50% from baseline. in three patients, snfl levels increased >20%. clinical history could not reveal any reason for these increases. figure 2: reduction of snfl levels in progressive ms patients treated with hdcs a: snfl levels at baseline, within 2 weeks after baseline hdcs cycle and ≥ 20 weeks after baseline hdcs cycle are presented. the median snfl levels in pg/ml and the interquartile range are shown. the dashed line indicates the median snfl level of the age-matched healthy control cohort. significantly lower levels of snfl were observed ≥ 20 weeks after baseline hdcs treatment (p=0.03; wilcoxon test), but not at earlier time points (<2 weeks, p=0.7). b: snfl level at baseline, before the second cycle of hdcs and at last follow-up are depicted. the median snfl levels in pg/ml and the interquartile range are shown. the dashed line shows the median snfl level of the age-matched healthy control cohort. a significant reduction in snfl levels compared to baseline was observed before the next hdcs cycle (p=0.02) and at last follow up (p=0.01; wilcoxon test). the median interval from baseline to the subsequent hdcs cycle was 26 weeks and to the last follow-up 28 weeks. c: snfl change in percent from baseline to last follow-up shown for individual patients. the majority of patients shows a decrease in snfl levels. five patients showed an increase of snfl level, which was not accompanied by clinical disease activity. the dashed line indicates a 20% decrease or increase of snfl levels, possibly reflecting natural fluctuations of snfl values [8]. snfl= serum neurofilament light chains; hdcs= high-dose corticosteroids in accordance with these findings, correlation and logistic regression model analyses showed a negative correlation between snfl levels and the time that passed after baseline hdcs therapy (r=-0.2, p=0.03; median time interval from baseline 24 weeks, range 0.9-55.0 weeks) (table 2). table 2: correlation of snfl levels with clinical parameters clinical parameters correlation coefficient spearman p-value number of values time after hdcs therapy -0.2 0.03 103 age 0.3 0.003 103 gender -0.2 0.5 25 treatment with dmts -0.2 0.4 25 edss 0.1 0.6 49 disease course 0.0 1.0 25 disease duration -0.1 0.8 25 fatigue -0.4 0.1 24 cognitive deficits 0.1 0.6 25 psychiatric symptoms 0.3 0.1 25 current smoker 0.0 1.0 24 former smoker 0.2 0.3 24 cardiovascular risk factors -0.3 0.2 25 hdcs equivalent dose per day -0.4 0.1 21 stable edss after hdcs therapy the edss score remained stable during a median follow-up time of 26 weeks (range 13-51 weeks) after baseline hdcs therapy (median edss 6.5 at baseline and before second hdcs cycle). confounding factors for snfl levels as described in previous studies, snfl levels showed a positive correlation with patient age (r=0.3, p=0.003) (figure 3a). the gender had no influence on snfl levels. other factors such as relapse activity and disease-modifying drugs are also known to influence snfl levels, but relapse activity was not observed in our cohort of progressive ms patients. the following clinical parameters were analyzed and did not significantly influence snfl levels: treatment with dmts, disability level measured by edss, disease course and disease duration, presence of fatigue symptoms, cognitive deficits, psychiatric symptoms, past or present regular nicotine use and cardiovascular risk factors. no association was found between the dose of hdcs that patients received and snfl levels (table 2). we performed a multiple regression analysis including the age of patients, treatment with dmts and the time after baseline hdcs therapy to analyze the influence on snfl levels. the negative correlation of snfl levels with the time after baseline hdcs therapy was confirmed (adjusted r2= 10%, p=0.01), verifying a reduction in snfl levels after hdcs therapy (figure 3b). dmts showed no correlation with snfl and did not influence the level of snfl in the multiple regression model. however, this analysis was limited due to a small number of patients treated with dmts (n=5). the model is summarized in table 3. figure 3: partial regression plots of variables that contribute to snfl levels the dependent variable is the snfl level. independent variables are the age of patients at the time of blood sampling (a) and the time that passed after the baseline hdcs therapy (b). due to calculations with multiple variables, the scaling of axes differs from normal units and does not seem plausible. snfl levels correlate positively with the age of patients (a, p=0.003) and shows a negative correlation with the time that passed after the baseline hdcs therapy (b, p=0.03), indicating a reduction in snfl levels due to hdcs treatment. snfl= serum neurofilament light chains; hdcs= high-dose corticosteroids table 3: model to explain snfl levels in patients after hdcs therapy unstandardized coefficient standardized coefficient significance 95% confidential interval (lower and upper bound) time after hdcs therapy -0.003 -0.247 0.001 -0.005 -0.001 treatment with dmts 0.015 0.035 0.718 -0.067 0.097 age at time of blood sampling 0.007 0.237 0.015 0.001 0.012 snfl levels were defined as dependent variable. variables which possibly effect snfl levels were included in the model. the unstandardized coefficient, the standardized coefficient, the significance, and the confidence interval are shown. this model has been corrected for r2=10%. discussion we aimed to evaluate the efficacy of long-term treatment with hdcs in patients with progressive ms and used snfl as an established biomarker for disease activity and treatment response. in our cohort of 25 patients recruited from ongoing clinical routine care, we found that long-term repeated hdcs therapy for patients with progressive ms reduced snfl levels by 22% after 20 weeks. we observed a reduction of snfl levels in the majority of patients, so that these changes are most likely due to the steroid therapy and not to normal fluctuations as described by bridel et al. [8]. this implies that hdcs treatment may reduce ongoing diseaseactivity and neuroaxonal damage in a subgroup of progressive ms patients, which is the main cause of accumulation of clinical disability. in support of this, the edss score did not increase during the treatment period. hdcs are effective not only in relapsing-remitting ms but also in progressive ms hdcs are the first-line treatment for ms relapses [6, 35, 44]. this treatment typically provides a rapid clinical benefit, decreasing the severity of clinical symptoms and duration of the attack [18]. intravenous hdcs treatment of relapses was also associated with a lower risk of contrast-enhancing lesions to turn into black holes, indicating a reduction in destructive lesions [13]. here, we have shown that repeated hdcs cycles reduce snfl levels in a subgroup of patients with progressive ms without signs of active inflammation. early publications support our findings and describe an improvement in the edss score as well as a functional benefit from repeated hdcs cycles in progressive ms [5, 10, 25, 44]. even oral monthly administration of hdcs showed a benefit in progressive ms: the edss, ms functional composite test and t2 lesion volume showed a significant improvement after oral hdcs therapy [45]. however, a reduction in csf biomarkers of inflammation and neurodegeneration including nfl was not observed [45]. in our study, we assessed the treatment effect with the serum biomarker nfl and observed a reduction in the majority of patients (76%) after repeated intravenous cycles of hdcs. importantly, all of our patients except for one had received hdcs treatments before baseline blood sampling, so that a reduction of snfl might already have occurred before baseline blood sampling. thus, the effect of hdcs therapy on snfl levels might even be stronger than described here. functional improvement of neurological systems and mri parameters were not addressed in our study. snfl levels for hdcs therapy monitoring and long-term treatment effects snfl levels serve as a highly sensitive marker for neuroaxonal injury in different conditions such as alzheimer, parkinson’s, traumatic brain injury or amyotrophic lateral sclerosis [1, 22, 27, 48]. their concentration increases with age by an average of 2.2% per year in healthy controls [15]. an age-dependent increase in snfl levels could also be confirmed in our study. increased snfl levels reflect recent or ongoing neuroaxonal damage related to ongoing inflammatory activity in late-stage ms [7, 14]. a reduction in snfl levels indicates less neuroaxonal loss and thus beneficial treatment effects. in progressive ms patients, a reduction in snfl was observed after treatment with dmts as well as after autologous hematopoietic stem cell transplantation [3, 29, 33, 36, 53]. snfl levels typically decrease within 3-6 months after initiation of anti-inflammatory therapies in relapsing-remitting ms [32]. similar snfl dynamics were found in our study. a significant reduction in snfl levels was evident for the first time ≥20 weeks after baseline hdcs treatment, but not at earlier time points, and showed a positive correlation with the time that passed after hdcs treatment. long-term immunomodulatory effects of hdcs have already been described in previous studies. for example, treatment of the first optic neuritis episode with intravenous hdcs was found to reduce the development of clinical ms within two years [4]. brain atrophy and the development of t1 black holes in ms patients were reduced with prolonged treatment with intravenous hdcs [56]. a positive effect on the sustained progression of disability after hdcs treatment in progressive ms patients was also observed [25]. mode of action of hdcs in progressive ms corticosteroids are used in ms treatment due to their anti-inflammatory effect by inhibiting lymphocyte proliferation and the secretion of pro-inflammatory cytokines, mediated through specific corticoid receptors. however, the immediate and long-term mechanisms of steroid actions are complex and involve both non-genomic and genomic pathways [24]. interaction of steroids with the cellular membrane and membrane receptors is responsible for the rapid non-genomic mechanism of action, whereas binding of specific cytosolic receptors mediates delayed genomic effects [9, 24]. in progressive ms, the degree of inflammation declines with age and disease duration, but still drives neuroaxonal loss and thus the accrual of disability [20]. so-called smouldering lesions with slowly progressing demyelination and axonal damage at the edge of white matter lesions are a hallmark of progressive ms. these lesions are driven by pro-inflammatory microglial cells as well as by astrocytes with a characteristic and partially pro-inflammatory signature [2, 28]. next to white matter pathology, lymphoid structures in meninges orchestrate inflammation, demyelination, and neurodegeneration in underlying cortical tissue [40]. steroids can restore the blood brain-barrier (bbb) and reduce the contrast enhancement of ms lesions. they downregulate the expression of adhesion molecules reducing the extravasation of immune cells into the cns and improve the integrity of the tight junctions between endothelial cells [16, 23, 47]. however, this effect is short-term, as new contrast enhancing lesions can already be observed 4 weeks after treatment [19, 43]. in our cohort of inactive, progressive ms patients, we found no indication of a bbb impairment, and treatment effects were evident 20 weeks after hdcs treatment. we thus assume that the reduced snfl levels cannot be explained by steroid effects on the bbb, but indeed indicate a reduction in cns axonal damage. hdcs treatment reduces the level of the pro-inflammatory cytokines ifn-γ, tnf-α and il-12, and the effect on tnf-α may last up to several months after the therapy [12, 50]. the anti-inflammatory cytokine il-10 is also upregulated [26]. steroid treatment induces apoptosis of t and b cells [37, 49] and shifts the population of cd4+ t lymphocytes to the memory subpopulation [42]. chronic activation of microglial cells and astrocytes may also be influenced by hdcs: in an animal model of spinal cord injury, steroids could inhibit microglial activation and support the normal function of astrocytes [57]. hdcs treatment also inhibits proliferation of microglia/macrophages and reduces the production of nitric oxide, which is a main effector molecule of these cells [38, 51]. in conclusion, hdcs treatment may reduce inflammatory processes that characterize progressive ms pathology. limitations of the study although the present study is limited due to the small patient number, we were able to show a reduction in snfl levels with long-term hdcs treatment. the observational design of the study implies that patients were not randomized to treatment, and a non-treated control cohort could not be studied. the hdcs therapy was also not standardized, and dosage of steroids ranged from 300-2250 mg per day, depending on the individual clinical situation and patient preferences. however, statistical analyses did not show any steroid dosage effect. patients included in the study did not experience any relapse activity 3 months prior to the study. we can thus assume that the observed reduction in snfl was indeed associated with hdcs therapy and was not a consequence of a typical snfl reduction observed within the months after a relapse had occurred. edss score and clinical disease activity were outcome parameters in the study. due to the absence of clinical symptoms, mris were not indicated for routine hospital evaluation and thus not available for analysis. hence, “silent” mri activity cannot be completely excluded. although we did not document steroid side effects in a standardized manner, no severe side effects were observed during the observational period. conclusion in conclusion, we have shown that treatment with repeated cycles of hdcs 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international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. an optimized filter trap assay for detecting recombinant authentic tau fibrils feel free to add comments by clicking these icons on the sidebar free neuropathology 5:15 (2024) methods an optimized filter trap assay for detecting recombinant authentic tau fibrils allison r. balaj1, thomas l. rothstein1, hiroaki kaku1 department of investigative medicine, western michigan university homer stryker m.d. school of medicine, kalamazoo, mi, usa corresponding author: hiroaki kaku · department of investigative medicine · western michigan university homer stryker m.d. school of medicine · kalamazoo, mi · usa hiroaki.kaku@wmed.edu additional resources and electronic supplementary material: supplementary material submitted: 23 april 2024 accepted: 14 july 2024 copyedited by: georg haase published: 02 august 2024 https://doi.org/10.17879/freeneuropathology-2024-5497 keywords: truncated tau fibrils, filter trap assay, sarkosyl, tauopathies, protein aggregation abstract the development and optimization of the filter trap assay (fta) for the detection of authentic tau fibrils in vitro mark a pivotal advancement in the realm of tauopathy research, particularly by addressing the limitations of using polyanion-induced tau fibrils, which structurally differ from those isolated from tauopathy patients. recently it has been shown that truncated tau fragment (297-391), also termed dgae, can form authentic tau fibrils in the absence of polyanions. this study introduces a refined protocol that reliably detects authentic tau fibrils in a physiologically relevant framework, utilizing nitrocellulose membranes to achieve heightened sensitivity. our investigation highlights the superior efficacy of sarkosyl, an anionic surfactant traditionally used to prepare protein lysates from brains and cultured neurons, in preserving the aggregated state of tau dgae fibrils in vitro, underscoring its potential for further exploratory studies. by offering a user-friendly and economically feasible approach, this technique enables a broad range of laboratories to measure the presence of authentic tau fibrils. this methodological enhancement propels our understanding of tauopathies forward and bridges the gap between basic research and advanced structural analyses, enriching the scientific community's methodologies for studying neurodegenerative disorders. introduction the microtubule-associated protein tau forms disease-specific filamentous aggregates in tauopathies, including alzheimer’s diseases (ad). recent advances in cryo-electron microscopy (cryo-em) have made it a powerful tool for determining the 3d structure of various proteins, including tau aggregates. the structure of aggregated tau from ad patients was determined: tau filament cores are made of two identical protofilaments comprising residues 306-378 of tau, which adopt a combined cross-β/β-helix structure and define the seed for tau aggregation1. to model tau aggregation in vitro, polyanions, such as heparin, have been widely used to mimic tau aggregation2,3. however, the 3d structure of heparin-induced tau fibrils is quite different from those found in patients. unlike the uniform structure observed in disease-related tau, heparin-induced tau fibrils are a mixture of three distinct structures: snake, twister, and jagged4. thus, heparin-induced tau aggregates may not represent a physiological model for in vitro studies. this raises important considerations about the relevance and accuracy of using heparin-induced models in tau aggregation research. to overcome this issue, methods to generate tau aggregates without the need for polyanions were identified using the aggregation core region, such as truncated tau 297-391, termed dgae tau5,6. this breakthrough offers new avenues for studying tau aggregation more physiologically. the filter trap assay (fta), also known as the filter retardation assay, is a widely recognized, user-friendly in vitro method for detecting protein aggregates including polyanion-induced tau aggregates. given the structural differences between polyanion-induced tau aggregates and those extracted from tauopathy patients, work on the aggregation of the dgae tau variant is expected to increase. a common approach to assess dgae tau aggregation involves the use of thioflavin t (tht) fluorescence assays. however, these fluorescence-based assays sometimes yield inconsistent results, necessitating alternative methods to verify findings. in this context, fta, which utilizes a distinct mechanism for detecting tau aggregates, emerges as an essential complementary technique. while fta detection methods for polyanion-induced tau aggregates have been previously established, the methods for detecting authentic tau fibrils remain largely unexplored7. the notable structural differences between these fibrils suggest distinct biochemical characteristics. herein, we present an optimized fta protocol for analyzing dgae tau aggregates. this protocol is designed to provide more reliable and reproducible results than previous protocols, thereby enhancing the study of tau aggregation dynamics in vitro and contributing to a deeper understanding of tauopathies. results initially, we followed the fta protocol published in 20087. according to this protocol, tau protein samples are diluted in 2 % (w/v) sodium dodecyl sulfate (sds) and the fta is conducted using three types of membranes: nitrocellulose, cellulose acetate, and polyvinylidene fluoride (pvdf)7. our findings indicated that the nitrocellulose membrane yielded the highest sensitivity. notably, the sensitivity of dgae tau fibrils in the fta diminished when diluted in 2 % sds (figure 1a). subsequently, we explored fta using various solutions, such as phosphate buffered saline (pbs), 1 % (w/v) sarkosyl, and assembly/hepes (4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid) buffer, all of which have been previously documented for suspending whole tau fibrils6,8,9. tau dgae fibrils maintained their aggregated form more effectively when diluted in 1 % sarkosyl (figure 1a). no significant difference in sensitivity was observed between nitrocellulose membranes with pore sizes of 0.2 μm and 0.45 μm (figure 1a). as little as 7.8 ng of dgae fibrils could be detected using a 0.45 μm pore size nitrocellulose membrane when diluted in 1 % sarkosyl (figures 1b and 1c). figure 1. optimal sensitivity in detecting dgae tau fibrils is achieved using a 1 % sarkosyl solution in conjunction with a nitrocellulose membrane a. dgae tau protein (100 ng) was applied in duplicate per well (50 μl/well) to nitrocellulose membranes and pvdf membranes pre-treated with methanol for 30 seconds using a 96-well dot-blot apparatus and visualized with an anti-dgae antibody. similar results were obtained from at least three independent experiments. b. different amounts of dgae tau protein were processed similarly, representative of three independent experiments. c. densitometry analysis was performed using bio-rad imagelab 6.1, with results normalized to the highest tau fibril concentration (1000 ng) in 1 % sarkosyl from three independent experiments. using sds-page we found that the majority of dgae tau aggregates were reduced to monomers when treated with laemmli sample buffers containing sds or lithium dodecyl phosphate (lds), regardless of heat denaturation and reducing processes (figure 2a). to corroborate our observations, we subjected protein samples, prepared in pbs, sds, and sarkosyl, to native-gel page followed by western blotting. both the monomeric and fibrillar forms of tau dgae protein, when in pbs, remained in the wells, presumably due to the high basic nature of tau dgae, which has a theoretical isoelectric point of 9.59 and thus would be positively charged in the absence of an anionic surfactant10. when subjected to anionic detergents, the dgae tau proteins displayed distinct behaviors: in sds, the fibrils predominantly were broken down into monomers, whereas in sarkosyl, they retained their fibrillar integrity (figure 2b). figure 2. the majority of dgae tau aggregates are reduced to monomers when treated with sds a. dgae tau protein (100 ng/lane) was treated with laemmli buffer containing sds or lithium dodecyl sulfate (lds), with and without 100 mm dithiothreitol (dtt), and processed both unboiled and boiled for 10 minutes. proteins were then subjected to sds-page and visualized using coomassie brilliant blue staining. b. samples in pbs, 2 % sds or 1 % sarkosyl (100 ng/lane) were analyzed using native page and western blotting, detecting tau protein with an anti-dgae tau antibody. data shown are representative of two independent experiments. taken together, these data imply that for effective fta detection, tau dgae samples should be diluted in sarkosyl that preserves their aggregated state. discussion the introduction of truncated tau variants such as dgae tau offers a more representative in vitro model for studying tau aggregation as compared polyanion tau aggregation. we optimized a protocol for the fta using dgae tau aggregates, representing a notable stride towards more accurate and physiologically relevant in vitro models. the use of nitrocellulose membranes in the fta emerged as a key factor in achieving high sensitivity in detecting tau aggregates, thereby providing a robust alternative to tht fluorescence assays, which are prone to inconsistency. moreover, our exploration of different solvents such as pbs, 1 % sarkosyl, and assembly/hepes buffer for fta further refines the approach to study tau aggregation. although sarkosyl has traditionally been used to prepare lysates from brain tissues and from cultured neurons to detect tau aggregates, the effectiveness of sarkosyl in maintaining the aggregated state of tau dgae fibrils in vitro presents a promising avenue for future studies. the underlying mechanism for the enhanced efficiency of sarkosyl in detecting tau dgae aggregates through fta compared to pbs and assembly/hepes buffer remains elusive. we speculate that the unique physicochemical properties of sarkosyl may interact with tau aggregates in a manner that is less denaturing than sds, or promotes the stabilization of tau aggregates, thus facilitating their capture on the membrane. further investigations are required to delineate the specific molecular interactions and biophysical changes induced by sarkosyl, which may offer insights into its preferential efficacy and may potentially uncover novel aspects of tau aggregation dynamics. thus, the assay we developed in this study is especially relevant given the need for more reliable and reproducible methods in tauopathy research, particularly in view of its simple, efficient, user-friendly and cost-effective approach. this ease of use and affordability make fta an accessible tool for laboratories with varying resources, democratizing research in neurodegenerative diseases. this method offers a practical alternative to more complex and expensive techniques, such as em. while cryo-em provides unparalleled detail in structural analysis, its high cost and technical requirements can be prohibitive. in contrast, the fta protocol, especially with the use of nitrocellulose membranes, presents a straightforward and economical solution for initial screening and analysis of authentic tau aggregates. this approach not only facilitates routine laboratory investigations but may also serve as a critical preliminary screening step before employing more sophisticated methods. the capacity to detect minimal quantities of dgae fibrils (as low as 7.8 ng) using affordable materials underscores its practicality for widespread application. the flexibility of the method, demonstrated by the effective use of different solvents like sarkosyl, adds to its appeal in diverse research settings. this study therefore positions the optimized fta protocol as an excellent tool in the arsenal of in vitro cell-free tauopathy research, enabling more laboratories to engage in meaningful investigation of tau aggregation. it paves the way for the use of more intricate techniques such as em to further elucidate the complex mechanisms underlying tauopathies. by providing a cost-effective and user-friendly method, we bridge the gap between basic research and advanced structural analysis, fostering a more comprehensive understanding of tauopathies. author contributions conceptualization, h.k.; software, h.k.; investigation, a.r.b. and h.k.; resources, h.k.; data curation, a.r.b. and h.k.; writing original draft preparation, h.k.; writing review and editing, a.r.b., t.l.r. and h.k.; visualization, h.k.; supervision, h.k.; project administration, h.k.; funding acquisition, t.l.r. and h.k. acknowledgments we are grateful to our colleagues for helpful discussions and technical assistance throughout the course of this study. this work was supported by the national institute on aging of the national institutes of health (nih) under award number r03 ag072148. data availability statement the datasets generated and/or analyzed during the current study are available on demand. conflicts of interest statement the authors declare no conflicts of interest. funding statement this work was supported by the pilot research project grant awarded by the western michigan university homer stryker m.d. school of medicine, and public health service grant ag072148 awarded by the national institutes of health. references 1. fitzpatrick, a. w. p. et al. cryo-em structures of tau filaments from alzheimer's disease. nature 547, 185-190, https://doi.org/10.1038/nature23002 (2017). 2. friedhoff, p., schneider, a., mandelkow, e. m. & mandelkow, e. rapid assembly of alzheimer-like paired helical filaments from microtubule-associated protein tau monitored by fluorescence in solution. biochemistry 37, 10223-10230, https://doi.org/10.1021/bi980537d (1998). 3. montgomery, k. m. et al. chemical features of polyanions modulate tau aggregation and conformational states. j am chem soc 145, 3926-3936, https://doi.org/10.1021/jacs.2c08004 (2023). 4. zhang, w. et al. heparin-induced tau filaments are polymorphic and differ from those in alzheimer's and pick's diseases. elife 8, https://doi.org/10.7554/elife.43584 (2019). 5. al-hilaly, y. k. et al. tau (297-391) forms filaments that structurally mimic the core of paired helical filaments in alzheimer's disease brain. febs lett 594, 944-950, https://doi.org/10.1002/1873-3468.13675 (2020). 6. lovestam, s. et al. assembly of recombinant tau into filaments identical to those of alzheimer's disease and chronic traumatic encephalopathy. elife 11, https://doi.org/10.7554/elife.76494 (2022). 7. chang, e. & kuret, j. detection and quantification of tau aggregation using a membrane filter assay. anal biochem 373, 330-336, https://doi.org/10.1016/j.ab.2007.09.015 (2008). 8. greenberg, s. g. & davies, p. a preparation of alzheimer paired helical filaments that displays distinct tau proteins by polyacrylamide gel electrophoresis. proc natl acad sci u s a 87, 5827-5831, https://doi.org/10.1073/pnas.87.15.5827 (1990). 9. desantis, m. e. et al. operational plasticity enables hsp104 to disaggregate diverse amyloid and nonamyloid clients. cell 151, 778-793, https://doi.org/10.1016/j.cell.2012.09.038 (2012). 10. gasteiger, e. et al. expasy: the proteomics server for in-depth protein knowledge and analysis. nucleic acids res 31, 3784-3788, https://doi.org/10.1093/nar/gkg563 (2003). copyright: © 2024 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. ex situ perfusion fixation for brain banking: a technical report feel free to add comments by clicking these icons on the sidebar free neuropathology 3:22 (2022) original paper ex situ perfusion fixation for brain banking: a technical report andrew t. mckenzie1,2,3,4, emma woodoff-leith1,2,3, diana dangoor1,2,3, alessandra cervera1,2,3, hadley walsh ressler1,2,3, kristen whitney1,2,3, kristen dams-o’connor5,6, zhuhao wu1,7,8, elizabeth m. c. hillman9, alan c. seifert10, john f. crary1,2,3 1 department of neuroscience, icahn school of medicine at mount sinai, new york, new york, usa 2 friedman brain institute, departments of pathology, neuroscience, and artificial intelligence & human health, icahn school of medicine at mount sinai, new york, new york, usa 3 neuropathology brain bank & research core and ronald m. loeb center for alzheimer's disease, icahn school of medicine at mount sinai, new york, new york, usa 4 department of psychiatry, icahn school of medicine at mount sinai, new york, new york, usa 5 department of rehabilitation and human performance, icahn school of medicine at mount sinai, new york, new york, usa 6 department of neurology, icahn school of medicine at mount sinai, new york, new york, usa 7 department of cell, developmental, and regenerative biology, icahn school of medicine at mount sinai, new york, new york, usa 8 black family stem cell institute, icahn school of medicine at mount sinai, new york, new york, usa 9 laboratory for functional optical imaging, departments of biomedical engineering and radiology and the mortimer b. zuckerman mind brain behavior institute, columbia university, new york, new york, usa 10 biomedical engineering and imaging institute, department of diagnostic, molecular and interventional radiology, and graduate school of biomedical sciences, icahn school of medicine at mount sinai, new york, new york, usa corresponding author: john f. crary · department of pathology · icahn school of medicine at mount sinai · icahn building 9th floor, room 20a · 1425 madison avenue · new york, ny 10029 · usa john.crary@mountsinai.org submitted: 15 august 2022 accepted: 20 september 2022 copyedited by: georg haase published: 28 september 2022 https://doi.org/10.17879/freeneuropathology-2022-4368 keywords: brain banking; perfusion fixation; perfusion pressure; postmortem interval; tissue morphology; rnascope; ex vivo neuroimaging abstract perfusion fixation is a well-established technique in animal research to improve preservation quality in the study of many tissues, including the brain. there is a growing interest in using perfusion to fix postmortem human brain tissue to achieve the highest fidelity preservation for downstream high-resolution morphomolecular brain mapping studies. numerous practical barriers arise when applying perfusion fixation in brain banking settings, including the large mass of the organ, degradation of vascular integrity and patency prior to the start of the procedure, and differing investigator goals sometimes necessitating part of the brain to be frozen. as a result, there is a critical need to establish a perfusion fixation procedure in brain banking that is flexible and scalable. this technical report describes our approach to developing an ex situ perfusion fixation protocol. we discuss the challenges encountered and lessons learned while implementing this procedure. routine morphological staining and rna in situ hybridization data show that the perfused brains have well-preserved tissue cytoarchitecture and intact biomolecular signal. however, it remains uncertain whether this procedure leads to improved histology quality compared to immersion fixation. additionally, ex vivo magnetic resonance imaging (mri) data suggest that the perfusion fixation protocol may introduce imaging artifacts in the form of air bubbles in the vasculature. we conclude with further research directions to investigate the use of perfusion fixation as a rigorous and reproducible alternative to immersion fixation for the preparation of postmortem human brains. introduction a detailed examination of the brain in various settings, including educational, clinical, and research domains, is critically dependent upon the quality of brain preservation. human tissue-based research, which plays a significant role in studying brain diseases, relies on the brain banking process to sufficiently preserve the postmortem brain. for example, in the study of brain disorders such as alzheimer’s disease, it is essential to have well-maintained histoarchitecture and biomolecular structure to reliably distinguish the features of brain donors with and without the disorder (lucassen et al., 1997). the goal of the initial preservation procedure is to interrupt postmortem degradative processes in a way that prepares the brain for long-term storage, while minimizing damage resulting from the preservation process itself. with access to intact, well-preserved human brain tissue, investigators can conduct a diverse set of next-generation brain mapping studies, including high-resolution ex vivo neuroimaging (pallebage-gamarallage et al., 2018), connectomics studies using high-throughput serial section electron microscopy (shapson-coe et al., 2021), and volumetric 3d histologic imaging of the brain’s cellularand molecular-level organization (patel et al., 2022). all existing brain preservation procedures face trade-offs in the extent and type of tissue preservation achieved and the consequent degree of information loss. in non-human animal studies, perfusion fixation is widely considered to be the state of the art for achieving the highest-quality morphologic preservation of the whole brain (bodian, 1936; karlsson and schultz, 1965; mcfadden et al., 2019). in human tissue brain banking, immersion fixation is the most common preservation method employed. a key conundrum in immersion procedures is that the outer regions of the brain are liable to be over-fixed and the inner regions of the brain are liable to be under-fixed, both of which have the potential to limit optimal and consistent antigen preservation. while sectioning tissue prior to immersion may allow fixative to penetrate inner brain regions earlier, this technique can damage tissue morphology (adickes et al., 1997). perfusion fixation is a potential solution for this challenge and may more consistently preserve tissue morphology and antigenicity than immersion fixation. the perfusion technique may allow for a shorter fixation period, without requiring pre-sectioning, by penetrating inner brain regions more quickly. perfusion fixation is also expected to limit tissue autolysis, a postmortem change that inevitably occurs in the inner brain regions before the immersion fixative can reach those areas (beach et al., 1987; mcfadden et al., 2019). perfusion can either be performed via an intrathoracic approach in the case of smaller animals or via an intracarotid approach in the case of larger animals such as pigs or elephants (manger et al., 2009; musigazi et al., 2018). the two classes of methods to perform perfusion fixation are ex situ methods, where the brain is extracted from the skull prior to perfusion, and in situ methods, where the brain remains inside the skull during perfusion (mcfadden et al., 2019) (figure 1). in situ approaches often involve cannulation of one or both carotid arteries, with or without also cannulating the vertebral arteries. ex situ approaches often involve cannulation of the internal carotid arteries and the vertebrobasilar artery system. figure 1. two major approaches for performing perfusion fixation example approaches of human brain perfusion fixation using an in situ (brain inside of the skull); a) or ex situ (brain removed from the skull); b) approach. while only an ex situ perfusion protocol is described in this report, we include an illustration of the in situ method as well to demonstrate this alternative approach. also, while the use of surgical sutures is an alternative approach that is illustrated here, in our protocol, hemostats are used to clamp cannulae in place. arrows represent the direction of perfusate flow during perfusion. scm = sternocleidomastoid muscle. illustrations by jill k. gregory, certified medical illustrator, fellow of the association of medical illustrators. the purpose of this technical report is to describe our use of perfusion fixation for human brain banking as a quality improvement project. we used an ex situ approach, which can be more easily integrated into the autopsy suite workflow and which is compatible with the anatomical donation of the brain alone, as opposed to a full-body donation. our aim is to describe our experience in implementing this procedure to provide pertinent information for the research community. there have been several previous descriptions of ex situ approaches to perfusion fixation in brain banking described in the literature (adickes et al., 1997; beach et al., 1987; de oliveira et al., 2012; grinberg et al., 2008; halliday et al., 1988; insausti et al., 1995; waldvogel et al., 2006; welikovitch et al., 2018). we recognize that there are likely many other approaches. we also suspect that perfusion fixation methods in brain banking will likely evolve in the future as more researchers engage with the procedure. materials and methods patient samples all specimens were obtained and de-identified at the icahn school of medicine at mount sinai in accordance with its policies, regulations, and institutional review board recommendations. the use of the perfusion fixation protocol was developed as part of an ongoing quality improvement project in the neuropathology brain bank & research core. the goal of the project is to see whether it is possible to decrease fixation times and thereby mitigate antigenicity loss of formalin-sensitive antigens in intact brains. the specimens were a convenience sample of brains processed at the neuropathology brain bank & research core, with no specific exclusion criteria. perfusion fixation methods we used the same perfusion circuit setup throughout the development of the protocol, with or without an in-line pressure sensor. details of the procedure, including the use of different reagents, were iterated upon over the course of the study. in our perfusion circuit, fluid in a beaker, which consists of either washout or fixative solution, is pumped through silicone tubing via a peristaltic pump with a variable pump drive speed, which mediates the flow rate (see table 1 for details of equipment used). downstream of the pump, there is a pulse dampener that acts to prevent significant pulsations in fluid flow. the next component in the circuit is the pressure sensor, which connects to a digital pressure monitor. finally, there is a y connector that splits the circuit into two circuits, each of which connects to a catheter that can be used to cannulate one blood vessel. following cannulation, the cannulae are clamped in place with hemostats in all cases. if it is preferred to perfuse through only one of the two catheters at a time, the perfusion tubing following the y connector for the other catheter can be clamped with a hemostat to stop the flow through that component of the circuit. all perfusion procedures are performed at room temperature. † catheter bevels are filed to have blunt ends prior to use in perfusion. we performed this procedure on two types of specimens, termed either ‘whole brain’ or ‘hemibrain’. the term hemibrain refers to one of the two cerebral hemispheres, which is obtained after a hemi-sectioning procedure designed to retain enough of the circle of willis for adequate vascular access. three hemi-brains (cases 1-3) and two whole brains (cases 4-5) were subjected to perfustion fixation. in case 1, the hindbrain was not removed prior to the perfusion procedure; however, in all other cases, the hindbrain was detached following transection through the upper midbrain to vascular access. thus, whole brain specimens have both cerebral hemispheres, but some brainstem structures removed. there were a couple of differences in how perfusion fixation was performed for hemibrain and whole brain specimens. first, for hemibrains, a plastic container with muslin cloth was used to suspend the specimen without immersing it in fixative, allowing the perfusate to drain into the bottom of the container during the procedure. for whole brains, there was a focus on volumetric analysis with ex vivo neuroimaging. as a result, for the whole brain cases, the specimen was suspended in a formalin bath, because suspending the brain in fluid was expected to less shape deformation during the procedure. second, for whole brain perfusions, fixation was accelerated after the initial procedure by injecting fixative into ventricular spaces in the brain using a 50 ml syringe. we employed this additional fixation step to mimic how fixative more readily diffuses into the ventricular system for hemibrains, assuming that the septum pellucidum does not act as a barrier to diffusion, which is expected to be the case following brain hemisection. following perfusion, the brains were fixed via additional immersion in 10% (w/v) neutral buffered (sodium phosphate) formalin for approximately two weeks. in the perfusion fixation literature, this subsequent immersion step is referred to as postfixation (mcfadden et al., 2019). the two whole brain specimens were imaged with mri as described below. then the brains were dissected coronally into slabs 3-5 mm thick in the anterior to posterior direction. brains were systematically sampled for neuropathologic postmortem diagnosis and placed into cassettes for processing. the samples were embedded in paraffin and 5-7 μm thick sections were cut serially on a microtome, mounted on glass slides, deparaffinized, and stained with hematoxylin and eosin (h&e) or luxol fast blue counterstained with h&e for microscopic examination. the slides for cases 1, 2, and 3 were viewed and photographed using a nikon eclipse ci microscope. the slides for cases 4 and 5 were imaged using a philips ultra fast scanner. rna in situ hybridization rna in situ hybridization was performed using the fully automated rnascope 2.5 lsx reagent kit-red (advanced cell diagnostics, catalog #322750) according to the manufacturer’s instructions for formalin-fixed paraffin-embedded (ffpe) brain tissue. 5 µm ffpe tissue sections prepared from perfusion fixation brain tissue were baked for 1 hour at 70° c, and the rnascope assay was run on the leica bond rx automated slide stainer platform (leica biosystems). target retrieval was performed for 20 minutes at 95° c followed by 15 minutes protease iii treatment at 40° c and amplification and detection steps according to the manufactures protocol. a probe for the housekeeping gene ubiquitin c (hs-ubc, catalog # 312028) was used to assess mrna detection. slides were visualized using a nikon eclipse brightfield microscope with a nikon ds-fi3 camera and nis elements software. ex vivo whole brain mri imaging following perfusion, both of the two ex vivo whole brain specimens were placed in a custom-built imaging container (boonstra et al., 2021), immersed in fluorinert (3m, saint paul, mn), and exposed to vacuum for 15 minutes to dislodge air bubbles (stram et al., 2022). the container was sealed, and the specimen was imaged using a 7 tesla whole-body mri scanner (magnetom 7t as, siemens, erlangen, germany) equipped with a 32-channel radiofrequency head coil (nova medical, wilmington, ma). after acquisition of localizer images and main magnetic field shimming by 3d field mapping, the specimens were imaged with a 3d multi-echo gradient-echo pulse sequence with the following parameters: repetition time = 38 ms, echo times = 5, 10, 15, 20, 25, 30 ms, flip angle = 18°, resolution = 0.38 mm isotropic, in-plane acceleration by grappa (griswold et al., 2002) with acceleration factor r=2, readout bandwidth = 260 hz/px, bipolar readouts, non-selective excitation. individual echo images were co-registered by fsl flirt (jenkinson et al., 2002; jenkinson and smith, 2001), and combined into a single image by root-sum-of-squares of individual echoes. results implementation of the perfusion fixation protocol we report our deployment of the ex situ perfusion fixation procedure on five brains in the form of individual case summaries. in all instances, we used a perfusion circuit with the same core design and evaluated different variations of the procedure. for each case, we describe operational parameters, case-specific factors of the procedure, and metrics recorded regarding the quality of the perfusion fixation for that case (table 2). * semicolons denote vessels cannulated separately and sequentially. † volume perfused approximate. ‡ flow rate is mediated by pump drive speed. with our circuit, 50 rotations per minute (rpm) corresponds to approximately 38 ml/min and 60 rpm to 50 ml/min. § not available. case 1. the perfusion fixation procedure was performed with a hemibrain specimen from a 90+-year-old man with a clinical diagnosis of sepsis who had died of pneumonia. the postmortem interval (pmi) was 52 hours prior to the procedure. grossly, the blood vessels were assessed as having moderate atherosclerosis. the internal carotid and posterior cerebral arteries were cannulated with a 21g catheter. the vessels were first perfused with 0.5 l of phosphate buffered saline (pbs) with sodium azide (0.01% wt/vol) as a washout solution for 15 minutes. during this washout process, the blood was not found to substantially clear on visual inspection of the surface of the brain. the vessels were next perfused with 1.5 l of fixative solution, consisting of 10% (w/v) formalin and 1% (w/v) glutaraldehyde. qualitatively, at the end of the fixation procedure, palpation revealed that areas near the perfusate entry were found to be stiffer than other areas of the brain, suggestive of rapid fixation in those areas. case 2. this procedure was performed with a hemibrain specimen from a 66-year-old woman with a clinical diagnosis of sepsis who had died of cardiac arrest. the pmi was 44 hours. for most of the pmi, the brain was stored at refrigerator temperature (4° c). the anterior carotid artery and middle cerebral artery were cannulated with 19g catheters. with this specimen, it was more difficult to cannulate the vessels as only partial remnants of the original vessels remained after brain removal. the vessels were first perfused with 0.5 l of washout solution at a pump speed of 50 rpm, consisting of pbs with mannitol (4.5% wt/vol) and sodium nitrite (1% wt/vol). mannitol was employed to assist with opening of the blood brain barrier, while sodium nitrite was used to assist with vasodilation (ikeda et al., 2003; palay et al., 1962). during this washout process, the apparent clearance of blood from some blood vessels was visualized. the vessels were then perfused with about 1 l of fixative, consisting of 10% formalin and 1% glutaraldehyde, at a pump speed of 60 rpm. physical stiffening of the hemibrain post perfusion was not appreciated, possibly suggesting inadequate perfusion in this case, although we did not use precise measurements of stiffness. case 3. the perfusion fixation procedure was performed with a hemibrain specimen from a 77-year-old man with a diagnosis of diffuse lewy body disease who had died of pneumonia. the pmi was 12 hours. grossly, the blood vessels were assessed as having mild atherosclerosis. the middle cerebral artery and posterior cerebral artery were cannulated with 19g catheters. to streamline the procedure, washout solution and glutaraldehyde were omitted. the vessels were perfused with 2 l of 10% (w/v) formalin at a pump speed of 60 rpm for 30-40 minutes. at the end of the procedure, the anterolateral aspect of the perfused hemibrain had significant swelling. the reason for the swelling was unclear, but the shorter pmi of the specimen in case 3 may have led to a lower resistance to perfusate flow than in cases 1 and 2, leading to a higher effective perfusion pressure, potentially resulting in blood brain barrier disruption (schwarzmaier et al., 2022). case 4. this perfusion fixation procedure was performed on a whole brain specimen from a 90+-year-old woman who was positive for covid-19 at the time of death. the pmi was 24 hours. the blood vessels were cannulated with 23g catheters in three different ways. no washout solution was perfused. we first cannulated the bilateral internal carotid arteries and perfused approximately 250 ml of 10% formalin at an initial pump speed of 50 rpm that was subsequently raised to 60 rpm. during this process, it was possible to visualize blood being removed from an artery in the right frontal cortex. the inferior frontal gyrus and inferior temporal gyrus appeared to stiffen. however, there was a concern that the bilateral anterior frontal poles were becoming slightly edematous, although the gross effects of this could not be clearly seen post-perfusion. additionally, at the beginning of the perfusion, the pressure monitor was at 1.5 psi (77.6 mmhg), and it increased to approximately 2.1 psi (108.6 mmhg), suggestive of a possible blockage or buildup of fluid. as a result, the perfusion through the bilateral internal carotid arteries was stopped. we next cannulated and perfused two other blood vessels to assess whether the same swelling phenomenon would occur. after dissection to remove the cerebellum, it was possible to visualize the circle of willis, which was intact. we cannulated and perfused through the anterior cerebral arteries bilaterally at a pump speed of 50 rpm. arteries in the right hemisphere temporal cortex were visualized being drained of blood. however, there was a concern that the vessels may have become clogged because an unintentional pressure increase was detected in the pressure sensor. we then switched to perfusing through the basilar artery at a pump speed of 50 rpm. after a few minutes of perfusion through the basilar artery, the fixative appeared to be accumulating near the circle of willis rather than penetrating through the blood vessels into the deeper parts of the brain, so this perfusion was stopped. leakage from the superior cerebellar arteries, which are severed upon removal of the cerebellum, may have contributed to the observed accumulation of fixative around the circle of willis. for injection of fixative, we suspended the whole brain by tying a string around the circle of willis and placing the specimen into a container filled with formalin. to access the third ventricle, we directly inserted a syringe through the floor of the third ventricle at the posterior aspect of the tuber cinereum and injected 25 ml of 10% formalin. we then inserted a syringe into the cerebral aqueduct and injected 25 ml of 10% formalin there. the brain was then placed on a rocker at refrigerator temperature for postfixation. the gross anatomic condition of this brain shows that the brain grossly appears more pale, suggestive of less remaining blood in vessels, over the course of the preservation process (figure 2). figure 2. serial gross images of a donated human brain during ex-situ perfusion the gross anatomic condition of the brain (case 4) is shown via a high-resolution photograph after extraction from the skull but prior to perfusion (a), following perfusion fixation and further dissection of the circle of willis but prior to immersion fixation (b), and following both perfusion fixation and post-perfusion immersion fixation (c). note that the cerebellum has been removed from the brain specimen between steps (a) and (b). additionally, the frontal pole has been removed at step (c). case 5. the perfusion fixation procedure was performed on a whole brain specimen from a 64-year-old woman with a clinical diagnosis of metastatic adenocarcinoma. the pmi was 7 hours. the blood vessels were cannulated with 19g catheters in three different ways. we first cannulated the bilateral middle cerebral arteries, then we cannulated one of the anterior cerebral arteries, and then we cannulated one of the vertebral arteries leading into the basilar artery. after each of these vessel cannulations, we perfused 10% formalin for 5-10 minutes, with no washout solution. the posterior communicating arteries were small and there was no evidence of intact collateral circulation through the circle of willis. when perfusing through each of the blood vessels systems in this case, there was clearing of blood appreciated in the proximal areas of the tissue next to the cannulated blood vessel. manual palpation revealed stiffening of areas of brain tissue distal to the cannulated vessels. the pressure monitoring showed a range of 1.5 psi (77.6 mmhg) to 2 psi (103.4 mmhg) when perfusing through two blood vessels. higher pressures were found, even up to 3 psi (155.1 mmhg), when perfusing through just one blood vessel. at one point, we also noticed an air bubble to be present in the perfusion tubing, although it is unclear what impact this had on the perfusion quality. after perfusion, we used a syringe to perform injection fixation of the brain. fixative was introduced into the third ventricle, the fourth ventricle via a superior approach through the anterior corpus callosum, and each of the lateral ventricles after sectioning through the midbrain and cerebellum. for each injection, we used 25-50 ml of 10% formalin. we infer that the injection was made into the ventricles rather than into the brain parenchyma because a partial flow of fluid could be seen coming out of the perforation in the third ventricle during injection of fixative into the lateral ventricles. additionally, there was no evidence upon sectioning that the striatum was disrupted, as might have been expected from parenchymal injection. however, because we could not directly visualize the injection into the ventricles, we cannot completely exclude that some of the fixative may have directly entered the brain parenchyma. histology and neuroimaging data histology data from the perfused brains showed that the expected tissue morphology and cell shape were present, which was similar to typical findings from immersion-fixed brains (figure 3). there were no clear alterations due to the edema observed in the perfused brains. figure 3. routinely histology stainings of tissue sections from perfused brains representative histology sections were luxol fast blue, hematoxylin and eosin (lh&e)-stained in case 1 (a-c) or hematoxylin and eosin (h&e)-stained from case 2 (d), case 3 (e), case 4 (f-g), and case 5 (h-i). sections were derived from the striatum (a-b), corpus callosum (c), temporal cortex (d), white matter of the temporal pole (e), substantia nigra (f), hippocampus – dentate gyrus (g), mid-frontal cortex (h), and striatum (i). tissue morphology shows expected findings, suggesting that the perfusion process did not adversely affect tissue morphology. red blood cells were observed in some vessels, indicating absence of complete perfusion. all scale bars 100 μm except for sub-figures d (1000 μm), g (50 μm), and h (200 μm). to assess the distribution of single rna molecules we performed rna in situ hybridization analysis in one of the perfused brains (figure 4). specifically, we utilized the rnascope in situ hybridization (ish) assay, which is a commercial rna ish technology that detects individual mrna molecules on a single-cell level, in the superior frontal region of case 5 (wang et al., 2012). we observed a strong and robust ubiquitin c signal in all cell types including neurons, glia and blood vessels, suggesting that mrna species can be detected in perfused brains (figure 4), as expected from previous results using the rnascope in immersion-fixed human brain tissue (baleriola et al., 2014). figure 4. robust detection of ubiquitin c rna by in situ hybridization in a perfusion-fixed brain representative low (a) and high (b) magnification images of the rnascope assay in the superior frontal region of case five. strong and robust signal of ubiquitin c mrna is detected in all cell types including neurons (black arrow), glia (black arrowhead), and blood vessels (red arrow). scale bars 1 mm (a) and 50 µm (b). we also performed ex vivo mri scans following perfusion of the two whole brains. while pure immersion-based diffusion of formalin has the potential to create an artifact on ex vivo mri scans corresponding to the fixation front, in the mri scans of perfusion-fixed brains there were no prominent rim artifacts (i.e. variations in signal intensity in superficial versus deep regions) related to inhomogeneous fixation (nazemorroaya et al., 2022). however, the perfused brains contained numerous small hypointensities seen on multi-echo gradient-echo with root-sum-of-squares echo recombination images, which are not present in comparative immersion-fixed brains scanned with the same protocol (figure 5). these hypointensities are consistent with susceptibility-induced signal loss due to small air bubbles distributed in the vasculature. therefore, one of the potential downsides of the current perfusion protocol may be the inadvertent introduction of air bubbles into the tissue. these hypointensities are less likely to be related to perivascular spaces, which typically appear as hyperintensities on these mri contrasts. however, the effect of these small hypointensities on histological features is uncertain and we have not detected any parenchymal air bubble artifacts in the preliminary histology data reviewed so far. figure 5. perfusion-fixed brains contain numerous small hypointensities on mri scans multi-echo gradient-echo with root-sum-of-squares echo recombination images of a comparison immersion-fixed brain (a) as well as two perfusion-fixed whole brains: case 4 (b) and case 5 (c). numerous small hypointensities are visualized in the two perfusion-fixed brains but not in the immersion-fixed brain imaged with the same protocol. discussion in its simplest form, perfusion fixation can be thought of as an extension of immersion fixation, except that fixative solution extravasates from the blood vessels rather than penetrates from outside brain surfaces. this thereby increases the surface area by which fixatives can penetrate the tissue by orders of magnitude and homogenizes its distribution. however, introducing additional variables causes other potential alterations to the perfused brains that might contribute to artifacts or batch effects in comparison to immersion-fixed brains. glutaraldehyde, for instance, is not typically used in brain banking and there was a theoretical concern that it might affect the results of downstream immunolabelling assays, introducing the risk of batch effect (mrini et al., 1995). for this reason, we removed glutaraldehyde from the fixative solution. in further cases, we also removed the washout solution from the protocol. because formaldehyde takes a significant time to fix tissue, it is unclear if a washout process is necessary to prevent fixation of debris within blood vessels when perfusing with formaldehyde, while using a washout solution adds to procedural complexity and creates a possible batch effect. in our view, the value of perfusing washout solution prior to perfusing fixative remains an open question, with both potential upsides and downsides (mcfadden et al., 2019; tao-cheng et al., 2007). a limitation of this technical report is that the recorded outcome metrics were largely qualitative. because edema was often seen and red blood cells were observed in some vessels, we expect that perfusion fixation was incomplete. however, no quantitative analysis has been performed to determine whether there is a change in autolytic artifacts, blood vessel shape, or other histologic features in the perfused brains compared to brains preserved with the standard immersion protocol (frigon et al., 2022). as a result, we were unable to quantify the extent of partial perfusion fixation. in future studies, both operational and histologic perfusion fixation quality outcome metrics should be measured more quantitatively. for example, the use of an indentation device that measures both force and displacement could precisely measure tissue stiffness (elkin et al., 2011). additionally, the use of video monitoring during the perfusion fixation procedure could help to precisely record and quantify any changes in brain pigmentation or volume. more rigorous outcome metrics would also help to monitor and make any adjustments necessary during the procedure and to optimize the procedure for future cases. one major challenge in ex situ brain perfusion is the presence of air bubbles. consistent with this, mri images from our perfusion-fixed brains showed hypointensities that may correspond to small air bubbles generated or distributed in the vasculature during the perfusion procedure. because air bubbles will accumulate in sulci, ventricles, and blood vessels as soon as the brain is extracted from the skull, it is an immense challenge to completely avoid air bubbles during ex situ perfusion fixation (bolliger et al., 2018). air bubbles can also form in the perfusion circuit, which may introduce an unnecessary impediment to perfusion flow. when air bubbles are present in blood vessels, they can theoretically obstruct the flow of fixative. for future iterations of the procedure, we are currently working on a strategy using drip chambers in the perfusion circuit to decrease the potential for air bubbles to be perfused into the blood vessels. keeping the tubing of the perfusion circle at a vertical angle may also help bubbles to rise to the top rather than continuing through the perfusion circuit. when a catheter is placed into a blood vessel for cannulation, there is a need to secure it in place, or it will move due to the perfusate flow. here, we employed hemostats to secure the catheters. with this approach, the length of vessel required for cannulation is limited by the diameter of the hemostat. with the use of our mosquito hemostats, approximately 1 cm of blood vessel is required to adequately secure the cannula. the presence of atherosclerosis did not appear to affect the ability to seal the cannulated vessels. however, hemostats are unwieldy and can obstruct the field of vision. an alternative approach is to use surgical sutures; however, this requires significant skill and takes more time. we are currently evaluating alternative approaches to address the problem of securing catheters in the blood vessels. it is also important to note the necessity of clamping arteries that are downstream of the cannulated artery and that are not wished to be perfused. the failure to clamp severed superior cerebellar arteries may have contributed to the fixative accumulation that occurred during perfusion through the basilar artery in case 4. the two main methods for driving fluid in perfusion fixation are gravity-based systems and peristaltic pumps. with gravity, a constant pressure is provided with variable flow rate, whereas with a peristaltic pump, a consistent oscillating flow rate is provided irrespective of resistance (scouten et al., 2006). by monitoring the in-line pressure in our peristaltic pump-based perfusion circuit, we can detect whether there is a build-up of resistance to flow, as indicated by an increase in pressure. for a given cannula size, it is possible to control the pressure by varying the pump speed. therefore, if a pressure build-up is detected, we can either decrease the pump speed and/or cannulate other arteries (schwarzmaier et al., 2022; scouten et al., 2006). because the perfusion circuit will have a higher resistance when perfusing through one blood vessel as opposed to multiple, lower flow rates may need to be used when perfusing through only one vessel, to avoid raising the perfusion pressure to excessive values, as was seen in case 5. the ideal pressure value for perfusion fixation, or even the one that best simulates brain-specific physiologic perfusion pressure, is unclear. at lower perfusion pressures, the perfusion may be incomplete and hence fail to sufficiently extravasate into the tissue; while at higher perfusion pressures, the blood brain barrier could be disrupted or blood vessels could rupture (saliani et al., 2017; schwarzmaier et al., 2022). currently, we target an in-line perfusion pressure in the range of 70-100 mm hg based on theoretical expectations. further investigation is necessary to query the optimal perfusion pressure for the ex situ human brain. we speculate that the tissue swelling that we sometimes detected may have resulted from an overly high perfusion pressure (schwarzmaier et al., 2022). if tissue swelling is detected, suggesting increased resistance to flow, then an alternative approach may be to decrease the perfusion pressure and to perfuse for a longer period of time. related to perfusion pressure is the issue of postmortem interval. the postmortem intervals in our sample varied considerably, from 7 hours to 52 hours, which likely contributed to differences in tissue or blood vessel integrity between cases. we hypothesize that factors contributing to resistance to flow are likely to accumulate in brains that have undergone a more severe agonal state or longer postmortem interval prior to the procedure. in those cases, a higher perfusion pressure may be necessary to achieve sufficient perfusate flow throughout the brain. determining the optimal perfusion pressure range in brain banking and identifying ways to modify the perfusion pressure during the procedure are important areas for further research. in addition to uncertainties regarding perfusion pressure, the duration of perfusion fixation to provide adequate tissue fixation is also unclear. in rodent studies, as few as 5 minutes of perfusion fixation at optimal pressure has been found to be sufficient for proper tissue preservation (schwarzmaier et al., 2022). in brain banking studies, however, reported times of perfusion fixation have varied widely, from 5 minutes to 2 hours, with 15-30 minutes being a commonly reported time range (mcfadden et al., 2019). because of the potential for inadequate perfusate flow in human brains, methods that allow for precise monitoring during the perfusion procedure, such as ultrasound or mri, may help investigators to tailor the perfusion time for each brain, albeit at higher cost (vrselja et al., 2019). taken together, the amount of time required for adequate perfusion fixation and postfixation in brain banking that preserves tissue morphology while minimizing alterations of antigenicity is an important open research question. conclusion in our standard immersion fixation protocol, the whole brain or hemibrain is immersed in formalin for two to four weeks prior to further sectioning and storage procedures. based on previous data and biophysical principles, there are theoretical reasons to expect that preservation using the perfusion fixation protocol may yield improved histology quality (mcfadden et al., 2019). however, we do not have any systematic outcome data comparing our perfusion procedure to an immersion procedure. we also encountered several challenges in implementing the perfusion fixation procedure, including the intermittent occurrence of tissue swelling and the potential for introducing air bubbles into the vasculature. the added value to preservation quality using a perfusion fixation approach may depend in part on the agonal phase and the postmortem interval, during which thrombi may accumulate and blood vessels undergo autolysis, thereby restricting perfusate flow (hansma et al., 2015). further research and development are needed to improve the rigor and reproducibility of perfusion fixation methods and to assess the value of the procedure in improving preservation quality. acknowledgements the authors would like to thank helmut heinsen, henry waldvogel, kátia cristina de oliveira, maglóczky zsófia, ricardo insausti, and thomas beach for helpful advice in setting up the perfusion fixation system. we gratefully acknowledge the following funding sources: nih grants rf1mh128969, r01ag062348, rf1ns115268, r01ag054008, r01ns095252, r01ag060961, r01ns086736, an alzheimer’s disease research center (adrc) developmental project funding award to a.t.m. (p30ag066514), the rainwater charitable foundation, and an alexander saint-amand fellowship to j.f.c. abbreviations pbs: phosphate buffered saline; pmi: postmortem interval; mri: magnetic resonance imaging; rpm: rotations per minute. author contributions a.t.m., k.d.-o.c., a.s., z.w., e.m.c.h., and j.f.c. contributed to the original conception of the study; a.t.m., e.w.-l., a.c., h.w., and j.f.c. performed perfusion fixation studies; e.w.-l. and d.d. performed histology studies; k.w. performed rna in situ hybridization studies; a.s. performed mri imaging and data analysis; a.t.m. and j.f.c. wrote the original draft of the manuscript. all authors read and approved the manuscript. conflict of interest the authors declare that they have no competing financial interests. data availability all histology and mri data presented is available to the academic community upon request. references adickes, e.d., folkerth, r.d., sims, k.l., 1997. use of perfusion fixation for improved neuropathologic 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(berl.) 136, 901–917. https://doi.org/10.1007/s00401-018-1922-z copyright: © 2022 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. soluble brain homogenates from diverse human and mouse sources preferentially seed diffuse aβ plaque pathology when injected into newborn mouse hosts feel free to add comments by clicking these icons on the sidebar free neuropathology 3:9 (2022) original paper soluble brain homogenates from diverse human and mouse sources preferentially seed diffuse aβ plaque pathology when injected into newborn mouse hosts brenda d. moore1,2,3, yona levites1,2,3, guilian xu1,2,3, hailey hampton1,2, munir f. adamo1,2, cara l. croft1,2,3, hunter s. futch1,2, corey moran1,2, susan fromholt1,2, christopher janus1,2,3, stefan prokop2,3,4,5, dennis dickson6, jada lewis1,2,3, benoit i. giasson1,2,3, todd e. golde1,2,3,7, david r. borchelt1,2,3 1 department of neuroscience, college of medicine, university of florida, gainesville, fl 32610, usa 2 center for translational research in neurodegenerative disease, college of medicine, university of florida, gainesville, fl 32610, usa 3 mcknight brain institute, college of medicine, university of florida, gainesville, fl 32610, usa 4 department of pathology, university of florida, gainesville, fl 32610, usa 5 fixel institute for neurological diseases, university of florida, gainesville, fl 32610, usa 6 department of neuroscience, mayo clinic, jacksonville, fl, usa 7 department of neurology, college of medicine, university of florida, gainesville fl 32610, usa corresponding author: brenda d. moore · 1275 center dr · po box 100159, 352-273-7868 · gainesville, fl 32610 · usa brendar@ufl.edu submitted: 18 january 2022 accepted: 28 february 2022 copyedited by: henry robbert published: 23 march 2022 https://doi.org/10.17879/freeneuropathology-2022-3766 additional resources and electronic supplementary material: supplementary material keywords: amyloid-β, tau, α-synuclein, seeding, alzheimer’s disease, lewy body dementia abstract background: seeding of pathology related to alzheimer’s disease (ad) and lewy body disease (lbd) by tissue homogenates or purified protein aggregates in various model systems has revealed prion-like properties of these disorders. typically, these homogenates are injected into adult mice stereotaxically. injection of brain lysates into newborn mice represents an alternative approach of delivering seeds that could direct the evolution of amyloid-β (aβ) pathology co-mixed with either tau or α-synuclein (αsyn) pathology in susceptible mouse models. methods: homogenates of human pre-frontal cortex were injected into the lateral ventricles of newborn (p0) mice expressing a mutant humanized amyloid precursor protein (app), human p301l tau, human wild type αsyn, or combinations thereof. the homogenates were prepared from ad and ad/lbd cases displaying variable degrees of aβ pathology and co-existing tau and αsyn deposits. behavioral assessments of app transgenic mice injected with ad brain lysates were conducted. for comparison, homogenates of aged app transgenic mice that preferentially exhibit diffuse or cored deposits were similarly injected into the brains of newborn app mice. results: we observed that lysates from the brains with ad (aβ+, tau+), ad/lbd (aβ+, tau+, αsyn+), or pathological aging (aβ+, tau-, αsyn-) efficiently seeded diffuse aβ deposits. moderate seeding of cerebral amyloid angiopathy (caa) was also observed. no animal of any genotype developed discernable tau or αsyn pathology. performance in fear-conditioning cognitive tasks was not significantly altered in app transgenic animals injected with ad brain lysates compared to nontransgenic controls. homogenates prepared from aged app transgenic mice with diffuse aβ deposits induced similar deposits in app host mice; whereas homogenates from app mice with cored deposits induced similar cored deposits, albeit at a lower level. conclusions: these findings are consistent with the idea that diffuse aβ pathology, which is a common feature of human ad, ad/lbd, and pa brains, may arise from a distinct strain of misfolded aβ that is highly transmissible to newborn transgenic app mice. seeding of tau or αsyn comorbidities was inefficient in the models we used, indicating that additional methodological refinement will be needed to efficiently seed ad or ad/lbd mixed pathologies by injecting newborn mice. abbreviations ad alzheimer’s disease, lbd lewy body disease, ad/lbd alzheimer’s disease/lewy body disease, aβ amyloid-β, αsyn α-synuclein, app amyloid precursor protein, pa pathological aging, gfap glial fibrillary acidic protein, mapt microtubule-associated protein tau, ndc nondemented controls, wt wild type, caa cerebral amyloid angiopathy, elisa enzyme linked immunosorbent assay, sds sodium dodecyl sulfate, fa formic acid, thio-s thioflavin s. background the amyloid cascade hypothesis posits that deposition of the aβ peptide is an initiating factor in alzheimer’s disease (ad) with downstream effects of increased inflammatory response, altered neuronal homeostasis, altered tau phosphorylation leading to tangle formation, neuronal dysfunction, and cognitive decline (1, 2). the interaction between aβ and tau pathology has been modeled in transgenic animals that co-express human amyloid precursor proteins (app) with mutations associated with familial ad (fad) and wild-type or mutant human tau (3-12). multiple studies report that the presence of aβ pathology exacerbates the severity of tau pathology, leading to increased tau phosphorylation or increased levels of neurofibrillary tangles (3, 6, 8, 10, 11). interactions between aβ and tau pathology have also been modeled in mice by utilizing the prion-like capacity of aβ and tau aggregates to seed accelerated ad pathology. aβ containing seeds, derived from human ad and pathological aging (pa) brain lysates, have been shown to accelerate the time-course and severity of aβ deposition in amyloid precursor protein (app) transgenic models as well as impact the morphology of the seeded deposits (for review see (13)). seeding preparations can consist of human brain homogenates, app transgenic mouse brain homogenates, enriched preparations derived from such homogenates and synthetic aβ fibrils (13). in most of these published studies, seeds are injected into adult app mice that are a few months away from developing aβ pathology on their own and in such cases the inherent pathological tendencies of the app host model can contribute to the pathological outcome. partially purified tau aggregates from ad brains were found to seed enhanced neuritic tau pathology in the 5xfad mouse model and the appnl-g-f/human mapt double knock-in model (14); however, neither of these models produced pathology resembling neurofibrillary tangles. in appswe/ps1de9 and appnl-f/nl-f mice crossed to humanized htau mice, the presence of aβ pathology did not produce significant enhancement of tau seeding (15). these latter studies illustrate the need to refine seeding approaches towards producing models that reproduce a broader spectrum of ad-related pathology. recent studies have begun to use seeding approaches to create models of mixed ad or lewy body disease (lbd) pathology. in parkinson’s disease and lbd, the presence of aβ plaques is associated with cortical αsyn pathology (16, 17). similar to studies of aβ/tau interactions, investigations of aβ and αsyn interaction in transgenic mice have suggested a possible synergistic relationship (18, 19). in seeding models involving the injection of αsyn preformed fibrils, aβ deposits accelerated αsyn pathogenesis and spread throughout the brain of 5xfad mice (20) and in bigenic mice co-expressing appswe/ps1de9 and wt αsyn (21) . one of the goals of our study was to further explore the potential to use seeding as a means to generate novel mouse models of mixed ad and lbd pathology. a subset of cognitively normal aged individuals exhibit substantial aβ pathology at autopsy (22). these individuals, which we use the designation of pathologic aging (pa), may represent prodromal, presymptomatic ad (23-26). the amyloid pathology of pa brains has typically been described as diffuse in nature whereas ad brains contain both diffuse and compact/neuritic deposits of aβ (26-28). cognitively normal individuals with pa generally have very low levels of tau pathology (26). rigorous biochemical examination of aβ from the pre-frontal cortex of pa and ad brain revealed extensive overlap in aβ levels, peptide profiles, solubility, and sds-stable oligomeric assemblies (25). thus, pa brains provide a source of human brain-derived aβ seeds that can be compared to human ad brain preparations that contain both aβ and tau seeds. nearly all of the published studies involving injection of aβ, tau, or αsyn seeds into the brains of transgenic mice have followed a protocol of injecting the seeds stereotaxically into the brains of young adult mice. in this study, we have examined the efficacy of intracerebroventricular injection of homogenates prepared from ad, pa, and ad/lbd donors into newborn app, app/tau, or app/αsyn host mice. in prior studies, we had established that injection of recombinant adeno-associated virus into newborn mice was an effective approach to obtaining widespread dispersion of injected virus (29, 30). we therefore hypothesized that misfolded seeds of aβ, tau, or αsyn might similarly be more widely dispersed to more effectively seed pathology. in fact, we observed efficient seeding of αsyn pathology in newborn mice, expressing a53t human αsyn (m83 line), by injecting brain lysates from patients with multisystem atrophy (31). here we performed brain lysate injection studies at p0 in the lateral ventricles of host transgenic mice that co-express humanized mutant app with either mutant human tau-p301l or wild-type human αsyn (m20 line). importantly, the recipient transgenic mice used in the current studies develop pathology at late ages or not at all; enabling sensitive evaluation of potential seeding. we observed that injecting newborn app, app/tau, or app/αsyn mice with homogenates from diverse pathological specimens, including pa brains, produced robust diffuse aβ deposition. though the human homogenates used contained coexisting αsyn and tau seeds, we were not able to directly induce lbd or tau pathology in recipient models tested here. we compared the data from human lysates to lysates prepared from transgenic mouse donors finding again that diffuse aβ deposits were preferentially seeded when these preparations were injected in newborn mice. app mice seeded with ad brain lysates, exhibiting diffuse aβ pathology, also showed no significant impairments in a contextual fear paradigm of cognitive performance. our findings indicate that the injection of human brain homogenates from ad and ad/lbd donors into newborn app mice preferentially induces diffuse aβ pathology. interestingly, pa brains induce the same pathology implying that ad and ad/lbd brains contain amyloid seeding entities that are shared with pa brains. methods description of ad, pa and ndc cases for this study we utilized a subset of the human brain samples that our group previously analyzed (25). tissue sections and frozen pre-frontal cortex (ad = 2, pa = 4, and ndc = 2) were obtained from the mayo clinic brain bank with informed consent, in accordance with the mayo clinic institutional review board, using previously described acquisition and diagnostic analyses (26, 32, 33). samples were initially cryo-pulverized to allow for multiple studies. we previously analyzed the aβ peptide profile, quantity, and solubility in these brain samples (25). in this study, we analyzed two brains from ad patients (both aged 84), four pa brains from subjects (age range = 78 to 83) without clinical evidence of dementia, and two brains with rare or no ad lesions from elderly individuals without clinical evidence of a neurological illness (ages 75 and 78). in addition, human brain samples were also obtained from the university of florida neuromedicine human brain tissue bank following institutional regulations and previously described classification (34-36). we analyzed tissue sections and frozen pre-frontal cortex from two ad patients (ages 82 and 86), two cases with ad/lbd (ages 81 and 83), and two control individuals without pathology (ages 52 and 82). table 1 summarizes the cases studied in this report. table 1. list of human brain samples used in this study. pathological diagnosis (pathdx), lewy body disease (lbd) classification, amyloid plaque score (thal phase), braak stage, consortium to establish a registry for alzheimer’s disease (cerad) protocol, 2% sds and 70% formic acid (fa) solubilized aβ40 and 42 (25), cerebral amyloid angiopathy (caa), apolipoprotein e (apoe) genotype, gender, and age are reported. ad, alzheimer’s disease; pa, pathological aging; ndc, non-demented controls; oa, optic atrophy; tr, transynaptic degeneration; gba, gross brain atrophy; x, below detection levels; nd, not determined, f, female; m, male. transgenic mice the transgenic mice used in this study have been described previously and are summarized in table 2. the prp.appsi mice express mouse app-695 cdna with a humanized aβ sequence and fad mutations (appswe/ind) using the moprp.xho vector, which was co-injected with a vector to express egfp in skin (first described in (37)). the itau-p301l mice express human 0n4r tau-p301l using a tet-regulated vector that includes a minimal cmv promoter (38). tau levels were assessed by western blotting. pbs lysates of 2.5-6 month old p301l mice were heated at 95°c for five minutes in the presence of denaturing sds sample buffer, were separated on a 4-12% bis-tris gel (bio-rad, hercules, ca) in 3-(n-morpholino)propanesulfonic acid (mops) running buffer (bio-rad, hercules, ca) and transferred onto pvdf membrane. the membrane was blocked in casein blocking buffer and incubated overnight at 4°c with primary antibodies to tau, tau-13 (anti-human tau2-18; covance) and 3026 (rabbit polyclonal antibody raised against full length 0n3r human tau and also reacts with 0n4r human tau (39, 40)), and anti-β-actin (sigma-aldrich, st. louis, mo) before incubation with secondary antibody, fluorophore conjugated alexafluor 680 anti-mouse igg (thermo fisher scientific, waltham, ma). protein bands were detected and quantified using the multiplex odyssey infrared imaging system (bio-rad, hercules, ca). table 2. mouse strains used in this study. caa, cerebral amyloid angiopathy; na, not applicable. the m20 mice express wild type αsyn under the control of the prion promoter and do not develop αsyn pathology (41). prp.huaβ/ps1 mice express human appswe and human ps1de9. prp.moaβ/ps1 mice express mouse appswe mutation and human ps1de9, and tet.moaβ mice expresses inducible mouse appswe (37). these mice were bred in-house. prp.appsi and m20 were maintained on hybrid c57bl6/c3h backgrounds, following a breeding scheme in which transgene-positive males were bred to nontransgenic f1 b6/c3 female mice. the itau-p301l mice we used had been backcrossed to the fvb/ncr mouse strain for more than 10 generations. appsi/tau-p301l and appsi/αsyn mice were generated by intercrossing mice that where hemizygous for the respective transgenes. prp.appsi mice were genotyped by visualizing gfp expression, which is possible because these mice were co-injected with a transgene that expresses gfp in the skin integrated next to app transgene. prp.appsi/itau-p301l mice were genotyped for app by illumination and visualization with special filter goggles (bls ltd, budapest, hungary) and for tau by pcr of tail dna. itau-p301l and m20 mice were genotyped by pcr of tail dna. prp.appsi mice destined for behavioral studies were backcrossed one generation onto c57bl6. all animals were housed 1-5 per cage with unlimited access to food and water with a 14-hour light and 10-hour dark cycle. all experiments involving mice were approved by the university of florida institutional animal care and use committee (iacuc) and conducted in accordance with nih guidelines. human brain lysates human and mouse brain lysates were prepared as previously described (42). briefly, frozen tissue was homogenized at 10% (w/v) in sterile pbs, subjected to vortex and sonication (3 x 5 sec) and then centrifuged 3000 x g for 5 min at 4°c. lysates were immediately aliquoted and stored at -80°c until needed. as previously described (25), the aβ levels and solubility in the lysates was assessed by western blotting. briefly, lysates were heated at 50°c for three minutes in the presence of denaturing sample buffer, were separated on a 4-12% bis-tris gel (bio-rad, hercules, ca) in 1x2-(n-morpholino)ethanesulfonic acid (mes) running buffer (bio-rad, hercules, ca) and transferred onto nitrocellulose membrane (bio-rad, hercules, ca). the membrane was boiled in pbs for 5 min, blocked in starting block (thermo fisher scientific [formerly thermo scientific], waltham, ma) and incubated overnight at 4°c with primary antibody ab5 (human aβ1-16 specific; t.e. golde) before incubation with the secondary antibody, fluorophore conjugated alexa fluor 680 anti-mouse igg (thermo fisher scientific, waltham, ma). protein bands were detected using the multiplex odyssey infrared imaging system (bio-rad, hercules, ca). brain seeding with neonatal cerebral ventricle injections injections of human brain lysates and lysates from a 27mo prp.appsi mouse, a 25mo prp.huaβ/ps1 mouse, a 24mo tet.moaβ mouse, and a 24mo prp.moaβ/ps1 mouse were performed as described previously with recombinant adeno-associated virus (43). briefly, p0 pups were cryoanesthetized and 2 ul of human or mouse brain lysates were bilaterally injected into the cerebral ventricle using a 10 ul hamilton syringe with a 30-inch needle (hamilton company, reno, nv). pups were placed on a heating pad for recovery and returned to their home cage. cohorts of mice were euthanized at 6, 9, 12, and 18 months for analysis. both male and female mice were analyzed (see tables s1 and s2 for description of cohorts). mouse brain tissue collection mice were anesthetized with isoflurane and perfused transcardially with 20 ml of cold pbs. the brains were cut sagittally through the midline, and one hemibrain was drop-fixed in 4% paraformaldehyde in pbs (ph 7.5) for ~48 hrs at 4°c followed by processing and paraffin embedding. the other hemibrain was snap frozen with isopentane on dry-ice and then stored at -80⁰c until it was thawed and homogenized in preparation for elisa measurements of aβ peptide levels. histology and immunochemistry paraffin sections (5 μm) were used for all the histology and immunochemistry studies. campbell-switzer silver (44) and thio-s (37) tissue staining methods were performed as previously described. immunochemistry followed standard protocols described previously (37). to assess amyloid pathology, embedded sections were immunohistochemically stained with a biotinylated pan-aβ antibody ab5 (1:500; t.e.g.) and developed using vectastain elite abc kit (vector laboratories, burlingame, ca) followed with 3,3’-diaminobenzidine (dab) substrate (vector laboratories, burlingame, ca). to evaluate tau pathology, embedded sections were immunohistochemically stained with 7f2 (1:10,000; ben giasson (39)), cp27 (1:1000; peter davies), at8 (1:1000; thermofisher scientific), and mc1 (1:1000; peter davies). to assess αsyn pathology, sections were stained with 9c10 (1:1000; ben giasson) (31). after development by 3,3’-diaminobenzidine (dab) (vector laboratories, burlingame, ca) substrate and counterstaining with hematoxylin, the slides were coverslipped and images were taken using an olympus bx60 microscope or scanned by aperio® xt system (leica biosystems, buffalo gove, il, usa) or zeiss microscope (carl zeiss, oberkochen, germany) and analyzed using the zen 2.6 program (carl zeiss, oberkochen, germany). blinded observers then reviewed the images and scored the burden of aβ pathology based on the following criteria: “+++” = heavy aβ burden with too many deposits to count. “++” = abundant pathology with >30 deposits per section. “+” = consistent pathology with > 5 deposits per section. “+/-” = inconsistent pathology with <3 deposits per section. brain amyloid extraction human brain samples were originally analyzed by moore (25). briefly, frozen pre-frontal cortex tissue was cryo-pulverized in liquid nitrogen and then sequentially extracted with tris-buffered saline (tbs), radioimmunoprecipitation buffer (ripa), 2% sodium dodecyl sulfate (sds), and 70% formic acid (fa) containing protease inhibitor cocktail (roche, indianapolis, in, usa). after harvesting the mouse brain, the left hemisphere was flash-frozen in isopentane. the frozen cortex was sequentially extracted with protease inhibitor cocktail (roche) containing tris-buffered saline, ripa buffer, 2% sds, and 70% formic acid (fa) as described previously at a concentration of 150 mg/ml (25). elisa aβ levels from the 2% sds and 70% fa extracted samples were quantified by sandwich elisa as described previously (25). total aβ was captured with mab ab9 and detected by hrp-conjugated mab 4g8 (human aβ17-24; covance, princeton, nj, usa); aβ1-40 was captured with monoclonal antibody (mab) ab9 (human aβ1-16; t.e. golde) and detected by horseradish peroxidase (hrp)-conjugated mab 13.1.1 (human aβ35-40 specific; t.e. golde); aβ1-42 was captured with mab 2.1.3 (human aβ35-42 specific; t.e. golde) and detected by hrp-conjugated mab ab9. elisa results were analyzed using softmax pro software (molecular devices). contextual fear conditioning transgenic prp.appsi and nontransgenic littermates, seeded with ad cases 1 and 2, and ndc case 7, were aged to 12 months and subjected to contextual fear conditioning as previously described (45, 46). briefly, in the contextual fear conditioning test, mice learn the association between the training chamber, which represents an initially neutral conditional stimulus (cs) and an aversive, brief electric foot-shock, unconditional stimulus (us), which takes place in the training context. we have previously established that 0.45 ma electric current elicits robust avoidance response and results in strong freezing response after 2 cs-us pairings (45). during the training session, mice were allowed to explore the training chamber for 120 sec, with a 2 sec foot shock immediately following a 30 sec tone (80 db) (day 1; see fig. 4a). the mice recover for 60 sec and then receive another 2 sec foot shock following a 30 sec tone. the final post-us period is 60 sec. for the context test session, on day 3, mice were exposed to the same training context and the freezing presentation during the 300 sec was recorded by an image analysis system (freezeframe, actimetrics). on day 4, mice experienced the tone session, and were placed in a modified chamber (altered inserts and smell), during the first 180 sec the mice were allowed to explore the new environment. during the second 180 sec period the tone was delivered and the percent freezing was recorded. no shock was applied during context or tone session on days 3 and 4. results characterization of ad/lbd, ad, pa, and ndc cases in this study, we selected a subset of ad, pa, and ndc donors that were previously analyzed (25) for examination of seeding activity in app, app/tau, and tau transgenic mice (table 1, ad 1 and 2, pa 1, 2, 3, 4, and ndc 1 and 2). all 8 of these donors exhibited some degree of lewy body pathology (lbd). two of the donors selected for the study were sub-categorized with cerebral amyloid angiopathy (caa); pa 3 had a high abundance of vascular amyloid with a caa score of 2+-3+ while ad 1 had moderate levels of vascular aβ deposition with a score of 1+ (table 1). as expected, the two ad brains had significant accumulation of tau, indicated by braak staging (5.5 and 6) (table 1). the pa and ndc brains had lower abundance of tau than the ad brains, but were similar to each other (2, 2.5, 3, 2, and 2, 3, respectively) (table 1). in this set of cases, all also exhibited incidental, diffuse, αsyn pathology. to these 8, we added 6 additional donors that included two ad (no lbd), two with lewy-body variant ad (ad/lbd), and 2 additional controls that were free of all types of pathology (table 1, ad 3 and 4, ad/lbd 1 and 2, and ndc 3 and 4). these brains were used in seeding app, app/α-synuclein (αsyn), and αsyn mice so that we would be able to compare ad and ndc that lacked αsyn pathology to ad/lbd brains with high levels of αsyn pathology. to characterize the pathology in the ad and pa brains used to seed the app/tau, app, and tau mice, sections were stained with a pan-aβ antibody (fig. s1a), thioflavin s (thio-s) (fig. s1b), and with a phospho-specific tau antibody (fig. s1c). we observed widespread aβ deposition of both diffuse and compact amyloid in both ad and pa (fig. s1a, b), with a subset showing striking caa. pa 3 had numerous, thio-s positive, vascular amyloid deposits, consistent with the assessment of caa as 2+-3+ (fig. s1a, b). similarly, we observed thio-s positive amyloid staining surrounding several vessels within ad 1, consistent with a caa score of 1+ (fig. s1a, b). ad 2 contained several cored thio-s positive deposits while the compact deposits in pa 1, 2, and 4 had little to no thio-s staining. as expected, ad 1 and 2 contained both substantial phosphorylated tau in form of dystrophic neurites, neuropil threads and neuronal inclusions while all four of the pa were negative for tau deposits (fig. s1c). ndc 1 and 2 were negative for aβ and tau deposits by both immunostaining and thio-s staining. cerebral injection of brain lysates into newborn app/tau and app mice results in widespread, robust amyloid deposition to gain a better understanding of the type of aβ pathology that unseeded prp.appsi mice produce, we harvested breeder mice at various ages to assess phenotypic variation (fig. s2a and b) (37). at 12 months of age, when deposition begins to occur in the brains of prp.appsi mice, any given section through the cortex and hippocampus may exhibit 1 or 2 diffuse tufted deposits and/or cored deposits (score +/-). at this age, aβ deposition also begins to appear in the meninges surrounding the cerebellum (fig. s2a). approximately 50% of prp.appsi mice at 11-13 months of age exhibit no aβ pathology or show only meningeal deposition in the cerebellum (fig. s2a). to concurrently examine whether tau pathology could be augmented by seeding from these lysates, the prp.appsi mice were crossed to a line of mice that express human tau p301l (itau-p301l) (table 2) (38, 47). the itau-p301l mice express mutant human tau at levels 2-3 fold higher than nontransgenic mice (fig. s3). to compare the relative ability of homogenates prepared from ad and pa cases to seed aβ deposition in these mice, lysates from the pre-frontal cortex of the ad, pa, and ndc were injected into the cerebral ventricles of newborn mice at p0 (31, 42, 43). we confirmed that the lysates contained aβ by immunoblotting and elisa (fig. s1d and table 1). aβ was detectable by immunoblot in each of the lysates used for injection (fig. s1d), and enzyme-linked immunosorbent assay (elisa) measurements confirmed high levels of sds-soluble and formic acid (fa) soluble aβ42 in each brain lysate (table 1). we hypothesized that injection of these lysates in p0 mice would result in widespread dispersion of the aβ or tau seeds, and potentially offer the best chance to extensively alter the type of pathology that would be induced by seeding. to assess amyloid pathology, hemibrains of seeded mice were stained with a pan-aβ antibody and thio-s. in mice injected with either ad or pa lysates, we observed significant induction of aβ deposition, comprised primarily of diffuse deposits throughout the cortex and hippocampus, with some vascular deposition in the pia surrounding the cortex and within the hippocampal fissure (fig. 1b, c). mice injected with homogenates from ndc 1 and 2, and mice that were not injected, had little aβ pathology by 12 months of age (fig. 1b, c). examples of cases with the most severe pathology are shown in figure 1, while images of animals with the least pathology are shown in figure s4. in all cases, however, the induced aβ pathology exhibited a diffuse, thio-s negative morphology (fig. s5). the levels of induced aβ pathology in mice that co-expressed appsi and tau-p301l were similar to that of mice that expressed only appsi (fig. 1b versus c, s4a versus b). mice that were transgenic for only tau-p301l, or were non-transgenic, showed no evidence of aβ deposition (data not shown). to compare the data across all of the mice examined, aβ pathology was qualitatively scored by three independent observers (fig. 1d, e). at 12 months, only pa 4 was scored as having a level of aβ pathology that approached mice injected with the ad cases; both of which exhibited widespread, diffuse pathology (fig. 1b, c). aβ pathology in mice seeded by pa 1, 2, and 3 was scored as less abundant than in mice seeded by the ad cases (fig. 1d, e). we confirmed the high seeding activity of ad 1 and 2, and pa 4 by assessing aβ pathology at 9 months post-injection. although the severity of pathology varied, multiple animals injected with each of these lysates exhibited aβ deposition (fig. s6a). notably, dilution of the brain lysates by 10-fold prior to injection greatly diminished seeding activity (fig. s6b). to further examine the relative seeding activity of pa lysates, we injected newborn prp.appsi mice with each of the 4 pa lysates and ndc 1, and analyzed pathology at 18 months post-injection. although the mice injected with the pa lysates appeared to have more severe aβ deposition, we also observed some significant aβ pathology in animals injected with the ndc lysate (fig. s7). whether this outcome was due to some small amount of aβ seed in the ndc 1 will require further study. collectively, these studies demonstrated that ad and pa brains contain misfolded forms of aβ that preferentially seed diffuse aβ pathology. fig. 1 cerebral injection of brain lysate at p0 induces widespread, robust amyloid deposition. overall schematic of experiments (a) images of prp.appsi/tau-p301l (b) and prp.appsi (c) mice injected with ad, pa, or ndc lysate at p0. brain sections (hemibrain) stained with biotinylated anti-aβ mab ab5 (anti-aβ 1-16) and counterstained with hematoxylin. cases with the most abundant amyloid pathology are shown. scale bar: 100 μm. qualitative analysis of amyloid deposition in seeded prp.appsi/tau-p301l (d) and prp.appsi (e) mice. three observers independently scored representative slides from each seeded mouse. the amyloid pathology in each mouse was categorized on a scale from (-) as no pathology to (+++) as abundant pathology. each point represents a rating score by an observer (3 observations per animal). the number of seeded mice for each scale of amyloid burden by the three observers is reported. a subset of mice injected with ndc lysate were rated as + based on 3-5 plaques per section, and thus we set this rating as baseline (marked by a dash line). only the two ad cases and pa 4 consistently scored above +. the number of prp.appsi/tau-p301l or prp.appsi mice injected with each lysate, respectively, were as follows: ad 1: n=4, n=3; ad 2: n=4, n=3; pa 1: n=3, n=3; pa 2: n=3, n=5; pa 3: n=5, n=4; pa 4: n=2, n=5; ndc 1: n=3, n=3; ndc 2: n=5, n=5; uninjected: n=3, n=3. two of the donor cases were identified as having caa pathology, ad 1 and pa 3, and were scored 1+ and 2+-3+, respectively (table 1). to determine if caa pathology was seeded in the recipient mice, we stained sections with thio-s and searched for evidence of vascular amyloid. in mice seeded with pa 3, we observed thio-s positive, vascular pathology in 6 of the 7 recipient mice; however, the incidence of this pathology was limited (fig. s8). although ad 1 also contained caa pathology, we did not observe thio-s positive, vascular pathology in mice seeded by this homogenate. these findings suggest that it may be possible to selectively increase caa using seeding, but enhancing such pathology may require purification of cerebral vessels before preparation of the seeds. we biochemically confirmed our histological data by analyzing aβ levels from sequentially extracted brain lysates using c-terminal specific antibodies in sandwich elisas to measure aβ40 and aβ42 specifically. in our aβ elisas the detection limit was approximately 0.04 pmol/g. in the brains of all seeded mice, the levels of aβ40, both sds-soluble and fa, were 10-100-fold lower than that of aβ42 (fig. 2a, b). consistent with the presence of abundant diffuse aβ pathology, we detected elevated levels of aβ42 in sds-soluble fractions from mice seeded with ad 1 and 2, and pa 4 (fig. 2c). somewhat surprisingly, the levels of aβ42 in the fa-soluble fractions were similar to that of the sds fraction despite the absence of cored aβ deposits (fig. 2c and d). notably, there was considerable variation in the levels of aβ in these seeded animals, which rendered relatively few indications of statistical differences (fig. 2e-h). for aβ40 measures, the only instance in which the seeded mice had levels that were higher than uninjected controls, or mice injected with ndc lysate, were mice seeded by ad 1 (fig. 2e, f). for measurements of sds-soluble aβ42, the brains of mice seeded with ad 1, 2, and pa 4 were the only examples in which the levels were statistically higher than the levels in mice seeded with ndc 1 and 2, or uninjected mice (fig. 2g). in the fa-soluble fractions, only the ad lysates possessed higher levels of aβ42 than the brains of mice seeded with the two ndc lysates or uninjected mice (fig. 2h). collectively, these findings demonstrated that both ad and pa brains have the potential to seed diffuse aβ pathology, with the ad brains appearing to be slightly more potent. fig. 2 biochemical analysis of sequentially extracted aβ42 and aβ40 levels by end-specific sandwich elisa. prp.appsi/tau-p301l and prp.appsi mice were seeded by ad, pa, or ndc lysate at p0 and aged 12 months. (a, e) 2% sds-extracted aβ40, (b, f) 70% formic acid aβ40, (c, g) 2% sds-extracted aβ42, and (d, h) 70% formic acid aβ42. data plotted as scatter dot plot of prp.appsi/tau-p301l (square) and prp.appsi (circle) ± standard error of the mean. the number of prp.appsi/tau-p301l or prp.appsi mice injected with each lysate, respectively, were as follows: ad 1: n=5, n=4; ad 2: n=6, n=5; pa 1: n=5, n=4; pa 2: n=4, n=5; pa 3: n=5, n=4; pa 4: n=3, n=5; ndc 1: n=5, n=4; ndc 2: n=5, n=5; uninjected: n=5, n=3. aβ42 and aβ40 levels were quantified with corresponding one-way anova with multiple comparisons test (ns, p > 0.05; *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001; ****, p ≤ 0.0001). to determine whether the injection of these lysates into the brains of newborn prp.appsi/itau-p301l mice also induced tau pathology, we stained the brains of the seeded animals with antibodies specific for phosphorylated tau (at8) and misfolded tau (mc1). immunostaining with the antibody cp27, which is specific for human tau, confirmed the presence of human p301l tau in the bigenic mice, but we observed no obvious reactivity with at8 or mc1 antibodies (fig. s9). to determine if the induction of amyloid would secondarily induce αsyn pathology, we examined the seeding activity of individuals with ad and lbd pathology. to characterize the pathology of these ad, ad/lbd, and ndc brains, we stained sections with a pan-aβ antibody (fig. 3a), thio-s (fig. 3b), αsyn antibody (fig. 3c), and a phospho-specific tau antibody (fig. 3d). both ad 3 and 4 contained numerous amyloid deposits that were compact and thio-s positive (fig. 3a, b), had widespread tau positive inclusions (fig. 3d), and lacked αsyn pathology (fig. 3c). ad 3 showed striking thio-s positive amyloid staining surrounding vessels, consistent with a caa score of 3+ (fig. 3b, table 1). we also observed caa staining with ad/lbd 1 (fig. 3b). the ad/lbd cases showed modest aβ pathology with some αsyn pathology and sparse tau deposition (fig. 3a, c, d). ndc 3 contained some amyloid pathology, but both lacked αsyn and tau pathology (fig. 3). fig. 3 characterization of ad, ad/lbd, and control cases. representative image from the cortex of 2 ad cases, 2 ad/lbd cases, and 2 ndc cases stained with (a) biotinylated anti-aβ mab 33.1.1 (anti-aβ 1-16). scale bar: 100 μm, (b) thio-s. scale bar: 50 μm, (c) anti-αsyn mab 9c10 (anti αsyn 2-21). arrowheads depict lewy bodies. scale bar: 100 μm, and anti-ptau mab 7f2 (pthr205). scale bar: 150 μm. prp.appsi/m20 and prp.appsi mice injected with ad brains developed widespread, robust aβ pathology by 12 months post-injection (fig. 4a and b). aβ deposition was primarily diffuse and thio-s negative (fig. s10). there was no difference in aβ seeding capacity between prp.appsi/m20 and prp.appsi mice. interestingly, ad/lbd 1 and 2 also promoted deposition of aβ in mice expressing appsi with and without αsyn (fig. 4), indicating that the ad/lbd brains contained considerable levels of aβ seeds despite a much lower burden of aβ pathology (fig. 3). none of the injected animals developed appreciable αsyn pathology (data not shown). although two of the donor cases contained significant caa pathology, caa was not seeded in the recipient mice (fig. s10). fig. 4 cerebral injection of ad and ad/lbd brain lysates results in widespread amyloid deposition and caa. overall schematic of the experiment (a). newborn p0 mice were injected with ad, ad/lbd, and ndc brain lysates and aged 12 months. representative brain sections (hippocampus, cortex, and meninges) of (b) prp.appsi/line m20 and (c) prp.appsi mice stained with biotinylated anti-aβ mab ab5 (anti-aβ 1-16) and counterstained with hematoxylin. n=4-8. scale bar: hippocampus: 250 μm, cortex and meninges: 50 μm. seeding diffuse aβ deposition does not impair performance in a fear-conditioning cognitive task to assess whether the diffuse aβ pathology seeded by ad brain produces cognitive deficits, an additional cohort of prp.appsi mice were seeded with ad 1 and 2, or ndc 1, and then were aged 12 months before behavioral testing in a fear-conditioning paradigm. control groups were mice that were not injected with brain lysate and nontransgenic littermate mice that were either injected with the same lysates or uninjected. the performance of the animals in the contextual fear memory test was conducted twice. in the first round of testing, the performance of the seeded mice was not statistically different from that of the control uninjected mice (fig. 5a). overall, in this first round of testing, the percentage of time animals that exhibited freezing behavior was relatively low. in the second round of testing, we expected to reinforce memory of the adverse cue (context or tone) and we observed that re-tested mice showed increased levels of freezing behavior (fig. 5b); however, there was still no statistically significant difference in the performance of the seeded mice relative to any control. these findings indicate that the diffuse aβ pathology seeded by these homogenates of human ad brain does not produce a meaningful impairment in the systems involved in fear-conditioning memory tasks. fig. 5 diffuse amyloid seeding does not cause cognitive impairment in fear-conditioning. prp.appsi mice seeded by ad and ndc lysates, were aged 12 months and subjected to contextual fear conditioning. mean percentage freezing ± standard error of the mean exhibited by prp.appsi and nontransgenic (ntg) littermates injected with ad cases 1 or 2 and ndc case 7. uninjected mice were the control group. (a) context and tone paradigm. (b) a subset of mice were re-tested the following week, context and tone test. n = 6-8/tg, n = 12-16/ntg mice per group. assessment of aβ seeding efficiency in appsi mice to examine the relative seeding efficiency of different types of aβ pathology in prp.appsi mice, we used an approach in which homogenates from the brains of 4 different lines of aged app and app/ps1 mice were used to seed accelerated aβ deposition (described in table 2). at these advanced ages, each of the mice used to produce inoculum had substantial aβ pathology (fig. s11). following the strategy used above, we injected the homogenates containing aβ seeds into newborn mice on neonatal day 0, which were then aged to 9, 12, or 15 months before euthanasia and neuropathological analysis by campbell switzer silver stain and thio-s staining. the type of pathology noted and the relative abundance score are noted at the bottom of each column of images in figure 6. in all of the prp.appsi mice injected with brain homogenate seeds from mice that primarily exhibited cored plaques, we observed an obvious shift in the type of aβ deposits in recipient mice to match the pathology found in the donor mice used for seeding (fig. 6). self-seeding of newborn prp.appsi mice with brain homogenates from aged prp.appsi mice produced diffuse deposits (fig. 6), which was the predominant form of aβ pathology in the prp.appsi animal used to generate the seeding homogenate (fig. s11). similarly, the predominant pathology in the ~25 month old tet.moaβ animal used to prepare seeding homogenate was diffuse aβ deposits (fig. s11)(48), and brain homogenates from this animal efficiently seeded the deposition of human aβ in the prp.appsi mice to produce diffuse deposits (fig. 6). prp.appsi mice seeded with homogenates from donors that had primarily cored deposits had lower aβ burden scores, but importantly we observed a shift in the neuropathologic features of the recipient mice to a much higher incidence of cored deposits (fig. 6). interestingly, the morphology of deposits in the seeded prp.appsi mice was not quite identical to the source prp.huaβ/ps1 mice in that the seeded deposits were smaller and appeared to be more compact than the donor line. these small dense deposits were thio-s positive (fig. 6). similar small dense core deposits that were thio-s positive were observed in the cortex of prp.appsi mice injected with brain homogenates from the prp.moaβ/ps1 mice (fig. 6). collectively, these findings demonstrate the relative ease with which diffuse aβ pathology can be induced in prp.appsi mice. fig. 6 comparison of seeding activity between transgenic mice that exhibit diffuse versus cored aβ pathology. we selected brains from four sources that exhibit either diffuse or cored neuritic aβ deposits (see fig. s11). newborn prp.appsi mice were injected with homogenate from each of the four sources identified at the top of the figure. compared to uninjected mice, prp.appsi mice injected with brain homogenates from any source showed an induction of aβ pathology. inoculum from aged prp.appsi and tet.moaβ mice, which primarily exhibit diffuse aβ pathology (37, 48), robustly seeded diffuse pathology in the injected prp.appsi mice (severity of pathology and number of animals indicated at the bottom of the figure). inoculum from aged prp.huaβ/ps1 (a.k.a. appswe/ps1de9) and prp.moaβ/ps1 mice, which primarily exhibit cored neuritic deposits (37, 48), induced the deposition of a limited number of cored aβ deposits in injected prp.appsi mice with little or no diffuse deposits. d = diffuse aβ pathology. m = mixed diffuse and cored deposits. c = cored, neuritic deposits. discussion we investigated whether injection of neonatal app, app/tau, or app/αsyn mice with ad or ad/lbd brain homogenates could be used as a paradigm to generate mice that model the mixed pathology associated with each disease. despite the widespread induction of diffuse aβ deposition by injection of four different ad brain lysates, and the presence of tau seeds in these lysates, tau pathology was not induced. for comparison, we also injected pa brain lysates, finding a similar induction of diffuse aβ deposition without induction of tau pathology. similarly, injection of ad/lbd brain lysates induced diffuse aβ deposition, but αsyn pathology was not induced. in all cases, the induced aβ pathology exhibited a diffuse, thio-s negative, morphology. prp.appsi mice injected with ad brain lysates that develop diffuse aβ pathology showed no significant impairment in a fear-conditioning cognitive task. our findings suggest that seeding the brains of neonatal transgenic mice with ad, ad/lbd, and pa brain homogenates can efficiently induce diffuse aβ deposition but neither ad nor ad/lbd brains seeded concurrent tau or αsyn pathology. the most common route of administering aβ seeding preparations is by stereotaxic injection into the hippocampus of adult app transgenic mice (reviewed in (13)). in host mice that develop amyloid pathology at relatively young ages, the induced pathology created by seeding may be localized to the site of injection and overlying cortex (13); whereas, in models that do not develop deposits until late in life, the induced pathology may be more wide-spread (reviewed in (13)). the prp.appsi host we used in the present study develops pathology on its own between 12-14 months of age. in testing the approach of injecting aβ seeds into newborn mice from this model, we aspired to attain a widespread distribution of seeding material and induce aβ, tau, or αsyn pathology throughout the brain. this approach replicates previous studies where p0 injection of adeno-associated virus resulted in widespread distribution in the brain (49). injection of newborn hamsters with scrapie prions has been shown to accelerate onset of prion disease (50). additionally, we have observed that intracerebral injection of newborn a53t αsyn mice with brain lysates from multiple system atrophy donors induces αsyn pathology and motor impairment (31), and that intraspinal injection of newborn mice expressing mutant superoxide dismutase (sod1) can accelerate the onset of paralysis and pathology (51, 52). thus, in performing newborn injections our goal was to initiate pathological cascades as early as possible in order to determine whether there may be distinct synergies between aβ and tau, or aβ and αsyn, which can be elaborated by seeding. we recognize that there are a large number of potential mouse models that could have been used for these studies. the app mouse model we used for these studies was selected because prp.appsi mice do not inherently develop aβ pathology until 12-14 months of age and because this model can exhibit a full spectrum of aβ pathology including cored-neuritic, diffuse, and vascular deposition (see supplemental fig. s2). the tau model we chose to use expresses human p301l tau at relatively low levels and does not develop tau pathology on its own. when paired with mice that express the tetracycline transactivator in the rtg4510 model these develop a robust tau pathology (38). in paradigms in which tau expression in rtg4510 mice is induced early and then suppressed by doxycycline, the low level of “leaky” tau expression in this model is sufficient to sustain neurofibrillary tangle pathology (38). in our paradigm, we asked whether neonatal seeding of this tau model could induce a sustained pathology in the same way that early expression of the transgene at high levels produced sustained pathology. alternatively, we were interested to determine whether we could detect any synergy between concurrent aβ pathology and tau seeding. the αsyn model we chose to use expresses human wt αsyn at levels that do not cause pathology (41). wt αsyn (m20) mice can be seeded when injected with high levels of purified αsyn fibrils, but are not easily seeded by human brain homogenates (31). our goal in choosing the m20 wt αsyn mice was to develop a model of human αsyn pathology and assess whether concurrent aβ pathology could synergize αsyn seeding. our method of seed preparation followed commonly used protocols where we injected a soluble fraction; frontal cortex homogenized in pbs (10% w/v) followed by sonication and low speed centrifugation, as previously described (42, 53, 54). by this method, we expected sonication to fragment all types of seeds with a mixture of seeds remaining in suspension after low-speed centrifugation. as we have observed here, the most common outcome of aβ seeding with similar preparations is induction of diffuse aβ pathology (reviewed in (13)). in order to efficiently seed mixed pathology, it may be necessary to optimize preparations of each type of seed independently. interestingly, injection of the supernatant and pellet of fractionated app23 brain homogenate resulted in morphologically different aβ deposits, with the supernatant fraction seeding diffuse, congo red negative aβ and the pellet seeding deposits similar to the total lysate, a mixture of diffuse and punctate, congo red positive deposits (55). several studies have shown induction of tau pathology after injection of synthetic tau fibrils, brain extract from mutant p301s tau mouse, or human ad, corticobasal degeneration, and progressive supranuclear palsy brain lysates (56-62). in these studies, the human brain lysates were enriched for tau seeds by sequential fractionation with the addition of sucrose and/or sarkosyl, creating fractions that contain potent tau seeds. similarly, injection of the fractionated homogenate from brain lysates of individuals with lbd resulted in the induction of αsyn, indicating that with enrichment it is possible to seed αsyn pathology directly from human brain (20, 63, 64). because one of our goals was to determine whether we could detect synergy between aβ and tau or αsyn pathology, we chose to avoid enriching for any particular type of seeding activity. the absence of tau or αsyn pathology in our seeded models indicates that the type of aβ deposition we generated did not synergize to induce, or exacerbate, tau or αsyn pathology. we demonstrated that the aβ seeding activity of brain lysates from pa cases was similar to ad and ad/lbd cases. these findings agree with a previous study that reported brain homogenates from a human pa case seeded diffuse aβ deposition in tg2576 app mice (65). although mice seeded by pa brains scored as having less amyloid positivity at 12 months post-injection than mice injected with ad brain lysates, the difference between pa and ad brain seeding activity was less evident by 18 months post-injection. our study was not powered or designed to determine whether homogenates from ad and pa brains have quantitative differences in seeding activity. all three of the pa cases tested were able to seed amyloid pathology, supporting the hypothesis that the aβ pathology in pa is similar to ad (25). interestingly, several of the brain lysates we used were from tissues that exhibited relatively high levels of caa pathology (see table 1). pa 3 and ad 3 were both scored as having the highest levels of caa, with significant caa in ad/lbd 1 and 2. in mice injected with pa 3, we observed modest seeding of parenchymal caa pathology in the host mice but in all other mice the only obvious vascular deposition was meningeal. augmented caa pathology has been observed in app23 mice, which develop caa, by intraperitoneal injection of app23 lysates (66). caa pathology was also induced in crnd8 mice by injection of synthetic aβ42 oligomers, generated in the presence of anionic micelles composed of fatty acids (67). recent studies have documented early onset cerebrovascular aβ pathology in individuals receiving dura mater transplants and cadaveric pituitary-derived growth hormone, suggesting that caa may be transmitted iatrogenically (68, 69). collectively, these results suggest that caa could be a distinct conformer of aβ that may be independently propagated. not all aβ conformers seeded as proficiently as vascular aβ. tissues from ad and ad/lbd brains that were used to prepare these lysates contained both diffuse and compact aβ deposits, with significant levels of formic acid extractable aβ. yet, even in mice aged to 18 months of age, diffuse aβ deposits were the dominant form of pathology. these results mirror similar studies in which injection of human brain homogenates resulted in induction of diffuse aβ pathology, with relatively few thio-s or congophilic compact deposits (49, 65, 70, 71). the lack of conversion of the diffuse deposits to cored plaques indicates that these types of amyloid deposition are not freely interchangeable, supporting the idea that each type of aβ pathology arises from different amyloid strains with distinct seeding capabilities. this conclusion is also supported by our studies of aβ seeding in which transgenic mice were used as the seed source. newborn prp.appsi mice injected with transgenic mouse brain homogenates prepared from mice that exhibit high levels of diffuse aβ pathology produced a robust induction of diffuse aβ deposition by 12 months post-injection. by contrast, brain homogenates prepared from mice that predominantly produce cored, thio-s positive, aβ deposits induced minimal aβ deposition by 12 months. these results are consistent with previous studies with app/ps1 and app23 mice. injection of appps1 mice that typically develop compact, punctate plaques, with app23 seeds results in a mixture of diffuse, filamentous aβ as well as compact plaques (42, 72). injection of app23 mice that typically develop mixed pathology, both cored and diffuse plaques, with appps1 homogenate results in plaques that are more diffuse than the punctate deposits seen in appps1 mice injected with appps1 homogenate (42, 72). thus, although it is possible to seed cored deposits in app mice (42, 55, 73-75), it appears that diffuse aβ pathology is more easily seeded. together these results suggest that aβ plaques differ in their morphology, seeding ability, and impact on cognitive function. current amyloid therapies aim to reduce amyloid deposition without much consideration of the type of plaque that is being targeted. reduction of diffuse amyloid may at first appear to be beneficial, since it appears to be the dominate seeding strain, however, it may not be the most pathological strain. cognitive behavior studies of the seeded appsi mice reported here, and of a non-seeded bri-aβ42 model that also shows primarily diffuse aβ pathology (76), failed to associate diffuse amyloid with reduced performance in a fear-conditioning memory task. if diffuse aβ pathology is less damaging, then therapies that only reduce this pathology may not produce therapeutic benefit in ad. conclusions in conclusion, we assessed the type of pathology induced when neonatal app transgenic mice were injected with brain lysates prepared from ad, ad/lbd, and pa brains, finding robust and widespread induction of aβ pathology. the induced aβ pathology was diffuse, which has been reported in other seeding studies where adult animals were the recipients of injections (13). our results are consistent with earlier reports in finding that diffuse aβ is easily seeded by crude homogenates of human brain. the diffuse aβ pathology induced in these models was not accompanied by secondary tau or αsyn pathology in bigenic mice co-expressing app/tau or app/asyn. seeded mice with diffuse aβ pathology were not impaired in a fear-conditioning memory task. the seeding approach described here may be useful in producing mice that model aspects of non-ad pathological aging, providing a useful comparison model to mice that exhibit pathological features more similar to ad and ad/lbd pathology. ethics approval human brain tissue was obtained from the mayo clinic brain bank and university of florida neuromedicine human brain tissue bank with informed consent following institutional regulations. all animal procedures were approved by the university of florida institutional animal care and use committee. consent for publication not applicable. availability of data and materials all data generated and analyzed during this study are included in this manuscript. competing interests the authors declare that they have no competing interests. funding this work was supported by funding from the national institute of health; grant numbers r21ag046125, r21ag055113, u54ns110435, p50ag047266, p30ag066506. authors’ contributions study concept and design: bdm, yl, jl, big, drb. pathological analysis: sp and dd. acquisition of data: bdm, yl, gx, hh, mfa, clc, hsf, cm, sf, sp, dd. statistical analysis: bdm and drb. analysis and interpretation of the data: bdm, yl, gx, cj, jl, big, teg, drb. drafting of the manuscript: bdm and drb. acquisition of funding: jl, big, teg, drb. all authors read and approved the final manuscript. acknowledgements the authors would like to thank the patients and their families that donated 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normal cognition in transgenic bri2-abeta mice. mol neurodegener. 2013;8:15. copyright: © 2022 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. molecular fragment characteristics and distribution of tangle associated tdp-43 (tats) and other tdp-43 lesions in alzheimer’s disease feel free to add comments by clicking these icons on the sidebar free neuropathology 4:22 (2023) original paper molecular fragment characteristics and distribution of tangle associated tdp-43 (tats) and other tdp-43 lesions in alzheimer’s disease keith a. josephs1, shunsuke koga2, nirubol tosakulwong3, stephen d weigand3, nha trang thu pham4, matt baker2, jennifer l. whitwell4, rosa rademakers2,5, leonard petrucelli2, dennis w. dickson2 department of neurology, mayo clinic, rochester, minnesota, usa department of neuroscience, mayo clinic, jacksonville, florida, usa department of quantitative health sciences, mayo clinic, rochester, minnesota, usa department of radiology, mayo clinic, rochester, minnesota, usa department of biomedical sciences, university of antwerp, antwerp, belgium corresponding author: keith a. josephs · mayo clinic · college of medicine and science · 200 first street s.w. · rochester, mn 55905 · usa josephs.keith@mayo.edu submitted: 15 november 2023 accepted: 03 december 2023 copyedited by: georg haase published: 08 december 2023 https://doi.org/10.17879/freeneuropathology-2023-5192 keywords: tar dna binding protein 43, tmem106b, cluster analysis, tat, hippocampal sclerosis abstract tar dna binding protein 43 (tdp-43) pathology is a defining feature of frontotemporal lobar degeneration (ftld). in ftld-tdp there is a moderate-to-high burden of morphologically distinctive tdp-43 immunoreactive inclusions distributed throughout the brain. in alzheimer’s disease (ad), similar tdp-43 immunoreactive inclusions are observed. in ad, however, there is a unique phenomenon of neurofibrillary tangle-associated tdp-43 (tats) whereby tdp-43 intermingles with neurofibrillary tangles. little is known about the characteristics and distribution of tats, or how burden and distribution of tats compares to burden and distribution of other ftld-tdp-like lesions observed in ad. here we characterize molecular fragment characteristics, burden and distribution of tats and assess how these features compare to features of other tdp-43 lesions. we performed tdp-43 immunohistochemistry with anti-phosphorylated, cand n-terminal tdp-43 antibodies in 20 high-probability ad cases and semi-quantitative burden of seven inclusion types within five brain regions (entorhinal cortex, subiculum, ca1 and dentate gyrus of hippocampus, occipitotemporal cortex). hierarchical cluster analysis was used to analyze the dataset that consisted of 75 different combinations of neuropathological features. tats were nonspherical with heterogeneous staining patterns and present in all regions except hippocampal dentate. all three antibodies detected tats although n-terminal antibody sensitivity was low. three clusters were identified: cluster-1 had mild-moderate tats, moderate-frequent neuronal cytoplasmic inclusions, dystrophic neurites, neuronal intranuclear inclusions and fine neurites, and perivascular and granular inclusions identified only with the n-terminal antibody throughout the brain; cluster-2 had scant tats in limbic regions and cluster-3 mild-moderate tats and mild-moderate neuronal cytoplasmic inclusions and dystrophic neurites throughout the brain and moderate fine neurites. only 17% of cluster 1 cases had the tmem106b gg (protective) haplotype and 83% had hippocampal sclerosis. both features differed across clusters (p=0.03 & p=0.01). tats have molecular characteristics, distribution and burden, and genetic and pathologic associations like ftld-tdp lesions. abbreviations ad alzheimer’s disease, dn dystrophic neurites, erc entorhinal cortex, ftld frontotemporal lobar degeneration, fn fine neurites, gn granular inclusions, hca hierarchical cluster analysis, hpscl hippocampal sclerosis, nci neuronal cytoplasmic inclusions, nft neurofibrillary tangle, mapt microtubular associated protein tau, nii neuronal intranuclear inclusions, tat tangle associated tdp-43, tdp-43 tar dna binding protein of 43 kda, tmem transmembrane protein, part primary age-related tauopathy, pv perivascular inclusions, otc occipitotemporal cortex, ctdp-43 c-terminal fragment of tdp-43, ntdp-43 n-terminal fragment of tdp-43, ptdp phosphorylated tdp introduction tar dna binding protein 43 (tdp-43) is a pathological protein strongly linked with frontotemporal lobar degeneration (ftld) (1, 2). in ftld-tdp there are widespread, characteristic histopathological lesions, including neuronal cytoplasmic inclusions (ncis), dystrophic neurites (dns), fine neurites (fns) of the hippocampus, granular (sometimes referred to as small discrete ncis)/globular inclusions (gns), perivascular inclusions (pvs) and neuronal intranuclear inclusions (niis) (3-7). each lesion type in ftld-tdp has a signature molecular pattern of phosphorylated tdp-43 defined by relative amounts of c-terminal fragments to full-length tdp-43 (8-11). since the discovery of tdp-43 in ftld (1, 2), we and others have also found tdp-43 immunoreactive inclusions in the brains of patients with alzheimer’s disease (ad) (12-16). in ad, as in ftld-tdp, there are ncis, dns, fns of the hippocampus, gns, pvs and niis. in ad, however, we have identified a distinct inclusion whereby tdp-43 in the amygdala is found intermingled with the neurofibrillary tangle (nft) (12, 17). subsequently, others have confirmed our finding (18). this nft-associated tdp-43 (tats) is found in over 55% of ad cases with tdp-43, and hence could play an important role in ad pathogenesis. at present, little is known about tats. it is unknown whether tats are only deposited in the amygdala or are more widely distributed, whether the burden of tats differs across different brain regions, and how the distribution and burden of tats in different brain regions compare to the distribution and burden of other ftld-tdp lesions found in ad. lastly, the molecular characteristics of tats is unknown, nor how its molecular characteristics compare to the molecular characteristics of other ftld-tdp inclusions found in ad. in this study we aimed to assess the molecular characteristics, distribution and burden of tats and compare these features to those of the other ftld-tdp inclusions found in ad. we hypothesize that tats will show a pattern of widespread distribution given the distribution of nft in ad, and that tats will show similar molecular fragment characteristics to ncis. material & methods case selection the database of the brain bank at mayo clinic, jacksonville, florida was queried to identify 20 consecutive cases, between january 2018 and july 2021, that received a neuropathologic diagnosis of high probability ad with tats in the amygdala. a diagnosis of high probability ad was based on the national institute on aging-alzheimer's association guidelines for the neuropathologic assessment of ad (19). high probability ad was selected given the widespread distribution of nfts in these cases to ensure that we would capture the true distribution of tats in ad. to be included in the study, all cases had to also have available paraffin blocks for additional molecular pathological analyses. demographic characteristics of the 20 cases in the cohort are shown in table 1. all but two cases (90%) were caucasian and nine (45%) were male. the median age at death in the cohort was 82 years (range: 76-100 years) and median disease duration was 11 years (range: 5-19 years). fifty percent of the cases had a family history of a neurodegenerative disease. all but one case (95%) had a final clinical diagnosis of an alzheimer’s spectrum dementia diagnosis; one case (case 5) had a frontotemporal dementia diagnosis. neuropathologic methods all cases had undergone neuropathologic assessment by a single expert neuropathologist (dwd) and had standardized tissue sampling and semi-quantitation of ad pathology. thioflavin-s fluorescent microscopy was used for the evaluation of the distribution of senile plaques and nfts which were then used to determine the thal β-amyloid phase (20, 21) and the braak nft stage (22), respectively. immunohistochemistry was performed on all 20 cases with an antibody to alpha-synuclein (nacp, 1:3000 rabbit polyclonal, mayo clinic antibody (23)) and with a phosphorylated tau antibody (phf-1, 1:1000 mouse monoclonal, gift from dr. peter davies). hippocampal sclerosis (hpscl) was assessed on hematoxylin-eosin stained sections and was diagnosed based on the presence of neuronal loss and gliosis occurring in the subiculum and/or ca1 sector of the hippocampus (24). the cohort had a median thal phase (21) of 5 (range: 4-5) and a median braak nft stage (22) of vi (range: v-vi) (table 1). eleven cases (55%) had amygdala-only lewy bodies (25) and eight cases (40%) had hpscl (24). table 1: demographic and clinical features of the 20 cases case cluster sex race/ethnicity family history disease duration (yrs.) age at death (yrs.) final clinical diagnosis brain weight (gms.) thal phase braak stage josephs stageω lewy bodies hpscl 1 1 m caucasian no 9 80 alzheimer's dementia 1020 5 vi 3 none yes 2 1 f caucasian no 15 93 alzheimer's dementia 880 5 v 3 amygdala yes 3 1 m caucasian yes 10 85 logopenic variant ppa & caa 1120 4 v 3 amygdala no 4 1 f caucasian no 10 78 alzheimer's dementia with subdural hematoma 920 5 vi 6 none yes 5 1 f caucasian no 11 76 dementia with behavioral change¥ 920 5 vi 4 amygdala yes 6 1 m caucasian yes 10 82 alzheimer's dementia 1140 5 v 3 none yes 7 3 m caucasian yes 10 98 alzheimer's dementia 1060 5 v 3 none no 8 3 m caucasian yes 8 77 alzheimer's dementia 1280 5 vi 3 none no 9 3 f caucasian no 16 98 alzheimer's dementia 860 5 vi 6 amygdala yes 10 3 m caucasian no 5 82 alzheimer's dementia 1120 5 v 3 none no 11 3 f caucasian yes 5 100 alzheimer's dementia familial 960 5 vi 3 none no 12 3 f caucasian no 13 84 alzheimer's dementia with psychosis 760 5 vi 3 amygdala yes 13 3 m caucasian yes 14 84 alzheimer's dementia 1020 5 v 3 amygdala no 14 3 f caucasian no 12 81 alzheimer's dementia 920 5 vi 3 none yes 15 3 f caucasian no 14 87 alzheimer's dementia 1160 5 vi 3 amygdala no 16 2 f caucasian yes 10 77 alzheimer's dementia 820 5 vi 3 amygdala no 17 2 m caucasian no 6 79 alzheimer's dementia 1220 5 vi 1 amygdala no 18 2 f hispanic yes 11 82 alzheimer's dementia 980 5 vi 1 amygdala no 19 2 f caucasian yes 19 89 alzheimer's dementia 960 5 vi 1 none no 20 2 m asian/pi yes 18 81 alzheimer's dementia 960 5 vi 1 amygdala no caa = cerebral amyloid angiopathy; f = female; hpscl = hippocampal sclerosis; m = male; pi = pacific islander; ppa = primary progressive aphasia ¥ suggestive of frontotemporal dementia; disease duration and age at death are shown in years. ω staging was determined prior to the study and was based only on ptdp-43 immunohistochemistry. tdp-43 immunohistochemistry & semi-quantitation for all 20 cases we performed serial sectioning and tdp-43 immunohistochemistry with three different tdp-43 antibodies: a commercially available phosphorylated tdp-43 (ptdp-43) antibody (ps409/410, 1:5000 mouse monoclonal, cosmo bio co., ltd) that recognizes tdp-43 with phosphorylated epitopes; anti tdp-43 antibody that recognizes a neoepitope in the c terminal fragment of cleaved tdp-43(ctdp-43) (mc2085, 1:2500 rabbit polyclonal, gift from leonard petrucelli, mayo clinic) (8); and anti tdp-43 antibody that recognizes an epitope in the amino terminus (ntdp-43) (mc1079; 1:2500 rabbit polyclonal, gift from leonard petrucelli, mayo clinic) (8). antigens for both non-commercial antibodies, as well as detailed biochemistry regarding both antibodies have been previously published (8, 26-28). antibodies targeting phosphoserine 409 and 410 have been shown to have very strong immunoreactivity to tdp-43 inclusions in ftld-tdp (9, 29). n-terminal tdp-43 fragments are believed to be rapidly degraded (30, 31) and as expected mc1079 binding represents detection of full length tdp-43(8). in all cases, immunohistochemistry was performed using a dako autostainer (universal staining system, carpinteria, ca). for this study, two investigators (kaj & dwd) reviewed all 20 cases together and performed regional semi-quantitation of tdp-43 pathology with the three antibodies (ptdp-43, ctdp-43 & ntdp-43) in five regions: 1) entorhinal cortex (erc); 2) ca1 sector of the hippocampus; 3) subiculum of the hippocampus; 4) dentate gyrus of the hippocampus and 5) occipitotemporal cortex (otc). lesions were considered positive, independent of the intensity of lesion staining. hence, lesions showing slight/mild staining with one antibody were given equal weight to being positive to lesions with robust intensity with another antibody. that is, both staining patterns were assumed positive. this accounts for differences in affinities of the three different antibodies. for each region, tdp-43 deposition was assessed independently for seven different types of lesions: (1) small discrete ncis; (2) short/comma-like dns; (3) fns in the ca1 region of the hippocampus as described by hatanpaa et al (4); (4) pvs (typically bilobular and located next to capillary basal lamina) described by lin et al (7); (5) tats described by amador-ortiz(12); (6) grainand globular-like intraparenchymal inclusions (gns) and (7) niis. the burden of all lesions except for niis were scored semi-quantitatively based on a 5-point scale to be able to compare across lesion type: 0 = no inclusions identified, 0.5 = scant number of inclusions (1-3); 1= mild inclusions (4-10), 2= moderate inclusions (11-30) and 3 = marked/frequent number of inclusions (>30). for niis, we documented the presence/absence since when present, they were rare. for each lesion type we assessed the burden detected with all three anti-tdp-43 antibodies independently: ptdp-43; ctdp-43 and ntdp-43. inclusion burden was assessed at a 200x magnification, with multiple visual fields of view, i.e., as many as necessary to examine the entire region of interest. any possible niis that were observed at 200x magnification was confirmed as an nii at a 400x magnification. hence, a total of 75 combinations of brain regions (n=5), inclusions (n=7) and antibodies (n=3) were analyzed (note: not every lesion was assessed across every region, e.g., fns were only assessed in ca1). confocal microscopy immunofluorescence double-staining with the combinations of phospho-tau (cp13; mouse monoclonal; 1:1000; from the late dr. peter davies, feinstein institute, north shore hospital, ny) and phospho-tdp-43 (rb3655, rabbit polyclonal, 1:1000; mayo clinic antibody) was performed by sk who has expertise in the technique (32). the deparaffinized and rehydrated sections were steamed in distilled water for 30 minutes. next, sections were blocked with protein block plus serum free (dako) for 1 hour and incubated with primary antibodies diluted in with antibody diluent with background-reducing components (dako) overnight at 4°c. sections were washed three times with 1xpbs at room temperature, and then incubated with secondary antibodies alexa fluor 568 and alexa fluor 488 (1:500, thermo fisher scientific, inc.) diluted with antibody diluent with background-reducing components (dako) for 1.5 hours at room temperature in a dark chamber. sections were washed three times with 1xpbs at room temperature, incubated with 1% sudan black for 2 minutes, washed with distilled water and mounted with vectashield mounting media containing dapi (vector laboratories). representative images were taken with a confocal laser-scanning microscope (lsm 880; carl zeiss, jena, germany) using ec plan-neofluar 40x/1.30 oil dic m27 objective (carl zeiss). genetic screening genomic dna was extracted from the cerebellum of all twenty cases using the dneasy blood and tissue kit (qiagen sciences) and genotyped using taqman snp assays analyzed with the quantstudio 7 flex real-time polymerase chain reaction (pcr) system (thermofisher). genotypes were generated for apoe (rs429358 and rs7412) using taqman assay id c___3084793_20 and c____904973_10, grn (rs5848) using c___7452046_20, mapt (rs1052553) using c___7563736_10 and tmem106b (rs3173615) using c__27465458_10. the rs1052553 polymorphism defines the mapt h1/h2 haplotype. the rs3173615 variant in tmem106b encodes the amino acid coding change threonine to serine at position 185, which been shown to be a risk factor for ftld-tdp (39), and reduced tmem106b homozygote carrier status has been found to be associated with tdp-43 in ad (33). statistical methods hierarchical clustering analysis (hca) was performed using a total of 75 features (combinations of brain regions/inclusions/antibodies). burden of niis (12 features) were treated as binary variables, while the rest (63 features) were normalized into the range of 0 and 1. to perform hca, a distance between 2 data points was defined using "gower" (which is appropriate for a data set with different data types (numeric, binary, etc.)), with "ward's" (variance was minimized within clusters) as a cluster method. hca results are shown with a dendrogram; a tree-like diagram that shows the hierarchical relationship between all data points. height (y axis) represents the distance between two clusters that contain those data points (the higher the height, the less similar those data points are). we assessed clustering tendency of the case by visualizing a plot of the first two principal components of the data set, and used the hopkins statistics as a measure of spatial randomness. the hopkins statistic was 0.66 which shows that the data was likely to have meaningful clusters. we used the elbow, silhouette, and gap statistic to determine the optimal number of clusters. bootstrap resampling along with the jaccard similarity index were used to assess cluster stability. jaccard similarity index values close to 1 indicate stable clusters, values < 0.6 indicate cluster is unstable. demographic, genetic, and pathological characteristics were compared across clusters using kruskal-wallis rank sum test for continuous variables, and fisher's exact test for categorical variables. data shown are median (iqr) or n (%). statistical analyses were performed using r statistical software. results characteristics of tats tats were characterized by being nonspherical and slightly larger than ncis with a heterogeneous staining pattern whereby, within the inclusion, some areas show darker staining than others (figure 1). confocal microscopy confirmed that tats represent tdp-43 colocalized with paired helical filament tau (figure 2). tangle-associated tdp-43 was identified in all regions, except for in the dentate granular cells of the hippocampus, and was identified with all three antibodies, although sensitivity of detection with the ntdp-43 antibody that detects full length tdp-43 was low compared to the ctdp-43 and ptdp-43 antibodies (figure 3). burden findings across the cohort a summary of the lesion burden data by subject, region, inclusion type and antibody type are shown in figure 3. there were differences identified across the three antibodies. the ctdp-43 and ptdp-43 antibodies both similarly detected ncis, dns, tats, niis and fns, although the ctdp-43 antibody was slightly less sensitive to burden (figure 4). the ntdp-43 antibody, while less sensitive at detecting ncis, dns, tats, niis and fns compared to the other two antibodies (figure 4), was more sensitive at detecting gns and pvs (figure 5). there were some differences identified across the five regions that are worth highlighting (figure 3). first, the subiculum and ca1 were less likely to have ncis, and, when present, tended to have lower burden than the erc, dentate gyrus of the hippocampus and otc. of the four regions where tats were found (erc, ca1, subiculum and otc), tats were most common in erc and subiculum, followed by ca1 and lastly the otc. niis were only observed in erc and otc. gns, when present, were least common in the otc, while pvs, when present, where least common in the subiculum. tats were more common than ncis in the subiculum across all three antibodies but were less common than ncis in the otc, especially with the ctdp-43 antibody. tats were not identified in the dentate while ncis were common in this region. cluster analysis findings the elbow, silhouette and gap statistic suggested optimal number of clusters of 2 to 4. however, the jaccard similarity index for 3 clusters had the highest value (0.70), and the least instability (0.27), and hence we assessed 3 clusters in our data. the hierarchical cluster analysis dendrogram is shown in figure 6. cluster-1 consisted of six subjects, cluster 2 of five subjects and cluster 3 of nine subjects. cluster-1 was characterized by moderate-frequent burden of ncis and dns across regions, high burden of fns in ca1, mild pv and gn identified only with the ntdp-43 antibody and mild-moderate tats throughout the brain; cluster 2 had scant tats only; and cluster 3 mild-moderate burden of tats and mild-moderate ncis, dns and fns and scant-mild pv and gn identified predominantly with the ntdp-43 antibody (figure 7). niis were found in 83% of cluster 1 cases, 0% of cluster 2 cases and 33% in cluster 3 cases. table 2 shows the demographic, genetic and pathological features across the three clusters. no statistically significant differences were observed in sex, family history, disease duration, age at death or apoe genotype. there was a significant difference in tmem106b genotype for the rs3173615 snp, with 17% of cluster 1 cases have the protective (gg) allele, while 40% of cluster 2 and 33% of cluster 3 cases had the protective allele. there was also a significant difference in hpscl with the highest frequency observed in cluster 1 (83%) and the lowest frequency in cluster 2 (0%). figure 1: morphological characteristics of tangle associated tdp-43 (tats). lesions are shown with anti-ptdp-43 antibody given that this antibody showed the most robust staining of tats with different morphological characteristics seen in panels a, b, and c. arrows point to tats with different morphological characteristics. figure 2: confocal microscopy demonstrating overlap of phosphorylated tdp-43 and phosphorylated paired helical filament tau of tats. panels a and d show single labelling of tdp-43 immunoreactive inclusions. panels b and e show single labelling of tau immunoreactive inclusions. panels c and f show double labelling of tdp-43 and tau immunoreactive inclusions. yellow color in panels c and f represents the overlap of the two proteins (tdp-43 and tau). dapi (blue, single labelling) was used as a nuclear counterstain. figure 3: distribution and burden of tdp immunoreactive lesions across regions for each case. the relationships between the three antibodies (ptdp-43, ctdp-43 and ntdp-43) and the seven lesion types (ncis, dns, fns, niis, gns, pvs, and tats) across the five brain regions (entorhinal cortex (erc), subiculum, ca1 of the hippocampus, dentate gyrus of the hippocampus and occipitotemporal cortex (oct)) for each case. brighter shades of orange represent higher burdens of pathology. note niis were scored as present (1)/absent (0). figure 4: phospho (ptdp), c-terminal fragment (ctdp) & full-length (ntdp) tdp-43 immunoreactivity of lesion types in ad. labelling of neuronal cytoplasmic inclusions (ncis), tangle associated tdp-43 (tats) and dystrophic neurites (dns) with ptdp-43 (a, d, g), ctdp-43 (b, e, h) and ntdp-43 (c, f, i) in the entorhinal cortex (row 1), fine neurites (fn) in ca1 region of the hippocampus (row 2) and ncis in the dentate granule cells of the hippocampus (row 3). the arrow in panel 4c points to an inclusion that is detected with ntdp-43 antibody, in the entorhinal cortex. the inset shows a magnified view of an inclusion labelled in the entorhinal cortex that has been labelled with the ntdp-43 antibody. figure 5: full-length (ntdp) tdp-43 immunoreactivity of granular neurites and perivascular (pvs) inclusions. granular neurites (a) and perivascular inclusions (b) in the occipitotemporal cortex (otc) labelled with anti-ntdp-43 (full-length) antibodies. figure 6: hierarchical cluster analysis dendrogram using all 75 features and highlighting the three clusters. figure 7: distribution and burden of tdp immunoreactive lesions across regions for each cluster. the relationships between the three antibodies (ptdp-43, ctdp-43 and ntdp-43) and the seven lesion types (ncis, dns, fns, niis, gns, pvs, and tats) across the five brain regions (entorhinal cortex (erc), subiculum, ca1 of the hippocampus, dentate gyrus of the hippocampus and occipitotemporal cortex (oct)) for each cluster. brighter shades of orange represent higher average burdens of pathology within cluster. note niis were scored as present (3)/absent (0) to put them on the same scale as the other inclusions. table 2: participants characteristics cluster 1 (n=6) cluster 2 (n=5) cluster 3 (n=9) overall p-values demographic and clinical features female, n (%) 3 (50%) 3 (60%) 5 (56%) >0.99 family history, n (%) 2 (33%) 4 (80%) 4 (44%) 0.39 duration, yrs. 10 (10, 11) 11 (10, 18) 12 (8, 14) 0.78 age at death, yrs. 81 (78, 84) 81 (79, 82) 84 (82, 98) 0.23 genetic characteristics apoe ε4 carrier, n (%) 4 (67%) 4 (80%) 9 (100%) 0.14 24 1 (17%) 1 (20%) 1 (11%)   34 3 (50%) 2 (40%) 6 (67%)   44 0 (0%) 1 (20%) 2 (22%)   33 2 (33%) 1 (20%) 0 (0%)   grn rs5848, n (%)       0.63 cc 3 (50%) 4 (80%) 3 (33%)   ct 2 (33%) 1 (20%) 5 (56%)   tt 1 (17%) 0 (0%) 1 (11%)   tmem106b rs3173615, n (%)       0.03* cc 3 (50%) 3 (60%) 0 (0%)   cg 2 (33%) 0 (0%) 6 (67%)   gg 1 (17%) 2 (40%) 3 (33%)   mapt haplotype, n (%)       >0.99 11 4 (67%) 4 (80%) 6 (67%)   12 2 (33%) 1 (20%) 3 (33%)   pathological characteristics brain weight 970 (920, 1095) 960 (960, 980) 1020 (920, 1120) 0.94 vascular disease, n (%) 2 (33%) 4 (80%) 7 (78%) 0.23 hippocampal sclerosis, n (%) 5 (83%) 0 (0%) 3 (33%) 0.01† amygdala lbd, n (%) 3 (50%) 4 (80%) 4 (44%) 0.52 josephs tdp-43 stage (range) 3 (3, 6) 1 (1, 3) 3 (3,6) 0.03*† overall p-values for continuous variables are from kruskal-wallis rank sum test, and fisher's exact test for categorical variables. data shown are median (iq) or n (%) * cluster 2 is statistically different from cluster 3 (p = 0.009); † cluster 1 is statistically different from cluster 2 (p = 0.02); apoe = apolipoprotein e; mapt = microtubule associated protein tau; grn = progranulin; tmem106b = transmembrane protein 106b; lbd = lewy body disease discussion in this study, we focus on tat lesions that we described in the brains of patients with ad (17). we demonstrate that tats are a mixed inclusion, characterized by the intermingling of two proteins: tau and tdp-43. we show that morphological characteristics of tats are heterogeneous across lesions and that the burden of tats vary by region within individual brains, as well as in the same region across different cases. we found that the molecular characteristics of tats is identical to that of ncis in ad. lastly, we showed that tats can be limited to being present in the amygdala only in ad although they are more often widespread and associated with the presence of ncis, dns, niis, fns and other ftld-tdp characteristic lesions; a relationship that appears to have genetic underpinnings. neurofibrillary tangle-associated tdp-43 (tats) have only been described in ad (12, 17, 18), unsurprisingly, given that nfts are one of the two characterizing lesions in ad, the other being the senile plaque (19). tats have not been described in ftld-tdp, likely because nfts tend to be absent, or no more than scant and limited in distribution when present in ftld-tdp. with-that-said, we hypothesize that tats should also be observed in other diseases in which nfts are present, such as primary age-related tauopathy (part) (34) since tdp-43 has been reported in part (35-37). however, given the limited distribution and burden of nfts in part, one would not expect tats to be as frequent as in ad, and certainly uncommonly found outside of limbic regions given that braak and braak stage is < iv in part (34). we observed heterogeneous morphologies across tat inclusions, often having the appearance of a bitten apple, or flame shaped. under the light microscope, we also observed a heterogeneous staining pattern in individual lesions whereby different parts of the lesions had more and less robust (dark) staining. the reason for this is unclear but could be due to how much tdp-43 vs how much tau is being exposed on the surface that is being viewed. another reason for the heterogeneous appearance of the inclusion may be due to the relative amounts of the different molecular fragments that make up the tdp-43 inclusions. we found tats to be most robustly stained by ptdp-43 antibodies suggesting that phosphorylated tdp-43 is the main composition of tats. there was an almost equal staining observed with the ctdp-43 antibody suggesting that the main composition of tats are c-terminal fragments of tdp-43. interestingly, there was some staining, although much less robust, with the ntdp-43 antibody that recognizes full-length tdp-43. this suggests that tats, while consisting of predominantly phosphorylated c-terminal fragments of tdp-43, also consist of some full-length tdp-43. one of the interesting findings of this study was that the molecular characteristics of tats mirrored that of ncis. ncis were indeed smaller than tats, but, like tats, also showed the most robust staining with the ptdp-43 antibody followed closely by staining with the ctdp-43 antibody and much less staining occurring with the ntdp-43 antibody. we have previously found the identical molecular staining pattern in ncis in ftld-tdp (11). hence, ncis in ftld-tdp, ncis in ad and tats share similar molecular characteristics. what was interesting was the fact that although all cases in this study were braak stage v-vi with nfts extending into association and primary neocortices, tats were predominantly deposited in limbic regions (amygdala + erc and hippocampal subregions) although not in the dentate gyrus of the hippocampus. the burden of tats in the oct, a non-limbic region, was much less compared to the amount observed in limbic regions. this was different from what we observed for ncis, which in many instances showed an equal burden, or even higher burden in the oct compared to limbic regions. there were 75 different combinations of antibodies, lesions and regions which made it difficult to visually determine whether there were distinct relationships within the cohort. however, when we applied cluster analysis, we found three different clusters. the first (cluster 1, n=6) was characterized by a minimal burden of tats, as well as widespread deposition of ftld-tdp inclusions. all cases in this cluster had fns of the ca1 region of the hippocampus, and the majority (83%) were also found to have niis and hpscl. hence, cluster 1 has features that are highly reminiscent of ftld-tdp (3, 5) and, therefore, could be best summarized as ftld-tdp with tats. cluster 2 (n=5) was very different from cluster 1. in four of the five cases in cluster 2 there were no inclusions whatsoever identified with any of the three antibodies. the presence of tdp-43 was limited to the amygdala in four of the cases, with the fifth case only having scant tats in limbic cortex with a single nci identified in oct with the ptdp-43 antibody. this cluster, therefore, had no features suggestive of ftld-tdp and could best be summarized as one with focal amygdala tats. the third cluster was the largest (n=9) and consisted of a moderate-marked number of tats in limbic regions and in oct. there were also ftld-tdp inclusions present throughout the brain, but they had a lower burden compared to cluster 1. interestingly, niis and hpscl were also present, albeit in only 33% of the cases. hence, cluster 3 could be best summarized as having widespread tats with some features of ftld-tdp. therefore, overall, it appears that approximately 50% of this cohort of high probability ad cases, all of the cases in cluster 1 and for argument’s sake half of the case of cluster 3, have salient features of ftld-tdp. we found evidence for an association between cluster and the tmem106b alleles. tmem106b has been found to be associated with an increased risk of having ftld-tdp pathology (38-41). more specifically, the gg haplotype is associated with a decreased risk of ftld-tdp (40). only one of the five cases in cluster 1 (ftld-tdp with tats) had the protective gg haplotype while 40% of the cases in cluster 2 and 33% of the cases in cluster 3 had the gg haplotype. of note, approximately 20% of normal controls vs only 6% of ftld-tdp subjects have the gg haplotype (40). hence, the absence of the gg haplotype in cluster 1 is another characteristic of ftld-tdp. although the ptdp-43 and ctdp-43 antibodies were the best at staining ncis, dns, niis, fns of the ca1 region of the hippocampus, as we have previously shown in ftld-tdp (11), and tats, we found that pvs and gns were best identified with the ntdp-43 antibody. this suggests that pvs and gns have a different molecular composition from the other ftld-tdp-like inclusions in ad. the fact that pvs and gns were best observed with the ntdp-43 antibody that recognizes full-length tdp-43 suggests that pvs and gns may not be pathologic. another explanation could be that pvs and gns represent early inclusions that were not yet cleaved by caspases into c terminal fragments (8). there are strengths and limitations to this study that are worth mentioning. in terms of strengths, our sample was unbiased as we selected consecutive cases for the study; pathological determination of braak nft stage was performed by a single experienced neuropathologist, reducing variability, and tdp-43 inclusion assessments were performed by two experts to reach consensus on severity ratings; and we performed confocal microscopy to confirm that tats represent tdp-43 colocalized with paired helical filament tau. limitations of the study include the lack of racial heterogeneity in our sample which may limit our findings to caucasians, and the fact that we did not have volumetric head mri or [18f] fluorodeoxyglucose pet scans to determine whether our clusters had different antemortem signatures of atrophy or hypometabolism. in conclusion, we have identified key characteristics of an understudied pathological lesion that is common in ad, that we refer to as tats (tangle-associated tdp-43). it appears that the association between tats and lesions characteristic of ftld-tdp is not random but instead may have a genetic underpinning. declarations ethics approval and consent to participate the mayo institutional review board (irb) has deemed this study exempt from irb review and approval given that it utilizes postmortem brain tissue only. consent for publication not applicable availability of data and material the datasets generated and analyzed for the current study is available from the corresponding author on reasonable request. competing interests none funding the study was funded by national institute of aging grants r01-ag37491 and rf-ns120992. authors' contributions dr. josephs was responsive for generating tdp-43 burden data, obtaining study funding, and for drafting the manuscript. dr. koga was responsible for confocal microscopy and critical revision of the manuscript. ms. tosakulwong was responsible for performing the statistical analyses. mr. weigand was responsible for supervising ms. nirobul tosakulwong, and for critical revision of the manuscript. mr. baker was responsible for performing the genetic screening. dr. rademakers was responsible for supervising the genetic screening and for critical revision of the manuscript. ms. nha pham was responsible for creating the pathological figures. dr. whitwell was responsible for helping to draft the manuscript, supervising ms. nha pham and for critical revision of the manuscript. dr. petrucelli was responsible for antibody development and critical revision of the manuscript. dr. dickson was responsible for screening and identifying all cases, pathological assessments, generating tdp-43 burden data and 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carriers against frontotemporal dementia. acta neuropathol. 2014;127(3):397-406. doi: 10.1007/s00401-013-1240-4. 41. gallagher md, suh e, grossman m, elman l, mccluskey l, van swieten jc, et al. tmem106b is a genetic modifier of frontotemporal lobar degeneration with c9orf72 hexanucleotide repeat expansions. acta neuropathol. 2014;127(3):407-18. doi: 10.1007/s00401-013-1239-x. copyright: © 2023 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. blocking tgf-βand epithelial-to-mesenchymal transition (emt)-mediated activation of vessel-associated mural cells in glioblastoma impacts tumor angiogenesis feel free to add comments by clicking these icons on the sidebar free neuropathology 5:4 (2024) original paper blocking tgf-βand epithelial-to-mesenchymal transition (emt)-mediated activation of vessel-associated mural cells in glioblastoma impacts tumor angiogenesis luisa merk1, katja regel1, hermann eckhardt1, marietheres evers1, ali el-ayoubi1, michel mittelbronn2-7, marcel krüger8, jean-jacques gérardy3,7, andreas f. mack9, ulrike naumann1, 10 molecular neuro-oncology, department of vascular neurology, hertie institute for clinical brain research and center neurology, university hospital of tübingen, germany department of cancer research (docr), luxembourg institute of health (lih), luxembourg luxembourg centre of neuropathology (lcnp), luxembourg luxembourg centre for systems biomedicine (lcsb), university of luxembourg, luxembourg department of life sciences and medicine (dlsm), university of luxembourg, esch-sur-alzette, luxembourg faculty of science, technology and medicine (fstm), university of luxembourg, esch-sur-alzette, luxembourg national center of pathology (ncp), laboratoire nationale de santé (lns), luxembourg department of preclinical imaging and radiopharmacy, university of tübingen, tübingen, germany institute for clinical anatomy and cell analytics, university of tübingen, germany gene and rna therapy center (grtc), faculty of medicine university tübingen, germany corresponding author: ulrike naumann · molecular neuro-oncology · department of vascular neurology · hertie institute for clinical brain research and center neurology · university hospital of tübingen · hoppe-seyler-straße 3 · 72076 tübingen · germany ulrike.naumann@uni-tuebingen.de additional resources and electronic supplementary material: supplementary material submitted: 10 november 2023 accepted: 07 february 2024 copyedited by: joão gama published: 01 march 2024 https://doi.org/10.17879/freeneuropathology-2024-5188 keywords: glioblastoma, tumor vasculature, pericytes, tgf-β, emt abstract glioblastoma (gbm) is the most common malignant primary brain tumor in adults. gbm displays excessive and unfunctional vascularization which may, among others, be a reason for its devastating prognosis. pericytes have been identified as the major component of the irregular vessel structure in gbm. in vitro data suggest an epithelial-to-mesenchymal transition (emt)-like activation of glioma-associated pericytes, stimulated by gbm-secreted tgf-β, to be involved in the formation of a chaotic and dysfunctional tumor vasculature. this study investigated whether tgf-β impacts the function of vessel associated mural cells (vamcs) in vivo via the induction of the emt transcription factor slug and whether this is associated with the development of gbm-associated vascular abnormalities. upon preventing the tgf-β-/slug-mediated emt induction in vamcs, the number of pdgfrβ and αsma positive cells was significantly reduced, regardless of whether tgf-β secretion by gbm cells was blocked or whether slug was specifically knocked out in vamcs. the reduced amount of pdgfrβ+ or αsma+ cells observed under those conditions correlated with a lower vessel density and fewer vascular abnormalities. our data provide evidence that the slug-mediated modulation of vamc activity is induced by gbm-secreted tgf-β and that activated vamcs are key contributors in neo-angiogenic processes. we suggest that a pathologically altered activation of ga-peris in the tumor microenvironment is responsible for the unstructured tumor vasculature. there is emerging evidence that vessel normalization alleviates tumor hypoxia, reduces tumor-associated edema and improves drug delivery. therefore, avoiding the generation of an unstructured and non-functional tumor vasculature during tumor recurrence might be a promising treatment approach for gbm and identifies pericytes as a potential novel therapeutic target. introduction glioblastoma (gbm) is the most common malignant primary brain tumor in adults with an incidence of 2 to 5 cases per 100,000 people in north america and europe. gbm accounts for more than 50 % of primary malignant brain tumors. worldwide, the number of new cases per year can be estimated at around 250,000. the median age at diagnosis is 64 years and it is more common in men as compared to women (for review see [1]). its mean progression-free survival is just a few months. by standard therapy using optimal surgical resection, radiation and chemotherapy with temozolomide, or even using more recent additional treatment approaches such as tumor treating fields (ttf), there is invariably tumor recurrence. however, this combined treatment only slightly prolongs the median survival, but usually not more than up to 20 months [2]. several characteristics of gbm account for its poor prognosis: (i) gbms are highly invasively growing tumors which makes a complete surgical resection impossible. even after aggressive surgery, tumor cells that invaded healthy brain areas will remain and are the source of recurrence [3]. (ii) gbms show massive proliferation which makes them grow rapidly. the growing tumor mass is in turn the cause of several neurological problems such as seizures, impaired vision, drowsiness, changes in personality or massive headache, dependent on the location of the tumor in the brain [1]. (iii) gbms can be categorized as highly immunosuppressive tumors. several mechanisms such as the expression of immune checkpoint proteins like programmed cell death ligand 1 (pd-l1), the secretion of immunosuppressive cytokines such as transforming growth factor (tgf)-β by gbm cells or tumor vessel associated pericytes [4], the downregulation of major histocompatibility complex (mhc) class i molecules on gbm cells as well as metabolic changes in the tumor cells and the tumor microenvironment (tma) negatively influence the immune surveillance of gbms [5-8]. (vi) glioma is a highly treatment resistant type of cancer [9]. (vii) finally, gbms are highly vascularized tumors, and the interaction between gbm cells and blood vessels promotes neoangiogenesis, thereby facilitating tumor growth [10]. numerous mechanisms contribute to the robust formation of blood vessels within the tumor. this involves endothelial cell (ec) proliferation, which is dependent on hypoxia and hypoxia inducible factor (hif)-1 induced transcription and release of vascular endothelial growth factor (vegf) from gbm cells. this process leads to the sprouting of capillaries from pre-existing blood vessels. the release of further angiogenic factors from gbm cells recruits a variety of cells that may also participate in new blood vessel formation (for review see [11]). in gbm, the newly formed vessels often show abnormalities in shape, size and complexity, resulting in glomeruloid, garland-like or clustered bizarre vascular formations, which negatively influences the patient´s outcome [12]. however, not only ecs are involved in angiogenic processes and vessel formation. especially pericytes came into focus to be the responsible cell type for the chaotic vessel structure in gbm, since these cells are heavily modified under pathological conditions (reviewed in [13, 14]). pericytes are cells adjacent to ecs, wrapping around blood vessels. in the cns, pericytes are necessary for the formation and regulation of the blood-brain-barrier (bbb). in former studies we have demonstrated that pericytes are prominently involved in the formation of gbm-associated vascular proliferations and that those pericytes covering gbm-associated vessels can be distinguished from "normal" pericytes covering vessels outside the tumor core by their expression profile, especially by the expression of epithelial-to-mesenchymal transition (emt) factors such as slug or twist [15]. in vitro, gbm cells, by secretion of tgf-β, induce an emt-like program in human brain microvascular pericytes, drive these cells to change their growth morphology, shift them towards energy production by glycolysis, induce proliferation and increase cell motility, and impact the bbb integrity [16, 17]. in this study, we were interested in whether the tgf-β-mediated induction of the emt program in vamcs might have impact on the density, formation and structure of newly built blood vessels in the tumor core of gbm, particularly in a murine mouse model in vivo. material and methods cell culturing and testing primary murine brain vascular pericytes (mbvp) were purchased from ixcells (san diego, ca, usa) and were cultured in fully supplemented mouse pericyte growth medium (pm, ixcells). mbvps were used up to passage 5. gl261 mouse glioma cells (cellosaurus cvcl_y003), known to express tgf-β [18], were from the institute's repository. gl261 cells were created in 1970 via chemical induction with methylcholanthrene pellets implanted into the brains of c57bl/6 mice [19]. nih/3t3 cells were from atcc (#crl-1658). gl261 and nih/3t3 cells were cultivated in dulbecco's modified eagles medium (dmem; sigma-aldrich, taufkirchen, germany) supplemented with 10% fbs, 100 u/ml penicillin, and 0.1 mg/ml streptomycin (sigma-aldrich) at 37 °c, 5 % co2. all cell lines were regularly tested to be free of mycoplasma using the mycoalert mycoplasma detection kit (lonza, cologne, germany). generation and characterization of gl261 tgf-β1/β2 double knockout, mcherry expressing cells for the generation of gl261 tgf-β1 plus -β2 double knockout cells, the cells were transfected by electroporation with alt-r cas9 nuclease, atto 550 crispr-cas9 tracrrna, mouse tgfb1 guide rnas (ggcaguagccgcagccccgaguuuuagagcuaugcu and guacaaagcgagcaccgccuguuuu-aga-gcuaugcu; all from integrated dna technology, coralville, ia, usa) using the amaxa cell line nucleofactor™ kit v (lonza) as described [20]. guide rnas were designed using benchling (https://www.benchling.com). an in silico off-target binding prediction was performed using the online tool off-spotter (https://cm.jefferson.edu/off-spotter; [21]). after electroporation, transfected cells were sorted on a sh800 cell sorter (sony, stuttgart, germany). after expansion of cell clones, biallelic tgf-β1 knockout gl261 cell clones were identified by pcr on genomic dna on a ptc-200 pcr cycler (biorad, feldkirchen, germany) using the following detection primer: mtgfb1-frw gggttcccgctctccgaagtg and mtgfb1-rev gtccaccattagcacgcggg (sigma). tgf-β1-ko cells were subsequently transfected using mouse tgfb2 guide rnas (gcuccgcucgcgcucgcaggg-uuuuagagcuaugcu and gcgccaccgggaccagaugcguuuuagagcuaugcu; integrated dna technology) as described above. after sorting and clonal expansion as described above, the tgf-β2 knockout was analyzed by pcr on genomic dnausing tgf-β2 specific primer (mtgfb2-frw cgtttcttccttttaaaaacatg and mtgfb2-rev tgtcgattttataaacctccttg; all from sigma). further validation of the double knockout was performed by sequencing on an abi sequencer using bigdye® terminator v 3.0 sequencing reaction mix (applied biosciences, carlsbad, germany). gl261 as well as gl261-tgf-β-ko cells were grown up and were transduced twice with lenti-mcherry (addgene; #36084). gl261mcherry (par) and gl261mcherry-tgf-β1/2-knockout (tgf-β-ko) cells were subsequently characterized in terms of their proliferation and migration capacity. for this, 2.000 cells were seeded in microtiter plates and allowed to attach. 4 h after seeding and subsequently every 24 h later, cell density was determined by staining the cells with crystal violet as described [22]. to determine differences in cell motility, the transwell boyden chamber migration assay was used as described [23]. briefly, 25.000 par or tgf-β-ko cells were treated for 3 h with mitomycin (0.5 µg/ml, sigma) to block proliferation, were washed to remove residual mitomycin, then seeded in the top chamber of an 8 µm pore migration cassette (corning, kaiserslautern, germany), and were allowed to migrate for 24 h. migrated cells on the bottom side were stained with crystal violet and were counted manually. as attraction medium, conditioned medium from nih/3t3 cells was used. identification of the murine rgs5 minimal promoter dna fragments of different lengths located in the mrgs5 promoter region (ensemble id grcm39:cm000994.3) were amplified by pcr from genomic dna of c57bl6 using different primer combinations (f1 and f4, r1-r4; suppl. table 1, suppl. fig. 1). nhei and xbai recognition sites were added to the 5’ and 3’ ends of the primer. subsequently, the fragments were cloned into pcr blunt ii topo using the zero blunt topo pcr cloning kit (thermofisher scientific, waltham, ma, usa) and sequenced. after sequencing, the fragments were recloned into pgl4.10[luc2] (promega, madison, wi, usa). to identify the murine rgs5 minimal promoter, mbvps or gl261 cells were transfected with the appropriate plasmids expressing the luciferase gene under the control of one of the mrgs5 promoter fragments (mrgs5-pro1, 2, 3, 11, 12, 14, 15), and a luciferase assay was performed 48 h after transfection using the luc-pair™ firefly luciferase hs assay kit (genecopoeia, cat# lf007). lentivirus cloning and preparation to knockout slug in mural cells in vivo, we generated three different lentiviruses using the plenticrispr v2 (addgene cat# 52961) system in which the murine slug specific oligonucleotide that serves as sgrnas as well as the cas9 enzyme, under control of the rgs5 promoter, are encoded on one plasmid [24]. therefore, we removed the ef1α promoter in front of the cas9 gene in plenticrispr v2 and integrated the identified murine rgs5 minimal promoter fragment at this position. in a second step, we inserted three different oligos (suppl. table 2) that serve as sgrnas for the knockout of murine slug. slug/snai2 genomic dna and cdna sequences were obtained from ensembl (ensmusg00000022676), the crispr guide design tool was from benchling, in silico off-target binding prediction was performed using off-spotter. the cloning strategy is shown in suppl. fig. 2. lentiviral particles were generated by transfection of hek293ft cells with the modified plenticrispr v2 plasmids in combination with plp1, plp2 and plp-vsvg (the latter three from invitrogen, darmstadt, germany) using the mirus transit-lenti reagent (thermo fisher). viral particles collected from supernatants were concentrated and purified using peg 6000 precipitation as described [25]. virus titers were determined using quicktiter™ titration kit (cell biolabs inc., san diego, usa) and were stored in aliquots at -80 °c. animal experiments animal work was performed in accordance with the german animal welfare act and its guidelines (e.g. 3r principle) and was approved by the regional council of tübingen (approval n05/17). rgs5-gfp pericyte reporter mice, which express gfp under the control of the rgs5 promoter, were from the karolinska institute (stockholm, sweden) and are described in detail in [26]. the mice were bred in the animal facility of the institute under spf conditions and used at an age of 3 9 months. the specificity of gfp positivity to vamcs in the brain was determined by immunofluorescence for gfp in parallel with the staining of ecs using a cd31 specific, fluorochrome coupled antibody. to determine the growth of gl261 parental (par) and tgf-β-knockout (tgf-β-ko) tumors, under anesthesia and analgesia the cells were stereotaxtically implanted into the right striatum of the mice at the following coordinates (1 mm rostral, 2 mm lateral of the bregma, 3 mm in depth, using the mouse brain atlas coordinates; the mouse brain atlas (gaidi.ca)). the mice were intensively monitored to avoid and reduce pain. magnetic resonance imaging (mri) analyses using intravenously applied gadovist (600 mg/kg of body weight) were performed thrice to determine tumor growth. to investigate alteration in tumor angiogenesis induced by the knockout of tgf-β in gbm cells, either 100.000 par or tgf-β-ko cells were intrastriatally implanted as described above. the mice were sacrificed when the tumors reached a median size of 1.5 2.5 mm as determined in a preliminary experiment. brains were collected and processed for further analyses. to knockout slug in vamcs in the tumor region, either 3x105 infectious particles of lenti-crispr v2 (control virus) or a mixture of three lenti-slug-ko viruses were intrastriatally injected 3 days before par cells were implanted using the same brain coordinates as described above. histology and immunofluorescence brains were collected and fixed in 4 % paraformaldehyde (pfa) for 20 hours. after subsequent dehydration via sucrose gradient, the cells were embedded in tissue tek (sakura finetek, alphen aan den rijn, the netherlands) and were stored frozen at -80 °c. sectioning (10 µm slices) was performed on a leica cm3050 cryotome using superfrost™ plus slides (r. langenbrinck gmbh). for if staining, samples were permeabilized in pbs containing 0.2 % triton® x-100 and blocked in 5 % bovine serum albumin (bsa)/ 0.5 % triton® x-100. the respective primary antibody dilutions were used for overnight incubation at 4 °c: αsma, 1:100; cd140b, 1:100; cd31, 1:100; gfp, 1:100 (all from invitrogen, carlsbad, ca, usa). excess primary antibodies were removed by washing the slides thrice in pbs. the following fluorescence labeled antibodies were used for incubation at room temperature for 90 minutes: goat anti-chicken alexa fluor™ 488, 1:1000; goat anti-rabbit alexa fluor™ 405, 1:1000; goat anti-rabbit alexa fluor™ 647, 1:1000; goat anti-rat alexa fluor™ 647, 1:1000 (all from invitrogen). after washing three times in pbs, the sections were mounted either with dako mounting medium or alternatively with vectashield® antifade mounting media containing dapi in case no af405 secondary antibody was used for staining and were stored at 4 °c. all stained tissue samples were evaluated for the proteins of interest using the zeiss axio imager with apotome2 (carl zeiss, jena, germany). images were taken in different magnifications (10x, 25x, 40x and 63x) with fixed exposure settings and were processed using the zen blue 3.6 software (carl zeiss). subsequent quantification analyses were performed using image j (fiji, [27]). double and triple positive cells were counted manually. clarity brains were fixed and incubated in cold hydrogen monomers (hm) solution contain 4 % acrylamide, 4 % paraformaldehyde and 0.25 % va-044 initiator (wak0 fisher scientific, waltham, ma, usa) for 1-2 days. afterwards, polymerization was performed under vacuum for 2-3 h at 42 °c as previously described [28, 29]. fixed brains were cut into 2-3 mm coronal slices. for the lipid removal, the brain slides were put in 8 % sds/pbs and were incubated at 50 °c with continuous movement in a rotator by changing of sds once a week until the tissue had reached a homogeneous transparency. tissue transparency was assessed by visualization of high-contrast signals through the tissue and was reached after approximately 4 weeks. samples were washed thrice in pbst before they were put in pbs overnight. samples were incubated for 2 days at 37 °c in pre-incubation solution consisting of 4 % goat-serum, 1 % bsa, 0.25 % triton and 0.01 % sodium azide. subsequently, the brains were incubated for 5 days with the following primary antibodies diluted in pre-incubation solution at 37 °c: anti-cd31 (1:25-1:50; abcam, cambridge, uk) and anti-mcherry (1:500; novus/biotechne, minneapolis, mn, usa). after washing the samples thrice for 2 hours in pbs containing 0.1 % triton x, they were incubated for 2 days at 37 °c in the following secondary antibodies: goat-anti-rabbit alexa 488, 1:200; goat-anti-chicken alexa 546, 1:200 (both from invitrogen) and draq5 for nuclear staining (1:500, thermo fisher scientific, waltham, ma, usa). afterwards, the samples were washed three times in pbst for 2 hours each followed by washing with either pbs containing 0.01 % sodium azide or pbst overnight. after further washing in pbs for 2 hours, samples were transferred in 80 % glycerol at 37 °c to match the refractive index within the hydrogel [30]. confocal microscopy was performed on a zeiss lsm 510 followed by image processing using the zen black software (carl zeiss) and image j. subsequent analysis was performed using imaris (oxford instruments, oxford, uk). statistical analyses all in vitro experiments were performed at least thrice if not mentioned otherwise. for in vivo experiments, the group and sample size are indicated in the figure legends. to assume a gaussian distribution, all data received from in vitro experiments passed a normality test (shapiro-wilk and tukey’s multiple comparison tests). further statistical analyses were done with a two-tailed student’s t-test or one-way anova using graphpad prism 7.0 (graphpad inc., san diego, ca, usa) or excel (microsoft corp, redmond, wa, usa), followed by bonferroni correction. the results are represented as mean ± standard deviation (sd). p-values of < 0.05 are considered as statistically significant (ns: not significant; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001). results characterization of the tgf-β knockout in gl261 cells in vitro and in vivo the knockout of tgf-β1 and -β2 was verified by pcr and sequencing and was found to be complete in the gl261 tumor cells, since no full length tgf-β mrna was detected in tgf-β-knockout (tgf-β-ko) cells (suppl. fig. 1). as tumor angiogenesis is correlated to tumor growth and invasion, it is of importance to determine the growth rate of tgf-β-ko cells compared to that of parental (par) cells. in this regard, and to later on avoid effects of different tumor size of par and tgf-β-ko tumors on angiogenesis in our mouse gbm model, we determined proliferation as well as cell motility of the cells. as shown in fig. 1. tgf-β-ko cells have a significantly lower capacity to proliferate and also showed lesser cell motility than par cells. figure 1: characterization of gl261 tgf-β knockout cells indicates lower proliferation and cell motility upon knocking out tgf-β. a. cell growth of parental (par), tgf-β1 knockout (β1-ko), tgf-β2 knockout (β2-ko) and tgf-β1 + β2 double knockout gl261 cells (β1/2-ko; n=3, sd, **** p<0.0001). b. transwell migration of the cells indicated in a (n=4, sd, * p<0.05, ** p<0.01, n.s.: not significant). c. cells on the bottom side of the transwell migration membrane after migration, one picture is exemplary shown. identification of the murine rgs5 minimal promoter used to knockout slug in tumor-associated vamcs to knockout slug in vivo in our mouse gbm model, and as the murine, vamc specific rgs5 promoter [31] has not been described in detail so far, we firstly had to identify the minimal rgs5 promoter that can be used to induce gene expression. for this, by pcr we amplified different fragments of the murine rgs promoter, cloned overlapping fragments in pgl4.10 [luc2] and determined luciferase activity in mbvps. fragment 14 covers the entire region from the enhancer, ending at the start of exon 1 of the murine slug gene. fragment 12 additionally includes exon 1, and fragment 15 contains 597 bp directly upstream of exon 1 (fig. 2a). fragments 12 and 14 showed highest luciferase activation, whilst luciferase induction by fragment 15 was only half (fig. 2b). to determine specificity of the rgs5 promoter regions, we measured luciferase activity also in gl261 gbm cells. for fragment 14, luciferase activity in mbvps was 25 times increased compared to the empty vector construct whilst we only saw a weak and not significant induction in gl261 cells (appr. 2-fold). for fragment 15, induction of luciferase activity was about 12x in mbvp and 4x in gl261 cells (fig. 2c). therefore, we chose to use fragment 14 as this fragment seemed to be the most specific part of the murine rgs5 promoter. fragment 14 was cloned upstream of the cas9 gene of plenticrispr v2 to specifically express cas9 in rgs5 expressing cells (plenti-rgs5-crispr). the plenticrispr v2 system that allows the expression of cas9 and guide rnas from the same vector (suppl. fig 2a) was used to knockout slug in tumor-associated mural cells in our mouse model. three specific guide rnas (sgrna # 1, 4 and 5) were designed using the tool provided by benchling (san francisco, ca, usa). off-target analyses (off-spotter; [32]) indicate no further targets outside of slug for the designed guides. the slug oligos were cloned into plenticrispr v2 (cas9 under control of the ef1α promoter) and target efficacy of the slug knockout was tested by infection of gl261 cells with these lentiviruses either alone or in combination, followed by sequencing. using a combination of the three sgrnas, the total editing efficiency was notably higher than for single sgrnas and reached 85 %. indels were found at the target sites for all three sgrnas (suppl. fig. 2e). we therefore cloned sgrnas# 1,4 and 5 into plenti-rgs5-crispr, creating lenti-slug-ko #1, #4 and #5, and used a combination of these three viruses to specifically knockout slug in gbm adjacent vamcs in our mouse in vivo model. figure 2: identification and specificity of the murine rgs5 promoter. a. structure of the mrgs5 promoter region (e1: exon 1). numbered lanes indicate fragments that have been cloned into pgl19 [luc2] and used for luciferase assays. b/c. luciferase reporter assay for the murine rgs5 promoter fragments. mbvps (b/c) or gl261 cells (c) were transfected with pgl4.10[luc2] containing the promoter fragments as indicated in a. luminescence was measured 48 h post transfection and normalized to the mean luminescence measured in cells transfected with empty pgl4.10[luc2] (rfu: relative fluorescence units; n=3-4, sd, *p<0.05, **p<0.01, n.s. not significant). knocking out tgf-β in gl261 gbms reduces slug, αsma and pdgfrβ expression in tumor-associated vamcs and is associated with reduced tumoral vamc and ec density in our previous studies we observed that in human brain microvascular pericytes slug and αsma expression was induced by tgf-β. this observation correlates with the finding that in human gbms, slug expression is associated with pdgfrβ and αsma [16]. therefore, in our mouse gbm model we sought to investigate whether the knockout of tgf-β in gl261 cells also influences the expression of these proteins. as tumor angiogenesis correlates with tumor size and growth, we determined the growth rate of par and tgf-β-ko cells and tumors in a preliminary experiment to avoid artifacts generated by different sizes of par and tgf-β-ko gbms. in a preliminary experiment using mri analyses, we observed that tgf-β-ko gl261 cells showed a delayed tumor growth also in vivo. therefore, we determined the time point when the tumors reached a size of 1.5 to 3 mm in diameter. material for analyses was collected when tumors were in that range of size (day 29 after implantation for par tumors and day 59 after implantation of tgf-β-ko-tumors (suppl. fig. 3a). even if in some mice par tumor grew very fast (suppl. fig. 3c), we did not observe any significant differences in the size of par and tgf-β-ko tumors in the collected brain tissues in retrospective analyses (suppl. fig. 3 d/f). we also determined the specificity of gfp positivity in mural cells of rgs5-gfp reporter mice by cd31 and gfp staining. as indicated in suppl. fig. 4, gfp was only present in cells wrapping around cd31+ endothelial cells. in tgf-β-ko-gbm bearing mice, in the tumor core the number of gfp positive cells was reduced 2-fold (p < 0.01), accompanied by a highly significant reduction of slug (3.2 x), pdgfrβ (2.0 x) and αsma (3.3 x) expressing cells. notably the number of gfp+/pdgfrβ+/slug+ (7.4 x) and gfp+/pdgfrβ+/αsma+ cells (2.26 x), indicating activated vamcs, was significantly reduced (figs. 3 and 4). similar results, though not as pronounced, were observed in the tumor infiltration zone (suppl. figs. 5 and 6). figure 3. gfp+ vamcs expressing slug and/or pdgfrβ were significantly reduced in tgf-β-ko gbms. a. immunofluorescence of vamcs (green), slug (blue), pdgfrβ (dark red) in the tumor core. in the merged picture (second right) gfp/slug/pdgfrβ triple positive cells are indicated by white arrows. in the merged picture (right) mcherry+ gl261 tumor cells are also shown. (one picture each is exemplary shown; 63x magnification; bars: 10 µm). b. overview of gfp, slug, pdgfrβ and mcherry positive cells in the tumor area (25x magnification; bars = 50 µm). c-e. quantification of gfp+ cells (c), slug+ (d) and pdgfrβ+ (e) cells (n=3 mice per group, 9-60 slices per group). f. quantification of slug+/pdgfrβ+/gfp+ triple positive cells (n=3 mice, 3 sections/mouse). c-f: sem, t-test, * p<0.05, ** p<0.01*** p<0.001, **** p<0.0001. figure 4. gfp+ vamcs expressing αsma and/or pdgfrβ were significantly reduced in tgf-β-ko gbms. a. immunofluorescence of vamcs (green), αsma (blue), pdgfrβ (magenta) in the tumor core. in the merged picture (second right), gfp/αsma/pdgfrβ triple positive cells are indicated by white arrows. in the merged picture (right) mcherry+ gl261 tumor cells are also shown (one representative picture is shown; 63x magnification; bars: 10 µm). b. overview of gfp, αsma, pdgfrβ and mcherry positive cells in the tumor area (25x magnification; bars = 50 µm). c. quantification of αsma+ and d. of αsma+/pdgfrβ+/gfp+ triple positive cells (n=3 mice, 9-40 sections per mouse, sem, t-test, **** p<0.0001). as gbm is a highly vascularized tumor and pericyte coverage provides vascular stability whereas tgf-β negatively influences vessel architecture, we determined vessel density in par and tgf-β-ko tumors. compared to the brain parenchyma of the non-tumor bearing contralateral hemisphere, tumor areas of both, par and tgf-β-ko mice showed a massive enrichment in the density of cd31 cells. cd31 was used to demonstrate the presence of endothelial cells (ecs) and to determine vessel density. however, in tgf-β-ko mice, the vessel density in the tumor core was half that observed in par mice (fig. 5). figure 5. cd31+ ecs were significantly reduced in tgf-β-ko gbms. a. merged pictures showing immunofluorescence of gbm cells (red), gfp+ vamcs (green) and cd31+ endothelial cells (magenta) in the tumor core, transition and infiltration zone (one picture is exemplary shown; 25x magnification, bars: 50 µm). b. immunofluorescence of gbm cells (red), gfp+ vamcs (green), cd31+ ecs (magenta), cd31 and gfp+ cells (second right) and merged picture (right; one picture is exemplary shown, 63x magnification, bars: 10 µm). c. staining for cd31 in the tumor area of par and tgf-β gbms as well as in the non-tumor containing contralateral hemisphere (normal brain; bars = 100 µm). d. quantification of cd31+ cells (n=3 mice per group, 24-70 slices per group, sd, t-test, **** p<0.0001). abolishing the gbm-mediated induction of slug expression in vamcs reduces pdgfrβ and αsma levels and lowers vessel density in the tumor area in a former study we identified tgf-β as an inducer of emt transcriptional regulators such as slug/snai2 in human primary brain microvascular pericytes (hbvp). slug was responsible for the elevated proliferation and migration capacity of these cells, pushing hbvps towards a more activated phenotype [16]. in this regard, we wondered whether the tgf-β-mediated effects on vamcs and ec density observed in our mouse gbm model could be prevented by inhibiting the induction of slug in tumor associated vamcs. for this, we intrastriatally injected a mixture of 3 lentiviruses that express cas9 under the control of the rgs5 promoter as well as sgrnas specific for murine slug (lenti-slug-ko #1, 4 and 5; suppl. fig. 2) three days prior to implantation of par cells using the same stereotactic coordinates and collected the brains 29 days after tumor cell implantation (suppl. fig. 3b). to avoid artifacts that might be induced by differences in the tumor size after knocking out slug in mural cells in the tumor environment, we firstly analyzed the tumor size in the mice that received either the empty control lentivirus (lenti-v2) or the mixture of lenti-slug-ko viruses. however, we did not observe any significant differences in tumor size (suppl. fig. 3 e/g). after collecting the brains, we determined slug, αsma and pdgfrβ levels in the tumor core, its transition zone, defined by the border zone between the tumor core and brain parenchyma infiltrating gbm cells, as well as in the more distant area of gbm cell infiltration. in lenti-slug-ko injected mice, no slug expression in the tumor area was detectable anymore (suppl. fig 7), indicating that the slug-ko in vamcs is functional. compared to the knockout of tgf-β in gl261 cells, we observed a significant reduction, even at a higher magnitude, in the number of pdgfrβ+ (4.9-fold) and αsma+ (10.7-fold) cells, as well as in slug+/pdgfrβ+/gfp+ (15.9-fold) and αsma+/pdgfrβ+/gfp+ (11.4-fold) cells within the tumor core, the transition zone, and the infiltration zone of mice with knocked-out slug in tumor-associated vamcs (fig. 6, 7, suppl. fig. 8, 9). figure 6. gfp+ cells expressing slug and/or pdgfrβ were significantly reduced in mice injected with lenti-slug-ko. a. merged pictures showing immunofluorescence of gbm cells (red), vamcs (green), pdgfrβ+ (magenta) and slug+ cells in the tumor core, transition and infiltration zone (one picture is exemplary shown; 25x magnification, bars: 50 µm). b. immunofluorescence of slug (blue), pdgfrβ (magenta) and gfp+ vamcs (green). the merged pictures (right) also show gbm cells (red). one picture each is exemplary shown, 63x magnification, bars: 10 µm. c-f. quantification of gfp+ mural cells (c) slug+ (d), pdgfrβ+ (e) and slug+/pdgfrβ+ double positive cells (f). n=3 mice per group and 24-70 slices per group have been used for quantification (sd, t-test, **** p<0.0001). figure 7. gfp+ vamcs expressing αsma and/or pdgfrβ were significantly reduced in mice injected with lenti-slug-ko. a. merged pictures showing immunofluorescence of gbm cells (red), vamcs (green), pdgfrβ+ (magenta) and αsma+ cells in the tumor core, transition and infiltration zone (one picture is exemplary shown; 25x magnification, bars: 50 µm). b. immunofluorescence of αsma (blue), pdgfrβ (magenta) and gfp+ mural cells (green). the merged pictures (right) also show gbm cells (red). one picture each is exemplary shown, 63x magnification, bars: 10 µm. c. quantification of αsma+ cells. d. quantification of αsma +/pdgfrβ+ double positive cells (n=3 mice per group and 24-70 slices per group have been used for quantification; sd, t-test, **** p<0.0001). this indicates that the activation of vamcs (identified by the expression of pdgfrβ and/or αsma) in the tumor core is provoked by tgf-β released from gl261 cells and the subsequent induction of slug expression in vamcs located adjacent to gbm cells. furthermore, vessel density was also significantly decreased due to the slug knockout in all regions of the tumor, although it did not reach the baseline levels of ec density observed in non-tumor-bearing brain regions. (fig. 8). figure 8. cd31+ ecs were significantly reduced in mice injected with lenti-slug-ko. a. merged pictures showing immunofluorescence of gbm cells (red), vamcs (green) and cd31+ ecs (magenta) in the tumor core, transition and infiltration zone (one picture is exemplary shown; 25x magnification, bars: 50 µm). b. immunofluorescence of gbm cells (red), gfp+ vamcs (green) and cd31+ ecs (magenta) and merged pictures. one picture each is exemplary shown (63x magnification, bars: 10 µm). c. detection of gfp+ mural cells (green) and cd31 positive cells (magenta) in the normal mouse brain (bars: 100 µm). d. quantification of cd31 positive cells in the tumor area of lenti-v2 and lenti-slug-ko injected mice as well as in the contralateral hemisphere (normal brain; n=2 mice per group, 24-48 slices/group, sd, t-test, **** p<0.0001). knocking out tgf-β in gbm cells or slug in vamcs attenuates pathological alterations of the intratumoral vasculature vessels of par cell derived gbms clearly displayed abundant structural alterations consisting of vascular tangles or glomeroid-like structures. they exhibited a more interconnected, "net-like" appearance and were positioned in closer proximity to each other compared to the vessels found in tgf-β-ko tumors or when slug induction in vamcs was inhibited by lenti-slug-ko injection. the vasculature in par cell derived gbms not only showed a higher vessel density, but also greater variations. vessels in tgf-β-ko tumors as well as in those tumors where slug was knocked out in vamcs exhibited less pronounced structural vessel abnormalities and a reduced number of these cells. however, the intratumoral vessels were still chaotically organized compared to the vascular structure of the brain in the contralateral, tumor-free hemisphere of the same animal (suppl. fig. 10). for further detailed analysis, we conducted tissue clearing of the brain of one mouse per experimental group and performed 3d reconstruction of the tumor vasculature. the 3d vessel reconstruction strikingly shows that mouse brains bearing par tumor not only displayed a high vessel density but also several structural alterations such as vascular tangles or glomeroid-like structures instead of the straight vessel course seen in the contralateral brain hemisphere. we further observed that this altered and chaotic vessel structure was clearly attenuated after knocking out tgf-β in tumor cells or slug in vamcs (fig. 9; suppl. fig. 11). figure 9: three-dimensional reconstruction of tumor-associated vessels indicates a normalized vessel structure in mice harboring tgf-β-ko gbms as well as in mice with par tumors that received lenti-slug-ko. 3d reconstruction after image improvement using imaris. ecs (cd31+, green) were shown within the tumor region in rgs5-gfp reporter mice bearing either parental gl261 derived gbms (red) without further treatment (par, upper panel), of an animal bearing a tgf-β-ko gbm (tgf-β-ko, middle panel), and of a mouse bearing a par tumor and that have been intrastriatally injected with lenti-slug-ko (slug-ko, lower panels). black/white pictures (right side) showing vascular alterations (triangles exemplary show glomeroid-like vessel structures and arrows normal branches). all images are taken at 20x magnification (n=1 mouse per group; scale bars = 50 µm). discussion gbm is a highly vascularized tumor showing altered vascular morphology with chaotically organized vessels, for which anti-angiogenic therapies were considered as promising therapeutic option. however, until now these therapies lack a remarkable increase in patient overall survival [33, 34]. rather, vascular remodeling induced by anti-vegf treatment results in hypoxia, is associated with excessive tumor growth with simultaneous insufficient vascularization after an initial transient vascular "normalization", but finally favoring invasiveness [35]. for a long time, it has been thought that irregular vessel development in tumors depends on the activation of ecs resulting in proliferation, migration and chaotic vascular structures. more recently, increased attention has been paid to vessel-covering pericytes and their interaction with ecs during vessel formation processes in tumors, including gbm. brain pericytes are known to be crucial for angiogenesis, they regulate perfusion and maintain vessel structure as well as bbb integrity [36-38]. in addition, they can facilitate cerebral inflammatory processes, control the interaction between neurons and vascular cells to assist the energy demand of the brain and are an inherent part of the neurovascular unit [39]. in the healthy brain pericyte coverage is heterogeneous and dependent on the cell´s subtype. in humans, brain pericytes are classified into two subtypes with either elevated expression of transmembrane transporters (t pericytes) or of extracellular matrix (ecm) regulation genes (m pericytes). in mice, studies found two major subtypes of pericytes which either express αsma (ensheathing pericytes) or not (mesh pericytes), the latter ones mainly located on small capillaries [40, 41]. however, within gbm, the tumor vasculature displays structural and functional abnormalities with increased and irregular pericyte coverage, that is associated with worse prognosis and accelerated tumor recurrence [42]. in our study, after knocking out tgf-β in gbm cells or slug in vamcs, we saw a significant reduction of the total number of gfp+ (fig. 3 and 6) as well as of αsma+ cells (fig. 4 and 7). as in our mouse model, we cannot distinguish between ensheathing and mesh pericytes, we therefore cannot clarify to the end whether lesser numbers of the one or other subtype of pericytes were observed. one further limitation of the rgs5-gfp mouse model we used in our study is that we cannot clearly distinguish between pericytes and vascular smooth muscle cells (vsmc) as also vsmcs express rgs5 and might therefore be positive for gfp [41]. in gbm, there is a pathogenic crosstalk between tumor cells and pericytes. gbm cells modify the contractile activity of pericytes, resulting in the cooption of existing blood vessels, by this supporting the expansion of the tumor [43]. in addition, glioma vessel associated pericytes support tumor growth by the induction of an immunosuppressive microenvironment [5]. due to these tumor promoting function of pericytes, these cells have been recently identified as novel targets for the treatment of gbm [44]. in our recent studies we found that vamcs express emt-transcription factors like slug, indicating that, induced by gbm cell secreted tgf-β, these cells undergo an emt-like "activation" process [15]. this observation was underpinned by our data that in vitro tgf-β treatment of human primary microvascular pericytes induced proliferation, cell motility and morphological changes [16] and mitigates the integrity to the bbb [17]. in our study, we were interested whether this emt-like "activation" of glioma-associated vamcs might also be associated with vascular alterations in vivo. in a syngeneic mouse gbm model that allows to track both gbm cells as well as rgs5 positive vamcs, we prevented the induction of this emt -like activation by knocking out tgf-β in the tumor cells (suppl. fig. 1) on the one hand, or by knocking out slug in tumor adjacent, rgs5 expressing vamcs (suppl. fig. 7) on the other. as tumor angiogenesis is correlated to tumor size and growth, we firstly determined the growth rate of par and tgf-β-ko cells and tumors to avoid artifacts generated by different sizes of par and tgf-β-ko gbms. after knocking out tgf-β in gl261 cells these cells showed a diminished proliferation rate and cell motility in vitro (fig. 1), and also a delayed tumor growth in vivo. however, by using mri we determined the time point the tumors reached a size of 1.5 to 3 mm in diameter, and therefore material for analyses was collected when tumors were in that range of size, which was also retrospectively validated in the collected material (suppl. fig. 3). nevertheless, this might be a limitation of this study because even if we analyzed the vascular structure of gbms when par and tgf-β-ko tumor reached the same size, we cannot completely exclude that a delayed tumor growth also influences its vascularity. for the slug-ko in glioma-associated vamcs and to avoid artifacts that might be induced by knocking out slug in other cell types within the tumor area, we examined the expression of slug in the tumor area of par gbms and found it to be colocalized invariably with gfp, but not with the tumor cells. in contrast, slug was absent in gfp+ vamcs in those mice we injected with lenti-slug-ko (suppl. fig. 7). however, even if cas9 expression in lenti-slug-ko was driven by the rgs5 minimal promoter, which significantly induced luciferase activity in mbvps, but not in gl261 gbm cells (fig. 2) we cannot completely exclude that minimal slug levels (below the immunofluorescence detection limit) in other cell types will be also reduced by intrastriatally applied lenti-slug-ko. previous in vitro data showed an upregulation of αsma and pdgfrβ in hbvps in response to tgf-β or conditioned medium from gbm cells in vitro as well as a correlation of αsma and pdgfrβ with slug in human gbms [15, 16]. in accordance with those findings, strong signals of αsma and pdgfrβ have been detected in mice brains bearing tgf-β secreting par tumors (figs. 3, 4, 6, 7). during fibrosis, αsma and pdgfrβ are considered as pericyte activation markers, and in renal carcinoma, perivascular αsma and pdgfrβ expression is correlated with poorer survival [45, 46]. this suggests that in vascularized high-grade glioma, glioma-associated vamcs are pathologically activated. in parallel with the high levels of pdgfrβ and αsma we observed in tgf-β secreting gbms, we detected slug that was nearly virtually invisible in tgf-β-ko gbms (fig. 3, suppl. fig. 5). after knocking out tgf-β in the tumor cells, or slug in glioma-associated vamcs, both pdgfrβ and αsma positive cells were markedly and significantly reduced. in accordance with our previous in vitro data [16], this observation reinforces our assumption, that also in vivo in gbms the induction of a slug dependent emt-like program especially in glioma-associated vamcs is conveyed by gbm cell secreted tgf-β. nevertheless, apart from gbm cells, several other cell types in the cns have been shown to secrete tgf-β, including ecs and microglial cells, which could also stimulate vamcs to a certain amount [47]. moreover, additional influence on this stimulation mediated by different cytokines cannot be excluded. particularly the hypoxic microenvironment in gbm is known to be a potent inducer of emt and morphological alterations [48]. until now it remains unclear whether vegf signaling also influences pericytes or other mural cells. it is known that vegf primarily signals proliferation, survival and migration. however, a via the vegf receptor (vegfr)-2 leading to ec vegf-mediated activation of vegfr-1 has recently been shown to be not only involved in the recruitment of mural cells which is required for maturation and stabilization of neo-vessels, but also to affect vascular permeability [49, 50]. as pericytes are known to express vegfr-1 particularly under hypoxic conditions [51], vegf-mediated changes of pericytes towards a more mesenchymal phenotype enabling their recruitment might be considered. by preventing the emt mediated "activation" of glioma-associated vamcs, we observed a significant reduction of gfp+ cells in all tumor areas (fig. 3, 6, suppl. fig. 5). in this regard, the source of pericytes on gbm vessels remains controversial as it has been postulated that pericytes might also originate from glioma stem like cells [52]. it has been published that gl261 cells grown as neurospheres under reduced fcs concentrations contain a population of cells with stem cell characteristics [53]. to avoid stemness, in our study gl216 cells were grown in 10% fcs before implantation. however, we cannot exclude that in vivo in the growing tumor, stem cell characteristics were induced in the tumor cells. as gl261 cells do not express gfp and therefore gl261 derived pericytes cannot be detected by our approach, we cannot exclude that a small population of gl261 with stem cell characteristics might develop into pericytes. however, we did not observe any cells that are double positive of mcherry and a mural cell marker such as αsma or, in tgf-β-secreting par tumors, positive for slug (fig. 3 and 4). besides, it has been published that in gl261 tumor bearing mice the vast majority of pericytes within gbms are definitely endogenous, host-derived cells originating from either mesenchymal or neural crest cells [54]. hence, we believe that our mouse gbm model is suitable to investigate the tgf-β/slug mediated modulation of glioma-associated vamcs in vivo. vascularization not only involves ec proliferation, migration, branching and anastomosis, it also requires adequate pericyte coverage of vascular sprouts for vessel stabilization and maturation (for review see [55]). at least in gl261 tumors, the knockout of tgf-β or the prevention of a slug-mediated emt-like activation of vamcs in the tumor environment, reduces the number of gfp+ cell numbers as well as that of cd31+ cells, suggesting that not only ecs, but also vamcs are main cells that constitute the multiplicity and altered morphology of tumor microvessels. physiologically, pericytes constitute a monolayer surrounding ecs (for review see [56]). however, even if the vessel coverage with gfp+ vamcs in the tumor area was heterogeneous and areas with multilayers of overlapping gfp+ cells can be distinguished from vessels that display almost no coverage, the irregular covering correlated significantly with elevated slug, pdgfrβ and αsma levels. this underpins that the shift towards a more mesenchymal phenotype of glioma-associated vamcs and their subsequent activation might contribute to the development of vascular alterations in gbm. unlike the tight association of gfp+ vamcs and ecs that can be observed in normal vessels, cells with elevated pdgfrβ and αsma levels display an abnormal separation, but also multi-layered coverage of vessels (suppl. fig. 10). as the direct contact and communication between pericytes and ecs are critical for maintenance of cerebrovascular stability and blood-brain-barrier function, the observed loose association of gfp+ cells to tumor vessels might result in vascular dysfunction. aberrations in pericyte-ec signaling are already known to contribute to tumor angiogenesis [57, 58]. apart from abnormal pericyte coverage, tumor vessels displayed various structural abnormalities. the observed high amount of "emt-activated-state" gfp+ glioma-associated vamcs in par tumors was paralleled by an increased vascular density and vascular abnormalities which suggests an important role of activated pericytes in glioma-associated neoangiogenic processes. tumor blood vessels displayed inconsistent diameter and uneven shape with abnormal bulges, blind ends and glomeroid-like structures (fig. 5, 8, 9, suppl. fig. 10, 11). in gbms, structural and functional abnormalities of peritumoral vessels are often associated with an inconsistent, disrupted bbb that may constitute a main hurdle for targeting gbm by limiting adequate drug delivery. pericytes, being important contributors to the establishment and maintenance of bbb integrity by regulating tight and adherens junctions as well as transcytosis across endothelial cells [59], can be functionally modified by gbm cells [60]. via the tgf-β-mediated induction of emt-factors in vamcs, gbm cells are able to induce proliferation and cell motility [15, 16] and notably change the metabolic behavior of pericytes. schumacher et al. demonstrated that tgf-β treatment of hbvps results in bbb disruption at least in vitro. additional metabolomic and transcriptomic analyses underscored that the tgf-β-mediated functional and metabolic changes in pericytes are closely connected with their role during angiogenic processes [17]. in combination with the lower mural cell activation state and the less chaotic vessel structure, we observed after preventing the induction of slug expression in glioma associated vamcs in vivo, our findings suggest that the bbb integrity might also be affected. however, this has to be further evaluated for example by measuring the uptake of contrast agents using small animal mri. conclusions induced by gbm cell secreted tgf-β, microvascular brain pericytes undergo an emt-like "activation" process, indicated by an induction of the emt-associated expressional regulator slug. in vitro, this phenomenon is associated with elevated proliferation, cell motility and morphological changes. in our study, we demonstrate that an emt-like "activation" of glioma-associated, rgs5 expressing vamcs is also associated with vascular malformation. both the knockout of tgf-β in glioma cells and the prevention of slug-induction in glioma-associated vamcs mitigated the amount of activated pericytes or vamcs in the tumor core, as shown by a reduced expression of activation markers such as pdgfrβ or αsma. in addition, the prevention of this activation diminished the total amount of gfp+ vamcs covering the tumor vasculature and also, at least partially, led to a restoration of the chaotic vessel structure towards a more normal vascular morphology. in summary, our findings suggest that the induction of an emt-like program in glioma-associated vamcs is the prominent cause of vessel abnormalities frequently observed in gbm. acknowledgment we would like to acknowledge dr. olga oleksiuk of the hih-cin imaging cluster for her support in all microscopy and microanalysis experiments. we also like to thank ulrich mattheus for his support in the clarity experiments. michel mittelbronn would like to thank the luxembourg national research fond (fnr) for the support (fnr pearl p16/bm/11192868 grant). authors' contributions un has designed and supervised the study, has full access to all data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. un and lm wrote the manuscript. mm performed critical review of the manuscript. lm, kr, he, me, aea, mm, jjg and mk performed experiments and analyzed data. institutional review board statement all research meets ethical guidelines and adheres to the legal requirements of the study country. animal work was performed in accordance with the german animal welfare act and its guidelines and was approved by the regional council of tübingen (approval n7/17). data availability statement the datasets and material generated and/or analyzed during the current study are available on demand. conflict of interest the authors declare no conflicts of interest. all coauthors have reviewed and approved the contents of the manuscript and that the 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unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. title feel free to add comments by clicking these icons on the sidebar free neuropathology 4:11 (2023) obituary vale emeritus professor byron a kakulas ao, 1932 – 2023 clive harper1, colin l masters2 neuropathology, dept. of health sciences, charles perkins centre, university of sydney, australia florey institute and the university of melbourne, australia corresponding author: colin l masters md · laureate professor of dementia research · florey institute and the university of melbourne, australia c.masters@unimelb.edu.au submitted: 17 may 2023 accepted: 17 may 2023 published: 22 june 2023 https://doi.org/10.17879/freeneuropathology-2023-4820 keywords: obituary, neuropathology australia, inclusion body myositis, muscular dystrophy vale emeritus professor byron a kakulas ao, 1932 2023. it is with great sadness that we reflect on the recent passing of our friend and colleague, emeritus professor byron kakulas ao. byron kakulas was born on march 29, 1932 in perth, western australia. like many west australians seeking to join the medical profession at that time, he completed his of bachelor of medicine, bachelor of science at the university of adelaide in 1956. he commenced his medical career as a resident medical officer at royal perth hospital in 1957 and went on to complete his specialist clinical neurology training in 1963. byron married valerie patsoyannis in 1961 before they moved to boston. they have three children: arthur phillip, felice anne and carolyn rose. professor kakulas' move to boston was to continue his training in neurology/neuropathology at the massachusetts general hospital, harvard medical school (1963-1965). he worked closely with professors raymond d adams and e pearson richardson. they remained friends and colleagues for a lifetime and visited perth on several occasions. living in perth, byron had the opportunity of visiting the lovely island of rottnest, famous for its small marsupial, the rottnest island quokka. he observed that many of these cute little animals developed a paralytic disease when placed in captivity. he decided to make this the subject of his doctoral thesis and found that the disorder was due to breakdown of muscle resulting from vitamin e deficiency. by treating this deficiency in the quokkas, he showed for the first time that skeletal muscle had the ability to regenerate. this was a momentous breakthrough since it demonstrated the potential for some muscle diseases including muscular dystrophy to be curable. byron communicated his studies very effectively to the wider community, and became well known on television with the annual fundraising activities of telethon. this original work eventually led to a treatment for sufferers of duchenne muscular dystrophy, which received accelerated approval by the fda in 2016. his research contributions were very extensive covering a variety of fields especially childhood and adult muscle disease. he showed that paralysis in muscular dystrophy was the result of continuous cycles of necrosis and regeneration. he was the first to identify inclusion body myositis as an entity. in the early 1990s, he introduced molecular genetics and dna technology in the investigation of muscle diseases. other highlights were the development of in vitro models for polymyositis (in collaboration with roger l dawkins) and the pathology of kuru and experimentally transmitted prion infections (in collaboration with carleton d gajdusek and michael p alpers). the neuropathology of spinal cord injuries was a major lifelong interest, for which his pioneer work gained much international acclaim, setting the scientific basis for better treatments. in 1967, he founded the muscular dystrophy association of wa and, soon after, the australian neuromuscular research institute (anri) becoming the medical director of both. he held these posts until 2010. in 2017, the anri became the perron institute for neurological and translational science. professor kakulas was appointed as head of the department of neuropathology at the royal perth hospital in 1967. he was made the foundation professor of neuropathology at the university of western australia in 1971 and was dean of the faculty of medicine from 1976-1978. clive harper (emeritus professor of neuropathology, university of sydney) recalls that he met byron at the international society of neuropathology in budapest in 1974. byron asked clive what he planned to do after working in switzerland in neuropathology. byron said, "i can offer you the position of assistant neuropathologist in perth". clive flew into perth from switzerland in 1975. he started work at the royal perth hospital immediately. byron had set up the biggest and best neuropathology department in australia. it was big, even by international standards. clive recalls: "there were excellent facilities and large numbers of secretarial and technical staff. there were two neuropathology registrars and several overseas trainees. the registrars, peter blumbergs and tony tannenberg and myself worked closely together for several years until they took up senior neuropathology positions in adelaide and brisbane, respectively". all three have remained good friends to this day. colin l masters started with byron's research team in 1967 when, as a medical student, he helped with the analysis of the brains of prion-inoculated non-human primates sent to perth by carleton gadjusek. he continued postgraduate studies in neuro-virology in byron's department. colin later took up the position of professor of pathology at the university of melbourne in 1989. clive worked with byron until 1985 when he was invited to take up the foundation chair in neuropathology at the university of sydney and royal prince alfred hospital. hence, byron was responsible for the training of most of the senior neuropathology staff in queensland, nsw, victoria and south australia. the royal perth hospital department of neuropathology was responsible for most of the forensic and hospital neuropathology studies and, each week there were reviews of cases in the laboratory. these were attended by many of the hospital clinicians and were considered by trainees and senior staff as great learning experiences. professor kakulas held many important positions in a range of scientific, medical and other societies. these included membership of the science committee for the international spinal research trust (1983). he was a fellow of the royal australasian college of physicians, the royal college pathologists australia and the royal college of pathologists (united kingdom). he was a founding member of the australian and new zealand society for neuropathology and president from 1985 to 1988. professor kakulas played an important role in many national and international neuroscience societies and conferences. he was on the executive committee of the world federation of neurology and was vice president for the international congress in muscle disease in 1971, 1974 and 1978. byron was vice president for the fifth international congress on neuromuscular diseases in 1982. he was on the board of management at sir charles gairdner hospital from 1977-1980. byron was president of the australian brain foundation from 1981-1985. professor kakulas received many honours and awards including officer of the order of australia, an honorary doctorate of the university of athens, the gaetano conte prize of the naples conte academy and a lifetime achievement award by the world federation of neurology. he was also a paul harris fellow in rotary. written by prof clive harper & prof colin l masters on behalf of anzsnp. copyright: © 2023 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. memoirs of a neuropathologist who was unfit to be a clinician feel free to add comments by clicking these icons on the sidebar free neuropathology 4:7 (2023) reflections memoirs of a neuropathologist who was unfit to be a clinician hitoshi takahashi professor emeritus (neuropathology), niigata university, japan corresponding author: hitoshi takahashi · niigata university · brain research institute · department of pathology · 1-757 asahimachi · chuo-ku · niigata 951-8585 · japan hitoshi@bri.niigata-u.ac.jp additional resources and electronic supplementary material: supplementary material submitted: 27 march 2023 accepted: 28 march 2023 copyedited by: couger jimenez jaramillo published: 19 april 2023 https://doi.org/10.17879/freeneuropathology-2023-4741 keywords: neuropathology, brain research institute niigata, reflections, autobiography i am very honored to be invited as a contributor to the “reflections” series for free neuropathology. as my english speaking and writing skills are limited, i was initially very hesitant about documenting “my life and my neuropathology career” in english, but i feel it would be a worthwhile task to write about my past without flinching. in undertaking this task, perhaps my main starting point is that i was born into a family completely unrelated to medicine. origin and childhood i was born into a farming family in sendai, miyagi prefecture, japan, in june 1952. the treaty of san francisco came into force in april of that year. my father served in southeast asia during world war ii (wwii). after returning to his family home, he found a national civil service job nearby and took care of his parents. he acknowledged japan’s responsibility for wwii but did not say much about his wartime experience. my mother, who was also a farmer’s daughter, had experienced the sight of b29 bombers flying over, heading for downtown sendai when working on their farm. according to my mother, when i was little, i could not stop for a while once i started crying, but did so as soon as i was given a little apple. since i was a very active and restless baby, my mother left me with my grandmother and went to work on their farm. there was a kindergarten about 600 m from my house on the other side of the railroad crossing. at the age of 5, i went to the kindergarten and got an application for admission by myself, surprising my parents. i remember quite a lot of the year i spent at the kindergarten to which i walked. there were not many cars in those days. i grew up well and healthy. at the age of 7, i enrolled in elementary school. new children were obligated to undergo a chest x-ray examination in accordance with the school health act, and this revealed a small shadow on my right lung. an additional gastric juice test for mycobacterium tuberculosis was negative; this test was not as painful for me as it looked. eventually, however, i was diagnosed with asymptomatic primary lung tuberculosis and received penicillin injections and oral isonicotinic acid hydrazide (inh) for about one year. a school nurse told me that i should always avoid strenuous exercise. another piece of advice that stuck me at the time was not to touch the handrails of the stairs unnecessarily. thereafter, my annual chest x-ray examination revealed no changes in the small lung shadow, and the gastric juice test also remained negative. finally, the lung lesion was interpreted as an old inflammatory lesion. throughout my elementary school days, there was no distinction between myself and my classmates in terms of performance in physical education class; i was the fastest runner in my class. my mother had said from the beginning that the small lung lesion was the result of a time i had fallen onto a dirt floor, hitting my chest. in my 5th and 6th grades, i enjoyed playing baseball every sunday as a member of a local children’ baseball team called the falcons. one day, a gym teacher asked me whether i knew that fédération internationale de football association (fifa) had more members than the united nations (un). i underwent two minor surgeries, appendectomy at the age of 9 (fig. 1) and inguinal hernia repair at the age of 12. lastly, according to a friend who is a pulmonologist, my mother’s explanation of my lung lesion could not be ruled out, although it no longer matters. figure 1: my mother and i in a patient bedroom in a surgical clinic (1961). education in my junior high school days, i maintained excellent grades. i liked mathematics and english. my father told me that when he was young, he had learned english from a crystal radio set. however, english language education, which attached importance to grammar, was simply a subject to prepare for high school and college/university entrance examinations. influenced by my gym teacher’s story about fifa, i began to play football as an extracurricular activity. however, my school’s football team was very weak, and proved unable to score even a single goal in the several games we played. in 1968, i passed the entrance examination for high school (3-year course). the high school that i attended was a public school for boys, and one of the leading preparatory schools in the prefecture. i believe at that time that there were no coeducational high schools in the prefecture. the monthly tuition fee was about 3 us dollars. at my high school, all of the teachers were male – if a few female teachers were present, they escape my memory – and really unique individually. a japanese teacher told us bluntly during class that he had no responsibility for our willingness to learn, or for our future. i fondly recall the school motto that was expressed by two lines of four “kanji” characters. although i cannot explain in detail the meaning in english, it made me imagine the way of the scholar rather than that of the samurai. the following year, inspired by the university student movements that had started the previous year ago at the university of tokyo, my high school began a movement for abolition of school uniforms and caps. many students, including myself, joined the abolition side of the movement. debates were held between the students and the teachers several times in the gym, and finally we achieved our goal. a mathematics teacher, who was in charge of my class in the 1st and 3rd grades, was on the “conservative” side. i was able to eventually understand his viewpoint; he thought of the students’ parents, who had to worry about what kind of clothes their children should wear. in fact, my mother was a case in point. in the middle of the 3rd grade, my homeroom teacher began worrying about whether i would be able to pass the entrance examination for so-called elite universities. apparently, i entered the high school with excellent grades. however, even at that point, i was still undecided about my future as the oldest son of a farmer. or, rather, i began to think about taking a year off from school to study in a preparatory school to enter the university and/or faculty that i wished to attend. in our boys’ high school, there was an attitude that “3 (play well for 3 years) plus 1 (study well for 1 year) equals 4 (a 4-year course high school)”. i liked mathematics, english and world history in particular. however, i have no memory of studying any of those subjects hard during my 3 years. on the other hand, i was a good forward on my school’s football team and enjoyed the games a lot; the football team was really strong and ranked among the top 3 in the prefecture (fig. 2). figure 2: my high school football team (spring 1970). front row center: ht with the trophy. shortly after graduation, my father told me that i had often received care from doctors as a child, and suggested that i might like to become a doctor. i readily seized upon his suggestion without any great thought, but on the condition that i was able to enjoy my university life away from home; i considered that eventually i would have to take care of my parents at home. therefore, i decided to spend a year preparing for my entrance examinations in a new environment, tokyo, where my aunt lived. medical school – studies and encounters in april 1972, i enrolled at niigata university school of medicine. at that time, the tuition fee for one year was about 135 us dollars at all national universities, without any difference between the faculties. the japanese medical education system was quite different from that of the us, consisting of two courses: 2 years for the medical preparatory course and 4 years for the medical (2-year basic and 2-year clinical) course. in the former, i studied physics, chemistry, biology and other subjects. although german was a compulsory subject, i was convinced that it would be english from now on in the field of medicine. i finished the 2-year course in 3 years, ending simply in earning enough credits to move on. i joined a football club at medical school, but quit in less than a year because it felt like a small, limited group. i started the 2-year basic course in 1975 and developed a great interest in morphology, including anatomy and pathology. for neuroanatomy, i finished reading a textbook written in english, although it was not thick and had many illustrations. about a year later, i read a book entitled “the cancer story” (iwanami paperback, 1st edn. 1965) written in japanese by dr. j. kamahora, in which i learned about dr. fusahiro ikuta’s research related to the development of cancer (j neuropathol exp neurol 1965; 24: 225-43). surprisingly, dr. ikuta was working at the brain research institute (bri), niigata university, where he was professor of pathology. from october to november 1976, there were seven neuropathology classes consisting of lectures and practice (gross and microscopic observation). i still remember the first time that i saw him standing in front of the blackboard in a white lab coat. my neuropathology report, his evaluation sheet with an a-rating and his comment “i wish you good luck” are still treasured. they are the only things remaining from my medical school days. in april 1977, my 2-year clinical course started with practical training. i had been interested in neurosurgery and neurology, but as the days went by i began feeling myself unfit to be a clinician. during my last summer vacation at home as a medical student, my mother told me that i had been originally timid and was perhaps unsuited to be a doctor involved in life and death. i replied to her, “certainly.” although most people might take this as a joke, my mother told me seriously, “you should be a dentist.” figure 3: dr. fusahiro ikuta in his room (august 13, 1976). he became a professor in 1973. ht took over this room in september 1995. several months before graduation, i visited dr. ikuta at his office in the department of pathology, bri (fig. 3), and consulted him about my future. i asked him whether i could make a living as a resident of neuropathology. he answered with a smile that i could make a salary as a pathology resident in the university hospital of the school of medicine doing locums in local clinics once a week. in addition, he kindly showed me around the department of pathology in the bri. less than a day later, i decided to become a resident under dr. ikuta. my parents had no objection, but asked me what neuropathology was. i graduated from medical school in march 1979, and obtained a medical license at the end of may (all medical license holders are equivalent to md in japan). neuropathology – a choice without hesitation i selected dr. ikuta as my teacher and neuropathology as job from which i would make a living. in may 1979, i joined the department of pathology at bri. the bri is an independent organization/facility within niigata university. the following month, i became employed as a resident in the pathology and laboratory medicine section of the university hospital. i was trained in dr. ikuta’s lab at the department of pathology, where he was a professor and also chair. i also worked in a clinic located in the nearby city every saturday (a kind of moonlighting) as an assistant to dr. s. nakashima (neurologist), my senior in dr. ikuta’s lab. thus, even though the income was low, i became financially independent, and i was happy with that. my residency training was basically on-the-job training in autopsy, brain cutting, biopsy (surgical neuropathology), and histological observation and diagnosis. i came to admire my three seniors, dr. e. ohama, dr. s. takeda and dr. nakashima, as mentors. case review meetings called neuropathology conferences (npcs) and brain cutting were held every thursday morning and afternoon, respectively. autopsy and biopsy case slides for histological observation were displayed one week in advance. both residents and graduate students indiscriminately learned diagnosis through autopsy and biopsy cases in the weekly npcs. clinicopathological conferences (cpcs) for autopsy cases were also held every month, which was challenging for us as there were many question and answer sessions with neurologists and neurosurgeons. i had a particular interest in the surgical neuropathology of central nervous system (cns) tumors. it was fun for me, like guessing in a quiz. in fact, my first autopsy patient provided me with a series of precious experiences, including brain cutting, gross observation, microscopic observation, diagnosis, presentation at npcs and cpcs with discussion, revision, report writing and evaluation. finally, the autopsy patient, a 9-year-old girl, was diagnosed with a brainstem ganglioglioma. a fortunate event about a year after becoming a resident, dr. ikuta told me that i should turn to something new in the form of electron microscope (em) observation. as if in response to his comment, i had already started em observation of the brainstem ganglioglioma mentioned above, and several months later i obtained many electron micrographs demonstrating interesting features of the constituent ganglion cells. i have to thank a technician, mr. s. egawa, for his guidance on the use of the em. early the following year, 1981, i registered an abstract of my ultrastructural study for the 22nd annual meeting of the japanese society of neuropathology (jsn) held in fukuoka in may of that year. not long after, dr. ikuta called me to his room, and suggested i study abroad in new york. initially, i had serious doubts as to whether i would be up to the task. he told me he had been asked by his old friend, dr. kinuko suzuki, to nominate a young research fellow to help her with em studies. she was professor of pathology (neuropathology) at albert einstein college of medicine (aecm) in the bronx, new york. i prepared a simple cv written in english. before the annual meeting of the jsn, i heard from dr. ikuta that i had been officially accepted as a post-doctoral research fellow in dr. suzuki’s lab. i was delighted and convinced again that english was the way to go from now on. in august 1981, i left for new york after a send off by many doctors and staff from our pathology department at niigata station. for some reason, dr. ikuta was not able to come; instead of him, mrs. ikuta came to see me off. neuropathology in new york within a few days of arriving in new york, i rented a two-bedroom apartment located on davenport avenue, new rochelle, near the atlantic coast under the guidance of dr. hitoshi nagara. i took over the role of dr. suzuki’s research fellow from dr. nagara, starting my 2-year period abroad. i first visited dr. suzuki with dr. nagara at the albert einstein rose f. kennedy center (director, dr. dominick p. purpura). there, i found that she and her husband, dr. kunihiko (“kuni”) suzuki, had adjoining labs on the same floor, and i met 3 (a little later, 3 became 4) japanese research fellows working under dr. kuni. i soon made friends with these japanese fellows, having conversation entirely in japanese. i believe that within a few days, dr. suzuki introduced me to her colleagues at the kennedy center, including dr. d.s. horoupian and dr. c.s. raine. for a while, people called me “hitoshi number 2”. dr. suzuki showed me “twitcher” mice, which were used as a murine model of human krabbe disease (globoid cell leukodystrophy), an autosomal dominant neurological disorder, and roughly explained the future research plan. looking back on those days, she gave me free access to the em facility (fig. 4). figure 4: ht dissecting a post-perfusion twitcher mouse with unfamiliar hands and dr. kinuko suzuki keeping her eyes anxiously fixed on the mouse (1981). in 1982, dr. suzuki took sabbatical leave in japan, and i was left in her lab with a technician, ms. grace yuk gong, for the first half of the year (fig. 5). during her absence, i spent almost every day except weekends in a dark room. after she returned to the lab, i began preparing my maiden paper in english in parallel with continued em studies. armed with an english-japanese dictionary, i wrote the manuscript on a typewriter. unlike present-day work using a personal computer, it was a difficult task for me. i truly recognized that there was a big difference between “i like english” and “i use english.” dr. suzuki submitted the paper after an intensive review of my repeated manuscript drafts, and it was finally accepted for publication. that night, i toasted alone with a sense of accomplishment in my apartment room. figure 5: neuropathology faculty, residents and fellows at albert einstein college of medicine; note the absence of dr. k. suzuki due to sabbatical leave in japan (may 12, 1982). front row, left to right: dr. shu-hui yen, dr. david katz, dr. robert d. terry, dr. ute traugott, dr. dikran s. horoupian, yvonne kress and dr. felicia gaskin. top row, left to right: dr. peter davies, ht, dr. james e. goldman, dr. anne b. johnson, dr. david armstrong, dr. cedric s. rain, dr. celia f. brosnan, dr. gary l. wenk and dr. peter pick. in september of that year, i attended the 9th international congress of neuropathology (icn) held in vienna, and gave an oral presentation: “an ultrastructural study of oligodendrocytes in the twitcher mouse”. dr. ikuta and his colleagues/students also attended the icn from niigata, japan. my senior dr. takeda also gave an oral presentation about “pigmented and non-pigmented neurons in the substantia nigra in parkinson’s disease (pd)”. one participant raised his hand, gave his name, and asked dr. takeda a question. it was at that moment i saw the face of dr. kurt a. jellinger, editor-in-chief of acta neuropathologica, for the first time. in the symposium on “brain edema”, dr. ikuta gave an excellent presentation about the mechanism of lesion repair in brain ischemia using time-lapse images to show the movement of cultured (reactive) astrocytes. i witnessed all of the participants give him a standing ovation at the venue. after the icn, i joined dr. ikuta’s group for a week-long trip around athens (fig. 6), rome, zurich, paris and london. finishing the trip, dr. ikuta’s group and i flew back from london heathrow to narita and new york john f. kennedy, respectively. the round trip was a kind vacation gift from dr. suzuki, who had also attended the icn. back in new york, i began preparing a paper on the topic i presented at the icn. the paper was accepted for publication through the same process as the previous one. figure 6: with the parthenon, athens in the background (after the 9th icn, vienna, september 12, 1982). front row, left to right: dr. kiyomitsu oyanagi, shigekimi egawa, dr. takao makifuchi, dr. yo oyake and dr. kazunori yamazaki. top row, left to right: dr. yasuji yoshida, ht, mrs. yoshiko ikuta, dr. fusahiro ikuta, dr. shigeru nakashima, tomio ichikawa, dr. eisaku ohama and dr. shigeki takeda. in dr. suzuki’s lab at the aecm, i wrote 5 papers as first author under the direction of dr. suzuki. i knew nothing about biochemistry/neurochemistry. i was very surprised to see dr. hideki igisu, a research fellow in dr. kuni’s lab, grinding the brains of twitcher mice using a homogenizer. however, when i asked him about the morphology of oligodendrocytes, i found that he knew almost nothing about that, either. i was relieved that we shared a similar ignorance of certain areas. from this viewpoint, dr. kuni treated me as the second author for a biochemical paper written by dr. igisu on the twitcher mouse. this allowed me to study a little about biochemistry/neurochemistry. figure 7: dr. k. suzuki at ht’s farewell party (april 1983). on returning to niigata at the end of april 1983, i remembered what dr. suzuki had said to me (fig. 7; also see fig. 5 in j neuropathol exp neurol 2014; 73: 175-87): “you do not necessarily need scientific papers if you are going to be a good clinician, but you need such papers if you want to be an academician”. i also made many good friends. however, i seldom met non-japanese foreign friends again, even at subsequent academic meetings. memories of the albert einstein rose f. kennedy center in addition to dr. suzuki, two neuropathologists were working at the albert einstein rose f. kennedy center for research in mental retardation and human development. one was dr. horoupian who was in charge of the clinical neuropathology service. the other was dr. raine, who was a researcher studying demyelinating diseases, especially multiple sclerosis (ms). fortunately, i was allowed to participate in the case review meetings held every monday morning at the rose f. kennedy center led by dr. horoupian. on occasion, he would gently ask a resident who was presenting a case, “why don’t you ··········?” i thought that such a remark beginning with “why” to each of the residents was one aspect of his cordial educational policies. dr. j.e. goldman at the forchheimer building was a regular member at the weekly meeting. he was a man of few words and always had a rather serious look on his face. i remember that when i passed him in the corridor after my oral presentation at the icn (vienna, 1982), he smiled at me and said, “nice talk.” dr. r.d. terry (department of pathology, chair) at the forchheimer building sometimes also attended the weekly meeting. one day, when a resident was showing a slide of a brain slice from an autopsy case, the diagnosis of which i do not remember exactly, possibly ms, he pointed to the lateral part of the ventricle on the slide and said, “do you know what here is called in german?” after a moment of silence, i answered, “wetterwinkel.” he seemed surprised by my answer. i speculated that dr. terry’s “wetterwinkel” came from his teacher, dr. harry m. zimmerman, a pioneer in neuropathology in the us as well as a founder of the aecm, who studied neuropathology under dr. walter spielmeyer in germany. dr. ikuta, dr. suzuki and dr. terry were all his pupils. while dr. suzuki was away, i was especially indebted to dr. raine for the use of the em. i also learned much about oligodendrocytes from him. i already knew that he was very famous in the field of ms. it came as a slight surprise that he was never an arrogant person, but rather a cheerful and somewhat youthful individual, for which i admired him. i saw dr. asao hirano only once or twice at the weekly meetings. however, i had a few opportunities to visit montefiore hospital, the university hospital for the aecm, where i met many japanese research fellows, including dr. imaharu nakano, dr. yoshio hashizume and dr. toshihiko kubota. dr. hirano kindly took me to dr. zimmerman’s room and introduced me to him as dr. ikuta’s pupil. of course, dr. hirano was also a pupil of dr. zimmerman. there are still a large number of dr. zimmerman’s second-generation pupils in japan, including myself. lastly, dr. igisu and his wife often invited me to cheese fondue dinner with wine and to the offer to stay overnight. when i woke up one morning, he told me that i had been talking in english in my sleep. i thought that the reason was either i still liked english or i was already afraid of it. irrespective, i myself have no memory of “sleep talking”. based on my experience, i concluded that sleep talking in english is not necessarily the same as speaking english in a dream. return to niigata in may 1983, i returned to work as a resident in the university hospital, and again i worked not in the local clinic, but in the psychiatric hospital every saturday. although i was employed as a doctor, i worked as a kind of hospital helper, specifically as an interlocutor for patients, through the courtesy of the director (dr. masaharu tanaka, a former member of our pathology department). this gave me the opportunity to see many elderly patients with various forms of dementia such as alzheimer’s disease (ad). not long before i started research for a phd degree, dr. nakashima was trying to develop an antibody against tyrosine hydroxylase (th) for a study of pd using immunohistochemistry (ihc). i became involved in his next project to develop an antibody against serotonin. finally, using the two antibodies against th and serotonin and with his help and advice, i began to study the distribution pattern of monoamine neurons in the brainstem of the human fetus using specimens stored at the brain disease research center (bdrc), the so-called institutional brain bank founded by dr. ikuta in 1971. i presented my thesis and obtained a phd degree from niigata university in 1985 (fig. 8). later, i was very happy to learn that my two papers, one about catecholamine neurons written in japanese with english abstracts, had been cited in the book the human nervous system (george paxinos, ed. 1990, elsevier). figure 8: the distribution pattern of serotonin-containing neurons in the midbrain at the level of the caudal portion of the oculomotor nucleus (brain dev 1986; 8: 355-65). after a while, i realized that i had one important task remaining: i had promised myself to report the findings i had obtained from my neuropathology autopsy study of young girl in new york. when that paper, entitled “ultrastructural alterations of neuronal cells in a brain stem ganglioglioma”, was accepted for publication in 1987, i felt that i could finally thank her. later, i worked on an autopsy case of hereditary dentatorubral-pallidoluysian atrophy (drpla), a clinicopathological entity that had been established by dr. haruhiko naito and dr. shinsaku oyanagi at niigata university. this autopsy case was the second one in the same family with drpla, and the report on the two autopsy cases from different generations provided an opportunity to promote later collaboration between the departments of neurology and neuropathology at bri. i had also developed a great interest in progressive supranuclear palsy (psp) through the em work, which had revealed 15-nm-wide straight tubules in various regions of the brain. another aspect of great importance was learning to carry out quantitative analysis of neurodegenerative diseases from my senior dr. k. oyanagi, who kindly involved me as a collaborator for his research on ad, psp, and other diseases. i knew that dr. terry had emphasized the importance of quantitative morphometric analysis, including cell counts, in a study of senile dementia of the alzheimer type (annual meeting of american association of neuropathologists (aanp), philadelphia 1982), but i had not been interested in such methodology at that time. my experiences up to that point had only involved autopsy and biopsy, in a sense the patients concerned were largely faceless, and merely provided me with an opportunity to write papers. therefore, i was convinced that i was unfit to be a clinician who had to work with living patients suffering from illness. moreover, i became interested in neurodegenerative diseases such as pd, psp and ad, in addition to surgical neuropathology as routine work (fig. 9). my experiences at the psychiatric hospital may have contributed to the change in my academic interest. figure 9: brain cutting at the brain research institute (december 19, 1985). from left: dr. e. ohama, dr. f. ikuta and ht. with my junior fellows after returning to niigata, i came to have junior fellows working around me. they were mostly graduate students studying for their phd under dr. ikuta, and came to see me as an “elder brother” in our pathology department. i had various discussions with them about our future, specifically about the need to report our achievements in english if possible, regardless of whether it involved case studies or original work. they were excellent individuals, and all imbued with the spirit of “learn well and play well”. at night after work, we sometimes talked together about our research while drinking at the pub. the pub owner often said to us, “what’s so funny about ‘talking shop’ while drinking?” my junior fellows were kazuhiko watabe, shinji ohara, mitsunori yamada (fig. 10), kensuke kawai, koichi wakabayashi (fig. 11) and akiko furuta. i have two memorable episodes from my second residency when i was focused on neuropathology. i came to be involved indirectly in a study by my junior, dr. ohara, who was a neurologist with an interest in peripheral nerves, studying ultrastructural changes in perineurial cells in wallerian degeneration using mouse phrenic nerves. he often showed me electron micrographs and asked me to comment on the findings. he submitted a paper to a well-known pathology journal at my suggestion. one of the two reviewers requested an additional experiment for acceptance. i advised him that there was no need for such an experiment. when he received a letter of acceptance from the editor-in-chief, i was very relieved; the paper was accepted without difficulty in september 1985. it became my first collaborative study with a junior fellow. figure 10: good fellows (april 26, 1984). from left: dr. mitsunori yamada, dr. shinji ohara and ht. figure 11: dr. lysia k.s. forno and dr. koichi wakabayashi at the 13th icn in perth (september 8, 1997). all of us working in dr. ikuta’s lab experienced a very big event. dr. wakabayashi found lewy bodies (lbs) outside the brain in auerbach’s and meissner’s plexuses in the alimentary tracts of autopsied patients with pd – a discovery that overturned the conventional concept of the disease. i admired his observational skills, and this experience reaffirmed the maxim that seeing is believing. i spent a lot of valuable time with him in the paper-writing process. the paper was submitted at the end of 1987 and accepted after minor revision in february of 1988. i was very honored to be included as a co-author. faculty position in neuropathology by 1987, it had been 9 years since i started my life as a resident in the university hospital. at that time, i started to think about my future seriously from time to time, and always came to the conclusion that i should become a pathologist instead of a neuropathologist. even now, the former is in much higher demand nationwide. however, my feeling that i was unfit to be a clinician remained unchanged. then, in may 1988, there was a sudden change in our pathology department, and i obtained a full-time faculty position, assistant professor. later, in october 1991, i was promoted to associate professor under similar circumstances. in late may 1988, shortly after i had become an assistant professor, dr. ikuta arranged for dr. jellinger and dr. georg w. kreutzberg to visit our pathology department (see fig 7 in: free neuropathol 2020; 1: 25). at that time, i was preparing to submit a paper on “neuroendocrine markers in central nervous system neuronal tumors” with an office worker. as if peeking from behind, dr. jellinger said to me, “why not send it to acta?” in the evening, he delivered a lecture on “neuropathology of rett syndrome.” i asked him a question about the substantia nigra lesion, but unfortunately i do not remember the details of either my question or his answer. the above paper was accepted for publication in acta neuropathologica on july 28, 1988. regardless of my faculty position, i continued my studies freely. in fact, it was always of great interest for me to observe autopsy and biopsy cases. at some point, i encountered an autopsy case that determined a future research subject. my diagnosis for that case was “atypical motor neuron disease” (mnd): it did not matter to me whether the disease was called mnd or amyotrophic lateral sclerosis (als). the point was that neuron loss was also evident in the non-motor neuron systems. in addition, bunina bodies (bbs) were detected not only in the lower motor neurons but also in subthalamic neurons. thereafter, i also had an opportunity to report the clinicopathological features of two familial neurological disorders. one was familial als with sod1 mutation; it is a good example of the rebirth of an old case that had slept for a long time in the bdrc under the diagnosis of familial als with posterior column involvement. the other case was autosomal-recessive juvenile parkinsonism in a family, with neuropathological findings for one individual; the familial disease was later found to be linked to park2 (parkin). it was my pleasure to receive requests for joint research from both inside and outside the bri. in this context, i would like to mention three intra-institutional collaborative studies in my early professional career. the first was a clinicopathological study on “cerebral glioblastoma with cerebrospinal fluid dissemination” proposed by dr. kiyoshi onda in the department of neurosurgery. the paper was published in 1989 with favorable comments from dr. lucien j. rubinstein. dr. rubinstein, together with dr. scott r. vandenberg, visited to our pathology department in may 1984. the second was an experimental study of “remote astrocytic response of the prefrontal cortex to lesions in the nucleus basalis of meynert" using rats. it was designed by dr. katsuhiko yanagisawa in the department of neurology, and was of great interest to me when considering the pathological picture of cholinergic deafferentation in ad. the last was a genetic study, “causative gene analysis of hereditary drpla”, led by dr. shoji tsuji in the department of neurology, in which unstable expansion of a cag repeat in chromosome 12 was identified as being responsible for the familial disease. like the case of familial als with sod1 mutation, the aspect of greatest significance for me was that frozen brain tissues from autopsy-proven patients stored in the bdrc were also used in the genetic analysis. working with a chinese friend in our pathology department, there was a chinese neurologist named mu-yi wang from china medical university in shenyang, who joined us in october 1980 and studied neuropathology under dr. ikuta. he was a government-sponsored international student from china in the era of reform and opening up. we soon became good friends, and often talked about not only neuropathology but also chinese history. he was a sincere and kind gentleman who always had a smile on his face. in late 1981, i received an airmail letter with attached massages from all the lab members, in which dr. ikuta said “make a lot of good friends” and dr. wang said “we will meet again”. dr. wang left for china in march 1982 when i was still in new york. an opportunity came for me to meet dr. wang again when dr. ikuta received a grant-in-aid for his scientific research entitled “joint study on demyelinating disorders between japan and china” from the ministry of education, science and culture (monbusho). dr. ikuta, dr. oyanagi and i met dr. wang again at beijing airport in september 1989, the year of the tiananmen square incident. he greeted us with a warm smile, saying “nice to see you again,” in japanese. during the trip, he helped us as an interpreter of chinese (fig. 12). figure 12: with the forbidden city in the background (september 15, 1989). from left: dr. k. oyanagi, dr. f. ikuta, ht and dr. mu-yi wang at jingshan park in beijing. dr. oyanagi and i accompanied dr. ikuta on research trips in september 1989 and october 1991. we visited the medical schools in shanghai, chongqing, changchun and shenyang in 1989, and those in suzhou, nanjing, hefei, wuhan, xian, beijing and harbin in 1991. the cases selected and presented by the chinese side allowed us to observe many typical examples of baló’s disease. when visiting norman bethune university of medical science in changchun in 1989, i met two young men, dr. mu su and dr. da-lin yao, both of whom wanted to study aboard. dr. su joined our pathology department in bri as a monbusho scholarship student in may 1992 and finally obtained a phd degree from niigata university in 1997. since then, he has worked as a pathologist in the us for many years. the name da-lin yao appeared as the first author in a paper on “concentric sclerosis (baló)” published in 1994. i still believe that it was him i met in changchun. he must have studied neuropathology under dr. h.d. webster at national institutes of health (nih). in 1991, we met dr. ke-wei huang and dr. lu-ning wang at the chinese people’s liberation army general hospital. it was said that dr. huang was a pioneer of neuropathology in china and that dr. wang was his favorite disciple. in deep sorrow about a year after i was promoted to associate professor, a tragedy fell upon me. when i came home from my off-campus work in the evening and opened the front door of my house, the phone rang. when i picked up the phone, my junior colleague, dr. yamada, informed me that my wife shizue had been hit by a car on the way home and taken to the hospital by ambulance. she developed a subdural hematoma and underwent emergency surgery. thereafter, almost every day, i spent my lunch breaks and about an hour after 5:00 p.m. by her bedside talking to her, but she never regained consciousness. people around me used to say that they could not be like me. i always replied that it was easier for me to act the way i did. my love for her was why i was able to devote the rest of my time to neuropathology. years later, i recall trying to look her in the eyes and blink my own eyes three times. it was not so long before she began responding to me by blinking her own eyes three times. as a neuropathologist, i still do not fully understand her behavior or reaction; rather, i have not yet found a satisfactory answer as to whether or not she regained any degree of consciousness. at the end of february 1999, about 7 years after the car accident, she passed away surrounded by myself and relatives. in the meantime, dr. ikuta retired from his faculty position at niigata university at the age of 65 in late march 1995 and became professor emeritus the following april. almost half a year later, in september 1995, i was elected as his successor and became professor in the department of pathology, bri, niigata university. i told my wife and parents about this. shizue seemed to smile a little. to my mother, it would not have meant much: rather, she might have been saddened by my decision to settle in niigata. so ultimately, i did not fulfill my parents’ expectation of caring for them at home, and my younger brother took over that role. professorship in japanese medical schools, one department generally has one professor, one associate professor and two assistant professors. the professor also serves as a chair to take responsibility for departmental management. this meant that i was no longer simply a neuropathologist/researcher in the department of pathology. at first, i confirmed that our neuropathology was based on autopsy and biopsy as a routine form of work, just as it had been before. needless to say, as far as possible, there was no limit to individual study subjects/topics and methods. this meant that there was no change in the daily work of the faculty. on the other hand, the responsibility of teaching residents and graduate students, and supporting their research, weighed heavily on my shoulders. to meet this responsibility, i had to do my best to obtain funds and keep them flowing. fortunately, in this respect, i remained successful until my retirement. overall, i was blessed with excellent human resources, including the faculty and staff as well as the residents and graduate students, in the wonderful setting of the bri inherited from our predecessors, including dr. ikuta, for which i was extremely grateful. chinese students and academic exchange with china shortly after i became professor, i communicated with dr. wang in shenyang, china, by phone. in return for his friendship, i wanted his pupil to study neuropathology as a graduate student in our pathology department. this resulted in his favorite disciple, dr. yue-shan piao, coming to niigata in september 1997 after completing a master course at the china medical school in shenyang, and then enrolling in a phd course in april 1998. my teaching career started with her. she had a strongly inquisitive mind and a competitive personality. thereafter, i accepted a number of chinese graduates in medicine as graduate students for a phd course or as short-term international students. all of these students studied neuropathology well and enjoyed living in japan. in may 2003, through dr. piao’s mediation, i was invited to china for the first time to celebrate the 25th anniversary of the founding of the neurological clinicopathological conference in beijing. this was the beginning of my academic exchange with china, and allowed me to became reunited with dr. l-n wang for the first time in 12 years. she had since become a leader in neurology and related neuropathology in china. she told me that it was very difficult to have an opportunity to obtain autopsy cases in china. i immediately surmised that this was related to confucianism. dr. piao returned to china in july 2004 and began a new path as a neuropathologist under dr. de-hong lu in the department of pathology, xuanwu hospital, capital medical university. thereafter, i was often invited to conferences related to neuropathology in china. in 2005, i was invited to china twice: beijing (fig. 13) and changsha in april, and beijing and shanghai in september. i have a fond memory of presenting a lecture on “the importance and fascination of neuropathology” to young participants at a chinese neuropathology education seminar held in beijing in september 2005. i could feel great vitality in the young listeners. figure 13: at xuanwu hospital, capital medical university in beijing (april 5, 2005). from left: dr. de-hong lu, ht and dr. lu-ning wang. since 2008, i attended the annual chinese neuropathology case conference, giving me an opportunity to make new friends in the chinese neuropathology group. so far, i have observed many interesting cns tumors directly by light microscopy. at the venue, young participants, including residents and graduate students, listened intently to each presentation and during the following discussion, and some of them took pictures of the projected images on smart phones. the leaders placed strong importance on discussion of the findings and diagnoses (fig. 14). at the dinner party, both young and old participants were very frank and friendly. when talking, they did not appear to care about each other’s age or status; the atmosphere was similar to what i had experienced in new york. figure 14: with leaders of the chinese neuropathology group in fuzhou (june 13, 2018). left to right: dr. d-h lu (beijing), dr. shi-zhu yu (tianjin), dr. xiu-wu bian (chongqing), ht, dr. sheng zhang (fuzhou) and dr. yin wang (shanghai). although not shown here, this group also has several female leaders. unfortunately for me, presentations of autopsy cases, including those of neurodegenerative diseases, were extremely rare at the conference. almost certainly, the leaders’ specialty was surgical neuropathology dealing with cns tumors and allied diseases. since 2015, dr. piao has been professor in the department of pathology, capital medical university in beijing, and she must be now one of the leading researchers of neuropathology in china. unfortunately, since 2020, the annual conference on site has been canceled due to the covid-19 pandemic. neurodegenerative diseases my study of neurodegenerative diseases began with an autopsy case that made me take a closer look. my passive attitude was the exact same as that of a clinician was waiting for a patient in the examination room. in fact, my high school classmates had often told me that if i were a clinician, i would have made a good practitioner. therefore, autopsy studies often ended with a single case report, although i still believe that such studies are very important for the teaching of residents and graduate students. occasionally, however, a single case report developed into a study subject or topic as similar autopsy cases accumulated in the bdrc. although i forget the specific situation, i saw gallyas-positive argyrophilic glial cells in the midbrain of a pd patient using light microscopy. i immediately confirmed the finding with dr. wakabayashi. my interest in pd then skyrocketed. in the following year, 1997, an α-synuclein mutation was identified as being responsible for familial pd with lbs. shortly afterwards, i had the opportunity to discuss the discovery with dr. makoto yoshimoto, with whom a joint research project was undertaken. i was very surprised to hear that he had anti-nacp (human α-synuclein) antibodies that he had developed him-self. another joint study was soon initiated, and this was reported as “nacp, a presynaptic protein, immunoreactivity in lbs in pd” in 1997. although our report fell a little behind another group, our interest in α-synuclein (nacp) did not end there. by the end of the year, our target had shifted to multiple system atrophy (msa). dr. wakabayashi’s contribution to pd and msa research was significant. since february 2000, he has been professor in the department of neuropathology, institute of brain science, hirosaki university school of medicine, hirosaki. it is worth mentioning that dr. k. iwanaga was the first to report the occurrence of α-synuclein-positive aggregates in the cardiac plexus of both patients with pd and those with incidental lb disease. the study of hereditary drpla in collaboration with dr. tsuji’s group also progressed smoothly; it started with ihc using anti-ubiquitin antibody and later developed to the use of monoclonal anti-polyglutamine antibody (1c2). finally, dr. hayashi (later toyoshima) and dr. yamada made significant contributions in the wider area of polyglutamine diseases beyond hereditary drpla. they never forgot the necessity of em observation in parallel with ihc. since april 2015, dr. yamada has been professor in the department of brain disease research, shinshu university school of medicine, matsumoto. i also carried out a study of drpla with the help of my colleagues, as the topic “neuronal nuclear alterations in drpla” seemed plain and uninteresting to them. however, a photograph used in the report graced the cover of brain research, where it was accepted for publication. i felt like i had received a medal and was very happy; a feeling shared by the other staff of our pathology department. another study was on four-repeat (4r) tauopathies, involving many colleagues, including graduate students. when i first had an autopsy case diagnosed clinically as psp, it was a fairly rare neurological disorder in japan. i remember that at the invitation of a neurologist, i observed a wheelchair-bound male patient with psp, and could see at a glance there was marked nuchal dystonia. at that time, i had been unfamiliar with the neurological term dystonia. at present, psp is known to demonstrate a wide pathological and clinical spectrum as a representative 4r tauopathy. to my surprise and great pleasure, dr. hayashi (fig. 15) found a novel tau gene mutation (exon1, arg5his) responsible for late-onset frontotemporal dementia. he had taught me how to operate a personal computer, and i believed that he had a flair for genetic research. globular glial tauopathy (ggt) is a new category within the 4r tauopathies; we were able to collaborate to establish and strengthen the disease concept; dr. piao, dr. fu, dr. tanaka and dr. toyoshima played an important role in this. figure 15: with the po river in the background at the 15th icn in turin (september 13, 2003). front row, left to right: ht, dr. shintaro hayashi and dr. akiyoshi kakita. top row, left to right: mrs. yuriko takahashi, dr. chun-feng tan, dr. chikanori inenaga and dr. koichi kawasaki. cns tumors as mentioned already, the diagnosis of cns tumors was intriguing and sometimes challenging for me. among works of my generation, there were several authorities on the diagnosis of cns tumors, including dr. rubinstein. i remember dr. rubinstein giving an oral presentation on “astroblastoma” with slides of elegant electron micrographs. surprisingly, he performed the presentation by reading the manuscript prepared. i felt that he was a noble gentleman rather than a well-known authority (annual meeting of the aanp, dallas 1989). i also admired dr. jacques hassoun and dr. bernd w. scheithauer for their sharp diagnostic eye for cns tumors. my sense of enthusiasm was often shared with my residents and graduate students as case reports, in which we also showed the ultrastructural features of individual cases. our case reports included those on a range of notable cns tumors, including ganglioglioma, pleomorphic xanthoastrocytoma, tanycytic ependymomas, melanotic cerebral astrocytoma, atypical teratoid/rhabdoid tumor, chordoid glioma of the third ventricle, and astroblastoma. for diagnosis of cns tumors by biopsy, i got into the habit of looking at the specimen first. for example, in glioblastomas in the thalamus, the patient’s age could often be inferred and confirmed later by the clinical history. this was also the case for brainstem (pontine) gliomas, although biopsy was very unusual in such cases. i was curious as to which gene determined the location (thalamus) as well as the age of onset (children/adolescent). the gene associated with such gliomas has already been discovered. research on cns tumors awaits the next development, although the same can be said for neurodegenerative diseases. minamata disease and brain development niigata is famous as the “snow country” described by yasunari kawabata (nobel laureate, 1968) in his novel. unfortunately, the name is also associated with minamata disease, a neurological disease caused by methylmercury poisoning. i was involved in “research on the effect of mercury on the nervous system, etc. (1996-2009)” commissioned by the ministry of the environment. our study group led by dr. kakita (fig. 15) conducted experiments designed to clarify the pathomechanism of minamata disease using rats treated with methylmercury, focusing especially on intrauterine and fetal brain development. joint studies with dr. mineshi sakamito at the national institute for minamata disease (nimd) (director, dr. komyo eto), minamata, also yielded new biochemical and behavioral data through similar experiments. i visited minamata several times for meetings with the faculty at nimd. although i already had knowledge of the disease, i was able to witness for myself the scale of the pollution damage on the population, which far exceeded that in niigata in terms of both number and degree. when i visited with chinese graduate students, i showed them the birthplace of minamata disease and the minamata disease municipal museum. i am sure that at the time, various pollution-related problems in china, including minamata disease, were receiving international attention. i have visited the nursing home for patients with childhood minamata disease, and i will never forget their haunting gaze; they were about the same age as myself. dr. kakita also developed research on the neuropathology of epilepsy, which is often associated with focal cortical dysplasia, in our pathology department. he supported me in every aspect of my professional career as a professor. since april 2018, dr. kakita has been my successor as professor in the department of pathology, bri, his alma mater. at present, he serves as president of the jsn. amyotrophic lateral sclerosis – final lecture by a retiring professor i think that there is a custom of german origin whereby a retiring professor in japan delivers a final lecture. it has been said that retiring professors tend to give lectures on the outputs of their research resulting from passion and hard work. the title of my final lecture was “amyotrophic lateral sclerosis (als): before and after the discovery of tdp-43.” it was not a story of hardship, however. in als, there are two characteristic neuronal cytoplasmic inclusions: bbs and skein-like and/or round spherical inclusions. the latter are clearly shown to be ubiquitin-positive inclusions (upis) by ihc. interestingly, upis are sometimes also demonstrable in the temporal lobe, being significantly associated with dementia. i experienced a crossroads in promoting joint research on als. considering the number of neurons bearing upis, i always maintained that upis in the temporal lobes, and not those in the anterior horns, were targets for identification of the pathological protein involved in als. however, a molecular biologist partner wanted to select bbs in the anterior horn cells. at that time, a technique for capturing and analyzing single cells from the brain sections had already been established. the large anterior horn cells bearing bbs were easy for my partner to focus on, as he had both the equipment and the technology for the task. i could understand his research spirit, because i was still keen to understand the nature and origin of bbs. however, the discussion of joint research came to end. in september 2006, i attended the 16th icn in san francisco. leaving the room with a smile after listening to a lecture on pd by dr. j. william langston, i was tapped on the shoulder from behind. when i turned around, i was told by my friend, dr. paul g. ince, that the protein component of the upis in als had been finally identified, and that the report was in press. on the plane on the way back to japan, i thought that a series of related studies might already be underway. soon afterwards, i read the paper on the discovery of the pathological protein, tdp-43. however, after waiting for a few months, no follow-up papers appeared. eventually, i called a graduate student, dr. tan (fig. 15), to my room and told her that although it might be too late to do this study, we should start it. as a result, we were the first to show the distribution pattern of tdp-43-positive neuronal cells, together with occasional glial cell involvement, in sporadic als. i do not have any particular objection to the theory/hypothesis – “conformational disease” and “prion-like pathomechanism” – of neurodegenerative diseases such as ad (amyloidopathy/tauopathy), pd (α-synucleinopathy) and als (tdp-43 proteinopathy). however, i consider that als is quite different from the other two diseases, ad and pd, concerning the pattern of the pathological protein propagation. at the end part of the lecture, i also mentioned ggt, which is a sporadic 4r tauopathy showing a feature of mnd/als, i.e. upper and lower motor neuron involvement. in our research on als and 4r tauopathies, dr. osamu onodera (department of neurology) and dr. takeshi ikeuchi (department of molecular genetics) were often important collaborators at the bri. our published papers covered in my final lecture were as follows.   1. takahashi h, ohama e, ikuta f, tokiguchi s. an autopsy case of atypical motor neuron disease with bunina bodies in the lower motor and subthalamic neurons. acta pathol jpn 1991; 41: 46-51. 2. takahashi h, oyanagi k, ohama e, ikuta f. clarke's column in sporadic amyotrophic lateral sclerosis. acta neuropathol 1992; 84: 465-70. 3. takahashi h, oyanagi k, ikuta f. the intermediolateral nucleus in sporadic amyotrophic lateral sclerosis. acta neuropathol 1993; 86: 190-2. 4. takahashi h, makifuchi t, nakano r, sato s, inuzuka t, sakimura k, mishina m, honma y, tsuji s, ikuta f. familial amyotrophic lateral sclerosis with a mutation in the cu/zn superoxide dismutase gene. acta neuropathol 1994; 88: 185-8. 5. piao ys, wakabayashi k, kakita a, yamada m, hayashi s, morita t, ikuta f, oyanagi k, takahashi h. neuropathology with clinical correlations of sporadic amyotrophic lateral sclerosis: 102 autopsy cases examined between 1962 and 2000. brain pathol 2003, 12: 10-22. 6. toyoshima y, piao ys, tan c-f, morita m, tanaka m, oyanagi k, okamoto k, takahashi h. pathological involvement of the motor neuron system and hippocampal formation in motor neuron disease-inclusion dementia. acta neuropathol 2003; 106: 50-6. 7. tan cf, eguchi h, tagawa a, onodera o, iwasaki t, tsujino a, nishizawa m, kakita a, takahashi h. tdp-43 immunoreactivity in neuronal inclusions in familial amyotrophic lateral sclerosis with or without sod1 gene mutation. acta neuropathol 2007; 113: 535-42. 8. nshihira y, tan cf, onodera o, toyoshima y, yamada m, morita t, nishizawa m, kakita a, takahashi h. sporadic amyotrophic lateral sclerosis: two pathological patterns shown by analysis of distribution of tdp-43-immunreactive neuronal and glial cytoplasmic inclusions. acta neuropathol 2008, 116: 169-82. 9. nishihira y, tan cf, hoshi y, iwanaga k, yamada m, kawachi i, tsujihata m, hozumi i, morita t, onodera o, nishizawa m, kakita a, takahashi h. sporadic amyotrophic lateral sclerosis of long duration is associated with relatively mild tdp-43 pathology. acta neuropathol 2009; 117: 45-53. 10. takeuchi r, tada m, shiga a, toyoshima y, konno t, sato t, nozaki h, kato t, horie m, shimizu h, takebayashi h, onodera o, nishizawa m, kakita a, takahashi h. heterogeneity of cerebral tdp-43 pathology in sporadic amyotrophic lateral sclerosis: evidence for clinic-pathologic subtypes. acta neuropathol commun 2016; 4: 61. 11. kimura t, jiang h, konno t, seto m, iwanaga k, tsujihata m, satoh a, onodera o, kakita a, takahashi h. bunina bodies in motor and non-motor neurons revisited: a pathological study of an als patient after long-term survival on a respirator. neuropathology 2014; 34: 392-7. 12. yokoseki a, shiga a, tan cf, tagawa a, kaneko h, koyama a, eguchi h, tsujino a, ikeuchi t, kakita a, okamoto k, nishizawa m, takahashi h, onodera o. tdp-43 mutation in familial amyotrophic lateral sclerosis. ann neurol 2008; 63: 538-42. 13. konno t, shiga a, tsujino a, sugai a, kato t, kanai k, yokoseki a, eguchi h, kuwabara s, nishizawa m, takahashi h, onodera o. japanese amyotrophic lateral sclerosis patients with ggggcc hexanucleotide repeat expansion in c9orf72. j neurol neurosurg psychiatry 2013; 84: 398-401. 14. kosaka t, fu yj, shiga a, ishidaira h, tan cf, tani t, koike r, onodera o, nishizawa m, kakita a, takahashi h. primary lateral sclerosis: upper-motor-predominant amyotrophic lateral sclerosis with frontotemporal lobar degeneration immunohistochemical and biochemical analyses of tdp-43. neuropathology 2012; 32: 373-84. 15. fu yj, nishihira y, kuroda s, toyoshima y, ishihara t, shinozaki m, miyashita a, piao ys, tan cf, tani t, koike r, iwanaga k, tsujihata m, onodera o, kuwano r, nishizawa m, kakita a, ikeuchi t, takahashi h. sporadic four-repeat tauopathy with frontotemporal lobar degeneration, parkinsonism, and motor neuron disease: a distinct clinicopathological and biochemical disease entity. acta neuropathol 2010; 120: 21-32 16. tanaka h, toyoshima y, kawakatsu s, kobayashi r, yokota o, terada s, kuroda s, miura t, higuchi y, otsu h, sanpei k, otani k, ikeuchi t, onodera o, kakita a, takahashi h. morphological characterization of glial and neuronal tau pathology in globular glial tauopathy (types ii and iii). neuropathol appl neurobiol 2020; 46: 344-58. 17. takeuchi r, toyoshima y, tada m, tanaka h, shimizu h, shiga a, miura t, aoki k, aikawa a, ishizawa s, ikeuchi t, nishizawa m, kakita a, takahashi h. globular glial mixed four repeat tau and tdp-43 proteinopathy with motor neuron disease and frontotemporal dementia. brain pathol 2016; 26: 82-94. leadership – domestic and international our bri is the only research institute at niigata university, established in 1967, for the study of theories concerning the brain and brain diseases (fig. 16). i served as director of the bri for about 12 years (2002-2014). a year before i became director, the 21st century center of excellence (coe) program was initiated as a project of the ministry of education, culture, sports, science and technology (mext). in a bri faculty meeting, i was asked to act as a leader of the project, supported by the university president. in response, i decided to apply for the coe project with the help of dr. tsutomu nakada, head of the center for integrated human brain science, bri. the aim of the project, entitled “center for brain pathology research and education (2002-2008)” was to form a world-class base for research and education in the field of neuropathology. in 2007, we held the neuropathology international symposium at the bri, and this was one of the great achievements of the mext-supported coe project. to learn about current research and practice in neuropathology, we invited 15 foreign experts to the symposium, who delivered lectures on their specialist topics (fig. 17). figure 16: brain cutting at the brain research institute (june 21, 1971). front left: dr. f. ikuta (42 years old). front row, right to left: dr. tatsuji ito (pathologist, 67 years old), dr. komei ueki (neurosurgeon, 57 years old) and dr. mizuho nakata (neurosurgeon, 78 years old). these three are dr. ikuta’s teachers. figure 17: neuropathology international symposium in niigata (october 27, 2007). 2nd row, left to right: drs. bernd w. scheithauer (usa), takanori hirose, gregory n. fuller (usa), markus glatzel (germany) and herbert budka (austria). 3rd row, left to right: drs. wieslawa grajkowska (poland), a. kakita, skipping one person and thong k. wong (malaysia), francoise gray (france) and tumtip sangruchi (thailand). 4th row, left to right: drs. m. yamada, k. wakabayashi, paul g. ince (uk), dennis w. dickson (usa), james c. vickers (australia), min-cheol lee (korea), chitra sarkar (india) and chin-chen m. lee (taiwan). 5th row, 2nd left to right, drs. d-h lu (china), yasuo sugita, yoshio hashizume, toru iwaki, skipping one person and ht, f. ikuta and e. ohama. 6th row, 4th from left, dr. yoichi nakazato and 1st from right, dr. shinya tanaka. except dr. f. ikuta, only speakers and chairs are named. the jsn was established in 1960 through the efforts of neurologists, psychiatrists and neuropsychiatrists working at various medical schools. i served as president of the jsn for a relatively long period (2010-2018). my greatest concern was that there were only a small number of departments and faculties related to neuropathology research and education at japanese medical schools. i have the impression that neuropathology is a subdivision of pathology, particularly when considering biopsy diagnosis of cns tumors. however, it still appears to be difficult to find any faculty members who deal with neuropathology in pathology departments. in this sense, there is concern that our society membership will gradually decline, despite the old slogan that “the 21st century is the age of brain science” in japan. i also served as editor-in-chief of our society journal neuropathology (2003-2011). although my memory is vague, when i accepted this position, the papers published in neuropathology were already searchable on pubmed, and probably already had an established journal impact factor (if). on my watch, the if exceeded 2.0 once or twice (cf. if 2021: 2.076). it was also a pleasure to receive manuscripts submitted from abroad. during my time, submissions from china and korea showed an increasing trend. i do not mind the high number of case reports in the journal. if it serves the role of an international neuropathology journal, i am glad. after i invited dr. min-cheol lee to niigata in 2007, i made several korean friends who were pathologists at the university college of medicine. on december 8, 2012, the “seoul neuropathology forum” was held under the sponsorship of seoul national university hospital (dr. sung-hye park). dr. park is a self-proclaimed neuropathologist belonging to the department of pathology and one of the leaders of the neuropathology study group of the korean society of pathologists. they are experts in the pathological diagnosis of cns tumors and allied diseases. i was very impressed by the forum entitled “neurodegenerative diseases – up to date”. their work confirmed that they were unmistakably neuropathologists (fig. 18). figure 18: seoul neuropathology forum in seoul (december 8, 2012). front row, left to right: drs. tetsuaki arai (tsukuba), kazuhiko watabe (tokyo), yeon-lim suh (seoul), shin kwang khang (seoul), yue-shan piao (beijing), ht (niigata), min-cheol lee (gwangju) and en kyung hong (seoul). top row, left to right (except for one on the left and one on the right): drs. kyung-wha lee (seoul), jang-hee kim (suwon), narae kim (seongnam), sung-hye park (seoul), se-hoon kim (seoul), ghee-young choe (seoul) and yeon-soo lee (seoul). i succeeded dr. herbert budka as president (2014-2018) of the international society of neuropathology (isn). i do not remember the date exactly, but i received an e-mail message from dr. francesco scaravilli saying that he was considering nominating me as president-elect of the isn. i believe he had a role as a society officer of the isn at that time. when i was in dr. suzuki’s lab in new york, i first became aware of his name because it was often on dr. kuni’s lips (later his and kuni’s names appeared in nature 305; 713-5, 1983). i felt a sense of intimacy with him, despite never having met him. finally, i accepted his offer, and replied to his e-mail with a brief document introducing my career in research and teaching. however, i still do not know why he chose me. during my presidency, one of my fond memories was going to hyderabad, india with dr. budka, dr. david w. ellison and dr. raj kalaria in december 2015 to celebrate the official establishment of the neuropathology society of india (npsi) as well as the first annual conference of the npsi (fig.19). we had a good time with npsi officers and participants, the latter including many young residents. i had a reunion with dr. chitra sarkar, and had a chance to take a walk around the town with dr. budka, who said, “hitoshi, beware of the dogs on the street”, to which i answered, “certainly”. figure 19: taking part in the 1st annual conference of the neuropathology society of india held in hyderabad (december 2015). left to right: dr. david w. ellison, ht, dr. raj kalaria and dr. herbert budka. we were the isn delegation of four. lastly, i would like to mention the asian oceanian society of neuropathology (aosn), which was officially established as an offshoot of the asian society of neuropathology (asn) on september 25, 2018, at the 4th asian congress of neuropathology held in conjunction with the 19th icn in tokyo (fig. 20). i was very happy to have been involved in the establishment of the asn (1st congress in 2008 in tokyo) from its inception (asian neuropathology workshop in 2007 in tokyo). i sincerely hope that the aosn will continue to develop further in the future. figure 20: asian society of neuropathology general assembly in tokyo (september 25, 2018). members present. front row, left to right: drs. glenda halliday (sydney), mari yoshida (nagoya), anita mahadevan (bangalore), catriona a. mclean (melbourne), yue-shan piao (beijing) and vani santosh (bangalore). top row, left to right: drs. ranil de silva (colombo), tarik tihan (san francisco, as a representative of turkey), lai siang hui (singapore), ho-keung ng (hong kong), shigeo murayama (tokyo), ht (niigata), takashi komori (tokyo), bishan dass radotra (chandigarh) and thong k. wong (kuala lumpur). members absent. dr. sung-hye park (seoul) and dr. maysa ai-hussaini (amman). thinking back to all these experiences, i must say that i really enjoyed my various roles with the kind help of many domestic and international colleagues. i have really enjoyed not only my work but also my interactions with my colleagues and friends. retirement and departure i retired from my faculty position at niigata university at the age of 65 in late march 2018. in fact, i left my university at the end of january 2020, and i have spent the last 6 years, including the concurrent appointment as a professor for the first 4 years, as executive vice-president for research at my alma mater (fig. 21). figure 21: ht desk-working at his personal computer (november 7, 2016). at the invitation of my old friend, dr. k. onda (neurosurgeon; director), i am currently working as head of laboratory medicine and head of research at niigata neurosurgical hospital, a major hospital in niigata city specializing in cerebrovascular diseases, while maintaining a relationship with the department of pathology, bri, as professor emeritus. i am still an active neuropathologist. fortunately, my senior colleague dr. takeda also works at this hospital as head of pathology. i have started studying the neuropathology of cerebrovascular diseases, which is an unfamiliar field to me. my teacher dr. ikuta passed away on may 26, 2021 at the age of 92. he was a pioneer of neuropathology in japan and had many pupils who are distributed widely from hokkaido in the north to okinawa in the south. he had worked as director of the brain research center affiliated with this hospital for a long period after his retirement (1995-2015). he told me that during his life he was very happy to have had so many colleagues and friends abroad. most of them have passed away now. time flies like an arrow. enjoying life together several months after my wife shizue passed away, dr. ikuta told me that it might be difficult for me to fulfill my mission alone as a professor. after that, when i returned to my parents’ home, my mother told me that although i was not “fat”, i was swollen. i suddenly started to worry about my health. some of my friends in my academic societies sent me e-mails, telling me to get married as soon as possible and that they would find a bride for me; i sent them my pictures right away. in the end, i remarried in june 2001 after being introduced to a lady named yuriko by a friend. dr. ikuta often told me that yuriko has made me what i am today. although i am not good at expressing my gratitude, i almost always took her with me to academic conferences at home and abroad. on each occasion, she spent a pleasant time talking with my young fellows and my friends’ wives (fig. 15). unfortunately, we have no children. however, we have 4 female chinese pupils, and yuriko is always happy to see photos of the growing children sent via e-mail by three of them (drs. piao, tan and fu). she looks upon them as if they were her own grandchildren, and also has motherly concern about when the fourth pupil (dr. zhang) will get married. she is a trained pianist and her hobby is ikebana (flower arrangement). she is a master of ikenobo ikebana and also a good tennis player. i do not have such talents. however, i enjoy self-produced greetings with ikebana that signal “i am home” and “welcome home” in the entrance hall. as to sports, i am still a football freak, although i no longer play. since retirement, i have come to enjoy wandering in fields observing butterflies as a sort of intellectual curiosity. we both enjoy this together on weekends, and intend to continue appreciating life together as much as possible. we cannot wait for the covid-19 pandemic to go away. concluding remarks i genuinely appreciate the fact i was able to meet my two teachers, dr. ikuta and dr. suzuki, though i can’t be certain whether i met their expectations or not. i was not necessarily an expert in any particular field, such as neurodegenerative diseases, or any particular disease, such as als, and i had no strong intention of becoming one. i suppose my specialty would be neuropathology, specifically diagnostic neuropathology, whereby even with a diagnosis, hidden new features may be found and a new disease may emerge. i have no doubt that i have been a teacher, but also always a trainee. i am very pleased that i have been able to go through a series of epochs in neuropathology characterized by the use of electron microscopy, ihc and molecular genetics. i have many colleagues to thank for their friendship, which continues to the present day. addendum during my tenure as a professor, i have genuinely enjoyed and practiced neuropathology with the faculty and staff, as well as residents and graduate students. i would like to leave a list of their names with gratitude. mitsunori yamada, koichi wakabayashi, akiyoshi kakita, koichi kawasaki, shintaro hayashi, keisuke iwanaga, mu su, yasuko hayashi (toyoshima), yue-shan piao, chikanori inenaga, chun-feng tan, tsutomu sugai, mari tada, kenji sakai, yasushi nishihira, koshun matsuo, masae ryufuku, misato yamazaki, hiroki kitaura, hiroshi shimizu, kenichi okazaki, ying-jun zheng, yoshiko sakuma, ming-wei zhu, yong-juan fu, takayuki kosaka, yasuhiro hoshi, manabu natsumeda, yukari miki, hiroaki miyahara, kenichi soma, makiko matsuda, atsushi shiga, ryosuke ogura, yuichi yokoyama, ryoko takeuchi, tadashi kimura, hai-shan jiang, hidetomo tanaka, naohiko seike, tomoe sato, rie saito, lu zhang, junko ito, akari takeshima, asa nakahara and takanori nozawa. the staff who have worked prolonged periods: shigekimi egawa, tomiyoshi hasegawa, yuko ota, chieko tanda, junko takasaki and shingo nigorikawa (technical), and mihoko machida and mari yoshida (office). copyright: © 2023 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. regression of multiple intracranial meningiomas after cessation of long-term synthetic progesterone (megestrol) medication: case report and autopsy feel free to add comments by clicking these icons on the sidebar free neuropathology 5:27 (2024) case report regression of multiple intracranial meningiomas after cessation of long-term synthetic progesterone (megestrol) medication: case report and autopsy tamadar a. aldoheyan1,2, marc r. del bigio1,3 department of pathology, max rady college of medicine, university of manitoba, winnipeg, manitoba, canada current appointment: department of pathology, king saud university, riyadh, saudi arabia pathology, shared health manitoba, winnipeg, manitoba, canada corresponding author: marc del bigio · health sciences centre winnipeg · department of pathology (ms4) · 820 sherbrook street · winnipeg, mb r3a 1r9 · canada marc.delbigio@umanitoba.ca submitted: 16 august 2024 accepted: 03 october 2024 copyedited by: georg haase published: 29 october 2024 https://doi.org/10.17879/freeneuropathology-2024-5813 keywords: autopsy, meningioma, megestrol, progesterone receptor, tumor involution abstract we report the history of a woman who developed four intracranial meningiomas during 11 years of therapy with the synthetic progesterone agonist megestrol. after discontinuation of the drug at age 75 years, she improved clinically and a ct scan showed near complete regression of the meningiomas by 78 years. autopsy was performed at 83 years of age following an accidental death. at the tumor sites, we found both collagenous tissue with small islands of low grade meningioma having strong nuclear immunoreactivity for progesterone receptor and lipomatous tissue. a literature review showed similar cases of radiologic meningioma regression following discontinuance of progestins. our case is the first one with histopathologic characterization of the end point. introduction meningiomas are twice as common in females as in males. these tumors are known to express progesterone receptor and to a lesser extent estrogen receptors [1, 2]. numerous case reports and population-based studies show increased risk for meningioma, tumor multiplicity, and tumor progression in women who receive synthetic progestins including cyproterone acetate, megestrol acetate (also called nomegestrol), and chlormadinone acetate [3–10]. progestin-associated meningiomas are often located at the skull base and have higher frequencies of pik3ca and traf7 mutations [11]. some tumors decrease in size after drug discontinuation [12, 13], although administration of the antiprogesterone agent mifepristone has not been shown to be efficacious for treatment of progestin-induced meningioma [14, 15]. here we report the autopsy findings in a woman who developed multiple intracranial tumors during long-term therapy with the progesterone agonist megestrol acetate. the tumors, presumed to be meningiomas, regressed almost entirely upon cessation of the drug. clinical summary at 64 years age, this woman underwent hysterectomy with bilateral salpingo-oophorectomy for low grade endometrial stromal sarcoma and was placed on progesterone therapy with megestrol 160 mg daily. regular imaging follow-up in the subsequent decade showed no evidence of local recurrence or metastatic disease. psoriasis had been treated with oral methotrexate intermittently from age 62 to 73 years, acitretin, a retinoid, for 1 year at age 74, and apremilast, a selective inhibitor of the enzyme phosphodiesterase 4 / pde4, from age 77 to 83. the past medical history of the woman also included diabetes mellitus type 2, hypothyroidism, and arterial hypertension for which she was treated with metformin, levothyroxine, nifedipine, and hydrochlorothiazide, respectively. ct scans of the head had been done at 56 and 66 years because of headaches; these showed minor ischemic changes in cerebral white matter and no evidence of intracranial tumor. at age 73 years, the woman presented with labile emotions and confusion. she had a mild resting tremor but no localizing signs. a ct scan of the head showed three dura-based tumors in the left parietal / posterior falx region (2.9 cm), the left frontal / anterior falx region (2.8 cm), and the right parasellar region (1.7 cm) (figures 1 and 2). measurements presented in figure 2 are from the ct scans. all tumors were isointense with gray matter on unenhanced imaging, and all were bright following contrast enhancement. the tumors were thought to be meningiomas. a single magnetic resonance (mr) study was incomplete because of claustrophobia. the brain tumors were isointense on the t1 sequence. approximately 1 year later at 74.8 years of age, the woman stopped eating and was unable to walk, whereupon she was admitted to hospital. a ct scan at the time of admission showed further enlargement of two tumors with significant local mass effect and surrounding vasogenic edema (figure 1). the woman was judged not to be a candidate for neurosurgical intervention. a ct scan two months after discontinuation of megestrol showed that the two largest tumors had decreased in size, whereas a new small en plaque tumor had appeared in the lateral part of the left frontal lobe. during a one year hospital stay without specific anti-neoplastic therapy, the neurologic status of the woman gradually improved. a ct scan at 75.7 years age showed that all tumors had decreased (figure 1 and figure 2). at age 76 years, the woman returned to independent living and regained her motor vehicle driving license. at age 77 years her tremor had increased slightly. ct scans at 78 and 82 years age showed mild diffuse cerebral atrophy and all intracranial tumors had disappeared by 82 years age (figure 1 and figure 2). the woman died after choking on food at 83 years. figure 1. ct scans with contrast enhancement of brain showing dura-based tumors in the right parasellar, left frontal, and left parietal regions in a woman at ages 73.5 and 74.8 years when taking megestrol, and at ages 75.7 years and 82 years, after megestrol had been discontinued. figure 2. line graph showing change in meningioma sizes on ct scans over time. tumor size (in cm) is reported for the greatest dimension only. the arrow shows the time when megestrol was started and the vertical line shows the time when megestrol was discontinued. complete autopsy was performed 3 days after death. the body was that of a thin female with no somatic evidence of malignancy, including in the pelvis at the site of the original endometrial sarcoma. pathological findings the brain weight was 1137 g. the inner surface of the skull had extensive lobulated hyperostotic areas involving both frontal bones. corresponding to the sites of the tumors identified on earlier ct scans, the dura mater along the skull base anterior to the left olfactory groove had a soft, yellow lesion (approximately 2 x 1 x < 0.3 cm) (figure 3a). the dura mater in the right parasellar region was thickened and yellow. the internal surface of the paramedian left parietal convexity dura was focally thickened (approximately 3 x 2 x 0.5 cm) with adhesion to the brain (figure 3b). the leptomeninges were unremarkable. coronal slices through the cerebral hemispheres demonstrated minimal focal atrophy in the left frontal lobe tip and more pronounced atrophy with cavitation of the subcortical white matter in the left medial parietal region. figure 3. a) photograph showing the anterior cranial fossa with left frontal bone hyperostosis and yellowish thick, firm tissue (arrow) reflected from the anterior falx cerebri at the time of autopsy. b) photograph showing the thickened dura mater near the superior sagittal sinus in the left parietal region after formalin fixation. c) photomicrograph showing the left parietal dura and fibrotic tumor with lipomatous meningioma (arrow). hematoxylin & eosin stain, original magnification 12.5x. d) photomicrograph showing a cluster of meningothelial cells in the left frontal tumor. h&e stain, original magnification 200x. e) photomicrograph showing the residual left parietal tumor with immunoreactivity for epithelial membrane antigen (ema) in brown with blue hematoxylin counterstain. original magnification 100x. f) photomicrograph showing the residual left frontal tumor with nuclear immunoreactivity for progesterone receptor (pr) in brown. original magnification 200x. microscopic features of the dura mater from the three identifiable previous tumor sites were all similar showing multiple small collections of meningothelial cells (figure 3d), fibrovascular hyperplasia, and focal lipomatous meningioma (figure 3c). the latter resembled adipose tissue and did not include meningothelial cells with foamy cytoplasm i.e. lipidized or xanthomatous change [16, 17]. immunostains for epithelial membrane antigen (ema) and progesterone receptor (pr) highlighted the meningothelial cell aggregates buried within the collagenous tissue (figures 3e and 3f). nuclei in the lipomatous component were predominantly negative for pr. the cells were negative for cytokeratin (ae1ae3), estrogen receptor (er), wt1, and cd10 although fibroblasts and lipomatous cells were positive for cd10. this excludes the remote possibility that these lesions represented metastatic endometrial stromal sarcoma. an immunostain for ki67 showed no cell proliferation in the meningothelial aggregates. based upon the histopathological features, we conclude that these lesions represent regressed meningiomas. based upon the history of regression, inflammatory pseudotumor was also considered, but the patient had been on no anti-inflammatory therapy and the residual tissue mass showed no features of inflammation. a small focus of extramedullary hematopoiesis was present in the parasellar region. the left frontal lobe tip and the left medial occipital parietal region at the sites of the previous tumors were atrophic. in addition to the intracranial tumors, there was an intermediate level of alzheimer disease type neuropathologic change (nia-aa score a2, b2, c2) [18] and there were extremely rare cortical lewy bodies. there was no evidence for brainstem neurodegenerative changes to explain her tremor or swallowing difficulty. discussion spontaneous regression of intracranial meningiomas is rare and the histopathology of regressed meningiomas is not well documented. in this case report, we describe a woman with multiple meningiomas that appeared after a seven year period of treatment with megestrol, a synthetic progesterone, and that vanished approximately three years after discontinuation of the drug. it has been reported that meningiomas rarely regress following administration of estrogen antagonists e.g. mepitiostane [19], post-partum [20], following menopause [21], or “spontaneously” [22]. several case reports and small series show radiologic regression of meningiomas after cessation of progestin therapies with chlormadinone acetate, megestrol acetate, and cyproterone acetate [23–29]. in one series, osseous hypertrophy at the sites of regressed tumors was documented [8]. notably, tumor growth and regression could not be attributed temporally to any other medication or disease process. recent reviews of biological therapies used for dermatologic diseases including psoriasis highlighted very rare cases in which meningiomas were diagnosed in patients receiving methotrexate, secukinumab or etanercept. the authors concluded that these drugs did not appear to cause, exacerbate, or shrink the tumors [30, 31]. one woman was prescribed apremilast, an inhibitor of phosphodiesterase 4 (pde4), for her psoriasis during the period of meningioma regression [32]. although pde4 is reported to be expressed in some meningiomas, there is no evidence that pde4 inhibition can treat meningiomas [33]. in our case, the residual meningothelial cells were strongly immunoreactive for progesterone receptor (pr), as is often reported for meningioma [1]. in cell culture, the presence of pr determines the responsiveness of meningothelial cells to progesterone [34]. other experiments showed that progesterone in the culture medium does not have direct mitogenic effects but increases the sensitivity of meningioma cells to other mitogenic stimuli [35]. specific molecular effects of the synthetic progesterone agonist megestrol have not been studied in detail in meningioma cells. seemingly paradoxically, megestrol inhibits proliferation of cultured breast cells that express pr [36]. meningiomas however differentially express several distinct steroid receptor coactivators [37], which might explain why their response to sex hormones is variable and unpredictable [38]. the present report is the first one to show the histopathologic endpoint associated with meningioma regression. small clusters of residual meningothelial tumor cells were present in a background of lipomatous tissue. the lipomatous variant of meningioma is a rare but well-recognized entity [16]. it should be noted that the tumors, at their apex, had specific imaging characteristics with isointensegray matter on ct and mr suggestive of more common forms of meningioma, in contrast to the features of lipomatous meningioma which are hypointense on ct scans and hyperintense on t1-weighted mr [39]. whether conversion of meningioma to lipomatous meningioma [16] necessarily accompanies regression is unknown. some authors have argued about the nature of this is phenomenon which may be regressive or metaplastic [16, 39, 40]. conflicts of interest statement the authors have no conflicts of interest to report. funding statement the authors acknowledge that they received no funding in support for this work. informed consent permission to publish this report was explicitly granted by the next of kin. the university of manitoba research ethics and compliance committee approved the case report (reference # hs25528). references 1. maiuri f, mariniello g, de divitiis o, et al. 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(2021) lipomatous meningioma: clinical-pathological findings, imaging characterisation and correlations of a rare type of meningioma. in vivo 35(6): 3031-3037. https://doi.org/10.21873/invivo.12598 40. bolat f, kayaselcuk f, aydin mv, erdogan b, ulusan s, zorludemir s (2003) lipidized or lipomatous meningioma, which is more appropriate? a case report. neurol res 25(7): 764-766. https://doi.org/10.1179/016164103101202147 copyright: © 2024 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. a historical look using virtual microscopy: the first case report of adrenomyeloneuropathy (amn) feel free to add comments by clicking these icons on the sidebar free neuropathology 4:18 (2023) flashback a historical look using virtual microscopy: the first case report of adrenomyeloneuropathy (amn) herbert budka division of neuropathology and neurochemistry (obersteiner institute, formerly institute of neurology / neurological institute), department of neurology, medical university of vienna, austria corresponding author: division of neuropathology and neurochemistry (obersteiner institute, formerly institute of neurology/neurological institute) · department of neurology · medical university of vienna · vienna · austria herbert.budka@meduniwien.ac.at submitted: 11 september 2023 accepted: 20 october 2023 copyedited by: georg haase published: 26 october 2023 https://doi.org/10.17879/freeneuropathology-2023-5115 keywords: adrenoleukodystrophy (ald), adrenomyeloneuropathy (amn), peroxisomal diseases, peroxisomes, neuropathology, history abstract the history of adrenoleukodystrophy (ald), adrenomyeloneuropathy (amn) and other peroxisomal diseases is exemplary for the stunning progress of scientific medicine within the past 50 years. like many breakthroughs in medicine, the detailed analysis of patients’ pathologically affected tissues was instrumental, resulting in stepwise systematic clarification of what had remained enigmatic until the 1970s. this flashback paper is a recollection of the first neuropathological description of a slowly evolving clinical phenotype, spastic paraparesis with adrenal insufficiency, in a young adult by budka et al. 1976 [3], using virtual microscopy of the original histologic slides. the clinico-pathological presentation derives from the classical cerebral ald phenotype in boys, where electron microscopy demonstrated the underlying pathological hallmark of characteristic lipid inclusions shared by both phenotypes. our report allowed the delineation of a new disease type almost simultaneously described in more cases as amn by griffin et al. 1977 [4] and schaumburg et al. 1977 [11]. moreover, our report indicated clinical heterogeneity in the ald disease group that, as shown later, extends further to females, to addison-only, and even to asymptomatic subjects. the gene underlying ald was discovered in 1993 as a defect in the abcd1 gene. yet, it has hitherto remained unclear how the gene defect causes the strikingly broad and unpredictable phenotypic spectrum of ald/amn.   it's not what you look at that matters, it's what you see. henry david thoreau (1817-1862) [16]   the history of what is now established as adrenoleukodystrophy (ald), adrenomyeloneuro-pathy (amn) and other peroxisomal diseases is exemplary for the stunning progress of scientific medicine/medical science within the past 50 years. this journey starts with the mere description of clinico-pathological phenotypes, follows up with the identification of a biochemical abnormality, and culminates by defining the molecular and genetic basis of the disease, by newborn screening, and by promising therapies, as recently reviewed [17]. instrumental, like in many breakthroughs in medicine, was the detailed analysis of patients’ pathologically affected tissues, resulting in step-wise systematic clarification of what had remained an obscure enigma until the 1970s. moreover, the history of ald and amn is exemplary for the multidisciplinary and international character of contributing studies as a prerequisite for success. this article recollects an early step in our knowledge of the disease group where neuropathology had a pivotal role. local background of the first case of amn the present (hi)story evolved more than 50 years ago, after i became a trainee at the renowned neurological institute, or obersteiner institute, in vienna from late 1971, right after my graduation as an md. kurt jellinger (born 1931) was there in charge of clinical neuropathology, made the preliminary neuropathological autopsy report of an interesting patient who died in late 1972, and suggested me to write up the story. however, i was extremely busy in learning all elements of classical neuropathology and doing clinical training in parallel to become specialist in neurology and psychiatry. moreover, i was not yet aware of the paramount importance of publishing in science. so i failed to give priority to this task. in addition, my early years were increasingly overshadowed by kurt’s leave from the institute in 1975, a severe blow to diagnostic neuropathology and a disaster for me, as i was suddenly expected to bear most of the diagnostic workload myself; i gave a glimpse on those difficult early years elsewhere [2]. so it was may 1976 when the article was finally submitted, and was printed in the september issue of the journal of neurology (formerly: deutsche zeitschrift für nervenheilkunde and zeitschrift für neurologie) [3] that then had a mixture of germanand english-written articles. my co-authors were elfriede sluga (1930-2008, fig. 1) and wolf-dieter heiss (born 1939, fig. 2), both much more senior than me (fig. 3). figure 1. elfriede sluga 1972 (source h. budka) figure 2. wolf-dieter heiss (source european academy of neurology) figure 3. herbert budka 1972 (source h. budka) elfriede (“fritzi”) had pioneered the diagnostic use of the electron microscope (em) at the neurological institute, mostly for neuromuscular biopsies, had a dominant clinical interest and later became professor and director of a department of neurology at a municipal hospital in vienna, in kurt jellinger’s footsteps who had done the same earlier. by then, i never had a chance to touch the em, so fritzi contributed the essential em diagnostics of the report. wolf-dieter was then a consultant neurologist at the neurological university clinic in vienna, responsible for a ward of some 20 hospital beds where the patient was admitted, and an impressive teacher during my clinical training. he oversaw clinical details of the report and later became professor at the university of cologne and director of the max planck institute for brain research in köln-merheim, and is now widely recognized as a top expert on cerebral hemodynamics, including a pioneering role in the establishment of stroke units. wolf-dieter, a giant in clinical neurosciences in his own right, originally went to cologne to succeed another giant, klaus-joachim zülch (1910-1988) who had combined clinical neurology with neuropathology as neuro-oncologist and stroke expert; he is now remembered as main author of the very first edition of the famous who blue book series on histological typing of tumours of the cns [18]. for the neurological institute in vienna, our report laid the foundation for a new research and diagnostic area that became important not only for the institute’s neuropathology branch, but still more so for its division of neurochemistry, led by hans (“hanno”) bernheimer (1930-2016), and mainly involving brunhilde molzer (born 1940) [7] with very long chain fatty acids (vlcfa) diagnostic biochemistry for ald/amn suspects. in the 1990s johannes berger (born 1964) joined the neurochemistry group and expanded its molecular and experimental studies; from 2000, he has done research on peroxisomes as professor in the newly founded center of brain research in vienna. the patient our propositus was a relatively healthy – well, not quite – man who succumbed quickly and rather unexpectedly in 1972 at the age of 24, after a slowly evolving disease with spastic paraparesis over 2 years. no definite diagnosis was made during lifetime. you can make yourself familiar with the (neuro)pathology at autopsy by viewing scanned slides of the right adrenal gland (fig. 4) and of the medulla oblongata (figs. 5 & 6). indeed, i strongly recommend you to stop here and do your virtual microscoping before you continue to read more details. there are of course different types of observers, but my favorite personal style has always been the detective-style approach, starting with a few basic data (sufficient here in the preceding paragraph), and aiming to have a more complete story evolving from the discoveries during microscoping (and earlier autopsy with brain cutting). if you read the more detailed description already at start, you might easily and quickly look at everything, but it may happen that you do not see in thoreau’s sense (see citation at the start of this article). moreover, the suggested early microscoping reflects the usual neuropathologist’s way of working: ultimately, you learn most from what you initially might not see. recapitulation of the case report in our report, we made a detailed description of the clinical course, laboratory findings, and results of a complete autopsy including histology and electron microscopy of various organs. there is nothing new that can be added since then. so an excerpt follows for those who are unable or unwilling to read the original paper. our propositus had a family history of an unusual dark skin pigmentation of his mother and was hyperpigmented since childhood. originally, this was an active young man who used to make bicycle tours. at age 22, progressive weakness was noted in both legs. subsequently, bladder and anal sphincter dysfunction developed; sexual functions were unimpaired. seven months before death, he presented at the neurologic clinic in vienna with a deeply bronzed skin, alopecia including axillary and pubic hairs, and a spastic gait with bilaterally exaggerated tendon reflexes of legs, bilateral babinski’s sign, and loss of abdominal reflexes. there was hypesthesia of both legs for all qualities, with distal accentuation. when wolf-dieter heiss first saw the patient, he immediately suspected addison’s disease, because of the bronzed skin. however, extensive involvement of endocrinology specialists did not result in such a clear-cut diagnosis during the lifetime of the patient. thereafter, laboratory results as detailed in our report were interpreted as compatible with primary adrenal insufficiency. after a visit to a sauna bath 2 weeks before death, the patient deteriorated rapidly, with collapse, vomiting, vertigo, and impairment of consciousness alternating between agitation and sopor. one week before death, hyperthermia and progressive metabolic and circulatory dysregulation developed. death was caused by cardiovascular failure. a complete autopsy at the institute of pathology in vienna was performed by the then famous prof. lothar (“rex”) kucsko (1912-1976) who contributed also otherwise to my career [2]. he described the adrenals as atrophic, firm, chocolate-brown and without discernible structure, but including two small nodules on the right side; his final diagnosis was so-called immune adrenalitis, with an acute addison crisis as cause of death. he was perfectly right on the latter, less so on the former. histology of various other organs was characterized by widespread scattered lymphocytic infiltrates. under the microscope: what you may see fig. 4 adrenal (he). the organ structure is disorganized, with intact medulla (bluish stained tissue) but only focal remnants of cortical tissue (reddish stained tissue) with large, sometimes bizarre and ballooned cells with occasional cytoplasmic “striations”. in addition, there is prominent fibrosis and focal lymphocytic infiltration. fig. 5 & 6 medulla oblongata (lfb, he). there is symmetric degeneration of pyramids, to a lesser degree of medial and lateral lemnisci, spinocerebellar and goll’s tracts. in addition to perivascular lymphocytic infiltrates, there are perivascular cuffs of histiocytes with epitheloid appearance and occasional cytoplasmic “striation” (i am not sure how obvious the striation is on 2-d virtual files that don’t allow a 3-d impression, in contrast to old-fashion microscoping with a glass slide when you can play with the focusing screw). figure 4. right adrenal (h & e) figure 5. oblongata (luxol fast blue & nuclear fast red) figure 6. oblongata (h & e) clicking into the picture will lead you to the full virtual slide https://doi.org/10.57860/min_dts_000022 medico-scientific background for decades, what is now called ald has been a rare and poorly understood, but rapidly evolving and fatal demyelinating disease of male kids, a combination of inflammatory diffuse sclerosis of the brain with adrenal insufficiency. haberfeld and spieler have been credited with the first clinico-pathological description of classical childhood ald [5], followed by articles by schilder who gave his name to what he called encephalitis periaxialis diffusa [13][14][15]. in the 1970s, most research on ald was concentrated at the albert einstein college of medicine in bronx, new york. the department of pathology there was a hotbed of neuropathology, with eminent researchers like robert d. terry and henryk m. wisniewski who contributed landmark studies in alzheimer disease, and john w. prineas and cedric raine who did the same in multiple sclerosis. a young trainee, james m. powers, teamed up with herbert h. schaumburg, an expert in neurotoxicology and peripheral nerves, ann johnson and kunihiko suzuki, to study a disease that just recently had been termed ald [1]; their work over the next few years elucidated most of its (neuro)pathology and biochemistry. powers and schaumburg demonstrated characteristic lipid inclusions in ald adrenals by em examination [9], followed by their description in brain [12] and other organs [10]. subsequent neurochemical studies showed a striking excess of vlcfa in brain cholesterol esters as substrate of ald inclusions [6], clearing the path to biochemical assays that subsequently have dominated diagnostics in samples of cultured skin fibroblasts, amniocytes and plasma. independently from our report, griffin et al. [4], and schaumburg et al. [11], published in 1977 a small clinico-pathologic series of patients with familial spastic paraparesis with addison’s disease, ald-like ultrastructure and biochemistry, and coined the term adrenomyeloneuropathy (amn). from the 1980s up to now, much of clinical ald/amn research originated from the kennedy-krieger research institute in baltimore, md, led by hugo moser (1924-2007), the unrivalled eminence in peroxisomal diseases, and his wife ann, resulting in establishment of methods for early diagnosis and culminating in identification of the genetic basis by defects of the aldp/abcd1 gene [8] encoding for a peroxisomal membrane protein and member of the atp-binding cassette (abc) transporter family. on top of that, kennedy-krieger contributed much more that is by now known in peroxisomal diseases. moreover, they had a major role in development and assessment of therapies such as “lorenzo’s oil“ of hollywood fame, bone marrow and hematopoietic stem cell transplantation, and gene therapy [17]. still more impressively, hugo and ann moser established an absolutely exemplary relation to their ald/amn patients and their families who returned their love and affection to these two outstanding advocates for persons suffering from neurological disabilities. amn (neuro)pathology the (neuro)pathology of amn is typical but not specific without appropriate clinical information including family history, vlcfa biochemistry and/or em findings. nowadays genetic testing for the aldp/abcd1 gene is the mainstay of definite diagnostics. neuropathology, as seen in the virtual slides, is characterized by two hallmarks: first, degeneration of long tracts of brainstem and spinal cord in a pseudosystemic distribution, and second, the presence within degenerating long tracts of perivascular sleeves of lipid-filled histiocytes that rarely may, or may not, give a “striated” impression. this striation might be the light microscopical appearance of the circumscribed packaging of lamellar lipid aggregates with clear clefts as seen by em. in parallel, such degenerative changes may also be present in the peripheral nervous system. the scattered lymphocytic infiltrates are to be interpreted as secondary to the adrenal insufficiency that may, or may not, be manifest in an otherwise typical amn. the pathology of the adrenal glands reflects the degree of adrenal insufficiency, with storage of lipids in enlarged cortical cells. originally, in addition to histology, em examination was important to document peculiar lipidic inclusions with a lamellar “leaflet” structure in perivascular histiocytes of the oblongata pyramids, as described and illustrated on two pages of our report, and found earlier as characteristic of ald by powers and schaumburg [9] and schaumburg et al. [12]. we suggested the ultrastructural inclusions in the cns to derive from degenerated myelin in the affected tracts, linking ald/amn to pathological storage of a myelin degradation product [3]. significance our study [3] allowed the delineation of what became a new disease type, a little bit later termed amn [4][11]. the presentation deviated from the classical cerebral ald phenotype in boys. however, histology and electron microscopy demonstrated the underlying pathological hallmark of characteristic inclusions to be shared by both phenotypes. moreover, as the first pathological description of slowly evolving spastic paraparesis with adrenal insufficiency in young adults, it indicated clinical heterogeneity in the ald disease group that, as shown later, extends further to females [7] with the same heterogeneity as in males, to addison-only, and even to asymptomatics [17]. neonatal ald is now considered another disease with different genetic defects. despite all progress, however, it has remained unclear how defects of the abcd1 gene cause the strikingly broad, and unpredictable, phenotypic spectrum of ald/amn. our publication has met moderate interest, at least in scientometric terms: by sept. 3, 2023, google scholar listed 125 citations. indeed, in early years after the publication citations were rather scarce, until hugo moser started to cite it in his reviews on the history of the field. it was a great pleasure and honor when he was visiting our institute in vienna a few times, and i was greatly impressed by his personality, openness and kindness, knowledge and willingness to co-operate on behalf of his patients. i was also happy to have met jim powers a few times, a kind and experienced colleague and now the foremost neuropathologist of ald/amn. we discussed our mutual interests, and it is clear that he – with herb schaumburg, cedric raine, jack griffin, peter spencer, john prineas and others – published their amn cases in the december 1977 issue of neurology [11] without being aware of our preceding publication. indeed there was no pubmed or internet at that time. anyway, our stories are another example that scientific progress may occur simultaneously and independently in distant locations and settings. references 1. blaw me: melanodermic type leukodystrophy (adreno-leukodystrophy). in: vinken pj, bruyn gw (eds): handbook of clinical neurology vol.10, pp. 128-133. amsterdam: north holland 1970 isbn 9780444100597, 0444100598 oclc 872408480 2. budka h: neuropathology through the ages – personal reflections: the golden era of neuropathology. free neuropathol 1: 15 (26 pp.) (2020). https://www.uni-muenster.de/ejournals/index.php/fnp/article/view/2817 3. budka h, sluga e, heiss w-d: spastic paraplegia associated with addison's disease: adult variant of adreno-leukodystrophy. j neurol 213 (3): 237-250 (1976). https://doi.org/10.1007/bf00312873 4. griffin jw, goren e, schaumburg h, engel wk, loriaux l: adrenomyeloneuropathy: a probable variant 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9241760214 oclc 567810677 copyright: © 2023 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. 64th meeting of the french society of neuropathology meeting abstracts , december 2nd. 2022 feel free to add comments by clicking these icons on the sidebar free neuropathology 4:5 (2023) meeting abstracts 64th meeting of the french society of neuropathology meeting abstracts december 2nd, 2022 centre hospitalier sainte-anne (paris) amphithéâtre de l'institut de psychiatrie et de neurosciences the french society of neuropathology was created in 1989, succeeding the french club of neuropathology set up in 1965.   submitted: 22 march 2023 accepted: 23 march 2023 published: 04 april 2023 https://doi.org/10.17879/freeneuropathology-2023-4776 keywords: french society of neuropathology, sfnp, meeting abstracts, 64th meeting dec. 2022   neuro-oncology   free neuropathol 4:5:2-3 clinico-pathological and epigenetic heterogeneity of diffuse gliomas with fgfr3:tacc3 fusion alice métais1,2, arnault tauziède-espariat1,2, jeremy garcia3, romain appay3,4, emmanuelle uro-coste5, david meyronet6,7,8, claude-alain maurage9, fanny vandenbos10, valérie rigaud11, dan christian chiforeanu12, johan pallud13,2, yann suhan senova14, raphaël saffroy15, carole colin4, myriam edjlali16,17, pascale varlet1,2, dominique figarella-branger3,4 and contributors of the biopathology renoclip-loc network18 ghu psychiatrie et neurosciences, site sainte-anne, service de neuropathologie, paris, france institut de psychiatrie et neurosciences de paris (ipnp), umr_s1266, inserm, université de paris, equipe ima-brain (imaging biomarkers for brain development and disorders), paris, france aphm, chu timone, service d'anatomie pathologique et de neuropathologie, marseille, france aix-marseille univ, cnrs, inp, inst neurophysiopathol, marseille, france department of pathology, toulouse university hospital, toulouse, france groupe hospitalier est, département de neuropathologie, hospices civils de lyon, bron, france claude bernard university lyon 1, lyon, france department of cancer cell plasticity – inserm u1052 cancer research center of lyon, lyon, france department of pathology, lille university hospital, lille, france department of neuropathology, hôpital pasteur, nice, france department of pathology, gui de chauliac hospital, montpellier university medical center, montpellier, france service d'anatomie et cytologie pathologiques, pontchaillou university hospital, rennes, france department of neurosurgery, ghu paris psychiatrie et neurosciences, paris, france departments of neurosurgery and psychiatry, assistance publique-hôpitaux de paris (aphp) groupe henri-mondor albert-chenevier, créteil, france department of biochemistry and oncogenetic, aphp, paul-brousse hospital, villejuif, france department of radiology, aphp, hôpitaux raymond-poincaré & ambroise paré, dmu smart imaging, gh université paris-saclay, u 1179 uvsq/paris-saclay, paris, france laboratoire d'imagerie biomédicale multimodale (biomaps), université paris-saclay, cea, cnrs, inserm, service hospitalier frédéric joliot, orsay, france biopathology reno-clip-loc network: a. rousseau (angers), c. godfraind (clermont-ferrand), g. gauchotte (nancy), k. mokhtari and f. bielle (paris), f. escande (lille), f. fina (marseille) gliomas with fgfr3::tacc3 fusion mainly occur in adults, display pathological features of glioblastomas (gb) and are usually classified as glioblastoma, idh-wildtype. however, cases demonstrating pathological features of low-grade glioma (lgg) lead to difficulties in classification and clinical management. we report a series of 8 gb and 14 lgg with fgfr3::tacc3 fusion. tert promoter mutation was recorded in all gb and 6/14 lgg. among the 7 cases with a methylation score >0.9 in the classifier (v12.5), 2 were classified as gb, 4 as ganglioglioma (gg) and 1 as dysembryoplastic neuroepithelial tumor (dnet). t-sne analysis and unsupervised hierarchical clustering showed epigenetic heterogeneity among fgfr3::tacc3 fused gliomas. relevant factors associated with a better prognosis were age <40, fgfr3(ex17)::tacc3(ex10) fusion type and lack of tert promoter mutation. among gliomas with fgfr3::tacc3 fusion, age, tert promoter mutation, pathological features, dna-methylation profiling and fusion subtype are of interest to determine patients’ risk.   free neuropathol 4:5:4 chromothripsis, one major genetic instability factor in glioblastoma, is rare in idh-mutant gliomas baptiste sourty1,2, laëtitia basset1,2, emmanuel garcion2, audrey rousseau1,2 département de pathologie, chu angers, 4 rue larrey, 49933 angers, france univ angers, nantes université, inserm, cnrs, crci2na, sfr icat, f-49000 angers, france introduction: chromothripsis (ct) and whole-genome duplication (wgd) lead to massive chromosomal alterations that characterize genomic instability. those events are well described in glioblastomas, but scarcely in idh-mutant gliomas. their better prognosis may be related to their genomic stability compared to glioblastomas. methods: pangenomic profiles of 301 gliomas were analyzed by snp array (196 glioblastomas; 105 idh-mutant gliomas). tumor ploidy and ct events were assessed through manual screening and bioinformatics. results: thirty-seven glioblastomas (18.8%) displayed ct versus 5 idh-mutant gliomas (4.8%, p = 0.0007) (all high-grade astrocytomas). wgd was detected in 18 glioblastomas (9.2%) and 9 idh-mutant gliomas of any subtype and grade (8.6%), preceding 75% of chromosomal losses. conclusion: ct is rare in idh-mutant gliomas compared to glioblastomas, contributing to the genomic stability of oligodendrogliomas and grade 2 astrocytomas. ct occurrence in high-grade astrocytomas may underlie aggressive biological behavior. wgd occurs early, as much in idh-mutant gliomas as in glioblastomas.   free neuropathol 4:5:5-6 molecular and clinical diversity in primary central nervous system lymphoma i. hernández-verdin1, e. kirasic1, k. wienand2, k. mokhtari1,3, s. eimer4, h. loiseau5, a. rousseau6, j. paillassa7, g. ahle8, f. lerintiu9, e. uro-coste10, l. oberic11, d. figarella-branger12, o. chinot13, g. gauchotte14, l. taillandier15, j-p. marolleau16, m. polivka17, c. adam18, r. ursu19, a. schmitt20, n. barillot1, l. nichelli21, f. lozano-sánchez22, m-j. ibañez-juliá23, m. peyre1,24, b. mathon1,24, y. abada22, f. charlotte25, f. davi26, c. stewart27, a. de reyniès28, s. choquet25, c. soussain29, c. houillier22, b. chapuy2, k. hoang-xuan1,22, a. alentorn1,22* institut du cerveau-paris brain institute-icm, inserm, sorbonne université, cnrs, f-75013 paris, france department of hematology and medical oncology, university medical center göttingen, 37075 göttingen, germany and department of hematology, oncology and cancer immunology, campus benjamin franklin, charité universitätsmedizin berlin, corporate member of freie universität berlin and humboldt-universität zu berlin, 12203, berlin, germany department of neuropathology, groupe hospitalier pitié salpêtrière, aphp, f-75013, paris, france department of pathology, chu de bordeaux, hôpital pellegrin, 33076, bordeaux, france department of neurosurgery, bordeaux university hospital center, pellegrin hospital, place amélie raba-léon, 33076, bordeaux and ea 7435 imotion, university of bordeaux, bordeaux, france departement of pathology, pbh, chu angers, 4, rue larrey, 49933 angers cedex 9, france and crcina, université de nantes université d'angers, angers, france department of hematology, chu angers, 4, rue larrey, 49933 angers cedex 9, france department of neurology, hôpitaux civils de colmar, colmar, france department of neuropathology, hôpitaux civils de colmar, 68000 strasbourg, france department of pathology, chu de toulouse, iuc-oncopole, 31300 toulouse and inserm u1037, cancer research center of toulouse (crct), 31100 toulouse, université toulouse iii paul sabatier, 31062 toulouse, france department of hematology, iuc toulouse oncopole, toulouse, france neuropathology department university hospital timone, aix marseille university, marseille and inst neurophysiopathol, cnrs, inp, aix-marseille university, marseille, france department of neuro-oncology, chu timone, aphm, marseille, france and inst neurophysiopathol, cnrs, inp, aix-marseille university, marseille, france department of biopathology, chru nancy, chru/icl, bâtiment bbb, rue du morvan, 54511, vandoeuvre-lès-nancy and department of legal medicine, chru nancy, vandoeuvre-lès-nancy and inserm u1256, university of lorraine, vandoeuvre-lès-nancy and centre de ressources biologiques, bb-0033-00035, chru, nancy, france department of neuro-oncology, chru-nancy, université de lorraine, nancy, france department of hematology, chu amiens-picardie, amiens, france department of anatomopathology, lariboisière hospital, assistance publique hopitaux de paris, university of paris, paris, france pathology department, bicêtre university hospital, public hospital network of paris, le kremlin bicêtre, france department of neurology, université de paris, ap-hp, hôpital saint louis, 75010, paris, france department of hematology, institut bergonié hospital, bordeaux, france department of neuroradiology, sorbonne université, assistance publique-hôpitaux de paris, groupe hospitalier pitié-salpêtrière-charles foix, paris, france department of neurology-2, sorbonne université, assistance publique-hôpitaux de paris, groupe hospitalier pitié-salpêtrière-charles foix, paris, france department of neurology, perpignan hospital, perpignan, france department of neurosurgery, sorbonne université, assistance publique-hôpitaux de paris, groupe hospitalier pitié-salpêtrière-charles foix, paris, france department of pathology, aphp, hôpital pitié-salpêtrière and sorbonne university, paris, france department of hematology, aphp, hôpital pitié-salpêtrière and sorbonne university, paris, france broad institute of mit and harvard, cambridge, ma 02142, usa inserm umr_s1138 centre de recherche des cordeliers université pierre et marie curie et université paris descartes, paris, france hematology unit, institut curie, 92210 saint-cloud, france primary central nervous system lymphoma (pcnsl) is a distinct extranodal lymphoma presenting with limited stage disease but variable response rates to treatment despite homogenous pathological presentation. the likely underlying molecular heterogeneity and its clinical impact is poorly understood. we performed a comprehensive multi-omic analysis (whole-exome sequencing, rna-seq, and methyl-seq) in a discovery cohort of 147 immunocompetent pcnsls and a validation cohort of 93 pcnsls. these data were integrated and correlated with the clinico-radiological characteristics and outcomes of the patients, allowing us to identify four significant clusters within pcnsl with shared causative biologic factors of disease and outcome. we found evidence of the microenvironment playing a key role where in two clusters was associated with hypermethylation and in one with high proliferation and polycomb repressive complex 2 activity. meningeal infiltration was associated with a group enriched for hist1h1e mutations. functional analysis on proposed targets supports potential precision-medicine strategies in these pcnsl subtypes.   mini-symposium in memoriam pr charles duyckaerts pr danielle seilhean   free neuropathol 4:5:7 the role of environmental factors on sporadic creutzfeldt-jakob disease mortality: evidence from an age-period-cohort analysis angéline denouel, msc1, jean-philippe brandel, md1,2, danielle seilhean, md, phd1, jean-louis laplanche, phd3,4, alexis elbaz, md, phd5†, stéphane haik, md, phd1,2† cnrs umr 7225, inserm u1127, paris brain institute, sorbonne universités, paris, france ap-hp, centre national de référence des maladies de creutzfeldt-jakob, groupe hospitalier pitié-salpêtrière, paris, france département de biochimie et biologie moléculaire, hôpitaux lariboisière-fernand widal, paris, france inserm, umr 1144, “optimisation thérapeutique en neuropsychopharmacologie”, paris, france université paris-saclay, uvsq, univ. paris-sud, gustave roussy, inserm, u1018, team « exposome, heredity, cancer, and health », cesp, 94807, villejuif, france † these authors share senior authorship sporadic creutzfeldt-jakob disease (scjd) is the most common form of human prion diseases. its origin is still unknown and the role of exogenous factors remains possible. we aimed to study scjd mortality from data collected over 25-years (1992-2016) of active surveillance in france using an age-period-cohort (apc) model in order to better understand scjd origin. our study revealed that several factors influence mortality of scjd over time. indeed, apc analyses highlighted processes linked to aging through an age effect, improvement of surveillance system through a period effect, and, unexpectedly, showed a cohort effect supporting the role of unknown environmental risk factors in disease occurrence. in addition, an age-dependent gender effect was shown with a shift in men-to-women mortality ratio at the age peak. this approach was performed for all scjd cases and for patients associated with the most frequent strain of scjd (i.e. the m1 strain) with similar results.   free neuropathol 4:5:8 astrocytic permeability disorder in spheroid leukoencephalopathy with csf1r mutation teresa dot gomara1, sabrina leclère2,3, isabelle plu1,2,3, susana boluda casas1,2,3, danielle seilhean1,2,3 department of neuropathology, pitié-salpêtrière hospital, aphp, sorbonne university, paris, france neuroceb brainbank, france cnrs umr7225, inserm u1127, paris brain institute, sorbonne university, france adult-onset leukoencephalopathy with axonal spheroids and pigmented glia (alsp) is a rare, progressive neurological disease associated with mutations in csf1r (colony-stimulating factor-1 receptor) gene, known to control the production, differentiation and function of macrophages. we analyzed a series of seven cases of alsp with regards to microglial, astrocytic and axonal markers. we observed a progressive loss of microglia associated with a change in the shape and size of remaining cells. the distribution of astrocytic aquaporin-4 (aqp4) was significantly different in alsp compared to controls. in the relatively spared subcortical regions, an abundance of ramified iba1+ microglia was noticed as well as a strong expression of astrocytic aqp4. in contrast, in the most severely affected regions these markers were extinguished. these arguments provide evidence for the toxic role of microglia and disorders of water flow between cells as factors in the progression of lesions in the disease.   free neuropathol 4:5:9 neuropathological differences between down syndrome and familial alzheimer’s disease with app duplication: role of endothelial cells in cerebral amyloid angiopathy a. kasri1, s. boluda1, l. valay1, m. danjou1, v. montecalvo1, c. duyckaerts1, l. stimmer1, e. gkanatsiou2, g. brinkmalm2,3, y. vermeiren4,5, s. e. pape6, p. p. de deyn4,5, m. irmler7,8, j. becker7,8, h. zetterberg2,9, a. strydom6, m.-c. potier1 paris brain institute, icm, cnrs umr7225 inserm u1127 – upmc, paris, france institute of neuroscience and physiology, the sahlgrenska academy at the university of gothenburg, gothenburg, sweden sahlgrenska university hospital, clinical neurochemistry laboratory, mölndal, sweden department of biomedical sciences, neurochemistry and behavior, institute born-bunge, university of antwerp, antwerp, belgium department of neurology and alzheimer center, university of groningen, university medical center groningen (umcg), groningen, netherlands institute of psychology and neuroscience, king’s college london, 16 de crespigny park, london, united kingdom helmholtz zentrum münchen, neuherberg, germany technical university munich, tum school of life sciences, freising department of molecular neuroscience, ucl institute of neurology, queen square, london, united kingdom while amyloid plaques are common in all ad cases, caa is mainly found in familial ad with duplications of the app gene (dup-app), down syndrome (ds), and specific app mutations. mechanisms leading to these differences are not yet understood. we investigated the diversity of neuropathological phenotypes in sporadic ad (sad), dup-app, app mutations, ds with or without dementia (d), and control cases. in addition, we analysed endothelial cells derived from ipsc lines (ipsc-d-ecs) of patients to model the vessel wall forming the blood-brain barrier. aβ deposits in the parenchyma were numerous in sad, ds-d and app mutations, but less abundant in dup-app sections (12 sad, 7 dup-app, 3 ds, 10 ds-d and 9 app mutations cases). conversely, aβ deposits in the blood vessels (arteries and arterioles) were prominent in dup-app, less abundant in ds-d and scarce in sad and app mutations. only dup-app cases showed aβ deposits in the capillaries. despite striking differences in aβ pathologies, all cases with dementia had high tau pathology. ipsc-d-ecs secreted substantial amounts of aβ peptides. we identified changes in the morphology and tight junctions of ipsc-d-ecs with dup-app as well as specific gene expression dysregulations, suggesting intrinsic remodelling of ecs of the blood-brain barrier in dup-app. our study reveals new pathophysiological mechanisms involved in specific aβ production and deposition in the blood vessel wall of patients carrying dup-app involving ec. differences between dup-app and ds suggest the presence in the ds populations of protective factors against caa.   free neuropathol 4:5:10 genesis and plasticity of the als concept in research: what function for what history? anne fenoy1, danielle seilhean2, claire crignon3 umr 8011 snd, initiative humanités biomédicales, sorbonne université, paris, france inserm u1127, cnrs u7225, sorbonne université, institut du cerveau, paris, france & ap-hp, hôpital pitié-salpêtrière, département de neuropathologie, paris, france umr 7117, archives poincaré, université de lorraine, nancy, france the identification of als by jean-martin charcot in 1873 appears as a founding moment in the history of neuropathology and neurology. through the anatomical-clinical method, charcot was able to provide a first description of the main mechanisms of the disease by combining clinical and neuropathological observations. the history of knowledge of als is often reduced to the history of charcot, whose work is commemorated, notably by the use of the eponym "charcot's disease". reducing the story to a hagiographic approach can lead to a misguided view of how als research works and how it has evolved over time. a combined philosophical, historical, and observational field approach allows us to understand the evolution of the concepts and models developed. the comparison of representations questions the evolution of practices and models and their interpretation, from the original case study to the models, up to the use of animal models.   free neuropathol 4:5:11 neurofilament accumulations in amyotrophic lateral sclerosis patients’ motor neurons impair axonal initial segment integrity cynthia lefebvre-omar1, elise liu1, carine dalle1, boris lamotte d'incamps2, stéphanie bigou1, clément daube1, marc davenne1, noémie robil3, coline jost-mousseau1, françois salachas1,4, lucette lacomblez1,4, danielle seilhean1,5, christian s. lobsiger1, stéphanie millecamps1, séverine boillée1, delphine bohl1 sorbonne université, institut du cerveau – paris brain institute – icm, inserm, cnrs ap-hp, hôpital de la pitié-salpêtrière, paris, france université de paris, saints-pères paris institut des neurosciences, cnrs, paris, france genosplice technology, paris, france département de neurologie, centre de référence sla ile de france, assistance publique hôpitaux de paris (ap-hp), sorbonne université, hôpital de la pitié-salpêtrière, paris, france département de neuropathologie, assistance publique hôpitaux de paris (ap-hp), sorbonne université, hôpital de la pitié-salpêtrière, paris, france neurofilament (nf) levels in patient’ fluids have emerged as the prime biomarker of amyotrophic lateral sclerosis (als) disease progression, while nf accumulation in mns of patients is one of the oldest pathological hallmarks. however, the way how nf accumulations contribute to mn degeneration remains unknown. to assess nf accumulations and study the impact on mns, we compared mns derived from induced pluripotent stem cells (ipsc) of als patients carrying different mutations. our results show that the integrity of the mn axonal initial segment (ais), the region of action potential initiation and responsible for maintaining axonal integrity, is impaired in the presence of phosphorylated nf-m/h accumulations in mns. our results expand the understanding of how nf accumulation could dysregulate components of the axonal cytoskeleton and disrupt mn homeostasis. thus, preserving ais integrity could open new therapeutic opportunities for als.   free neuropathol 4:5:12 design of a customizable relational database to study clinic pathological correlations in autopsied series isabelle journe-mallet1, julien gouju1, frederique etcharry-bouyx2, valerie chauvire2, virginie guillet-pichon2,3,4, christophe verny3,4, franck letournel1, philippe codron1,4,5,6 laboratoire de neurobiologie et neuropathologie, centre hospitalier universitaire d’angers, angers, france centre mémoire de ressource et de recherche, centre hospitalier universitaire d’angers, angers, france centre de référence des maladies neurogénétiques, centre hospitalier universitaire d’angers, angers, france univ angers, inserm, cnrs, mitovasc, sfr icat, angers, france centre de référence des maladies neuromusculaires aoc, centre hospitalier universitaire d’angers, angers, france centre de ressources et de compétences sur la sla, centre hospitalier universitaire d'angers, angers, france the analysis of clinicopathological correlations in autopsy series remains a central method to improve our understanding of neurodegenerative diseases. however, this approach requires a wealth of information to be relevant, and the use of standard spreadsheet software to collect and manage such volume of data is hardly suitable. to overcome this constraint, we used an open-source database management systems (dbms) to design a customizable neuropathology form with 456 data entry fields. this approach allowed us to optimize the collection of clinical and pathological data from our brain collection, with an average filling time of about 10 minutes per patient. next, we could easily retrieve information from the generated database (22,885 data points) with multiple and conditional queries to study clinicopathological correlations and to quickly identify cases for both diagnosis and research purpose. the large amount of generated data in clinicopathological studies should encourage a more systematic use of dbms. copyright: © 2023 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. neurodevelopmental disorders: 2023 update feel free to add comments by clicking these icons on the sidebar free neuropathology 4:8 (2023) review neurodevelopmental disorders: 2023 update paulina carriba1,3, nicola lorenzón1, mara dierssen1,2,3 centre for genomic regulation (crg), the barcelona institute of science and technology, barcelona, spain universitat pompeu fabra (upf), barcelona, spain centro de investigación biomédica en red de enfermedades raras (ciberer), madrid, spain corresponding author: mara dierssen · systems biology program · crg-center for genomic regulation · c/ dr. aiguader, 88 · prbb building · 08003 barcelona spain mara.dierssen@crg.eu submitted: 28 february 2023 accepted: 30 april 2023 copyedited by: aivi nguyen published: 08 may 2023 https://doi.org/10.17879/freeneuropathology-2023-4701 keywords: centrosomes, clip: caudal late interneuron progenitor, human organoids, mcd: malformations of cortical development, stress granules, sudc: sudden unexplained death in childhood abstract several advances in the field of neurodevelopmental diseases (ndds) have been reported by 2022. of course, ndds comprise a diverse group of disorders, most of which with different aetiologies. however, owing to the development and consolidation of technological approaches, such as proteomics and rna-sequencing, and to the improvement of brain organoids along with the introduction of artificial intelligence (ai) for biodata analysis, in 2022 new aetiological mechanisms for some ndds have been proposed. here, we present hints of some of these findings. for instance, centrioles regulate neuronal migration and could be behind the aetiology of periventricular heterotopia; also, the accumulation of misfolded proteins could explain the neurological effects in covid-19 patients; and, autism spectrum disorders (asd) could be the expression of altered cortical arealization. we also cover other interesting aspects as the description of a new ndd characterized by deregulation of genes involved in stress granule (sg) assemblies, or the description of a newly discovered neural progenitor that explains the different phenotypes of tumours and cortical tubers in tuberous sclerosis complex (tsc) disease; and how it is possible to decipher the aetiology of sudden unexplained death in childhood (sudc) or improve the diagnosis of cortical malformations using formalin-fixed paraffin-embedded samples. abbreviations ad – alzheimer’s disease; adhd attention deficit/hyperactivity disorder; ai – artificial intelligence; asd autism spectrum disorder; bcs – balloon cells; ca – cornu ammonis; cd cluster of differentiation; cge caudal ganglionic eminence; clip caudal late interneuron progenitor; cns – central nervous system; dg – dentate gyrus; egfr epidermal growth factor receptor; eif2 eukaryotic initiation factor 2; fcd – focal cortical dysplasia; ffpe formalin-fixed paraffin-embedded; fs febrile seizure; gcs giant cells; go – gene ontology; hand – hiv-associated neurocognitive disorder; hco human cortical organoids; het – heterozygous; hiv – human immunodeficiency virus; id intellectual disability; ipscs induced pluripotent stem cells; ko – knockout; loh loss of heterozygosity; mcds – malformations of cortical development; mtor mammalian target of rapamycin; nsc – neural stem cells; orf – open reading frame; ph – periventricular heterotopia; prpf6 pre-mrna processing factor 6; rna-seq – rna sequencing; sars-cov-2 severe acute respiratory syndrome coronavirus 2; sgs stress granules; sudc sudden unexplained death in childhood; sudep sudden unexpected death in epilepsy; t-hco – transplanted human cortical organoids; ts – timothy syndrome; tsc tuberous sclerosis complex; wt wild-type. introduction for this new collection of the most relevant findings in neurodevelopmental disorders that appeared in 2022, our selection tried to encompass a wide range of aspects that we think could be of interest to neuropathologists. the topics chosen are: neurodevelopmental disorders and the proper space and time sequential events during brain neurodevelopment stress granule assemblies and neurodevelopmental disorders clip, a newly discovered interneuron progenitor, explains the divergent phenotype in tuberous sclerosis complex disorder improving the diagnostic of malformations of cortical development (mcds) diseases by dna methylation profile patterns deciphering the aetiology of sudden unexplained death in childhood (sudc) by proteomics transcriptomic dysregulation in asd occurs across the whole cerebral cortex and follows a regional gradient in vivo platform for the study of human neurodevelopmental disorders mitochondria participate in the neuron-glia crosstalk gaining insight on the role of hiv-1 in the cns novel mechanisms explaining covid-19 neurological anomalies along these subject matters we aim to discuss advances in different ndds, from brain malformations or classical neurodevelopmental conditions to more general aspects that a neuropathologist might face, such as paediatric neurological alterations associated with covid-19 or hiv-associated neurocognitive disorder (hand) in children. we also selected relevant findings regarding how formalin-fixed paraffin-embedded (ffpe) samples, the major form of stored brain samples, could be used for studying neurodevelopmental disorders, and how the use of artificial intelligence (ai) can improve the diagnosis of cortical malformations. some of the selected topics also provide new mechanistic insights, such as the newly discovered neural progenitor clip, which explains the divergent phenotypes in tuberous sclerosis complex (tsc) pathology. indeed, one of the most remarkable topics of 2022, which has acquired increasing relevance in recent years, is the importance of the spatial and temporal regulation of brain development. the coordinated and orchestrated series of cellular processes controlled by fine-tuned sets of genetic programs during neurodevelopment leads to immense cell diversity, with different features depending on their final fate, localisation, and properties distinctive from the cells from which they developed. those cells will be part of circuits that are adjusted, readjusted and refined by intrinsic and extrinsic signals (rubenstein and rakic, 1999; miyata et al., 2010; kwan et al., 2012; greig et al., 2013; wamsley et al., 2018; di bella et al., 2021; bonnefont and vanderhaeghen, 2021) in a precise spatial-temporal manner. within this choreographic arrangement, a single out-of-tune event in time or space may represent the inception of a neurodevelopmental pathological condition. finally, human-derived organoids continue to be a promising in vitro tool for modelling human physiological and pathological development (figure 1). in the last year, those systems gained popularity thanks to specific improvements in successfully modelling neurodevelopmental disorders, allowing the study of human neuronal function in an in vivo context. figure 1. schematic representation of some uses of human-derived organoids presented in this update. a) cerebral organoids derived from ipscs can be used for single-cell rna-sequencing (scrnaseq) to determine expression patterns for the study of mechanisms driving neurodevelopmental disorders. here presented in neurodevelopmental diseases and the proper space and time sequential events during brain neurodevelopment; clip, a newly discovered interneuron progenitor, explains the divergent phenotype in tuberous sclerosis complex disorder; and in transcriptomic dysregulation in asd occurs across the whole cerebral cortex and follows a regional gradient. also, cerebral organoids can be transplanted into a host brain circuit to study the functionality of human organoids, as introduced in the in vivo platform for the study of human neurodevelopmental diseases. b) new engineering organoids containing microglia have been developed last year to study novel mechanisms explaining covid-19 neurological anomalies. 1. neurodevelopmental diseases and the proper space and time sequential events during brain neurodevelopment although it has been suggested that out-of-tune events at specific time points or specific brain regions are crucial for understanding neurodevelopmental pathological conditions, few examples have established a concrete cellular process in which such timeand place-specific effects could be disentangled. o’neill et al. reported time-dependent dysregulation of the centrosome interactome at specific neuronal differentiation stages, which allowed studying the aetiology of neurodevelopmental diseases (o’neill et al., 2022). centrosomes, as anchor structures for the cell cytoskeleton, are involved in a number of cell functions, including mitosis and cell migration (wilsch-bräuninger and huttner, 2021; gönczy and hatzopoulos, 2019; vineethakumari and lüders, 2022; delgehyr et al., 2005; piel et al., 2000). to prove their time-dependent hypothesis, the authors derived neural stem cells (nsc) [15 days in culture], and differentiated neurons [40 days in culture] to forebrain identity, using human induced pluripotent stem cell (ipsc) lines. at these two stages, mass spectrometry (ms) of centrosome-associated proteins revealed large cell type-specificity, with around 60% of the neural centrosome proteins not being detected in the centrosome of other cell types. gene ontology (go) categorization indicated that, as expected, nsc centrosome-associated proteins are richer in proteins related to cell division, microtubule organization, and rna splicing; whereas in later stages, neuronal centrosome interactors are related to cytoskeleton and rna-interacting proteins. interestingly, the neural centrosome interactome is particularly enriched in rna-interacting proteins compared with other cell types. by overlaying the interactomes with published datasets of neurodevelopmental diseases with de novo variants (dnv) in autism spectrum disorder (asd), periventricular heterotopia (ph), intellectual disability (id), epileptic encephalopathy (ee) and polymicrogyria (pmg), the authors detected a clear disease association of the neural centrosome interactome. in asd, a pathological association was found for all datasets analysed, suggesting pan-cellular involvement of centrosome proteins in its aetiology. in ph, the authors identified the enrichment of the microtubule-anchoring pre-mrna processing factor 6 (prpf6). prpf6 is more abundant in the centrosome of nscs than of neurons. mutated prff6 recapitulated ph heterotopias in the periventricular cortex of early mouse embryos, along altered mrna splicing, that affected the centrosome associated brsk2 (brain-selective kinase 2) protein, involved in microtubule dynamic regulation and neural migration (barnes et al., 2007; kishi et al., 2005; nakanishi et al., 2019). indeed, rna dynamics play an important function during brain development (raj and blencowe, 2015). panagiotakos and pasca, in a perspective manuscript in neuron, remark how critical the moment and place of the events during brain development is for neurodevelopmental pathologies (panagiotakos and pasca, 2022). as an example, the temporal expression pattern of the voltage-gated sodium channels nav1.1, nav1.2, and nav1.3 isoforms explains developmental brain malformations. mutations in scn3a, encoding for nav1.3, which is elevated in immature progenitors and foetal brain neurons, can lead to abnormal neuronal migration and subsequent polymicrogyria (smith et al., 2018). instead, mutations in scn1a and scn2a encoding for nav1.1 and nav1.2, respectively (beckh et al., 1989; smith et al., 2018), which are elevated in postnatal neurons, are commonly related to infantile epilepsies (meisler and kearney, 2005). interestingly, these protein isoforms also display specific cell-type enrichment during brain development. parvalbumin (pv) cortical interneurons predominantly express nav1.1 channels during early life (yu et al., 2006), so that scn1a loss of function leads to postnatal epilepsy, which disappears in adulthood (favero et al., 2018). thus, it is relevant to differentiate the initial mechanism that triggers disease onset, from those contributing to chronic disease states. in fact, the individual variability of neuropathology onset or affectation may depend on the moment or place the alteration occurs. therefore, in addition to the proteomic and genetic information, understanding of neuropathology requires the understanding of cell-specific alterations, gene regulatory networks and protein interactomes and how they evolve and are regulated during the nervous system formation. 2. stress granule assemblies and neurodevelopmental disorders stress granules (sgs) are dynamic cytoplasmatic membrane-less compartments that assemble under a variety of stress conditions (anderson and kedersha, 2009; jain et al., 2016). a large number of sgs components and regulators have been described (jain et al., 2016; markmiller et al., 2018; yang et al., 2020), but the mechanistic dynamics of these assemblies are still unknown. accumulated data indicates that these cytoplasmatic compartments play important roles in the regulation of gene expression (buchan et al., 2008; arimoto et al., 2008; takahara and maeda, 2012; decker and parker, 2012; yang et al., 2020). sgs are detected where there are considerable pools of untranslated messengers and ribonucleoprotein particles (rnps) (protein-coding mrnas and non-protein-coding rnas, and rna-binding proteins) to shut down translation (guillén-boixet et al., 2020). thus, they seem critical for gene expression homeostasis (martin and ephrussi, 2009; wang et al., 2019), playing then relevant functions during brain development. last year, jia et al. reported a new ndd characterized by alterations in sg formation (jia et al., 2022). they detected disruptive variants of ubap2l, an essential regulator of sg formation (youn et al., 2018; cirillo et al., 2020), in patients with speech-language problems, id, motor delay, seizure, and with less prevalence in patients with adhd, asd, repetitive and aggressive behaviour, and anxiety, but without a defined ndd. the patients also presented morphological features such as facial dysmorphisms, visual impairment, hypotonia and hand and foot abnormalities. using skin fibroblast cell cultures from two patients, they showed reduced levels of ubap2l and fewer sgs under stressful conditions. the authors validated these observations in a cell line (hela) knockout (ko) for ubap2l. transfection of these ko cells with the ubap2l mutants detected in patients also led to a reduction in sgs formation. further experiments in ubap2l ko mice showed increased mortality in embryonic stages and reduced brain size compared with wild-type (wt) ubap2l+/+, and heterozygous (het) ubap2l+/littermates. ko mice showed anomalous neocortex lamination and reduction in neuronal progenitor proliferation possibly linked to altered sg dynamics during cortical neurogenesis. moreover, ubap2l+/animals showed impaired social novelty ability, abnormal spatial working memory, and more anxiety-like behaviour. all these data prompted the authors to analyse the enrichment of sg genes from published datasets of proteomics and high-throughput genome-wide screenings in curated ndd gene datasets, including from sfari (simons foundation autism research initiative) and ddg2p database (development disorder genotype phenotype database). they detected significant enrichment of sg genes, particularly sg core genes and rna-binding proteins. they also examined specific sg genes that could be related to ndd from previously reported de novo mutations (dnms), detecting 3410 variants in the coding regions of 843 sg genes. the statistical analysis showed enrichment of sg genes that clustered according to their network function, string database for protein-protein interaction (ppi), with some of the enriched genes that had not been previously implicated in ndds. although previous works have evidenced that stress conditions during embryonic stages increase the risk of ndds (kinney et al., 2008; babenko et al., 2015; fitzgerald et al., 2020; chui et al., 2020), this is the first study to identify alterations in sg assemblies as a common neuropathological feature of ndds with no defined aetiology. 3. clip, a newly discovered interneuron progenitor, explains the divergent phenotype in tuberous sclerosis complex disorder tuberous sclerosis complex (tsc) is a rare genetic condition that causes benign tumours in different parts of the body mainly the brain, kidneys, heart, skin, lungs and eyes. in the brain, tsc-associated lesions include subependymal tumours at the lateral ventricle and cortical dysplastic lesions, namely cortical tubers. both aberrant structures contain, among other cell types, giant cells (gcs), which are the histopathological hallmark of the disease (ruppe et al., 2014; gelot and represa, 2020; henske et al., 2016). these cells feature a large and central nucleus with peripheral chromatin and a prominent nucleolus, and nissl substance and neurofibrils in the cytoplasm (mizuguchi, 2007). the abnormally large size of gcs strongly indicates dysregulation of cell size control in tsc (mizuguchi, 2007). patients often develop tsc-associated neuropsychiatric disorders (tand) which include id, attention deficit/hyperactivity disorder (adhd), aggressiveness, difficulties with communication and social interaction (asd), epilepsy, seizures and psychiatric conditions (thiele, 2010). tsc is produced by the mutation of either tsc1 or tsc2. these two genes encode for proteins that inhibit mtor (mammalian target of rapamycin) signalling, which is the major regulator of cell growth. loss of regulation of this signalling pathway leads to abnormal cell development and differentiation. experimental data suggest that tsc is produced by a heterozygous germline mutation followed by somatic loss of heterozygosity (loh) in the other allele, due to loss-of-function mutations (crino, 2013; feliciano et al., 2011; feliciano et al., 2012). however, patient tissue analyses show that loh occurs only in tumours and not in dysplastic tubers (henske et al., 1996; chan et al., 2004; qin et al., 2010). moreover, mouse models with loh in either tsc1 or tsc2 cannot recapitulate the full spectrum of brain aberrations observed in patients. last year, eichmüller et al., solved the discrepancies owing to the discovery of a new interneuron progenitor (eichmüller et al., 2022). the authors found that cerebral organoids derived from patients with tsc2+/reproduced both histopathological features using different culture conditions; that is, brain tumours when cultured in high-nutrient medium, and dysplastic cortical tubers when cultured in low-nutrient medium. the characterization of the cellular composition by single-cell rna-sequencing (scrna-seq), along with exhaustive histological validation, allowed the authors to identify a specific interneuron progenitor population that gives rise to both the tumours and the cortical tuber lesions. comparisons with human foetal brain data revealed that this interneuron progenitor is first detected in the caudal ganglionic eminence (cge) during late mid-gestation, with manifest expansion and migration during late gestation. given their origin and embryonic stage, the authors called these interneuron progenitor cells clip, for “caudal late interneuron progenitor”. clip cells seem to be particularly sensitive to mtor levels, being disturbed upon loss of one copy of tsc1/2, which resulted in the over-proliferation of these progenitor cells. the authors determined that the tubers are generated from migrated clip interneurons while the tumours grow in the cge as a consequence of an additional aberration in the second allele, most probably produced by the over-proliferation of these clip cells and the contribution of other factors or cell types (figure 2). thus, although derived from the same altered progenitor clip, tuber cells do not show loh as a mechanism of action, while the tumour cells do show loh. figure 2. illustration depicting the mechanism described by eichmüller et al., 2022. the left shows normal development when neither of the two copies of tsc1/2 have mutations. on the right, when one copy of tsc1/2 is mutated, clip cells become sensitive to mtor levels, resulting in aberrant growth and expansion. clip neurons that migrate to the cortex develop into cortical tubers, and the remaining clip cells, through the participation of other additional alterations lose the other allele producing tumours. the manuscript shows how the same progenitor cell type diverges into two histopathological differential phenotypes. it also shows that clip cells depend on epidermal growth factor receptor (egfr) signalling, and that the inhibition of egfr regressed the organoid tumours, providing an alternative treatment therapy for this pathology. an interesting aspect of this manuscript is that the disease mechanism described is human-specific. indeed, human brain development encompasses the generation and/or expansion of cell types deriving large and gyrated cortices, which do not occur in small lissencephalic brains such as the rodent brain cortex. even postnatally there is extensive migration of interneurons from the cge into the cortex in humans (paredes et al., 2016; hansen et al., 2013; hodge et al., 2019), but not in mice (raju et al., 2018). the use of human organoids was key for this discovery. however, although human organoids are a powerful model system, this technology is still in its infancy. for example, the current lack of standardized protocols implies important variability among organoids from the same patient, which intrinsically puts the results in uncertainty. thus, further studies are necessary to validate clip cells and their functions. 4. improving the diagnostic of malformations of cortical development (mcds) diseases by dna methylation patterns malformations of cortical development (mcds) comprise various neurodevelopmental disorders that are a major cause of epilepsy (leventer et al., 1999), and medically stubborn childhood seizures (kuzniecky, 1995). mcds can be classified into three groups depending on their likely origin. in group i, derived from abnormal cell proliferation or apoptosis, there are hemi-megalencephaly, microcephaly, megalencephaly, and focal cortical dysplasia; in group ii, related to abnormal cell migration, we find tubulinopathies, lissencephalies and heterotopies; and in group iii polymicrogyria is produced by abnormal post-migrational development (desikan and barkovich, 2016). this heterogeneity of causes and phenotypic presentations with a broad range of symptomatology including cognitive deficits, id, and asd (barkovich et al., 2012; guerrini and dobyns, 2014), challenges neuropathologists in providing an accurate diagnosis and, consequently, an on-target prognosis and management of the affectation. as such, biomarkers to identify the type of mcd more precisely are a growing subject of research. however, biomarkers are available only for focal cortical dysplasia (fcd) type ii (d’gama et al., 2015; jansen et al., 2015; d’gama et al., 2017; baldassari et al., 2019) and mild malformations of cortical development with oligodendroglial hyperplasia (moghe) (schurr et al., 2017; bonduelle et al., 2021). currently, the diagnosis of mcd pathologies is based only on histopathological criteria providing, generally, an imprecise diagnosis. as an example, in the case of fcd type ii, two forms have been described fcdiia and fcdiib (blümcke et al., 2011), which differ in that only fcdiib contains balloon cells (bcs). bcs are enlarged cytoplasm cells that resemble gemistocytic astrocytes displaying multiple or convoluted nuclei without prominent nucleoli (mizuguchi, 2007). however, bcs histologically are very alike to gcs observed in tsc. fcdiib and tsc are both pathologies associated with dysregulation of the mtor pathway and display comparable histopathological features, particularly fcdiib and the cortical tubers, which suggests a closely related origin, although they are clearly different neuropathological entities (taylor et al., 1971; lee et al., 2022). in an attempt to improve the diagnosis of mcd pathologies, jabari et al. assayed a potential strategy based on dna methylation (jabari et al., 2022). dna methylation can be a reliable biomarker because it is preserved and, therefore, can be detected in archival human brains stored in ffpe (sahm et al., 2017; capper et al., 2018; wefers et al., 2020). moreover, the methylome manifests a combination of both the somatically acquired dna methylation alterations, and the molecular memory marks in response to environmental or pathogenic cues (kobow and blümcke, 2012; kobow et al., 2013; kiese et al., 2017; kobow et al., 2019; kobow et al., 2020). furthermore, dna methylation profile is widely used to classify cns tumours because of its reproducibility and sensitivity even in small samples (sahm et al., 2017; capper et al., 2018). thus, the purpose of this study was to find dna methylation patterns to accurately classify the different histopathological entities. the authors used surgical samples from patients with mcd and with a confirmed histopathological classification included 265 samples across all age groups and sex that demonstrated different pathological levels of the 12 major subtypes of mcd along with different controls. the authors performed a genome-wide dna methylation assay to correlate the dna methylation patterns with the histopathological classification. they used three different approaches: pairwise comparison, machine learning, and deep learning algorithms. the deep learning algorithm allowed for the most accurate discrimination providing a rationalized classification of the pathologies. then, they analysed the precision of the dna methylation-based mcd classification using a new cohort from different epilepsy centres. this test cohort contained 43 surgical ffpe samples, among which some previously underwent multiple rounds of histopathological evaluation from expert neuropathologists because of the difficulty of their classification. using the algorithm, the authors were able to accurately classify all samples from the test cohort. figure 3 depicts the flowchart the authors followed. thus, they demonstrate that dna methylation-based mcd classification is suitable across major histopathological entities and could be used to establish an integrated diagnostic classification scheme for mcd neuropathology. figure 3. flowchart of the process. top, generation of the algorithm by artificial intelligence (ai) of the correlation of the dna methylation profiles obtained from ffpe samples with confirmed histopathological mcds classification. bottom, validation of the algorithm to classify mcds based on dna methylation using a new set of ffpe samples. 5. deciphering the aetiology of sudden unexplained death in childhood (sudc) by proteomics sudden unexplained death in childhood (sudc) refers to the unexplainable death of children over 1 year of age. it is called unexplained because, after a complete review of the clinical history and the autopsy including toxicologic, genetic, metabolic and microbiology analyses, to cite some of the complementary studies, the cause of death is not determined. although the causes may be diverse, genetic variants are likely to be prone to sudc risk (crandal et al., 2020; halvorsen et al., 2021; harowitz et al., 2021; holm et al., 2012; narula et al., 2015). for example, among sudc cases, there is a high prevalence of individual or familial febrile seizure (fs) history (hefti et al., 2016; hesdorffer et al., 2015; mcguone et al., 2020). sudc shares some pathological similarities with sudden unexpected death in epilepsy (sudep) (devinsky et al., 2016; kinney et al., 2016), suggesting that they may share some coincident mechanisms that result in premature death. similar to sudc, the causes of sudep may also be diverse including genetic risk factors. since both pathological conditions show abnormalities in the hippocampus and cortex (ackerman et al., 2016; kinney et al., 2016; kon et al., 2020; mcguone et al., 2020), these brain regions, particularly the hippocampus, have attracted most sudc studies. however, no conclusive results are available to date (leitner et al., 2022a; roy et al., 2020). in this scenario, leitner et al., have defined differential protein abundance in several brain areas of sudc cases (19 cases), including the frontal cortex, hippocampal dentate gyrus (dg), and cornu ammonis (ca1-3). the study compared cases with and without febrile seizure history (sudc-fs and sudc-nofs) and without febrile seizure with control cases (n = 19) matched by age, sex, brain weight and post-mortem interval (leitner et al., 2022b). the authors micro-dissected the aforementioned brain regions from autopsy ffpe tissue to perform label-free quantitative proteomic analyses. the proteomic analyses revealed no differential hippocampal neuropathology between sudc-fc and sudc-nofc. instead, principal component analysis (pca) revealed a significant separation between sudc and controls in the frontal cortex, but not regarding fs history. differential protein abundance analysis showed significant differences between sudc and control cases in 660 proteins of the frontal cortex, while only 170 in the dg and 57 in ca1-3. pathway analysis revealed 238 signalling pathways in the frontal cortex, mainly involved in the activation of oxidative phosphorylation, inhibition of eif2 (eukaryotic initiation factor 2) signalling, and glutamate receptor signalling. in the dg, they mainly found pathways involving activation of the acute phase response and inhibition of reelin signalling, while in hippocampal ca1-3, the only involved signalling pathway was in the acute phase response associated with cellular stress response (liu and qian, 2014; brace et al., 2016; leitner et al., 2022b). the fact that they detected protein overlap in more than one signalling pathway supports the involvement of these signalling pathways. the authors also used weighted gene correlation network analysis (wgcna) to correlate the proteomic results with the clinical history. in the frontal cortex there were common affected enriched signaling pathways in sudc and sudep, some of which showed oppository effects in sudc and sudep (e.g., oxidative phosphorylation and eif2 signalling), while other proteomic pathways were similar(e.g., mitochondrial enzyme cox6b1). as a corollary, besides the relevant information provided, one of the most remarkable findings is that while the frontal cortex is not a studied region in sudc, it showed the most altered proteomic changes. hence, the authors put forward the relevance of the frontal cortex in this condition and the necessity of performing studies in this region to detect possible neuropathological signs associated with sudc to reduce and prevent the risk of this condition. 6. transcriptomic dysregulation in asd occurs across the whole cerebral cortex and follows a regional gradient asd is a prototypical example of neuropathology in which a definitive aetiology has not yet been determined. in the last years, comprehensive omics assessments have been conducted to determine risk genes (de la torre-ubieta et al., 2016) or differential patterns of splicing or gene isoform expression in asd (wu et al., 2016; sun et al., 2016; gandal et al., 2018). despite the heterogeneity of factors that drive to asd pathology, molecular profiling studies have found consistent patterns of transcriptomic and epigenetic dysregulation (ramaswami et al., 2020), involving upregulation of astrocytes, microglia, and neural immune genes; and downregulation of synaptic, neurite morphogenesis, and neuronal energy pathway genes accompanied by attenuation of gene-expression gradients in cortical association regions (voineagu et al., 2011; wu et al., 2016; parikshak et al., 2016; gandal et al., 2018). the work of gandal et al. explores whether these alterations are more widespread throughout the cortex and proposes that asd pathology is the physiological manifestation of altered cortical arealization (gandal et al., 2022). to support this hypothesis, they did bulk rna-sequencing (rna-seq) analysis to identify altered genes and alternative spliced gene isoforms. they used threefold more samples than previous works (voineagu et al., 2011; parikshak et al., 2016), analysing 725 post-mortem brain samples spanning 11 cortical areas from 112 individuals of both sexes and with ages ranging from 2 to 68, totalling 49 subjects with idiopathic asd and 54 neurotypical controls. they found transcriptomic changes across the cortex of asd patients with an anterior-to-posterior gradient, with the most remarkable differences in the primary visual cortex. in agreement with previous reports (walker et al., 2019), the greater differential expression detected was related to alternative splicing and differential isoform expression. thus, their findings indicate that molecular alterations, mainly in alternative splicing and isoform expression, in asd transcriptome extend beyond the associative cortex to broadly involve primary sensory regions. this may explain the altered sensory processing observed in individuals with asd. an interesting aspect is that the differences in gene expression that account for demarcating the different cortical regions, because they define the cytoarchitecture, connectivity, and function of a particular region, were attenuated in asd. this indicates that the cortical regions in asd patients are molecularly more homogeneous, and therefore, less differentiated and specialised. again, this attenuated expression followed a gradient pattern, which was more particularly affected in the posterior regions such as the primary visual cortex. the authors also determined whether the transcriptomic changes detected were reflected in the cell-specific type gene expression. by sn-rna-seq and methylation profiles, they determined a substantial differential expression profile in excitatory neuron classes and glia cells, once more, with a regional gradient more prominent in occipital and parietal cell types than in prefrontal cortex (pfc). this reiterative regional gradient may reflect a reminiscence of the buildout of the cortical cytoarchitecture, i.e., its patterning and connectivity, which depend on both cell intrinsic factors genetic and epigenetic regulatory programmes, and extrinsic signals, –such as morphogen gradients (cadwell et al., 2019). the data suggests that this process is altered in asd, indicating an early developmental alteration in cortical arealization, affecting local neuronal circuits, synaptic homeostasis, and leading to the asd manifestations. 7. in vivo platform for understanding the neuropathology of human neurodevelopmental diseases some years ago, several studies showed that it was possible to transplant human neurons into the rodent cortex that were able to establish connections with the rodent cells (espuny-camacho et al., 2013; mansour et al., 2018; real et al., 2018; linaro et al., 2019; kitahara et al., 2020; xiong et al., 2021), thus providing an in vivo platform to study human developmental disorders. although the hope was that those tools would allow uncovering circuit-level phenotypes from patient-derived cells and test therapeutic strategies, they had some problems. part of those have now been solved in the work of revah et al. (revah et al., 2022). to facilitate integration of the transplant they transplanted 3d human cortical organoids (hco) into the somatosensory cortex of immunodeficient rats, at early postnatal stage, in which corticocortical and thalamocortical innervation have not yet been completed, thus minimising the endogenous circuitry alteration (kichula and huntley, 2008). the novelty of this work is thus that they transplanted intact organoids, rather than a dissociated cell suspension; and at very early postnatal stages, rather than in adult rats. this strategy allows better synaptic and axonal integration of the human cells. hence, the transplanted hcos (t-hco) displayed more mature properties compared to not transplanted same-age hcos. the t-hco showed vascularization and the presence of microglia. snrna-seq revealed the canonical expression pattern of major cortical cell classes, even though t-hco did not present anatomical lamination. transplanted neurons showed a more mature morphological phenotype, i.e., larger somas, more dendrites, larger processes, and higher dendritic spine density, and more mature electrophysiological properties. the authors traced t-hco innervation with retrograde rabies showing integration of t-hco neurons in the rat brain's circuitry. fibre photometry, two-photon calcium imaging, electrophysiology, and sensory stimuli by deflecting the rat’s whiskers revealed the functionality of the integration, as t-hco stimulation evoked response in rat neurons indicating functional innervation. finally, by in vivo optogenetics they showed that activation of t-hco could modify the rats’ behavioural response. the authors used this system for studying timothy syndrome (ts), a severe genetic disorder caused by the mutation in the l-type voltage-sensitive calcium channel cav1.2 (ebert and greenberg, 2013). they compared the evolution of t-hco derived from control individuals and patients with ts with their non-transplanted counterparts (hco). ts t-hco neurons showed altered dendritic morphology with an extensive number of primary dendrites but with an overall reduction in the mean and total dendritic size. also, these neurons displayed increased synaptic spine density that impaired their electrophysiological properties. these phenotypes could only be detected in ts t-hco but not in non-transplanted organoids with the same differentiation stage, thus indicating that organoid transplantation allowed for better recapitulation of the disease phenotype that was elusive in non-transplanted organoids. 8. mitochondria participate in the neuron-glia crosstalk mitochondria play crucial roles in the regulation of cellular energy and metabolism; therefore, it is not surprising that this organelle is involved in all developmental stages with important functions in neuronal differentiation. for this reason, several mitochondrial disorders present an abnormal neuronal and neurological development (son and han, 2018). mitochondrial dysfunction has been widely associated with neurological and psychiatric diseases such as schizophrenia, bipolar depression, asd and rett syndrome (son and han, 2018). one example is leigh syndrome, which is caused by a mutation in mitochondrial dna, leading to dysfunctional mitochondrial complexes. the disorder usually manifests within the first year of life and leads to rapid degeneration of physical and mental abilities, ultimately leading to death within 2-3 years (murphy and craig, 1975). however, this close relationship between the mitochondria and the cns is not only applicable to neurons. in fact, it is astrocytic’ mitochondria that support neuronal development and function. in cases of neuronal damage, such as in brain ischemia, astrocytes release functional mitochondria into the extracellular medium via a mechanism that involves the activation of the cluster of differentiation 38 (cd38) and cyclic adenosine diphosphate (adp)-ribose signalling (hayakawa et al., 2016; english et al., 2020). this process facilitates neuronal recovery. at the same time, damaged neurons expel damaged mitochondria, which are in turn absorbed by the surrounding astrocytes to be recycled. in a paper published last year, gao et al. showed that the release of mitochondria might function as a signalling element between neurons and glial cells (gao et al., 2022). they also proved that, under physiological conditions, neurons release mitochondria into the extracellular medium. then, they tested different pathological conditions in vitro, such as acidosis, hydrogen peroxide or high levels of nmda (n-methyl d-aspartate) or glutamate, simulating brain ischemia. they detected a significant increase in the number of released damaged mitochondria by the neurons (gao et al., 2022), and proved that astrocytes uptake these mitochondria and trigger the rescuing response. hence, they speculate that, under stress conditions, mitochondria could serve as a “help-me” signal. if replicated, this discovery would represent a huge step forward in understanding the mechanisms behind neuronal stress response that could shed light on common neurodevelopmental/neurodegenerative disorders. 9. gaining insight on the role of hiv-1 in the cns since its discovery, the human immunodeficiency virus (hiv) has been associated with several neurological conditions. hiv-1 infection has long been known to have an impact on the nervous system, culminating in hiv-associated neurocognitive disorder (hand) (clifford and ances, 2013), as the brain, along with the bone marrow, is known to be a reservoir for the virus. an interesting aspect is how hiv-1 infection affects neurodevelopment. in the era of antiretroviral therapy (art), this question translates to how maternal infection affects offspring development. properly treated mothers give birth to uninfected children in almost 99% of cases (wedderburn et al., 2022). however, hiv-exposed children generally show a slower development of some neurological functions. in particular, a poorer expressive language and gross motor function compared to their hiv-unexposed counterparts (wedderburn et al., 2022). however, the mechanisms underlying this phaenomenon remain unclear. one hypothesis states that the virus could directly affect the development of the foetus, while other researchers think that it is the chronic systemic inflammation of the mother due to the infection what could affect the offspring. however, research has been hindered by the lack of a satisfactory model, owing to the complexity derived from the involvement of both the nervous and immune systems. for this reason, researchers have long been searching for a novel model to study this phenomenon. brain organoids, in fact, could provide an insight on neuronal development and in recent years, protocols for microglia-containing brain organoids have been developed, hence allowing for this technology to be applied to hiv research field (ormel et al., 2018). to prove so, a study in 2022 by gumbs et al. showed that the cluster of differentiation 4 (cd4) and cysteine-cysteine chemokine receptor 5 (ccr5)– expressing microglia were susceptible to hiv-1 infection in a human brain organoid model (gumbs et al., 2022). they tested this infection model on both microglia-containing organoids and organoid-derived microglia (omg), with positive results in both cases. albeit the obvious limitations of the organoid model, the results of this study represent a significant advancement in the hiv research field, as these organoids could help elucidate the mechanisms behind the effects of the virus on the brain and test novel therapies. 10. novel mechanisms explaining covid-19 neurological anomalies the severe acute respiratory syndrome coronavirus 2 (sars-cov-2) pandemic has had a huge impact on everyone's lives, causing millions of deaths worldwide and changing perpetually our society. although the disease was initially known to be hazardous to the respiratory system, soon several comorbidities with long-lasting effects were associated with the virus. it was soon discovered that the infection could lead to several significant neurological complications (chen et al., 2022). these effects vary largely from patient to patient, depending on factors such as age, weight, health status and pre-existing conditions. however, both shortand long-lasting neurological effects have been reported in all populations, from children to adults. regarding the effects of covid-19 on children, we should distinguish between the effects due to the pandemic and those due to the direct effects of the virus. a meta-analysis study by hessami et al. highlighted that infants born or raised during the pandemic showed a higher rate of communication impairment than the pre-pandemic cohort (hessami et al., 2022). given the nature of the study and the lack of other significant neurodevelopmental impairments in the general paediatric population, we can speculate that this communication impairment was an effect of the lack of social stimuli because of the lockdown rather than the virus itself. however, the direct neurological consequences of covid-19 infection have also been reported in both children and adults. these effects range from temporary anosmia and ageusia to memory loss, meningitis, stroke, and neurodegeneration (ledford, 2022). data on the possible long-term neurological effects of sars-cov-2 coronavirus are inconclusive. several studies have found associations with alzheimer’s disease (ad) risk, cardiovascular damage, or neurological sequelae. an analysis in the uk found a slight overall reduction of grey matter in the brains of recovered individuals. also, that the virus infects brain support cells and induces inflammation similar to parkinson’s or ad, and that severe covid-19 causes detectable brain ageing (douaud et al., 2022). despite the significant amount of research conducted on and the neurotropic nature of the virus, the molecular mechanisms linking covid-19 to these neurological symptoms are still unclear. a study published in 2022 by charnley et al. showed that some viral open reading frame (orf) proteins can assemble into amyloid-like aggregates and cause neurotoxicity (charnley et al., 2022). using an algorithm, they pinpointed two short regions of orf6 and orf10 as those responsible for the assembly. they then tested the peptides corresponding to these regions and showed that they self-assembles into amyloid-like structures. finally, these peptides were tested on neuroblastoma sh-sy5y cells, proving to be neurotoxic (charnley et al., 2022). we can presume that some of the neurological consequences of a covid-19 infection not only share symptomatology with some common neurodegenerative disorders (e.g., ad), but also share an accumulated misfolding protein-driven pathogenesis. it will be now important to test these mechanisms during development. funding the lab of m.d. is supported by the departament d'universitats, recerca i societat de la informació de la generalitat de catalunya (grups consolidats 2022), the agencia estatal de investigación (pid2019-110755rb-i00/aei/10.13039/501100011033), the european union’s horizon 2020 research and innovation programme under grant agreements nº 848077 (go-ds21) and under grant agreement nº 899986 (icod). horizon europe (horizon-hlth-2021-stayhlth-01-psych-strata). this reflects only the author's view, and the european commission is not responsible for any use that may be made of the information it contains. jerôme lejeune foundation (grant number 2002), nih (grant number: 1r01eb 028159-01), marató tv3 (#2022-12-30-31-32). the crg acknowledges the support of the spanish ministry of science and innovation to the embl partnership, the centro de excelencia severo ochoa, and the cerca programme/generalitat de catalunya. the ciber of rare diseases (ciberer) is an initiative of isciii. references ackerman mj, andrew ta, baker am, devinsky o, downs jc, keens t, kuntz j, lin 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credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. reference on copy number variations in pleomorphic xanthoastrocytoma: implications for diagnostic approach feel free to add comments by clicking these icons on the sidebar free neuropathology 4:19 (2023) original paper reference on copy number variations in pleomorphic xanthoastrocytoma: implications for diagnostic approach david e. reuss1,2, daniel schrimpf1,2, damian stichel1,2, azadeh ebrahimi3, chris dampier4, kenneth aldape4, matija snuderl5, david capper6, martin sill7,8, david t. w. jones8,9, stefan m. pfister2,8,10, felix sahm1,2,8, andreas von deimling1,2,8 department of neuropathology, institute of pathology, university of heidelberg, heidelberg, germany clinical cooperation unit neuropathology, german cancer consortium (dktk), german cancer research center (dkfz), heidelberg, germany department of neuropathology, dgnn brain tumor reference center, university of bonn, bonn, germany laboratory of pathology, national cancer institute, centre for cancer research, bethesda, md, usa department of pathology, new york university langone health and school of medicine, new york, new york, usa department of neuropathology, charité universitätsmedizin berlin, berlin, germany; german cancer consortium (dktk), berlin, germany; german cancer research center (dkfz), heidelberg, germany division of pediatric neurooncology, german cancer consortium (dktk) and german cancer research center (dkfz), heidelberg, germany hopp children’s cancer center heidelberg (kitz), heidelberg, germany pediatric glioma research group, german cancer research center (dkfz), heidelberg, germany department of pediatric oncology, hematology and immunology, heidelberg university hospital, heidelberg, germany corresponding author: andreas von deimling · department of neuropathology · university of heidelberg and clinical cooperation unit neuropathology · german cancer consortium (dktk) · german cancer research center (dkfz) · 69120 heidelberg · germany andreas.vondeimling@med.uni-heidelberg.de additional resources and electronic supplementary material: supplementary material submitted: 19 october 2023 accepted: 30 october 2023 copyedited by: shino magaki published: 13 november 2023 https://doi.org/10.17879/freeneuropathology-2023-5156 keywords: pleomorphic xanthoastrocytoma, pxa, copy number variations, cnv, methylation, classification, 7/10 signature, egfr, cdkn2a/b, amplification, homozygous deletion abstract pleomorphic xanthoastrocytoma (pxa) poses a diagnostic challenge. the present study relies on methylation-based predictions and focuses on copy number variations (cnv) in pxa. we identified 551 tumors from patients having received the histologic diagnosis or differential diagnosis pleomorphic xanthoastrocytoma (pxa) uploaded to the web page www.molecularneuropathology.org. of these 551 tumors, 165 received the prediction “methylation class (anaplastic) pleomorphic xanthoastrocytoma” with a calibrated score >=0.9 by the brain tumor classifier version v12.8 and, therefore, were defined the pxa reference set designated mcpxaref. in addition to these 165 mcpxaref, 767 other tumors received the prediction mcpxa with a calibrated score >=0.9 but without a histological pxa diagnosis. the total number of individual tumors predicted by histology and/or by methylome based classification as pxa, mcpxa or both was 1318, and these were designated the study cohort. the selection of a control cohort was guided by methylation-based predictions recurrently observed for the other 386/551 tumors diagnosed as histologic pxa. 131/386 received predictions for another entity besides pxa with a score >=0.9. control tumors corresponding to the 11 most common other predictions were selected, adding up to 1100 reference cases. cnv profiles were calculated from all methylation datasets of the study and control cohorts. special attention was given to the 7/10 signature, gene amplifications and homozygous deletion of cdkn2a/b. comparison of cnv in the subsets of the study cohort and the control cohort were used to establish relations independent of histological diagnoses. tumors in mcpxa were highly homogenous in regard to cnv alterations, irrespective of the histological diagnoses. the 7/10 signature commonly present in glioblastoma, idh-wildtype, was present in 15-20% of mcpxa, whereas amplification of oncogenes (likewise common in glioblastoma) was very rare in mcpxa (<1%). in contrast, the histology-based pxa group exhibited high variance in regard to methylation classes as well as to cnvs. our data add to the notion, that histologically defined pxa likely only represent a subset of the biological disease. introduction since its first description in 1979 (1), pleomorphic xanthoastrocytoma (pxa) has posed a notable diagnostic challenge. opinions on appropriate diagnostic parameters vary, resulting in debates on the defining characteristics of the entity itself as well as on the diagnosis of many individual tumors. the current who classification renders specific morphological features including pleomorphism, presence of multinucleated as well as spindle cells, xanthomatous cells and eosinophilic granular bodies as essential (2,3). molecular alterations including braf mutation, homozygous deletion of cdkn2a/b and the methylome profile of pxa (mcpxa) are currently considered as desirable criteria for confirming a diagnosis. a major problem is that only a fraction of tumors receiving the prediction mcpxa do exhibit the classical morphological features of pxa (and conversely, many tumors histologically called pxa show molecular features typical of other entities). the central dispute revolves around the question, how tumors with a methylation profile of pxa lacking the histological hallmarks should be classified. several studies have addressed this problem without coming to a consensus as for now (4-7). the present study has a very limited scope addressing only the three parameters: the initial histological diagnosis, the dna methylation-based tumor prediction and copy number variations (cnvs). thus, in 932 tumors with a heidelberg classifier prediction of mcpxa, the only additional parameters addressed are initial diagnosis and cnvs. conveniently, cnvs can be read out of array-based methylome data and allow for the determination of chromosomal losses and gains including gene amplifications and homozygous deletions. we determined the distribution of cnvs focusing on regions and genes known to be relevant in pxa and other morphological mimics. based on the large number of datasets included, the conclusions can be considered very robust despite the intentional limitations on scope. material and methods selection of study and control cohorts the basis for all tumors included in this study was presence of an illumina humanmethylation450 (450k) or epic dataset. there were 120800 tumors in the database of the department of neuropathology heidelberg and german cancer research center (dkfz) containing data from the heidelberg brain tumor classifier (www.molecularneuropathology.org) (date: 01. aug. 2023). of these, 932 tumors were predicted as belonging to the methylation class pleomorphic xanthoastrocytoma (mcpxa) by the heidelberg brain tumor classifier version 12.8 with a calibrated score >= 0.9. version 12.8 has been further developed from a previously published version 11b4 (8). in an independent query, we identified all tumors in the database for which the histological diagnosis or differential diagnosis pxa has been disclosed by the submitters. this yielded 551 tumors. one hundred and sixty-five tumors received both, the prediction of mcpxa and the diagnosis or differential diagnosis pxa and these were designated the mcpxa reference set (mcpxaref). thus, the central study cohort included 1318 cases. a graphical depiction of the study cohort is provided in figure 1. an overview of the classifier predictions for 551 histological pxa is given in supplementary table 1. all predictions with a calibrated score >=0.9 occurring at least twice were considered relevant differential molecular diagnoses of histological pxa. reference sets of 100 corresponding tumors for each class were compiled for eleven differential diagnoses. all tumors in the reference sets received a prediction with a calibrated score >=0.9 in the brain tumor classifier v12.8. this resulted in a reference cohort totaling 1100 methylation datasets. figure 1: graphical description of the study groups: of 932 tumors receiving a classifier prediction (calibrated score >=0.9), 767 did not receive a histological pxa diagnosis and the latter were designated as mcpxa. 165 of the 932 also were diagnosed histologically as pxa (or pxa was in the differential diagnosis) and these were designated as mcpxaref. a control group containing 11 mc with 100 tumors each was selected based on the most frequent predictions in tumors histologically diagnosed as pxa but with other classifier predictions. the reference cohort included: mc diffuse paediatric-type high grade glioma, rtk1 subtype, subclass a (mcpedhgg_rtk1a); mc ganglioglioma (mcgg); mc glioblastoma, idh-wildtype, atypical mesenchymal type (mcgbm_mes_atyp); mc glioblastoma, idh-wildtype, rtk1 subtype (mcgbm_rtk1); mc glioblastoma, idh-wildtype, rtk2 subtype (mcgbm_rtk2); mc glioblastoma, idh-wildtype, typical mesenchymal type (mcgbm_mes_typ); mc high-grade astrocytoma with piloid features (mchgap); mc infant-type hemispheric glioma (mcihg); mc infratentorial pilocytic astrocytoma (mcpa_inf); mc neuroepithelial tumour with patz1 fusion (mcnet_patz1) and mc supratentorial pilocytic astrocytoma (mcpa_cort). determination of cnv and summary cnv plots tsne analyses were performed using the r-package rtsne (https://github.com/jkrijthe/rtsne) employing the 20,000 most variable cpg sites according to standard deviation; 3000 iterations and a perplexity value of 10. cnv data were calculated from the output of the illumina 450k or 850k/epic platforms. cnv plots are based on the raw data subjected to analysis by the ‘conumee’ r package in bioconductor (https://www.bioconductor.org/packages/release/bioc/html/conumee.html). assessment of copy-number alterations was automated using the results from conumee after additional baseline correction. amplifications were called if the respective probes exhibited a value higher than 0,55 on a log2-scale. homozygous deletions were called if the respective probes exhibited a value lower than -0.4 on a log2-scale. statistics all data sets were collected retrospectively. distribution of nominal data was tested employing fisher’s exact test. results diagnoses in mcpxa tumors with a mcpxa prediction but without a pxa diagnosis (n=767) received another diagnosis in 399 instances. no diagnosis was provided for 368 cases. diagnoses were slightly simplified to allow for categorization. a list of given diagnoses is provided in supplementary table 2. tsne of mc pxa, mcpxaref, and reference sets using t-distributed stochastic neighbor embedding (t-sne) we analyzed all tumors of the study cohort and reference sets (figure 2). tumors receiving the classifier prediction mcpxa were annotated according to presence (mcpxaref) or absence (mcpxa) of the histological diagnosis (or differential diagnosis) of pxa. importantly, the distribution within mcpxa and mcpxaref is similar. this demonstrates that the morphological features defining histological pxa cannot readily single out a subpopulation within mcpxa associated with ‘canonical’ features. figure 2: tsne analysis of mcpxa, mcpxaref and reference sets. tumors from the mcpxa and the mcpxaref cohorts cluster together and are separated from the reference sets. parameters: tsne_cpgs_20000_perplexities_10_iter_3000_theta_0_eta_200_dim_2 summary cnv (sumcnv) plots of mcpxa, mcpxaref and reference sets for an overview, summary cnv (sumcnv) plots were generated for all cohorts. tumors with the classifier prediction pxa were divided in two subgroups, depending on whether the histological (differential) diagnosis was pxa (mcpxaref, n=165) or another histological diagnosis or no diagnosis was given (mcpxa, n=767). sumcnv plots of mcpxaref and mcpxa were virtually identical, indicating that the presence or absence of the histological diagnosis pxa does not associate with the frequency of any cnv (figure 3). the sumcnv profiles of the 11 reference sets of differential diagnoses were clearly distinct from the mcpxa and mcpxaref sumcnv profiles. notable differences can be observed between the 4 glioblastoma idh wildtype subgroups containing 100 samples each. most evident was the highest proportion of the 7/10 signature and an increased incidence of trisomies of chromosomes 19 and 20 in gbm idh wildtype rtk2. these differences will be followed up in larger cohorts. summary cnv profiles are provided in figure 3. figure 3: summary cnv plots of mcpxa, mcpxaref and reference sets. prevalence of the 7/10 signature in mcpxa, mcpxaref and reference sets combined gain of chromosome 7 and loss of chromosome 10, dubbed 7/10 signature, is a very frequent molecular feature of glioblastoma. the best parameters for defining the 7/10 signature have not yet been established. in this study, a previously published algorithm has been employed (9). presence of the 7/10 signature was scored if gain of at least 50% of the short or the long arm of chromosome 7 coincided with loss of at least 50% of the short or the long arm of chromosome 10. the 7/10 signature according to this guideline was not observed at all in infant-type hemispheric glioma. supratentorial pilocytic astrocytoma and ganglioglioma typically exhibiting frequent gain of chromosome 7 (figure 3) both presented with the 7/10 signature in 1% and 2% of the cases, respectively. likewise, the 7/10 signature occurred in only 1% of infratentorial pilocytic astrocytoma; however, this tumor group much more rarely exhibited chromosome 7 gains. only 2% of diffuse paediatric-type high grade glioma, rtk1 subtype, subclass a carried the 7/10 signature. among the glioblastoma subtypes we detected a presumably nonrandom difference in the frequency of 7/10 signature ranging from 38% in mcgbmmes_atypical to 92% in gbm_rtk2. these differences will be followed up in a larger cohort of glioblastomas. our study demonstrated the presence of the 7/10 signature in 24/165 (15%) mcpxaref and in 145/767 (19%) mcpxa. the difference in frequencies of the 7/10 signature in both groups was not significant (fisher’s exact test p >0.29). the distribution of numerical alterations on the chromosomal arms 7 and 10 in mcpxaref, mcpxa and the 11 control groups is shown in table 1. data for 2418 individual tumors are provided in supplementary table 3. group n 7p 7q 10p 10q 7/10     (gain>50%) (gain>50%) (gain>50%) (gain>50%) (signature) mcpedhgg_rtk1a 100 10 4 11 6 2 mcgg 100 29 30 3 3 2 mcgbm_mes_atyp 100 60 54 57 51 38 mcgbm_rtk1 100 82 80 82 86 75 mcgbm_rtk2 100 84 88 98 95 92 mcgbm_mes_typ 100 76 76 77 78 64 mchgap 100 5 6 28 7 5 mcihg 100 4 4 8 5 0 mcpa_inf 100 10 11 0 1 1 mcnet_patz1 100 5 6 8 10 1 mcpa_cort 100 28 26 1 0 1 mcpxa 767 268 244 291 257 145 (19%) mcpxaref 165 50 48 49 44 24 (15%) sum 2032           table 1: distribution of chromosomal 7 gains and 10 losses in mcpxa, mcpxaref and in reference sets prevalence of the cdkn2a/b homozygous deletion and gene amplifications in mcpxa, mcpxaref and reference sets a hallmark chromosomal numerical alteration in pxa is a homozygous deletion on chromosomal arm 9p including the cdkn2a/b locus (7,10). this alteration was seen in 147/165 (89%) mcpxaref and in 685/767 (89%) mcpxa. due to the high frequency of this alteration in gbm, pxa cannot be separated from gbm on these grounds. again, we observed different frequencies of homozygous cdkn2a/b deletions in gbm subgroups, ranging from 40% in glioblastoma, idh-wildtype, mesenchymal to 79% in glioblastoma, idh-wildtype, rtk2. the differences likely are non-random and will be followed up. as expected, high-grade astrocytoma with piloid features exhibited a high proportion of homozygous cdkn2a/b deletions (80%). gene amplifications affecting egfr, mdm2 and pdgfra were rare in mcpxa and pxa. highest incidences for egfr amplification were observed in glioblastoma, idh-wildtype, rtk2 (86%), for mdm2 amplification in glioblastoma, idh-wildtype, rtk1 (23%) and for pdgfra amplification also in glioblastoma, idh-wildtype, rtk1 (18%). the distribution of homozygous cdkn2a/b deletions in mcpxaref, mcpxa and reference sets as well as the distribution for gene amplifications is given in table 2. data for 2418 individual tumors are provided in supplementary table 4. group n egfr amp mdm2 amp pdgfra amp cdkn2a/b     (gain>5.5) (gain>5.5) (gain>5.5) (loss mcpedhgg_rtk1a 100 2 3 13 23 mcgg 100 0 0 0 0 mcgbm_mes_atyp 100 2 0 1 45 mcgbm_rtk1 100 14 23 18 52 mcgbm_rtk2 100 86 11 5 79 mcgbm_mes_typ 100 21 4 0 40 mchgap 100 0 2 0 80 mcihg 100 0 0 0 2 mcpa_inf 100 0 1 1 0 mcnet_patz1 100 0 0 0 3 mcpa_cort 100 0 0 0 0 mcpxa 767 2 4 1 685 (89%) mcpxaref 165 0 2 0 147 (89%) sum 2032         table 2: distribution of amplifications and homozygous cdkn2a/b deletions in mcpxa, mcpxaref and in reference sets predictions in tumors diagnosed as pxa in the 551 tumors which received the diagnosis or differential diagnosis pxa, 296 tumors received a score >=0.9. highest numbers for predictions (all scores / scores >= 0,9) were received for mcpxa (208/165), mcgbm_mes_typ (108/59), mcpa_cort (22/11), methylation class control tissue, reactive tumor microenvironment (mcctrlmicro) (30/8), mcgbm_rtk2 (13/6), mchgap (15/5), mcihg (5/4), mcgbm_rtk1 (4/3), mcpa_inf (3/3), mcnet_patz1 (4/3), methylation class astrocytoma, idh-mutant (mcaidhmut) (3/2), mc diffuse hemispheric glioma, h3 g34-mutant (dhgh3mut) (2/2), mcpedhgg_rtk1a (5/2), mcgg (12/2), mcgbm_mes_atyp (17/2) and mc malignant peripheral nerve sheath tumor (mcmpnst) (6/2). all predictions for the 551 tumors are listed in supplementary table 1. as expected, a substantial portion of tumors with a histological pxa diagnosis or suspicion received a prediction for glioblastoma. surprising was the distribution among the four adult glioblastoma subclasses. the mcgbm_mes_typ was predicted with high score in 62, mcgbm_mes_atyp in 5, mcgbm_rtk2 in 6 and mcgbm_rtk1 in 3 tumors. this is suggestive of shared histological features of pxa with gbm belonging to the mcgbm_mes_typ. pxa with predictions other than mcpxa and calibrated scores >0.9 clustered well with the respective reference groups (figure 4). pxa with predictions other than mcpxa and calibrated scores <0.9 exhibited a scattered distribution (data not shown). figure 4: tsne analysis of tumors diagnosed as pxa receiving a calibrated score >0.9, and reference sets. tumors not receiving a mcpxa prediction cluster with the respective reference groups. parameters: tsne_cpgs_20000_perplexities_10_iter_3000_theta_0_eta_200_dim_2 discussion one major weakness of the present study concerns the available data on histological diagnoses of the extended cohort (supplementary table 2). for 386/932 tumors with a mcpxa prediction no sufficiently specified diagnosis was given. submitters of data to the webpage may have been inexperienced, may have been not overly motivated to provide the (optional) information or may have uploaded data prior to finalization of the diagnosis. the webpage serving as a basis for the study does not allow for submitting a revised version of the diagnosis upon completion of methylation analysis. a review of the diagnoses is unfortunately not possible. therefore, we assume that many of the tumors which received a prediction mcpxa also would have received the histological classification pxa. this very large tumor series, however, allows drawing of two main conclusions: 399/932 tumors with a clear molecular pattern (dna methylation and cnv profile) of pxa received a non-pxa histological diagnosis suggesting a high morphological variance in mcpxa that precluded a histological pxa diagnosis in at least 43% and up to 82% of the cases (depending on scoring of the 386 tumors without a diagnosis at all). a conservative estimate would therefore estimate that ~50% of mcpxa will not be recognized by histology alone. the other conclusion is that the 165 tumors with both a pxa diagnosis as well as a mcpxa prediction is large enough for serving as a ‘standard’ reference set for subsequent comparisons with the 762 mcpxa not having received a pxa diagnosis. tumors with mcpxa prediction are highly homogenous in respect to grouping upon tsne analyses or cnv analyses independent of whether they were histologically diagnosed as pxa or as other tumors (figures 2 and 3, tables 1 and 2). this includes all methylation patterns, gains and losses of chromosomal arms as well as circumscribed amplifications or homozygous deletions. in contrast, tumors diagnosed as pxa on histologic grounds alone were heterogeneous upon tsne (figure 4) and cnv analyses (supplementary tables 3 and 4). histologically diagnosed pxa with non mcpxa classifier prediction exhibited copy number alterations corresponding to those seen in the respective methylation classes. this data constellation is compatible with a model assuming that a classifier-based approach predicting mcpxa is recognizing a tumor cohort more homogenous than a histology-based approach diagnosing pxa. based on the high homogeneity of mcpxa and mcpxaref this study provides a stable framework for cnv alterations. this is of relevance for separating pxa from gbm. the 7/10 signature has been proposed by who as a surrogate marker for gbm diagnosis in absence of necrosis or microvascular proliferation. the present data demonstrate that the 7/10 signature is present in 15% to 20% of mcpxa, suggesting caution is advised when considering this as a sole marker. this must be balanced against the clear value of the 7/10 signature especially in tissue specimens from the tumor edges or from stereotactic biopsies in elderly patients. amplifications of egfr, mdm2 and pdgfra occurred in 9/932 (< 1%) mcpxa (table 2). in contrast, gene amplifications are highly prevalent in gbm, although there appear to be methylation class specific differences in the frequency of distinct amplifications. the presence of these gene amplifications may be considered an argument against the diagnosis of pxa. the frequency of homozygous cdkn2a/b deletions in 932 mcpxa is close to 90% which exceeds the incidence of this alteration in gbm and hgap. while very characteristic for pxa, the high frequency of homozygous cdkn2a/b deletions does not distinguish from gbm. in conclusion, we are demonstrating a high homogeneity regarding cnvs and methylation pattern in tumors receiving the methylation-based prediction mcpxa, contrasted by a high heterogeneity in histologically diagnosed pxa. our cnv findings in mcpxa, orthogonal to the methylation class predictions, provide independent evidence for the existence of a coherent group of tumors that are unified not by their histopathology, but rather by their composite genomic and epigenomic signature. given the demonstrated morphological heterogeneity of mcpxa tumors, this work contributes to providing an alternative for defining pxa on molecular, rather than morphological grounds. disclosure m. snuderl, d. capper, m. sill, d.t.w. jones, s. pfister, d. schrimpf, f. sahm and a. von deimling are co-founders and shareholders of heidelberg epignostix gmbh. supplementary tables supplementary table 1 (download) predictions of heidelberg classifier v12_8 in 551 tumors with the diagnosis or differential diagnosis of pxa. reference groups selected for frequent predictions with a calibrated score >=0.9 are highlighted in green. astrocytoma, idh-mutant and diffuse hemispheric glioma, h3 g34-mutant were not included for their likely detection by mutation specific antibodies. malignant peripheral nerve sheath tumor (spinal or atypical type) was excluded and control tissue, reactive tumor microenvironment was also excluded. supplementary table 2 (download) diagnosies submitted to 767 methylation datasets receiving a heidelberg classifier v12_8 prediction of mcpxa with a calibrated score >=0,9. supplementary table 3 (download) crosstable chromosomal arms, 2418 tumors supplementary table 4 (download) crosstable genes, selected amplification and homozygous deletions, 2418 tumors references 1. kepes jj, rubinstein lj, eng lf. 1979. pleomorphic xanthoastrocytoma: a distinctive meningocerebral glioma of young subjects with relatively favorable prognosis. a study of 12 cases. cancer 44:1839-52. doi: 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genome-wide dna methylation profiling shows molecular heterogeneity of anaplastic pleomorphic xanthoastrocytoma. cancer sci 110:828-32. doi: https://doi.org/10.1111/cas.13903 7. vaubel ra, caron aa, yamada s, decker pa, eckel passow je, et al. 2018. recurrent copy number alterations in low-grade and anaplastic pleomorphic xanthoastrocytoma with and without braf v600e mutation. brain pathol 28:172-82. doi: https://doi.org/10.1111/bpa.12495 8. capper d, jones dtw, sill m, hovestadt v, schrimpf d, et al. 2018. dna methylation-based classification of central nervous system tumours. nature 555:469-74. doi: https://doi.org/10.1038/nature26000 9. stichel d, ebrahimi a, reuss d, schrimpf d, ono t, et al. 2018. distribution of egfr amplification, combined chromosome 7 gain and chromosome 10 loss, and tert promoter mutation in brain tumors and their potential for the reclassification of idhwt astrocytoma to glioblastoma. acta neuropathol 136:793-803. doi: https://doi.org/10.1007/s00401-018-1905-0 10. weber rg, hoischen a, ehrler m, zipper p, kaulich k, et al. 2007. frequent loss of chromosome 9, homozygous cdkn2a/p14(arf)/cdkn2b deletion and low tsc1 mrna expression in pleomorphic xanthoastrocytomas. oncogene 26:1088-97. doi: https://doi.org/10.1038/sj.onc.1209851 copyright: © 2023 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. malignant glioma in l-2-hydroxyglutaric aciduria: thorough molecular characterization of a case and literature review free neuropathology 5:11 (2024) case report malignant glioma in l-2-hydroxyglutaric aciduria: thorough molecular characterization of a case and literature review fleur cordier1,2, pieter wesseling1,3, bastiaan b.j. tops1, lennart kester1, pim j. french4, martin van den bent4, felix hinz5,11, eleonora aronica3, k. mariam slot6, floor abbink7, marjo s. van der knaap8,9,10, mariëtte e.g. kranendonk1 princess máxima center for pediatric oncology, utrecht, the netherlands department of pathology, ghent university hospital, ghent university, ghent, belgium department of pathology, amsterdam university medical centers (umc), university of amsterdam (ea) and vrije universiteit amsterdam (pw), amsterdam neuroscience, the netherlands department of neurology, erasmus medical center, rotterdam, the netherlands department of neuropathology, institute of pathology, heidelberg university hospital, heidelberg, germany department of neurosurgery, amsterdam university medical centers, amsterdam, the netherlands department of pediatric oncology, emma children's hospital, amsterdam university medical centers, amsterdam, the netherlands department of pediatric neurology, emma children's hospital, amsterdam university medical centers, amsterdam, the netherlands amsterdam neuroscience, amsterdam, the netherlands department of integrative neurophysiology, center for neurogenomics and cognitive research, vrije universiteit amsterdam, amsterdam, the netherlands clinical cooperation unit neuropathology, german consortium for translational cancer research (dktk), german cancer research center (dkfz), heidelberg, germany corresponding authors: marjo s. van der knaap & pieter wesseling · dept. of pediatric neurology & pathology · amsterdam university medical centers/vumc · de boelelaan 1117 · 1081 hv amsterdam · the netherlands p.wesseling@amsterdamumc.nl submitted: 15 february 2024 accepted: 23 april 2024 copyedited by: georg haase published: 3 may 2024 https://doi.org/10.17879/freeneuropathology-2024-5377 keywords: l-2-hydroxyglutaric aciduria, cns tumor, paediatric-type diffuse high-grade glioma, dna-methylation-classification, sequencing abstract l-2-hydroxyglutaric aciduria (l-2-hga) is a rare neurometabolic disorder characterized by accumulation of l2-hydroxyglutarate (l-2-hg) due to mutations in the l2hgdh gene. l-2-hga patients have a significantly increased lifetime risk of central nervous system (cns) tumors. here, we present a 16-year-old girl with l-2-hga who developed a tumor in the right cerebral hemisphere, which was discovered after left-sided neurological deficits of the patient. histologically, the tumor had a high-grade diffuse glioma phenotype. dna sequencing revealed the inactivating homozygous germline l2hgdh mutation as well as inactivating mutations in tp53, bcor and nf1. genome-wide dna-methylation analysis was unable to classify the tumor with high confidence. more detailed analysis revealed that this tumor clustered amongst idh-wildtype gliomas by methylation profiling and did not show the glioma cpg island methylator phenotype (g-cimp) in contrast to idh-mutant diffuse gliomas with accumulated levels of d-2-hg, the stereoisomer of l-2-hd. these findings were against all our expectations given the inhibitory potential of 2-hg on dna-demethylation enzymes. our final integrated histomolecular diagnosis of the tumor was diffuse pediatric-type high-grade glioma, h3-wildtype and idh-wildtype. due to rapid tumor progression the patient died nine months after initial diagnosis. in this manuscript, we provide extensive molecular characterization of the tumor as well as a literature review focusing on oncogenetic considerations of l-2-hga-associated cns tumors. introduction l-2-hydroxyglutaric aciduria (l-2-hga) is an autosomal recessive neurometabolic disorder.1 the disease is caused by pathogenic variants in the l2hgdh gene (14q22.1) encoding l-2-hydroxyglutarate dehydrogenase, a flavin adenine dinucleotide (fad)-dependent enzyme that normally oxidizes l-2-hydroxyglutarate (l-2-hg) to alpha-ketoglutarate. defective enzyme activity leads to l-2-hg accumulation with a myelinotoxic and oncogenic effect on the central nervous system (cns) and l-2-hg accumulation in urine, plasma, and cerebrospinal fluid.2 the disease poses a relatively consistent pattern of presentation, starting with delayed mental and motor development in the first years of life, epilepsy in around 60 % of the cases, continuing with a slowly progressive course eventually leading to ataxia and moderate to severe mental deterioration.2,3,4,5,6,7 by imaging, a unique pattern of abnormalities is seen, characterized by subcortical leukoencephalopathy with relative sparing of the periventricular white matter, corpus callosum, cerebellar white matter and the brainstem tracts. additional signal alterations in the basal ganglia and dentate nuclei are typical.2,8,9 a minority of patients also develop a cns tumor, usually later in life.2 because of the enhanced tumor risk, regular imaging for patients with l-2-hga is part of standard care, typically once per 1 or 2 years. here we present a teenager with l-2-hga who developed a glial neoplasm in the cerebral hemisphere. we hypothesized that the oncogenesis of this tumor would be in line with idh-mutant astrocytomas where d-2-hg (the enantiomer of l-2-hg) is considered the oncometabolite leading to the glioma cpg island methylated phenotype (g-cimp).10,11 however, the tumor of the l-2-hga patient did not show g-cimp and was challenging-to-classify, even using a combination of state of the art molecular diagnostic tools. additionally, we provide an overview of l-2-hga-associated cns tumors reported in the literature and discuss the similarities and differences with idh-mutant gliomas. case a 16-year-old girl, the sole child of a consanguineous couple, experienced a normal early development and could walk without support at 13 months. speech development was delayed, and she exhibited mild motor coordination issues. at 5 years age, an mri revealed extensive subcortical cerebral white matter abnormalities and signal abnormalities in the basal ganglia and dentate nuclei, characteristic of l-2-hga. the diagnosis was confirmed through elevated urinary excretion of l-2-hg and the identification of a homozygous variant in the l2hgdh gene (l2hgdh(nm_024884.2): c.339t > a p.(cys113*)).12 the parents were confirmed to be heterozygous for the same variant. over the years, the patient developed moderate cognitive impairment and mild cerebellar ataxia. starting at the age of 14, she experienced occasional epileptic seizures, treated with valproate. mri at the age of 14 years did not show evidence of tumor development. at age 16, a new mri, prompted by a new-onset subtle left-sided central paresis, revealed an augmentation in t2 hyperintensity and swelling in most of the right cerebral hemisphere. the border between cortex and subcortical white matter was blurred, particularly medial in the temporal lobe, where the cortex was thickened and had a slightly increased t2 signal (figure 1a). a similar tumorous area was identified in the right superior temporal gyrus (figure 1b). distinguishing pre-existing abnormal white matter related to l-2-hga from the new abnormalities within the white matter was challenging. the signal abnormalities extended into the right thalamus, right cerebral peduncle, pons, and were accompanied by subfalcine herniation and a midline shift to the left (figure 1a,b). post-contrast scans did not reveal abnormal enhancement. several small foci of diffusion restriction were present within the tumor. based on the findings in serial mri examinations a radiological diagnosis of a rapidly growing, likely high-grade glial tumor was made. a biopsy of the tumor was taken from the right temporal lobe near the superior temporal gyrus to confirm its nature. figure 1. preoperative mri. axial t2-weighted mr images with the red arrows indicating a space occupying lesion in the medial part of the right temporal lobe (a) and in the right superior temporal gyrus (b, i.e. the location of the biopsy), accompanied by midline shift and brain herniation (transtentorial in a, subfalcine in b). additionally, in both hemispheres t2 hyperintensity of the white matter is present, pathognomonic for l2-hga. histology histological analysis revealed a tumor consisting of small, poorly differentiated appearing cells with diffuse infiltrative growth in the preexistent brain parenchyma. the tumor cell nuclei were round to ovoid, showed substantial atypia and fine-grained chromatin with small nucleoli. mitotic figures were frequent. notably, the cells showed some degree of clustering with small areas of pre-existent neuropil in between clusters, but lacked true rosettes or evident perivascular pseudorosettes (figure 2a,b). florid microvascular proliferation and necrosis were absent. figure 2. histological and immunohistochemical characteristics of the tumor. (a,b) low and high magnification of the diffuse infiltrative high-grade glioma. (c-f) immunoreactivity for ki-67 (c), gfap (d), map2 (e) and ema (f). scale bars: 80 um (a,c,d); 50 um (b,e,f). immunohistochemically, the tumor cells were positive for vimentin, cd56, and map2, and in some areas for synaptophysin and gfap as well. dot-like ema staining was found in a small number of tumor cells. the tumor cells were negative in the following stainings: cam5.2, cd34 (with positive control in the vessels), neun (staining only pre-existing neurons), olig2, idh1 r132h and h3 p.k28m. furthermore, in the h3 p.k28me3, atrx, and ini-1 staining the nuclear expression was retained. the ki-67 labelling index was variable but locally >= 30 %. (figure 2c-f). molecular findings dna methylation analysis was performed using the infinium methylationepic beadchip (illumina), and explored using versions 11b4, 12.5 and 12.8 of the heidelberg classifier developed by dkfz (https://www.molecularneuropathology.org/mnp/)8 as well as the bethesda classifier.13 neither of these classifiers were able to classify the tumor with a high confidentiality score. the best classification possible was obtained with dkfz v12.8 which classified the tumor as pediatric-type diffuse high-grade glioma with a highest score of 0.83, diffuse paediatric type high grade glioma, mycn subtype (calibrated score 0.52). the bethesda classifier and umap (uniform manifold approximation and projection) suggested idh-mutant glioma with a score of 0.54. based on dna methylation-based t-distributed stochastic neighbor embedding (t-sne) clustering analyses, this tumor clustered most close to pediatric-type diffuse high-grade glioma, mycn-activated subtype (figure 3a). figure 3. dna methylation analysis of the tumor. (a) dna methylation-based t-distributed stochastic neighbor embedding (tsne) of the tumor of our patient with l-2-hga (marked as index, see arrow) compared to selected reference samples from the heidelberg database (see supplemental data for abbreviations). the tsne was run with following settings: cpgs analysed = 10.000, iterations = 3.000, perplexity = 5, theta = 0, dims = 2. our case was in close proximity to mycn-activated pediatric high-grade gliomas. samples are colored according to their methylation class. (b) copy number plot showing a rather complex profile with multiple chromosomal gains and losses. (c,d) methylation status in comparison to idh-mutant gliomas. unsupervised analysis using the 1000 most variable probes of a cohort of 435 idh-mutant and 215 idh-wildtype gliomas15 revealed that the tumor of our patient clustered amongst the idh-wildtype tumors (figure 3c, color-coding of the samples: idh-mutant, idh-wildtype and unknown samples are indicated respectively in green, red and grey. the index case is indicated in blue and by the red rectangle). (figure 3d, mean methylation beta value of the 1000 most variable probes shown in c. the idh-wildtype cases are colored in red, the idh-mutant cases in green and the index sample is highlighted with an orange triangle.) the copy number variation (cnv) plot obtained by the conumee package embedded in the methylome profiling analysis and whole exome sequencing (wes) revealed a complex cnv profile with multiple gains and losses, including partial loss of chromosome 10q. there was no high-level amplification of egfr or mycn, no chromosome 7 gain, no homozygous deletion of cdkn2a/b and no chromosome 1p/19q codeletion (figure 3b). the promoter of the o6-methylguanine-dna methyltransferase (mgmt) gene was found to be methylated. wes identified somatic mutations in tp53 (c.818g > a (p.arg273his)); 96.6 % vaf (including copy neutral loh), nf1:c.4977_4980delctct (p.lys1661glyfs*36); 95.1 % vaf (including copy neutral loh) and bcor:c.4438c > t (p.arg1480*); 93.7 % vaf (including copy neutral loh). furthermore, the known homozygous germline mutation in l2hgdh (c.339t > a p.(cys113*)) was detected in the tumor as well (100 % vaf). rna sequencing revealed no additional mutations or gene rearrangements. rna levels of l2hgdh in the tumor were comparable to or even lower than in other pediatric tumors in the reference cohort of the princess maxima center (supplemental figure 1). unfortunately, there was no tissue left for analysis of l2hgdh protein levels in the tumor. acknowledging that in idh-mutant tumors high levels of d-2-hg (the enantiomer of l-2-hg) result in hypermethylation and g-cimp9, 14, we investigated whether cimp was present in the tumor of our patient with l-2-hga as well. however, unsupervised analysis using the 1000 most variable probes of a cohort of 435 idh-mutant and 215 idh-wildtype gliomas15 revealed that the tumor of our patient clustered amongst the idh-wildtype tumors (figure 3c) and had relative low methylation levels (figure 3d). follow up given the dismal prognosis, which was partly attributed to the large size of the tumor, a restrained treatment policy was chosen. following the acns 0126 protocol, the patient began treatment with temodal two months after diagnosis, for stabilization of the tumor, in addition to dexamethasone for edema relief. unfortunately, in the course of the following months the condition of the patient rapidly worsened, leading to her death nine months after the diagnosis of the cns tumor. literature review of l-2-hga related cns tumors the association of l-2-hga and cns tumors has been documented in the literature, with an estimated rate of association of 5 % to 40 %2 according to available data (see table 1 for a compilation of cases in the literature). the spectrum of cns tumors reported to be associated with l-2-hga includes medulloblastoma in early childhood, anaplastic ependymoma predominantly in teenagers, lowand high-grade gliomas in both childhood and adults, and embryonal tumors (previously designated as “primitive neuroectodermal tumor”/pnets). apart from medulloblastomas (which by definition arise in the posterior fossa), l-2-hga tumors are often located in a cerebral hemisphere (frontal lobe, temporal lobe and/or thalamus), but rarely in the hippocampus or in the ventricles. however, there is some missing data in the cases described in the literature: in one case the tumor diagnosis is not described, in three cases the age is lacking, in four cases information regarding the sex is not reported and most cases do not entail survival data. it is worth noting that translating previously assigned diagnoses for cns tumors associated with l-2-hga (table 1) into labels conforming to who cns 5 criteria poses some challenges.16,17 for example, the tumors of patients 13 and 20 in table 1 were diagnosed as glioblastoma, while according to the who cns 5 classification they would likely be classified as diffuse pediatric-type high-grade glioma, h3-wildtype and idh-wildtype. additionally, no idh status was reported in the astrocytoma or oligodendroglioma cases, which is an essential piece of information for a correct diagnosis of diffuse gliomas nowadays. also, the case regarded as ependymoma and even the embryonal tumors may turn out to be high grade gliomas.18 all in all, it is therefore difficult to associate a specific tumor type with l-2-hga. discussion in this report, we present a teenager with l-2-hga who developed a temporal lesion, histomolecularly classified as diffuse pediatric-type high-grade glioma, h3-wildtype, and idh-wildtype according to the who cns 5 criteria. this tumor entity represents a group of malignant diffuse gliomas, primarily occurring in children or young adults that lack alterations in histone h3 genes, as well as in idh1 and idh2. the molecular alterations in this group of tumors are diverse and include tp53 mutations, mycn amplification, pdgfra, id2, nf1 or egfr alterations. by dna methylation profiling they comprise a distinct group with several molecular subgroups (rtk1, rtk2 and mycn).16,19 methylation array classification is based on “fingerprinting” the methylome of the tumor, and subsequently mapping this to a reference database.8 this methylome profile is a combination of both somatically acquired dna methylation changes as well as the methylation status of the cell of origin. noteworthy, high-grade gliomas based on a tumor predisposition syndrome such as mismatch repair (mmr) deficiency or li-fraumeni are often classified as diffuse pediatric-type high-grade glioma, h3-wildtype and idh-wildtype as well, although not always with high confidence. capper et al. described that the full spectrum of tumors in the context of a hereditary predisposition syndrome may not yet be reliably classified.20 in line with this, the tumor we present here could also not be classified with confidence. a possible explanation for classification difficulties might be that the effect of the germline alteration, which is present in all cells, together with the somewhat different oncogenesis alters the methylome of the cancer in such a way that it no longer fully reflects the methylome profile of the sporadic counterparts in the reference cohort. it remains to be seen if tumors that arise in the context of l-2-hga are (also on the epigenetic level) more similar to each other than suggested based on literature in the pre-molecular era (table 1). methylation analysis of tumors from additional l2-hga patients is necessary for further characterization of l2-hga-associated tumors, and for corroborating the absence of g-cimp that we observed in our case. table 1: reported cns tumors associated with l-2-hga case sex / age tumor location reported histological diagnosis molecular alteration methylation profile overall survival wilcken et al. 199335 1 f / 15 temporal pnet nd nd nm barbot et al. 199736 2 f / 10 thalamus right fibrillar diffuse astrocytoma nd nd 6 months wanders et al. 199737 3 f / 9 thalamus right low-grade astrocytoma nd nd 6 months ozisik et al. 200238 4 m / 3 posterior fossa medulloblastoma nd nd 6 months moroni et al. 20045 5 m / 13,5 temporal right pnet nd nd alive at 2 years fu 6 f / 26 temporal left glioblastoma loh chrom 1p nd nm 7 f / 18 temporal right nd nd nd 3 years 8 m / 12 frontal left low-grade astrocytoma nd nd few months topcu et al. 200539 9 nm / 3 nm medulloblastoma nd nd 6 months vilarinho et al. 200540 10 nm / nm nm astrocytoma nd nd nm 11 nm / nm nm astrocytoma nd nd nm 12 nm / nm nm astrocytoma nd nd nm haliloglu et al. 200841 13 m / 11 temporoparietal right glioblastoma nd nd nm aghili et al. 200942 14 m / 17 frontal anaplastic ependymoma nd nd nm patay et al. 20122 15 m / 23 hippocampus right anaplastic astrocytoma nd nd nm 16 m / 19 frontal right oligodendroglioma nd nd nm 17 f / 36 thalamus left low-grade glioma nd nd nm london et al. 201525 18 m / 29 both hemispheres, corpus callosum and brainstem gliomatosis cerebri nd nd 3 weeks patay et al. 201521 19 m / 22 temporal right anaplastic astrocytoma egfr amplification nd 2nd tumor after 40 months tan et al. 201832 20 f / 16 lateral ventricle left glioblastoma loss 1p, no mutation in exon 1 of the h3c2 or h3-3a genes nd no recurrence at 2 years fu ibrahim et al. 201843 21 m / 11 left hemisphere anaplastic oligodendroglioma nd nd nm current case 22 f / 16 temporal right high grade diffuse glioma l2hgdh, tp53, nf1, bcor diffuse pediatric-type high-grade glioma, h3and idh-wildtype (but with very low confidentiality score) 9 months m: male, f: female, nm: not mentioned, nd: not determined, pnet: primitive neuroectodermal tumor, fu: follow-up interestingly, idh-mutant diffuse gliomas exhibit elevated levels of d-2-hydroxyglutarate (d-2-hg), i.e. the enantiomer of the l-2-hg.21 this (onco) metabolite is a competitive inhibitor of alpha ketoglutarate-dependent dioxygenases, including the ten-eleven translocation (tet) enzymes which play a crucial role in the initial stages of dna demethylation. consequently, high levels of d-2-hg in idh-mutated tumors lead to hypermethylation and the manifestation of g-cimp. notably, d-2-hg alone is sufficient to induce such a cimp phenotype.9,14 similarly, l-2-hg inhibits tet1 and tet2 enzymes,2,11,22 and elevated levels of l-2hg were associated with hypermethylation of dna in clear cell renal cell carcinoma.23 even more, it has been shown that l-2-hg was a more potent inhibitor of both tet2 and tet1 than d-2-hg.24 based on these findings, we hypothesize that individuals with l-2-hga are at risk of developing diffuse glial tumors due to the pathological accumulation of l-2-hg in the brain, with a similar g-cimp impact on glial cells and maybe even clustering with the idh-mutant glioma cluster.2,21,25 however, in comparison with the methylation array data of 650 adult-type diffuse gliomas of the catnon database, the tumor of our patient showed relatively low mean dna methylation levels at cpg sites, and (in line with most methylation classifier and wes results) clustered with idh-wildtype samples. this unexpected observation illustrates the complexity of elevated levels of d-2-hg versus l-2-hg in oncogenesis. one possible explanation is that the mechanism underlying elevated levels of 2hg differs between l2hgdhor l2hgdh-associated disease compared to idh-mutant tumors. mutations in l2hgdh or d2hgdh lead to a generalized passive accumulation of 2hg throughout the body, whereas mutations in idh genes in tumors result in high levels of 2hg within the tumor cells themselves with therefore possibly stronger local effect on tet-enzyme activity and subsequent hypermethylation.26,27 of note, elevated d-2-hg levels can also occur in the context of neurometabolic disorders d-2-hga type 1 (due to a mutation in the d2hgdh gene) and type 2 (due to heterozygous mutation in idh2). in that context, increased d-2-hg levels do not lead to gliomas.28 this can be partly attributed to discrepancies in life expectancy, as patients with type 2 d-2-hga, based on an idh2 mutation, exhibit similarly elevated d-2-hg levels but have such a severe clinical course that they may not survive long enough for glioma development.29,30,31 another explanation can be sought in stereoisomerism. even though l-2-hg and d-2-hg are structurally similar molecules, studies have suggested that l-2-hg and d-2-hg may have differential inhibitory effects on certain enzymes, conceivably due to variations in the stereochemistry of l-2-hg and d-2-hg molecules. overall, while both l-2-hg and d-2-hg function as inhibitors in cellular metabolism, their specific effects and potency may vary depending on the context and the particular enzymes or pathways involved. this is further stressed by the observation that the level of l-2-hg does not per se correlate with risk of cns tumor development.32 therefore it seems that 2-hg has physiological roles that extend beyond being an oncometabolite by itself. notably, there appears to be no sex predisposition to the carcinogenic effect.2 also, so far, evidence for a common molecular mechanism downstream of the l2hgdh mutation is lacking. patay et al. (2015) identified an egfr amplification in their case of anaplastic astrocytoma (table 1, case 19) which lacked an idh mutation. they therefore proposed a potential connection between astrocytic tumors and egfr alterations as potential oncogenic driver mechanism in l-2-hg-induced tumorigenesis.21 however, in our case, and in all other cases presented in the literature, no egfr alterations were reported. possibly, the oncogenesis in l-2-hga is like a two-tiered oncogenesis system in which accumulated levels of l-2-hg form the first hit, and a mutation (which may arise in different pathways) forms the second hit eventually leading to tumor formation, with a different pathophysiology than in the idh-mutant tumor. besides the l2hgdh mutation, the tumor analyzed in this report also contained tp53, bcor and nf1 mutations, as well as numerous copy number changes. it is known that malignant progression of idh-mutant astrocytomas is associated with relative hypomethylation (reduced g-cimp status), a phenomenon which may be associated with (homozygous) cdkn2a/b loss or other cell cycle gene alterations in these tumors.33,34 however, in the tumor of our l-2-hga patient homozygous loss of cdkn2a/b was absent. we speculate that in our case other molecular alterations had an impact on l-2-hg levels, tet-enzymes or other epigenetic mechanisms resulting in absence of g-cimp. unfortunately, no tumor tissue was left for proper assessment of l-2-hg at the protein level. taken together, our findings shed unprecedented light on the unique developmental and epigenetic roots of a l-2-hga associated glioma. further studies are necessary to unravel how exactly the oncogenetic mechanism of elevated l-2-hg is different from that of its enantiomer d-2-hg. based on the literature, the prognosis for l-2-hga patients suffering from a cns tumor appears to be poor. among the documented cases, the patients experienced death with a mean survival of 10.8 months after diagnosis, although this is based on limited data. this poor outcome may partly be explained by delay in the diagnosis of these tumors, e.g., because on mri scans the tumors are often not clearly delineated and/or difficult to distinguish from diseased non-neoplastic brain tissue. additionally, among the reported patients, one individual (case 19) was described with a second tumor diagnosed 40 months after initial diagnosis and with the same histological and molecular characteristics as the first.21 conclusion this is the first report of a comprehensive molecular analysis of a challenging-to-classify cns neoplasm in a teenager known to suffer from l-2-hga. intriguingly, we found that elevated levels of l-2-hg resulted in a tumor with characteristics of idh-wildtype rather than of idh-mutant diffuse gliomas, including a lack of g-cimp in the tumor of our patient. our study thereby illustrates that the oncogenic mechanism of increased l-2-hg levels in diffuse gliomas is different from that caused by d-2-hg accumulation in idh-mutant diffuse gliomas. acknowledgments we kindly acknowledge prof. ken aldape and zied abdullaev for providing methylation classification results of the tumor of our patient using the bethesda (nih/nci) methylation classifier. author contributions fc, pw, pjf, msvdk, and megk established the study concept and design. fc, pw and megk performed the writing of the paper. pjf, ea, fh, kms, msvdk and megk contributed the figures. all authors contributed essential ‘building blocks’ for this paper and have read, edited and approved the final version of the manuscript. competing interests the authors declare no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. references 1. duran m, kamerling jp, bakker hd, van gennip ah, wadman sk. l-2-hydroxyglutaric aciduria: an inborn error of metabolism? 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universität berlin and humboldt-universität zu berlin, institute of virology, 10117 berlin, germany and german centre for infection research (dzif), partner site berlin, charitéplatz 1, 10117 berlin, germany charité universitätsmedizin berlin, corporate member of freie universität berlin and humboldt-universität zu berlin, department of neuropathology, 10117 berlin, germany department of internal medicine and clinical immunology, pitié-salpêtrière university hospital, 75013 paris, france centre for pathogen evolution, department of zoology, university of cambridge, cambridge, united kingdom department of pediatric neurology and centre for neuromuscular disorders in children and adolescents, center for translational neuroand behavioral sciences, university duisburg-essen, essen, germany department of neuropathology, sorbonne université, assistance publique-hôpitaux de paris, inserm, hôpital pitié-salpêtrière, france institute of pathology, universitätsmedizin greifswald, greifswald, germany * equally contributing co-first authors # jointly supervised this work corresponding author: josefine radke · institute of pathology · universitätsmedizin greifswald · friedrich-loeffler-str. 23e · 17475 greifswald · germany josefine.radke@med.uni-greifswald.de additional resources and electronic supplementary material: supplementary material submitted: 04 october 2023 accepted: 27 decembeer 2023 copyedited by: joão gama published: 04 january 2024 https://doi.org/10.17879/freeneuropathology-2024-5149 keywords: dermatomyositis (dm), interferon (ifn), viral signature, next generation sequencing abstract objective: to explore a possible connection between active viral infections and manifestation of dermatomyositis (dm). methods: skeletal muscle biopsies were analyzed from patients diagnosed with juvenile (n=10) and adult (n=12) dm. adult dm patients harbored autoantibodies against either tif-1γ (n=7) or mda5 (n=5). additionally, we investigated skeletal muscle biopsies from non-diseased controls (ndc, n=5). we used an unbiased high-throughput rna sequencing (hts) approach to detect viral sequences. to further increase sequencing depth, a host depletion approach was applied. results: in this observational study, no relevant viral sequences were detected either by native sequencing or after host depletion. the absence of detectable viral sequences makes an active viral infection of the muscle tissue unlikely to be the cause of dm in our cohorts. discussion: type i interferons (ifn) play a major role in the pathogenesis of both juvenile and adult dm. the ifn response is remarkably conserved between dm subtypes classified by specific autoantibodies. certain acute viral infections are accompanied by a prominent type i ifn response involving similar downstream mechanisms as in dm. aiming to elucidate the pathogenesis of dm in skeletal muscle tissue, we used deep rna sequencing and a host depletion approach to detect possible causative viruses. introduction although the pathogenesis of dm is not completely understood, it is well established that type i ifn plays a key role in both juvenile and adult dm. the ifn response can be defined by a specific up-regulation of ifn-stimulated molecules such as isg15 or mxa and is comparable to that found in lupus erythematosus (sle) and certain inherited interferonopathies, but also in viral infections [10]. in fact, parvovirus b19, coxsackie virus, polyomavirus, epstein-barr virus (ebv), influenza virus, human immunodeficiency virus (hiv), and sars-cov-2 have been associated with dm onset [3]. since the detection of specific autoantibodies, such as anti-mi-2, anti-tif-1γ, anti-nxp2, anti-sae, and anti-mda5, the spectrum of dm can be defined more precisely regarding prognosis and clinical course such as risk of cancer development [27]. dm subtype-specific investigation of type i ifn-regulated transcripts identified a set of significantly dysregulated genes in muscle biopsies derived from anti-tif-1γ+ patients [4]. moreover, mda5, rig1, and trim33 (tif1γ) are specifically involved in downstream signaling of viral infections [13]. mda5 is a key protein sensor for viral double-stranded rna (dsrna) motifs to induce expression of ifn1 in certain viral diseases [6], and trim33 inhibits endogenous retrovirus (erv) gene transcription [21]. recent studies have demonstrated that myositis autoantibodies, most commonly anti-tif-1γ, anti-nxp2, and anti-mda5, are also found in more than 50% of juvenile myositis patients, with different frequencies in different populations, but again associated with specific clinical manifestations and prognosis [12, 15, 22, 29]. it is well described, especially in children, that dm can occur with an acute onset of general viral infection-like symptoms such as fever, fatigue, and apathy [20]. hence, the aim of our project was the identification of any viral signatures (in terms of an active virus replication) in skeletal muscle samples using an unbiased high-throughput sequencing approach. in contrast to other targeted approaches such as virus-specific pcrs, we aimed at viral genome detection without prior restrictions to probable pathogens to investigate any including previously unknown possible viral species. material and methods patient cohort skeletal muscle biopsies were analyzed from treatment-naive patients diagnosed directly after symptom onset with juvenile or adult dm according to enmc diagnostic criteria [14]. we included n=22 dm cases from three institutions between 2008 and 2018, in which the serum of adult patients was either positive for autoantibodies against tif-1γ (n=7) or mda5 (n=5). the morphological muscle involvement was related to the underlying autoantibody with perifascicular atrophy and inflammation in tif-1γ+ patients and less severe muscle damage in mda5+ patients [1]. the serum of n=2 juvenile patients was positive for autoantibodies against npx2. the other juvenile patients were diagnosed before autoantibody test-ing was widely available and retroactive information on autoantibodies was not available. additionally, we investigated skeletal muscle biopsies (n=5) from non-diseased controls (ndc) with nonspecific complaints, without overt muscle weakness, absence of any morphologic abnormalities in the skeletal muscle biopsies, elevated creatine kinase (ck) levels, or laboratory evidence of any systemic inflammation including negativity of autoantibody testing. informed consent was obtained from all patients at each institution involved. procedures were approved by the official ethical standards committee (ea2/163/17) at the charité – universitätsmedizin berlin. the experimental setup is shown in figure 1. figure 1: experimental study design. the study cohort, including subgroups and information on autoantibodies, is given in the upper panel. histological work-up of dm skeletal muscle samples shows perifascicular atrophy (he, arrows). immunohistochemical staining of type i ifn-inducible proteins isg15 and mxa demonstrates a perifascicular staining pattern with siglec1-positive macrophages (brown) in close proximity to isg15 positive muscle fibers (red). deep sequencing of dm patients’ skeletal muscle biopsies only revealed viral reads derived from column-based extraction and library preparation kit contaminations. (adm: adult dermatomyositis, jdm: juvenile dermatomyositis, ndc: non-diseased controls, na: no autoantibody testing, yrs: years). high throughput-sequencing of rna total rna was extracted from fresh-frozen, cryopreserved (at -80°c) skeletal muscle specimens using the rneasy kit (qiagen, hilden, germany) according to the manufacturer’s recommendations. rna extraction was checked by testing for beta-2 microglobulin (b2m) mrna using a rt-qpcr as previously described [23]. for the detection of viral sequences two strategies were applied: (i) unbiased native sequencing of the nucleic acids and (ii) a more sensitive sequencing approach after the specific removal of ribosomal rna (rrna). the detailed steps are given in supplementary material and methods. statistics and hts data analyses for virus detection, we applied two approaches for viral read identification. first, a computational pipeline that consists of a collection of bash shell (c. ramey, gnu bash, available at https://www.gnu.org/software/bash/) scripts that invoke third-party and custom in-house programs. overall execution is coordinated by a slurm pipeline python package (t. c. jones, slurm pipeline, available at https://github.com/acorg/slurm-pipeline). second, we classified sequences with kraken2 (version 2.1.3) and the kraken2 [28] ‘standard’ database (available at https://benlangmead.github.io/aws-indexes/k2, revision date 10/9/23). the results were visualized using krona [19] and pavian [5] and independently analyzed by two virologists. no special cut-off value was applied, but rather each individual virus hit was examined and evaluated individually. the detailed steps for both analyses are given in supplementary material and methods. data availability the sequencing data (non-human reads) of the samples generated in this study have been deposited in the sequence read archive (sra) under the accession numbers samn39051134 samn39051187. results sequencing results before and after rrna removal successful rna extraction was confirmed by the detection of beta-2-microglobulin mrna in all samples (supplementary table 1). all samples were processed in the two sequencing approaches: using native sequencing, a total of 661.6 mio reads (range: 7.1-32.8 mio per sample, mean: 24.5 mio, median: 25.5 mio) were generated for all patients analyzed. roughly, 90% of the resulting reads could be mapped to rrna references (nr_003287.1, nr_145820.4) and less than 1% of the reads mapped against a globin reference sequence (hba1: nm_000558.5, hba2: nm_000517.6, hbb: nm_000518.5, hbd: nm_000519.4) (table 1). table 1. overview of samples, pcr, and sequencing results. the table includes number of generated reads and proportion of rrna and globin reads for native sequencing and sequencing after rrna depletion.   native sequencing rrna removal patient id cohort ⚥ age reads [r1+r2] reads against rrna* % rrna reads against globin** % globin reads [r1+r2] reads against rrna* % rrna reads against globin** % globin 1 mda 5 m 34 25,451,212 23,454,672 92.2 1 0.000004 23,752,226 700,27 2.95 2,965 0.012483 2 mda 5 f 32 16,964,910 15,449,997 91.1 100 0.000589 11,763,512 115,577 0.98 116 0.000986 3 mda 5 f 39 25,805,868 24,592,381 95.3 192 0.000744 23,635,252 349,242 1.48 54,525 0.230694 4 mda 5 m 31 27,640,030 26,338,410 95.3 97 0.000351 23,982,524 530,301 2.21 1,507 0.006284 5 mda 5 f 38 26,560,152 24,391,028 91.8 6.780 0.025527 12,321,160 142,752 1.16 269 0.002183 6 tif-1γ f 72 24,181,444 22,604,310 93.5 101 0.000418 32,040,902 1,823,768 5.69 388 0.001211 7 tif-1γ m 69 25,030,838 22,695,134 90.7 102 0.000407 32,634,054 839,161 2.57 1,214 0.003720 8 tif-1γ f 47 27,551,144 25,511,050 92.6 11 0.000040 37,285,322 877,105 2.35 225 0.000603 9 tif-1γ f 64 26,854,504 22,726,155 84.6 53 0.000197 38,565,498 969,885 2.51 3,156 0.008183 10 tif-1γ f 38 27,316,894 24,417,085 89.4 6 0.000022 19,416,276 233,351 1.20 293 0.001509 11 tif-1γ f 83 27,684,124 24,577,672 88.8 122 0.000441 38,328,838 681,366 1.78 548 0.001430 12 tif-1γ m 69 29,464,600 27,636,300 93.8 83 0.000282 21,102,676 388,385 1.84 22 0.000104 13 ndc f 70 30,284,074 26,373,635 87.1 55 0.000182 28,112,926 388,141 1.38 9,949 0.035389 14 ndc f 51 25,886,910 25,146,497 97.1 0 0.000000 25,898,230 711,277 2.75 332 0.001282 15 ndc m 53 22,971,962 20,643,167 89.9 590 0.002568 6,997,044 33,46 0.48 233 0.003330 16 ndc f 17 32,842,356 28,922,129 88.1 34 0.000104 20,534,850 363,574 1.77 107 0.000521 17 ndc f 32 19,838,176 18,580,234 93.7 532 0.002682 14,686,946 338,501 2.30 1,31 0.008919 18 jdm m 20 26,934,130 25,226,685 93.7 10 0.000037 10,881,868 421,412 3.87 257 0.002362 19 jdm f 15 24,941,254 22,494,587 90.2 98 0.000393 28,547,838 314,633 1.10 3,078 0.010782 20 jdm m 2 21,805,986 19,930,688 91.4 22 0.000101 5,877,104 17,646 0.30 13 0.000221 21 jdm f 2 22,330,238 20,058,021 89.8 284 0.001272 6,781,752 117,288 1.73 99 0.001460 22 jdm m 6 7,149,280 4,980,683 69.7 26 0.000364 5,332,966 236,384 4.43 119 0.002231 23 jdm f 5 23,297,262 21,965,327 94.3 62 0.000266 13,150,292 128,447 0.98 101 0.000768 24 jdm m 7 23,303,882 21,816,633 93.6 59 0.000253 11,615,248 31,92 0.27 111 0.000956 25 jdm m 10 20,889,528 19,197,167 91.9 13 0.000062 12,207,356 468,646 3.84 68 0.000557 26 jdm m 2 19,364,716 16,680,717 86.1 141 0.000728 32,487,164 53,126 0.16 2,411 0.007421 27 jdm f 5 29,292,446 27,326,249 93.3 816 0.002786 20,397,658 386,63 1.90 6,248 0.030631 * nr_003287.1, nr_145820.4 ** hba1: nm_000558.5, hba2: nm_000517.6, hbb: nm_000518.5, hbd: nm_000519.4 for removal of rrna and globin sequences, we initially applied the qiaseq fastselect -rrna and globin hmr kit for four samples. this resulted in a nearly complete removal of rrna (<1% remaining) and a similar reduction of globin sequences (data not shown). as the initial number of globin mrna reads was already low, we proceeded with the removal of only rrna for all samples. after rrna depletion, a total of 558.3 mio reads (range: 5.3-38.6 mio per sample, mean: 20.7 mio, median: 20.5 mio) were generated. a maximum of 5.69% rrna reads and well below 1% globin reads remained. as expected, kraken2 classification of reads from both approaches resulted in many reads classified as being of human, bacterial, or archaea origin (supplementary table 2). although not within the scope of the present study, which focuses on viruses, the detection of these reads can be used to ensure consistent quality and comparability of the sequencing data for all samples. the matched bacterial reads mainly correspond to environmental bacteria and are likely explained by contamination from the environment or the skin microbiome (e.g. cutibacterium acnes). matches against virus sequences for both sequencing approaches and both sequence classification approaches, no relevant human virus findings were detected. both classification approaches resulted in overall similar results (supplementary figure 1). a common finding were retroviral reads (murine leukemia virus) which were previously described as common contamination from library preparation kit reagents [8]. remarkably, after rrna removal, a higher number of previously described contaminations from extraction columns and library preparation kit reagents were found that were not detected in the native sequencing approach. these mainly included plantand algae-infecting viruses such as carrot red leaf virus, lake sarah-associated circular virus, and gokushovirinae. in one sample, viral reads against kadipiro virus were identified, which was also previously shown to be a potential kit contamination [11, 18]. additionally, we detected circovirus-like viral reads that were previously associated with rneasy minelute columns used for rna extraction [2]. in all depleted samples, including the control subjects, we found reads mapping against two short (<150 bp) genome regions of aav-5, which we therefore did not count as a virus finding. we were not able to identify the origin of these two short sequences, but an origin in the used kits or reagents are also likely. although only rna library preparation was performed, it was also possible to detect dna viruses, e.g., via transcripts or replication intermediates as shown by the detection of the dna virus gokushovirinae, cycloviruses, and chrysochromulina ericina virus. discussion the aim of the project was to explore a putative viral pathogenesis of dm using an unbiased methodology. this was based on obvious evidence concerning molecular links between relevance and presence of ifn-related gene expression, and known autoantibody functions such as mda5/rig1 and trim33, as well as epitope homology of trim proteins with specific viral species including poxviruses [16]. here, we detected viral sequences, especially after rrna removal, in most of the samples. nevertheless, the few viral reads concerning murine leukemia virus, carrot red leaf virus, lake sarah-associated circular virus, gokushovirinae, parvovirus nih-cqv, and kadipiro virus are very likely derived from column-based extraction and library preparation kit contamination [2] instead of being the cause of direct viral muscle infections, which have been reported for influenza viruses, enteroviruses, hiv, and hepatitis viruses [7, 17, 25] among others. the increase in such matches in the rrnadepleted samples indicates the higher sensitivity of this approach and that the sequencing depth was sufficient to detect potential causative virus sequences including rna transcripts from active replicating dna viruses, had they been present in the muscle samples. therefore, it can be assumed that no viral pathogens were present at the times and sites of sample collection. however, the absence of evidence of active viral infection in the muscle tissues at the time of sampling does not preclude previous viral infections. autoimmunity and the detection and accumulation of antibodies are well established to be at the root of the pathogenesis of dm [9]. viral infections and virus exposure in turn contribute to accumulation of virus-specific antibodies and generation of autoantibodies, which may lead to autoimmunity via several pathways including molecular mimicry, epitope spreading, and bystander activation even after complete viral clearance [9, 24]. multiple other factors such as genetic predisposition, host immune response, and viral strain may also play an important role in disease occurrence, progression, and prognosis. as antibodies recognizing viruses demonstrate a high abundance in dm patients, further studies using next generation sequencing techniques will be needed to investigate the viral exposure patterns in dm patients at different time points during the course of the disease [16, 26]. this may also help to detect new, previously unknown dm-specific autoantibodies to further prognostically stratify dm patients. author contributions werner stenzel, corinna preusse, victor corman, and josefine radke designed the study concept, performed data analysis, and wrote the manuscript. terry c. jones and julia melchert performed data analysis and revised the manuscript. randi koll performed data analysis. olivier benveniste, ulrike schara-schmidt, and sarah leonard-louis provided muscle biopsy samples and revised the manuscript. christian drosten and hans-hilmar goebel revised the manuscript and contributed to data analysis. funding this work was supported by the deutsche gesellschaft für muskelkranke (dgm) e.v. acknowledgements we gratefully thank p. matylewski and s. stefaniak for excellent technical assistance and advice. we thank nikolai zaki for help with kraken2 analyses. cartoon images were created with biorender.com. conflicts of interest the authors declare no conflict of interest. references 1. allenbach y, leroux g, suarez-calvet x, preusse c, gallardo e, hervier b, rigolet a, hie m, pehl d, limal n et al (2016) dermatomyositis with or without anti-melanoma differentiation-associated gene 5 antibodies: common interferon 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doi: https://doi.org/10.1186/s13059-019-1891-0 29. wu jq, lu mp, reed am (2020) juvenile dermatomyositis: advances in clinical presentation, myositis-specific antibodies and treatment. world j pediatr 16: 31-43. doi: https://doi.org/10.1007/s12519-019-00313-8 copyright: © 2024 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. from research to diagnostic application of raman spectroscopy in neurosciences: past and perspectives feel free to add comments by clicking these icons on the sidebar free neuropathology 3:19 (2022) review from research to diagnostic application of raman spectroscopy in neurosciences: past and perspectives gilbert georg klamminger1,2,3, katrin b. m. frauenknecht2,3, michel mittelbronn2,3,4,5,6,7, felix b. kleine borgmann2,3,1,5 1 saarland university medical center and faculty of medicine, homburg, germany 2 national center of pathology (ncp), laboratoire national de santé (lns), dudelange, luxembourg 3 luxembourg center of neuropathology (lcnp), dudelange, luxembourg 4 luxembourg centre of systems biomedicine (lcsb), university of luxembourg (ul), esch-sur-alzette, luxembourg 5 department of cancer research (docr), luxembourg institute of health (lih), luxembourg, luxembourg 6 department of life sciences and medicine (dlsm), university of luxembourg, esch-sur-alzette, luxembourg 7 faculty of science, technology and medicine (fstm), university of luxembourg, esch-sur-alzette, luxembourg corresponding author: felix b. kleine-borgmann · department of oncology (donc) · 84, val fleuri; l-1526 luxembourg · luxembourg felix.kleineborgmann@lih.lu submitted: 31 may 2022 accepted: 17 july 2022 copyedit and layout by: jerry lou published: 05 august 2022 https://doi.org/10.17879/freeneuropathology-2022-4210 keywords: raman spectroscopy, neurooncology, neurodegeneration, neurosurgery, neuropathology, machine learning abstract in recent years, raman spectroscopy has been more and more frequently applied to address research questions in neuroscience. as a non-destructive technique based on inelastic scattering of photons, it can be used for a wide spectrum of applications including neurooncological tumor diagnostics or analysis of misfolded protein aggregates involved in neurodegenerative diseases. progress in the technical development of this method allows for an increasingly detailed analysis of biological samples and may therefore open new fields of applications. the goal of our review is to provide an introduction into raman scattering, its practical usage and also commonly associated pitfalls. furthermore, intraoperative assessment of tumor recurrence using raman based histology images as well as the search for non-invasive ways of diagnosis in neurodegenerative diseases are discussed. some of the applications mentioned here may serve as a basis and possibly set the course for a future use of the technique in clinical practice. covering a broad range of content, this overview can serve not only as a quick and accessible reference tool but also provide more in-depth information on a specific subtopic of interest. introduction as one special method of various vibrational spectroscopic techniques, raman spectroscopy (rs) has been an integral part in neuroscience research for some time now, be it in neuro-oncology for tumor classification1 or for the biochemical description of various protein aggregates in neurodegenerative diseases2. currently it is making its way towards a clinical implementation3. looking at the numerous advantages of rs, the reasons for an increased use in research are obvious: it enables fast and user-friendly (easy to apply) analysis for the purpose of tissue identification (e.g., identification of different brain regions in three mice strains4) by observed changes in the vibrational level of the underlying biochemical and molecular composition. compared to other advanced molecular techniques, reproducible results can be obtained with few requirements regarding sample preparation. the insensitivity to water molecules predestines the technology for its use in a biomedical context. to date, the vast majority of studies using raman spectroscopy examine unprocessed native, or frozen tissue/cells few publications make use of formalin-fixed or paraffin-embedded (ffpe) tissue because raman measurements remain challenging due to the strong contribution of paraffin wax to spectral intensity, thin specimens, and a disruption of the molecular integrity, which is related to the preceding fixation process. the long-term archivability and the large number of available samples, however, suggest use of rs ffpe tissue in pathology is desirable, e.g., for the analysis of tumor heterogeneity, or identification of very small tumor fragments, which could escape diagnostic high throughput of histology samples. the following review and perspective paper is divided into three parts: a) the basics of rs and the most common forms of its application in medical research are presented, b) the use of rs in selected neuroscience disciplines is accentuated with the aim to present different research questions – but even more importantly – the most interesting findings discovered with the help of rs, c) a future outlook for potential application of rs in research but also in the daily clinical work is provided. at this point, the minireview by payne et al.5 needs to be mentioned; it describes in a clear way not only applications of rs in neuroscience, but also sets a special focus on the technical aspects and benefits of advanced spectroscopy-based techniques depending on the particular use case. by contrast, the following work places a special emphasis on topics that will inevitably become relevant to the practicing spectroscopist at some point, such as varying tissue sample requirements in different clinical settings (surgery department/pathology department) or common data processing methods, to name a few. whenever it serves expedient the attentive reader shall be referred to additional more in-depth reading. search for relevant literature a literature search (the search terms “raman”, “raman spectroscopy” were each combined alternately with the terms “brain”, “neuro”, “neuroscience”, “brain tumor”, “tumor”, “neurooncology”, “glioma”, “neurodegeneration”, “neurodegenerative disease”, “alzheimer’s disease”, “parkinson’s disease”, “huntington”, “amyotrophic lateral sclerosis”, “prion disease“, ”multiple sclerosis”, “myelin”, “demyelination”, “stroke” “brain ischemia”, “brain injury”, “muscular diseases”, “brain infections”, “meningitis”, “psychiatry”) was performed, and online databases pubmed central® and google scholar® were browsed for relevant reviews and original articles; other types of literature, such as congress papers, letters, comments e.g., were excluded. after search results were identified, they were hand-screened for eligibility (inclusion criteria: employment of rs on brain/peripheral nervous/muscle tissue, rs on extracellular components/cells of the nervous/muscular system, or rs in relation to neurological/oncological/psychological disorders; exclusion criteria: use of vibrational spectroscopic techniques other than rs) based on title/abstract. within the responsibility of the authors, the final selection of literature was conducted based on the article full text. finally, associated bibliographies of selected publications were searched for additional relevant sources that semantically met the search criteria. only english language literature was considered – even though japanese research groups describe an employment of raman spectroscopy in rat brains, and human brains / brain tumors as early as the 90s6–9. although references to historical developments are pointed out whenever a contemplation of the historical context seemed valuable special focus is set on literature of the years 2021 and 2022, reflecting ongoing research projects/groups such as spectroscopical examination of microglial changes due to sars-cov-2 exposure10 using rs in neuroscience. principle of raman scattering and general spectrometer set up the raman effect is the process of inelastic scattering of photons; this effect was first described in 1928 by c.v. raman, who examined the characteristics of scattered photons when applying a light source on different liquids11,12. for his discovery, the indian physicist won the nobel prize in 193013, but despite the discovery of the raman effect in the first half of the 21st century, it took until late 1960s before it was first used in a biomedical context14–17. the interaction of incident light with a molecule leads to changes in the vibrational state, so that the molecule falls into an excited virtual vibrational state. when returning to the ground state, the largest amount of the incident photons is elastically scattered, which means that the energy of the scattered photon is the same as that of the incident photon (=rayleigh scattering). only a minor part of the scattered light experiences a change in its energy compared to the incident light; in fact when the molecule ends up on a different state in comparison to the ground state, the photon is inelastically scattered. depending on the interaction between the molecule and the photon, inelastically scattered light can have a higher energy (anti-stokes effect) or a lower energy (stokes effect) than the incident light, whereas in practical application mainly stokes scatter is attributed to a resulting raman signal, due to its higher intensity.18 see figure 1 for a visualization of the vibrational states transitions. figure 1. occurring optical phenomena when irradiating a biological sample with a photon source (laser). left: vibrational states (v0, v1, v2) involved in rayleigh and raman scattering. in case of elastic scattering (raleigh scattering), incoming photons temporarily change the vibrational state of a molecule after this excitation, the molecule returns back to the initial vibrational state (v0). in the case of stokes raman scattering, a molecule gains energy due to the excitation process and finally ends up in a higher vibrational state (it rises from v0 to v1) – the scattered photon has lower energy than the incident light. in anti-stokes scattering the molecule ends up on a lower vibrational state after excitation compared to the ground state (it falls from v1 to v0) – therefore, the scattered photon gains energy. right: in contrast, the phenomenon of fluorescence occurs when a molecule absorbs light and thus is temporarily transferred to a higher electronic state (v’0, v’1, v’2). in order to be raman-active as a molecule, i.e., to emit inelastic raman scattering, a change in polarizability is required this already shows a difference to a related and often confused spectroscopic technique, infrared spectroscopy, in which an absorbed photon leads to a change in the dipole moment19. another phenomenon, also based on absorption and often observable as a disruptive factor in raman measurements due to its stronger signal is fluorescence; here the molecule, excited by energy of absorbed photons, leaves the ground electronic state and is transferred to a higher electronic state as soon as it returns to the ground state, energy is re-emitted as fluorescence light20. the interaction of photons with their target molecules resulting in an inelastic raman scattering with a distinct energy difference reflects specific chemical bonds and constitutions. this spectral fingerprint can indicate the identity of the target molecule. a spectrum can therefore be defined as a representation of the intensity values (based on the degree of change in polarizability) and the differing frequencies (raman shift) in a function18,20. the x-axis displays the raman shift in the unit wavenumber cm-1, thereby the wavenumber is reciprocal to the wavelength and thus directly proportional to photon energy19. the conventional experimental application of the process using the pure raman effect is so the called spontaneous raman scattering (sprs). additionally, there are several derivative methods allowing, for example, scattering with enhanced signal intensity or reduced background noise, thus lending themselves to different applications such as raman imaging (e.g., by coherent raman spectroscopy). table 1 gives an overview of the technical background and advantages of commonly used variants of rs in neuroscience. for a more detailed insight into the theoretical aspects of rs the interested reader may refer to cialla-may et al.21, who provides a comprehensive overview in the book “micro-raman spectroscopy: theory and application” by popp et al.22. additionally, hu et al.23, shi et al.24 and evans et al.25 give a good overview about stimulated raman spectroscopy (srs) and coherent anti-stokes raman scattering (cars); zheng et al.26 wrote an instructive review about surface-enhanced raman scattering (sers). table 1. summary of commonly used raman techniques, their physical background and the associated advantages and disadvantages. the exact structure of a raman spectrometer differs depending on the manufacturer and the technology used. only general components and their function are discussed below; additional components such as an additional laser or a special raman substrate are commonly required in spectrometer setups of advanced raman techniques (table 1). with a focus lens, emitted photons of a laser source are focused on the sample, and after interaction with the sample both the elastic and the inelastic scattered photons are collected by a collecting lens. the reflected and elastically scattered light is then separated from the remaining light, typically by a dichroic mirror. a prism or diffraction grating spatially separates the light according to wavelength, leading it to a detection system a photo paper was employed in the classical setup either simultaneously on a charge-coupled device (ccd) or through a monochromator on a photomultiplier tube (pmt) (figure 2). figure 2. schematic and simplified representation of a raman spectrometer set up. as excitation source, typically lasers, is used where the manner of photon generation as well as the wavelength differ. commonly employed excitation wavelengths within the biomedical field are 532nm, 785nm, 830nm, or 1064nm for practical application specific effects on the tissue type of interest as well as potentially induced background signals must be considered individually and adapted according to the experimental setup27. most employed lasers nowadays are diode lasers; with the advantage of portability and favorable energy efficiency, they have replaced the gas-based lasers (helium neon laser, argon-ion laser) that were often used in the past. the type of proton emission can be divided into continuous-wave lasers and pulsed lasers; the former being more common in sprs and the latter being necessary in srs18,28. it is necessary to bundle photons both in the suitable focus on the sample (focus lens) and to collect the scattered photons (collecting lens) after interaction with the sample. next, raleigh scattered photons are filtered by a dichroic mirror and separated according to their wavelength using a diffraction grating; depending on the sampling aperture (exit slits/pinholes) within the setup, a certain number of photons are detected in a final step by the sensitive detection system. while the classical “scanning spectrometer” employs a rotatable grid concentrating the photons on a narrow exit slit and a photomultiplier tube behind detecting rs, modern set ups usually use a ccd detector. this multichannel way of photon detection (a multichannel array chip consisting of several pixels) allows for simultaneous registration and display of all photons, i.e., the whole raman spectra18,28,29. regular wavelength calibration (process of transferring pixel hits on the ccd detector to distinct displayed wavenumbers) is recommended to receive reproducible spectra over the entire duration of the experiment28. raman spectra can be employed in various ways. in addition to the possibility of using them as raw spectra primarily for the identification of biochemical components of a sample, methods called raman microscopy/imaging use the assignment of colors to raman bands (only a limited number of wavenumbers is acquired or analyzed)30 over a scanned sample to generate contrast. when extended to focusing through the depth of the sample, three-dimensional raman images can be built16. raman microscopy/imaging techniques31,32 and computational image generation algorithms have been advanced to generate raman images of various brain pathologies, e.g., gliomas, stroke and demyelination25 or to image metabolism in the brain33–35. using this approach of data visualization, it is possible to obtain a similar look to traditional h&e-stained slides on unstained specimen, which enables histopathological diagnosis36. in raman mapping, the whole raman spectrum for each point of the desired area of the specimen is acquired (either point by point or with an excitation laser forming a line on the sample and measuring simultaneously); using computational analysis afterwards, a visualization of differences in the spectral properties of data points is achieved30. peak assignment raman peaks may occur at first sight in various forms with different characteristics. in addition to certain single peaks that appear narrow and can be assigned to exactly one corresponding functional group, an additive effect of several adjacent raman active molecules in the sample can also result in broad peaks. furthermore, the presence of several contributing components, and thus neighbor dependent changes in the vibrational mode in one specimen, may affect the actual peak in comparison to an isolated measurement20. the application of rs in the biomedical context often pays special attention of the regions within the wavenumbers 400-2000cm-1 and 2700-3500cm-1. these regions, often referred to as "biological fingerprint regions" in the literature, are characterized by a high proportion of raman peaks arriving from functional groups of a typical biological specimen28. an introduction to the use of rs for identification of different molecular functional groups can be found in pezzotti et al.37 (rs and cell biology) , czamara et al.38 (rs and lipids), rygula et al.39 (rs and proteins) and wiercigroch et al.40 (rs and carbohydrates). by using rs on biomolecules such as proteins, it is not only possible to identify molecular functional groups i.e., differentiate between different amino acids/proteins, but also spatial confirmations can be detected since the raman signal is influenced by aromatic/non-aromatic side chains and the backbone of a protein. distinct vibrations result in certain amide bands (amide band a, b, i-vii)41; for example carbonyl stretching modes, n–h bending or c–n stretching results in the widely used amid i (1600-1690 cm−1), amid ii (1480-1580 cm-1) and amid iii peaks (1230-1300 cm−1). they allow further examination of the peptide secondary structure. in larger unordered protein measurements a precise peak attribution may not be possible due its large number of contributors18,39,42–44. lipids are ubiquitous in biological specimen, as they form the membranes of cells and organelles. depending on the literature, spectral properties resulting mainly from the hydrocarbon chain and partly from the polar head group can be assigned to the regions 1050-1200cm-1 (c-c stretching), 1250-1300cm-1 and 1400-1500cm-1 (ch2, ch3 group activity) or also to the regions below 600cm-1 and between 1000-1150 cm-1 (opposite motion of carbon atoms of the hydrocarbon chain). consistently, an area within the high wavenumber region 2700-3500cm-1 (sometimes solely the range between 2800-3100cm-1 is considered in the literature) is reported and attributed in a large part to stretching of c-h groups. in-depth analyzes of peak intensity and distribution in the high wavenumber region allow conclusions to be drawn about the saturation status of fatty acids and the aliphatic/aromatic components of steroids18,38,45,46. an interesting contribution at this point may come from krafft et al.45, who in 2005 measured and characterized twelve brain lipids and further related occurring peaks to their functional groups and pezzotti et al.47, who employed rs to visualize single (phospho-)lipids in neuronal cells. carbohydrates and underlying c-c and c-h structures give rise to bonds in various areas within the raman spectrum18. for a long time, minor attention was paid to the investigation of carbohydrates. although specific peak assignment is possible, in comparison to protein and lipids it remains less specific40. about 30 raman peaks of nucleotides, distributed over several areas within the spectrum, are mostly attributed due to purine/pyrimidine ring modes and phosphate groups (especially peaks next to 800cm-1 and 1100cm-1). they are useful for characterization of inter alia dna, trna, and nucleic acid-protein complexes18,48. spectroscopic examination not only allows for examination of these specific functional groups enumerated above, but also to display their interactions, such as protein-protein / protein-lipid interaction. their changes in spectral property under different conditions can also be measured17. on that note, lee et al.49 have even managed to use srs as a tool in neurophysiology when examining the spectral properties of neuronal membrane potential. although specific raman peaks have been described for various molecules50–56, one should be cautious when actually assigning peaks to one's own sample. while peaks may be characteristic for a certain biochemical compound, they can also arise from different sources; viz they are not specific. in order to correctly assign peaks / detect them within a spectrum, it is essential to reduce potential confounders within the sample or the experimental set-up pre/post-experimentally. a potential way to assign distinct peaks with high evidence is direct observation: targeted manipulation of a sample can help to confirm the source of a peak. the vibrational spectroscopic experimental setup rs is a fast, non-destructive, user friendly, and easy to apply on tool providing molecular information with minimal sample preparation requirements in a reproducible manner. however, a routine use of rs-based tools in neuroscience has not yet been established. regardless of the numerous advantages certain limitations have to be considered not only pre-experimentally, but also during implementation of an experiment and afterwards when visualizing and processing the obtained data. the occurrence of the physically related phenomenon of (auto-)fluorescence (photons of the pump beam are absorbed by molecules of the sample which are raised to another energy level when returning to the basic energy level a photon is emitted, see also figure 1) is regularly observed and the expected intensity in this case is well above the intensity of the raman signals. to reduce wavelengthdependent autofluorescence, a distinct wavelength of the excitation source can be selected, or sers can be used57,58. although in contrast to other sophisticated laboratory techniques (e.g., genetic/epigenetic testing) there are less requirements for a correctly prepared raman sample. a few things need to be considered in order to avoid the occurrence of spectral background noise and spectral contamination: samples must be placed on a robust raman substrate so that the selected measuring point and the focus remain stable. depending on the experimental question as well as the expected background noise and the costs, various raman substrates are available. in addition to gold or aluminum-coated glass slides (as a function of the excitation wavelength glass alone exhibits a strong and broad fluorescence background signal in the “biological fingerprint region”), special slides (low-e slides, caf2 slides, quartz slides) can be considered28. these are characterized by a low spectral background or single peak attribution. fullwood et al.59 and kerr et al.60 examined the effect of substrate choice for spectral histopathology in more detail. it has been shown that caf2 slides (exclusive peak at 321cm-1 or 322cm-1 respectively, depending on the literature)61 have the least influence on the spectral background in comparison to low-e slides and spectrosil slides. the single background peak can either be ignored due to its irrelevant occurrence out of the important range of biological components within the raman spectrum, or can be subtracted via computational analysis afterwards. as a low-cost alternative aluminum foil can be used, which itself does not generate any significant background noise62–64. furthermore, the sample condition (most commonly native/frozen or formalin-fixed) needs to be considered pre-experimentally. although fresh tissue samples allow for a straightforward attribution of raman peaks to underlying biochemical components, they must be processed and analyzed within a certain time window and cannot be stored for a longer period of time. when working with fresh tissue, dehydration and associated denaturation of functional biochemical groups need to be prevented e.g., by keeping the specimen hydrated19,65. as an alternative, raman measurements of frozen biological samples allow longer storage and at the same time still give an insight in the biochemical composition of the biological sample. nevertheless, it should be noted that a reduction in certain peak intensities and significant alteration of raman signal in comparison to native tissue were described when using frozen sections66,67. the handling of formalin-fixed, methanol-fixed, or ffpe samples is routine during the pathological workflow; even though samples allow long archivability and are broadly available, this way of fixation damages the biological raman spectrum to a certain degree since the tissue undergoes an aggressive chemical procedure68–72. both formalin and methanol fixation reproducibly alter spectral tissue properties and affect raman bands assigned to lipids, proteins, and nucleic acids73. despite formalin-induced biochemical changes such as formation of cross-links in the structure of the amino acids, spectroscopic assessment and classification of formalin-fixed biological tissue is possible66; in contrast, methanol-fixation was reported to potentially hamper the detection of tissue malignancy72,74. the prominent spectrum of bound paraffin wax is reflected in certain points at 1063, 1133, 1296 and 1441cm-1, which make a manual or digital dewaxing process necessary and require a careful interpretation of the obtained spectra75. several conditions (aggressive chemical processing, required choice of special substrate and the fineness of the tissue) hamper spectroscopic examination when employing rs on ffpe tissue in the pathology department, although spatial orientation on the sample and proper identification of certain areas are a potential advantage. in the literature different approaches used rs on processed tissue; in any case they all face similar difficulties. huang et al.68 described the effects of formalin fixation on rs of cancerous human bronchial tissue, whereas draux et al.71 described the influence of formalin and air drying on single cancer cells and attributed spectral changes to affection of nucleic acids and proteins. even though not only a loss of the original chemical composition but also potential contamination due to the process of formalin-fixation in murine brain tissue was determined by hackett et al.76, several studies proposed formalin fixation as a sufficient and favorable method for subsequent spectroscopic diagnostic77,78. as a proof of concept, stefanakis et al.79 demonstrated the feasibility of vibrational spectroscopy on formalin-fixed malignant brain tissue. employing vibrational spectroscopy on ffpe tissue, an effect on the lipid content due to the dewaxing process was reported; nevertheless, raman bands related to cellular and extracellular proteins were successfully measured80. gaifulina and colleagues81 examined large intestine ffpe tissue from rats and analyzed biochemical signals obtained with label-free rs in the processed tissue. other groups examined ffpe tissue of rectal cancer to predict radiotherapy response82, to map/analyze cervical tissue83,84, or employed rs on healthy and malignant breast85–88/ovarian89/prostatic90 tissue in various fixation states. for a good overview on the influence of tissue processing on biological raman spectra the reader may refer to the work from faoláin et al.66. during spectroscopic examination, background noise due to a nearby photon source (e.g., room light) should be considered and reduced by performing the raman measurement in a darkened area or with dimmed operating room light91–94. additional methods of spectra quality control during intraoperative measurement have also been proposed95,96. by ensuring that the laser settings (wavelength and power, duration of acquisition) are optimized for the examined sample, the best signal-to-noise ratio can be determined, and thermal tissue decomposition can be prevented. this form of sample destruction can be detected by a burned area where the former focus area of the laser is located, as well as by the presence of an additional carbon band at approx. 1500cm-1 in the raman spectrum28. data processing and computational analysis after the measurement, the large amount of data97 should be sorted and stored in a structured manner (data annotation) to address the research question properly. it is good practice to start the data processing with an initial visualization of the data. in this way clear deviations from an expected result such as strong contamination or cosmic ray artifacts (randomly occurring electromagnetic radiation) and hot pixels (overresponse of a pixel on the ccd detector to an incoming photon) can be recognized and corrected28,98,99. for a more detailed reading on potential anomalies and artifacts that may occur, see bowie et al.100. during data preprocessing, a baseline correction can be applied to the data to minimalize residual background signal and autofluorescence101,102; a common way to model and subtract the background noise to obtain the intrinsic sample spectrum103,104. additionally, a common way to further reduce the noise in the data is a smoothing technique, such as savitzky-golay filtering28,105,106. both of the above-mentioned methods must not be used without proper caution as there is always the risk of producing artifacts, as well as equalizing significant data points. in order to correct confounders that result from the experiment setup itself (e.g., slightly different dryness or thickness of the specimens) data normalization methods, such as min-max normalization or z-normalization, usually precede the actual data analysis107. specialized spectroscopy software are commercially available and enable even the inexperienced spectroscopist to use the acquired data in a structured and comprehensive manner108. due to the large amount of data, several data reduction methods are used for quick explorative purposes, above all pca (principal component analysis) is widely employed. this unsupervised clustering technique can be used to determine principal components in a big data set, which explains a significant part of the variance and reduces noise41,109.< in the last step of computational analysis, classification algorithms and machine learning techniques110,111 are commonly used to classify the spectral data either according to pre-experimental defined groups (supervised clustering) or according to new groups based on similarities in spectral properties (unsupervised clustering)112. a widely used technique in unsupervised clustering is hierarchical cluster analysis (hca), in which the data is transferred to a higher-dimensional space, cluster in a certain proximity to one another based on similar properties. a number of cluster variables can be specified individually, which forms the selected number of similar clusters103. unsupervised clustering is beneficial for exploratory research questions since no prior knowledge of possible group properties is required28. common methods used for supervised clustering are trees/random forest classifications (several decision trees in a row) or support vector machines (search for a hyperplane to distinguish between classes)91. the groups determined a priori are referred to as "classes" and the gold standard histopathology often serves as ground truth. in general, the algorithm is trained with a training data set and tested with an external validation data set afterwards. to avoid overfitting (capability of good differentiation only on the specific training data set) a validation of performance e.g., k-fold cross validation or holdout validation is performed, and metrices of algorithm performance (e.g., sensitivity, specificity, f1-score, accuracy, auroc/aupr value) are calculated afterwards based on its output113. ralbovsky and colleagues provided an overview of machine learning algorithms and their functions in raman based cancer detection112. rs in neurooncology with a growing number of publications in the last years (zhang et al.114 and banerjee et al.115 described a change in spectroscopic properties of glioma cells in comparison to astrocytes already in the mid-2000s), the neuro-oncological field is one of the largest areas of research on rs, in which the therapeutical balancing act between maximum resection of normal-brain-resembling tumorous residues and minimal surgical disruption of healthy brain functions proves particularly difficult. on the subject of rs in (neuro)oncology reviews by auner et al.20 and hollon et al.116 give a comprehensive introduction to the respective topic; for further reading on implications and current progress of rs in oncology see also santos et al.117. at first sight, use of this spectroscopic technique mainly apply to two main research focuses: on the one hand a spectroscopic detection of malignancy118,119 which in a next steps allows precise, accurate diagnosis of the tumor entity intraoperatively without having to wait for further traditional tissue processing (pathological diagnosis on frozen sections)120,121, and on the other hand real time surgery guidance i.e., live feedback intraoperatively122,123 aiming for maximal tumor resection124–126. both topics merge and evolve at a certain point; this may result in new research questions, e.g., when aiming to determine tumor infiltration zone / resection margin or when aiming for detection of tumor genetics on various states of tumor tissue. moreover, also basic research questions in oncology can be addressed with this vibrational spectroscopic technique e.g., monitoring lipotoxicity in glioblastoma cells127, observing cell response of u251 glioblastoma cells after induced apoptosis128, examining the glycosylation pattern of proteins in medulloblastoma129, or observation of redox state of mitochondrial cytochromes130, just to name a few. most research groups use sprs20 as an easy to apply, label free method. more advanced raman techniques in neurooncology131 are used predominantly in animal models132–134 – where surface enhanced resonant raman spectroscopy (serrs) detection of tumor margins135 has shown prognostic benefits136, or cars was employed for detection of different human brain tumors in a mouse model137. rs for detection of tumor group, genetic alteration and histomorphology rs can distinguish between grey and white matter and (partly) other brain regions such as cerebellum, striatum, basal forebrain both macroscopically and on cellular resolution4,138–146,147. interestingly, one analysis of the mouse brain using sers revealed a different spectral fingerprint and thus also different biochemical composition between left and right hemisphere148. spectroscopically feasible discrimination between glioma tissue and brain tissue was reported in several studies3,149–153 as well as between dura mater and meningioma, which was demonstrated to be based in part on peaks corresponding to collagen and on the higher lipid content within tumorous tissue154–156. beside these binary classification models, several studies showed the potential of rs aiming for a multiclass classification to differentiate various tumor entities within one classifier119,157–166 or to determine the primary site of metastasis167,168. using raman mapping/imaging for brain tumor visualization116,169, even special morphological features of tumors (e.g., necrosis in glioblastoma, cell density or individual cell nuclei) could be identified170–172. even though areas of tumor necrosis are typically characterized by an increased presence of proteins such as phenylalanine (around 1032cm-1, among others) as well as cholesterol esters (1739cm-1)171,173, one group proposed two distinct spectral properties within the necrosis of glioblastoma cells: “highly necrotic”, showing an increase in plasma proteins and “peri-necrotic”, exhibiting a higher lipid content174. the histopathological heterogeneity of tumor tissue samples was addressed in fresh and frozen brain sections, although possible confusion between different tumor components (i.e., tumor hemorrhage and necrosis) is described36,173. the genomic heterogeneity in glioblastoma has also been successfully addressed175. other approaches make use of an alternative advanced raman technique named stimulated raman histology176–179 (srh), where distinct wavenumbers are used for image acquisition and virtual h&e-like images are generated after computational processing. with this approach in combination with deep convolutional neural networks, amongst others hollon et al. assessed (pediatric180) brain tumors intraoperatively1,181,182. in the scope of this imaging approach, also a traditional pathological diagnosis based on digital raman histology slides seems feasible183–185. rs could be used to identify brain edema186, tumor recurrence187 or tumor margins188–194 but also tumor infiltration zones.195,196 in general, infiltrative glioma cells showed significant spectral differences in the regions of phenylalanine and amide iii (around 1030cm-1 and 1230-1300cm−1), as well as the region assigned to c-c stretching lipids and nucleotides (around 1050-1100cm-1) – just to list a few wavenumbers of interest exemplarily197. ji et al.196 report the cellularity within a sample as well as the density of axons and the ratio of lipid and protein contents as the basis for the difference in spectral properties. even single tumor cells198 were detectable using rs, something alternative imaging methods struggle with. rs was also applied to observe glioblastoma tumor evolution199, to determine the molecular subtype of glioblastoma200, and to give insight in glioma biochemistry201. rs was shown to be superior in differentiation of brain tumor and glioblastoma in comparison to 5-ala-induced fluorescence202,203, and capable to detect idh mutations in gliomas – inter alia changes in the spectral protein profile are consistently reported in case of idh mutation204–206. it also showed diagnostic value in tumor discrimination when measuring small extracellular vesicles207, or potential when tracking/detecting metabolic changes208–210 in brain tumors/cancer cells, as well as drug delivery mechanisms211 and post-therapeutic changes212 in glioblastoma cells. spectroscopic classification of different grades of brain tumors is possible213. zhou et al.214 distinguished between different who grades of gliomas using raman bands of tryptophane (around 1588cm−1, among others) and carotenoids (1008cm−1, 1157cm−1, 1521cm−1, 2320cm−1, and 2667cm−1) as well as the peak intensity ratio between proteins and lipids in the high wavenumber region (2934cm−1/2885cm−1). the group of morais et al.215 and lilo et al.216 differentiated between different grades of meningiomas. zhang et al.217 associated an intensity ratio in the high wavenumber region with different meningioma grades. while gliomas/neuroepithelial tumors and meningiomas have been described218 and morpho-chemically analyzed219,220 extensively221, some work also exist on neuroblastomas. one group differentiated between different neural crest-derived tumors in fresh and frozen tissue222,223, and ricciardi et al.224 used rs to examine changes in the biochemistry of neuroblastoma cells after exposure to radiation. medulloblastomas225, biopsies of the pituitary gland209,226, seeds of retinoblastomas227, and carcinoma metastases228 have been spectroscopically studied as well. early, intraoperative, and neuropathological diagnostics using rs perioperative ex vivo tissue assessments allow for direct and early treatment decision, e.g., when examining smear brain tumor samples94 or discriminating between primary cns (central nervous system) lymphoma and glioblastoma based on biopsies229. rs can also be applied intraoperatively (in vivo) recently even in dogs230 using a hand-held probe for tumor classification231–237, where a real-time auditory feedback mechanism has been proposed to guide the neurosurgeon238. transcranial rs, leaving the skull intact, has been proposed and demonstrated in a mouse model239. using optical spectroscopy applied on ffpe tissue, devpura et al.240 and gajjar et al.159 examined a possible application of rs to various brain tumors already in 2012/2013. shortly after, fulwood et al.241 distinguished between glioblastoma, metastases and normal brain using immersion rs on ffpe samples. livermore et al. 204 have been able to carry out the above-mentioned analysis of the idh mutation detection in glioblastoma tumors also on ffpe tissue. different histological areas can be distinguished in glioblastoma in ffpe tissue, with a sound separability between the peritumoral area and the area of necrosis242. to enable early and non-invasive cancer diagnosis, some approaches aim for identification of meningioma243 and glioma244 patients based on serum samples and resulting spectroscopic behavior. using rs as an additive technique, le reste et al.245 combine spectroscopic data and transcriptomic data for machine learning analyses on glioblastoma subtypes and related clinical outcomes. rs in neurodegenerative diseases misfolded proteins and aggregates in various diseases246–248, e.g., alzheimer's (tau and amyloid), parkinson's (alpha-synuclein), huntington's (polyglutamine), are in general accessible to vibrational spectroscopic techniques249. usage of these techniques ranges from tracking and characterization of misfolded proteins41, to potential new diagnostic methods250,251, especially in biofluids252–254. studies on the pathological hallmarks of neurodegenerative diseases used a variety of rs techniques; most frequently employed techniques are sers, ters (tip-enhanced raman spectroscopy), as well as duvrr255,256 (deep uv resonance raman), where a wavelength in the range of uv (200nm) is used as excitation source which results in an increased intensity. another common technique named roa (raman optical activity) makes use of the principle that a chiral molecule scatters left and right handed polarized photons at different intensities and so is particularly useful to analyze protein aggregates257,258. furthermore, also ir (infrared)-spectroscopy and related/modified vibrational methods are common, and a combination of techniques could lead to an increased diagnostic ability and gain of knowledge2,259–262. several ways of increasing the detectability of a sample via rs have gained popularity in the neurodegenerative field. bringing in a labelled isotope into the backbone of a peptide shifts certain amid bands and enables a demarcation from the existing amide bands emanating from the unlabeled proteins in the sample, although an overlap of raman peaks of interests may occur263,264. another similar approach integrates external probes such as unnatural amino acids with vibrational potential into the sample, which can afterwards be traced by specific raman peaks, often in the range between 1900-2900cm-1 where the interference with other peaks of the specimen is minor264–266. for further reading, devitt et al.2 provides a detailed insight into the use of rs in the field of neurodegenerative diseases. around 20 years ago conventional rs was already capable of distinguishing between ad brain tissue and healthy control brain tissue (in 2022 machine learning algorithms are useful to do the same267) and to determine the presence of amyloid-beta-sheets in senile plaques268–270. shortly after, raman signals of the hippocampus of ad rats were proposed to aid diagnosis of ad271. kurouski et al.44 give an overview of the application of rs in the course of plaque formation and structure; wilkosz et al.41 provide a comprehensive list of wavenumbers associated with protein aggregation. detailed examinations of the (secondary)-structure of beta-amyloid in various experimental set ups have been carried out using duvrr272–275 or roa44. cunha et al.276 used a combination of raman techniques for amyloid plaque characterization. sers has been used to identify tau protein and (soluble) amyloid beta277,278, and to detect amyloid-beta1-40 monomers and amyloid-beta1-40 fibrils in solution279 as well as in brain tissue280. aβ40 and aβ42281 were shown to be distinguishable. ters was used to characterize natural aβ1-42 fibrils and identify toxic oligomeric forms282,283. rs was capable of visualizing amyloid in ad brains post mortem and of displaying neuritic plaques and neurofibrillary tangles284 – even though the latter findings were questioned and measurement of lipofuscin granulates instead of plaques was proposed285 raman imaging also determined the presence of hemoproteins in senile plaques286 and allowed for reconstruction of the evolution process of different types of amyloid beta plaques287. based on rs measurements, ad-associated astrogliosis288 and lipid deposits in vicinity of fibrillary plaques were identified and further morphologically described289. beside the identification of amyloid beta290–292, for example in the surrounding of neuronal spines293, raman imaging294,295 has been used to compare the concentration of aβ in hippocampal regions and eye lens tissue296 and to determine cholesteroland sphingomyelin-rich structures surrounding amyloid plaques, thought to represent dystrophic neurites297. another research group used cars to determine a higher content of lipid, collagen and amyloid fibers in alzheimer-affected brain samples298. searching for biomarkers as an early diagnostic tool in ad299–302, human tears303, saliva,304 cerebrospinal fluid305 (different states of amyloid beta confirmations could be detected in cerebrospinal fluid already in 2008306), retinal imaging307 and blood samples308–318 have been evaluated for a potential diagnosis of ad using spectral differences arriving from platelets319 or the concentration of the neurotransmitters glutamate (glu) and γ-aminobutyric acid (gaba)320. in the course of this approach, it has been shown that cortical cataract may not be a sufficient predictor of ad296. the detection of neurotransmitters using rs has been shown and further analyzed, by ardini et al.321, lee et al322, moody et al.323–325 (i.e. rs for detection of neurotransmitters through the skull), cao et al.326 / zhou et al.327 (neurotransmitter detection in serum), ciubuc et al.328 (rs for dopamine detection and analysis), silwal et al.329 (dopamine and dopamine transporter interaction), manciu et al.330 (dopamine – serotonin interaction) and shi et al.331 (quantification of norepinephrine). in addition, rs is also suitable to examine the interaction of beta-amyloid with metal ions332–337. interestingly, detection of tau335–338 and insulin342–345 has so far been studied to a lesser extent; ozone exposure as a known risk factor has been found to lead to spectroscopically measurable changes of the hippocampus in a rat model346. in parkinson’s disease (pd), a main focus of the application of rs is the characterization of the secondary structure of alpha-synuclein338,347–349 as well as the identification of alpha-synuclein aggregations, feasible not only in the brain but also in the gut350. mensch et al.351 used roa to examine the spectral properties of α-synuclein during transition to its secondary structure. another group spectroscopically characterized the striatal extracellular matrix in a pd mouse model352. since early loss of dopaminergic neurons is an early change in patients with pd, different approaches aim for detection of dopamine353–355, e.g., in striatum of mice356, or in blood samples of patients with antipsychotic drug-induced parkinsonism357. other efforts to establish early diagnostic tests for pd, such as examination of erythrocytes and blood coagulation in pd patients358, were carried out e.g., by carlomagno et al.359 using saliva of pd patients and schipper et al.360 who combined rs and nirs (near infrared spectroscopy) to distinguish between blood samples of pd patients and a control group through different spectroscopic properties correlated with oxidative stress. mammadova et al.361 used rs in a pd mouse model to detect pathological retinal changes as a method to distinguish between healthy and diseased samples. analyzing peripheral nervous tissue in als mice and autopsies of patients suffering from als, tian et al.362 showed that raman imaging was capable of visualizing and detecting early pathological changes. different approaches distinguish between altered lipids and proteoglycans in spinal cord tissue of als mice and healthy controls363, or test the prognostic value of sers in als patients364. in addition to the many approaches to diagnose ad and pd patients by rs, others focus on als as well. for diagnostic purposes, zhang et al.365 used sers on plasma samples to distinguish between als patients and a healthy control group; morasso et al.366 proposed vibrational spectroscopy and extracellular vesicles as a potential biomarker and another research group spectroscopically examined saliva from als, pd, and ad patients, showing differences in the spectral properties of each group367. in the context of huntington disease (hd), rs has been used for quantification and visualization of aggregated polyglutamine368 and for the assessment of its structure369,370. huefner et al.371 found significant changes in the spectra related to disease progression, as well as differences corresponding to genotype and gender in serum samples of hd patients and healthy controls. in another approach, membrane composition of hd-affected and control peripheral fibroblasts were separatable using rs, suggesting that cell membrane damage may serve as future diagnostic biomarker372. rs has also been used for research on prion diseases373–378; one research group employed the method to examine the diagnostic value when analyzing blood samples of sheep to detect the altering from of prpc to prpsc379. spectroscopic examination of myelin composition in the cns and in peripheral nerve tissue rs proves useful to gain a deeper understanding of the molecular myelin composition; pezzotti et al.380 examined the physical chemistry of cocultured neuronal and schwann cells. in addition, rs may be advantageous to detect pathological processes of demyelinating diseases in the cns or in peripheral nerve tissue. carmona et al.381 studied the spectroscopic hallmarks of lipid chains in myelin membranes as well as the secondary structure of associated proteolipid proteins (plp). some publications report the possibility of detecting myelin in vivo using raman microscopy382,383; huang et al.384 described different compositions of myelin structures, whereas wang et al.385 used cars microscopy to detect not only myelin but also axons, the node of ranvier, and the schmidt-lanterman incisure. fu et al.386 visualized fiber tracts in mice brain by imaging the myelin along the axons. in 2021 lucas et al.387 used cars to determine myelination deficits in a fragile-x-syndrome mouse model. out of pure academic interest the publication of poulen et al.388, in which raman scattering on spinal cord myelin distinguishes between three different species (human, mouse, lemur), shall be mentioned at this point. few raman experiments deal with multiple sclerosis (ms)389; the process of myelin degradation can be addressed with rs not only quantitatively390 but also qualitatively. to tackle alterations in the biochemical compositions in human brains postmortem, poon et al.391–393 measured various pathologic features and showed that even normal appearing white matter next to ms lesions included spectroscopically measurable changes. imitola et al.394 correlate the presence of microglia (on a side note: even the activation of microglia is traceable using rs395) and axonal injury/demyelination using cars microscopy. fu et al.396 applied the same method to examine different time points of experimental autoimmune encephalomyelitis in mice and gasecka et al.397 used cars to detect induced autoimmune demyelination in spinal cord of mice. another approach was carried out by the team of alba-arbalat et al.398; they detected spectral changes of defined molecules in the retina (even an in vivo use of rs applied on human retina is in line with laser safety regulations399) associated not only with different phases of ms, but also age-related in healthy patients. raman-based research of myelin composition and pathology is not limited to ms, it also extends to the study of demyelination and its biochemical changes in peripheral nerve tissue400 even pathological401 and age related402 changes. using different raman techniques the remyelination process in the spinal cord of rats after iatrogenic induced demyelination403, as well as remyelination in rat sciatic nerve404, and biochemical changes during nerve injury405,406 can be tracked. another approach used cars imaging to interpret the interaction of different macrophages (resident and recruited) after wallerian degeneration407. upcoming novel fields for rs from stroke to muscular diseases to psychiatry rs has been applied in combination with infrared spectroscopy and atomic force microscopy to characterize different types of thrombi in ischemic stroke408 or to characterize atherosclerotic plaques409,410. changes in fibrin concentration in a blood clot after zonal thrombolysis with urokinase411, or the metabolic regulation of artery tone412 were examined. other research groups investigated spectroscopic changes in the hippocampus due to cerebral ischemia-reperfusion413, or spectroscopic changes in the amount of cu+ and cu2+ ions in brain ischemia414. russo et al.415 used raman traceable cytochrome c to investigate effects of insulin on the hippocampus after transient ischemic brain conditions, yamazoe et al.416 used a self-developed raman approach to detect areas of an ischemic core area. the group of caine et al.417 used a combination of imaging techniques, amongst others raman imaging, to track biochemical changes in the peri-infarct zone after induced stroke in a mouse model. as an alternative way of infarction diagnostic, fan et al.418 proposed tear rs in combination with machine learning tools as a non-invasive technique. in context of brain hemorrhages, raman imaging has been used to detect microvessels and induced hemorrhage419, as well as to track the oxygen flow in brain vessels420. furthermore, rs was employed as a method in rat brains with striatal hemorrhages to evaluate the biochemical composition after rehabilitation treatment421. employing sers, the subarachnoid hemorrhage biomarker glial fibrillary acid protein can be detected422. sers can also be used to assess complications post subarachnoid hemorrhage, like vasospasm and hydrocephalus423. in tissue conditions of brain or spine injury, rs was applied to tissue of rat models424–426 and on retinae of mice after traumatic brain injury427. biochemical changes in affected areas arising from hem or divergent levels of cholesterol were discovered428 and compared to mri scans429. rs was capable of detecting injured motor cortex areas where certain spectroscopic properties were associated with cell death430. employment of sers-based methods allow for detection of neuron-specific enolase (nse), n-acetylasparate or s-100β in blood samples as biomarkers for brain injury431–435; aiming for intraoperative assessment of molecular changes one group developed a device for intracranial spectroscopy within brain injury436. changes in the biochemical and cellular composition of rat brain after gamma radiation have been addressed by kočović et al.437. for a further reading the reader may refer to stevens et al.438, who has recently reviewed the current deployments of raman spectroscopy in traumatic brain injury in a detailed way. even muscular diseases are accessible to rs: niedieker et al.439 used cars imaging to visualize morphological hallmarks such as glycogen storage and internalized nuclei in various muscular diseases; alix et al.440 reported different spectral properties of mitochondrial and non-mitochondrial muscular diseases; and gautam et al.441 showed the differences in the spectra of raman measurements from muscles of drosophila with certain mutations affecting the muscular system in comparison to healthy controls. sprs was used for in vivo identification of duchenne muscular dystrophy (dmd) affected muscles in a mouse model and human muscles affected with the same disease with ex vivo measurements showing similar raman peaks442. hentschel et al.443 evaluated the use of fibroblasts together with application of cars and other methods to study the etiology of neuromuscular diseases. blood sample testing for the diagnosis of dmd was proposed and successfully performed in a mouse model444; the comparison of spectral properties of the erythrocyte membrane in dmd patients and healthy controls demonstrated biochemical differences due to protein anomaly445. one of the potential domains of rs in the area of infectious diseases of the brain and meninges is the diagnostic detection of pathogens. it has already been capable of identifying viral strains446, changes in bacterial metabolism447, or differentiate/detect different types of bacteria related to meningitis448,449. although the diagnoses of tuberculous meningitis450 or neisseria meningitis451 as well as possible differentiation of blood cell types452 using rs on csf samples is reported, reliable detection of bacterial meningitis in csf was not yet sufficiently sensible; therefore, a combination of techniques was suggested453. another approach employs rs in neuroimmunology as a tool to monitor apoptotic changes in hippocampal progenitor cells454. rs has also been applied in psychiatric disorders; e.g. to visualize the drug mechanism of a serotonin reuptake inhibitor in mouse brain455 and to identify blood serum samples based on alterations in phospholipids and proteins of patients with affective disorders456–458. recently, chaichi et al.459 measured changes in brain lipidome spectroscopically in post-traumatic stress disorder (ptsd) rats, but also the vibrational spectroscopic properties within myalgic encephalomyelitis have been subjected to further analysis460,461. conclusions and outlook all studies and literature cited in this review focused on preclinical/clinical use of rs with the intention to provide the interested reader a general overview rather than a detailed account of each particular topic. before jumping into action and establishing rs as an additional research method in one’s own laboratory, taking a look on the methodological reviews by butler et al.28 (including concrete information about the general experimental setup and requirements for biological tissue), and guo et al.462 (analysis of raman data, machine learning algorithms) may prove useful. upcoming applications of rs potentially aim for in vivo prediction of progression risk463 or employment of vibrational spectroscopy for detection of epileptogenic brain regions464. advanced raman techniques such as spatially offset raman spectroscopy (sors)465 may potentially permit live insight into tissue biochemistry of deeper brain structures. alternatively, a future establishment of intraoperative raman imaging (in particular it may even be performed in vivo466) will potentially allow fast detection of both histomorphological features and tumor genetics; therefore producing an integrated diagnosis467 at an early stage of the diagnostic workflow468. extensive clinical studies aiming for approval of rs in neuroscience by regulatory authorities are still missing, even though a clinical need and a patient benefit has been demonstrated by a broad range of groups and laboratories. to translate promising results into clinical practice, several challenges should be considered. when vibrational spectroscopy is tested as a diagnostic method in a multicenter approach, experimental workflows of spectroscopic examination need to be standardized and facilitated; consensus within the spectroscopic community on a collaborative experimental setup and procedure prevents potential invariances due to different sample preparation protocols and hidden artifacts91. to maximize spectral output and enhance spectral intensity in a clinical setting, handheld probes / spectrometers with optimized design and in vivo parameters as well as a preferably low signal-to-noise and high signal-to-background ratio are currently under investigation by a growing number of companies stepping up their efforts in the interface of research and clinical implementation.117,469 since the use of rs on ffpe tissue allows direct comparison with the diagnostic gold standard of histology, rs is expected to expand its applications in neuropathological diagnostics in the future. upcoming studies will not only challenge the current use of rs on unstained ffpe tissue (is reliable diagnosis also achievable on h&e stained samples?) but also discuss a potential use of various raman substrates in a cost-oriented manner470. to reduce the cost factor (id est expensive substrates such as caf2 or low-e slides) future employment of rs on glass slides seems worthwhile; therefore, occurring autofluorescence during measurement needs to be addressed. within that approach, the use of a certain excitation wavelength or the detection of only a small spectral wavenumber range have been proposed60,471. in this sense, ibrahim et al.472 aimed to use glass as raman substrates by employing a digital processing method. in the field of neurodegenerative diseases, a major and highly anticipated impact of rs could be the early and non-surgical diagnosis of disorders in a reproducible manner. despite promising results, this application area is only beginning to develop. to maximize diagnostic reliability, a deeper understanding of raman features and their corresponding biochemical origin in biofluids is key. within the huge amount of obtained data, it remains necessary to address patient dependent spectral variation as well as variations related to a concrete experimental set up. close cooperation between different research groups and ensured data share470 potentially accelerate the development towards clinical implementation. an exemplary success story of clinical translation was reported in the field of dermatology, where rs had already been established as a diagnostic method for early detection of skin cancer; a handheld device was commercially produced in canada463,473,474. to speed up translation from research labs to commercialization and clinical use, several networks have been founded, e.g., international society for clinical spectroscopy (clirspec, clirspec.org) and raman4clinics (raman4clinics.eu), all aiming for exchange of expertise and creation of research collaboration117. to conclude, it highly likely that rs will continue to evolve as a method in the intersection of applied biophysics and medicine – and potentially make its way deeper into the field of life science, such as detection of plastic in zebrafish brain homogenates as a result of exposure to nanoplastic475 and even more clinical applications. where the journey will finally lead remains to be seen in the next years. acknowledgements figure 1 and figure 2 were created with biorender.com. mm thanks the fnr for funding support (fnr pearl p16/bm/11192868 grant). funding luxembourg national research fond, fnr (fnr pearl p16/bm/11192868 grant to m.m.) conflicts of interest we have no conflicts of interest to disclose. references 1. hollon tc, pandian b, adapa ar, et al. near real-time intraoperative brain tumor diagnosis using stimulated raman histology and deep neural networks. nat med. 2020;26(1):52-58. https://doi.org/10.1038/s41591-019-0715-9 2. devitt g, howard k, mudher a, mahajan s. raman spectroscopy: an emerging tool in neurodegenerative disease research and diagnosis. acs chem neurosci. 2018;9(3):404-420. https://doi.org/10.1021/acschemneuro.7b00413 3. jermyn m, mok k, 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distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. searching for a cut-off point for p53 immunohistochemistry as evidence of tp53 mutations feel free to add comments by clicking these icons on the sidebar free neuropathology 5:6 (2024) letter searching for a cut-off point for p53 immunohistochemistry as evidence of tp53 mutations ilay caliskan1, rufei lu1, cristine szu lyn ding1,2, francineide sadala de souza1,3, arie perry1, tarik tihan1 department of pathology, neuropathology division, ucsf medical center, san francisco, ca 94143, usa current address: division of anatomical pathology, changi general hospital, singapore, 529889, singapore current address: associação das pioneiras sociais, patologia cirúrgica, asa sul – brasília 70335901, df brazil, brazil corresponding author: tarik tihan · neuropathology division · room m551 · department of pathology · university of california san francisco · 505 parnassus avenue · san francisco · ca 94143 · usa tarik.tihan@ucsf.edu submitted: 23 january 2024 accepted: 29 february 2024 copyedited by: jerry lou published: 12 march 2024 https://doi.org/10.17879/freeneuropathology-2024-5337 keywords: p53, immunohistochemistry, tp53 mutation, glioma numerous publications and revised grading strategies reiterate the relevance of tp53 gene mutations for adult diffuse gliomas1–3. these studies identify tp53 mutation as a frequent alteration in idh-mutant astrocytomas along with other critical genes, such as idh1, idh2, and atrx. the 2021 who classification of central nervous system (cns)4 designates "strong" nuclear expression of p53 protein in greater than 10 % of tumor cells, which implies the presence of tp53 mutations4 as a desirable criterion for idh-mutant astrocytomas. other studies have also recognized similar percentages in adult diffuse gliomas as evidence of tp53 mutations. to incorporate tp53 alterations into the diagnostic work-up, these studies used immunohistochemistry as a viable alternative to provide the type and grade of diffuse astrocytomas5, and others recommended p53 protein immunohistochemistry as a reliable means of predicting tp53 mutations6,7,8. while a few studies report an association with treatment response, most studies do not find tp53 mutation status or positive immunohistochemical staining helpful in determining biological behavior or type of adult diffuse glioma8. contrary to the studies that expressed enthusiasm for p53 immunohistochemistry as a predictive marker for tp53 mutational status7, studies as far back as 25 years ago pointed to the discordance between p53 protein expression and tp53 mutations9. it was clear from these earlier studies that most gliomas with focal p53 nuclear immunopositivity do not harbor tp53 mutations9. certain alterations in the tp53 gene, such as exon 4 mutations or missense mutations, are more likely to lead to strong positive staining10, while truncating mutations (e.g., nonsense, splice site, or frameshift) are associated with absence or barely detectable p53 protein immunopositivity ("null phenotype")11. even among the few studies that propose p53 staining as a practical prognostic marker, there is little agreement on the optimal cut-off value. some studies presuming tp53 mutations based on p53 immunostaining simply used "positive" staining while others did not report definitions for p53 positivity, further confounding the literature11–13. one of the most recent comparative studies claimed 10 % positivity as a cut-off point to predict tp53 mutation based on receiver operating characteristic curve (roc) analysis12, quoted by the who 2021 classification4. to determine a practical value for p53 immunostaining in everyday neuropathology practice and to validate whether a 10 % cut-off can be reliably used to suggest tp53 mutation (as suggested by who 2021), we analyzed a group of diffuse gliomas for p53 staining and tp53 mutations. we identified all adult diffuse gliomas and diffuse midline gliomas for which analysis of p53 immunohistochemistry and tp53 mutation was performed during a five-year period. the inclusion criteria were: 1) diagnosis of adult diffuse glioma or diffuse midline glioma between 2015 and 2020, 2) available p53 (do-7 clone) immunohistochemistry staining, and 3) available analysis with next generation sequence analysis (ngs), referred to as "ucsf500 ngs" panel14, which includes a comprehensive assessment of all tp53 coding exons and most other genes known to be recurrently altered in cns tumors15. clinical and radiological features were obtained from electronic patient records. all cases were re-analyzed for p53 protein labeling index (li) to ensure uniform assessment based on strong nuclear staining (equal to or greater than the intensity of positive control tissue) of p53 protein in tumor cells (figure 1). the positive control tissue was validated by ucsf500 ngs for tp53 mutation at the ucsf histology and clinical cancer genomics laboratory. the p53 lis were determined by the available original reported li, which was achieved by capturing a digital image of the area with highest labeling and counting at least 500 cells in the captured image, followed by the re-assessment of the image by two of the authors independently to estimate the li within a 5 % maximum variance. genetic alterations involving the tumor and germline were determined as previously described15,16. statistical analyses were performed using r version 4.2.2. a receiver operator characteristic curve (roc) was analyzed using cutpointr r package (version 1.1.1). violin plot and pie chart were generated using ggplot2 r package (version 3.3.5). the study was approved by ucsf committee for human research (ucsf chr 10-01252). figure 1. representative p53 immunostaining patterns that were molecularly confirmed as tp53 wildtype (a), tp53 frameshift mutation (b) and tp53 missense mutations (c and d). our study group was composed of 99 patients with an average age of 44.5 years (range 1-81 years). there were 49 female and 50 male patients. tumor types and locations are presented in table 1. seventy-six (76) of the samples were from primary tumors (first resection) while 23 were from recurrent tumors. two glioblastomas had earlier biopsies at another hospital without additional treatment before the patient was transferred to our institution. seventy-two (72) of the primary tumors were cns who grade 4, six were cns who grade 3, and twenty-one tumors were cns who grade 2. there were 52 tp53-mutant and 47 tp53-wildtype tumors and the distribution of these mutations among tumor types is presented in table 1. forty-two (42) tumors had missense mutations, while three others had missense combined with frameshift mutations. there were 3 tumors with frameshift mutation-only (figure 2b). table 1. demographics, location of tumor, and final diagnosis     overall tp53 wildtype tp53 mutant p n   99 47 52   age, years (mean (so))   44.5 (19.1) 51.5 (18.6) 38.2 (17.4) <0.001 gender (%) female 49 (49.5) 28 (59.6) 21 (40.4) 0.088   male 50 (50.5) 19 (40.4) 31 (59.6)   site (%) brainstem 3 (3.0) 1 (2.1) 2 (3.8) 0.359   frontal 41 (41.4) 15 (31.9) 26 (50.0)     occipital 3 (3.0) 2 (4.3) 1 (1.9)     parietal 18 (18.2) 8 (17.0) 10 (19.2)     temporal 29 (29.3) 18 (38.3) 11 (21.2)     thalamic 5 (5.1) 3 (6.4) 2 (3.8)   final integrated diagnosis (%) astrocytoma, idh-mutant, cns who grade 2 11 (11.1) 0 (0.0) 11 (21.2) <0.001   astrocytoma, idh-mutant, cns who grade 3 6 (6.1) 0 (0.0) 6 (11.5)     astrocytoma, idh-mutant, cns who grade 4 8 (8.1) 0 (0.0) 8 (15.4)     diffuse midline glioma, h3 k27m-mutant, cns who grade 4 4 (4.0) 2 (4.3) 2 (3.8)     glioblastoma, idh-wildtype, cns who grade 4 60 (60.6) 37 (78.7) 23 (44.2)     oligodendroglioma, idh-mutant and 1p/19q-codeleted, cns who grade 2 10 (10.1) 8 (17.0) 2 (3.8) the median p53 immunohistochemistry labeling index for tumors harboring tp53 mutation was 60 % (range: 0-95 %) with an interquartile range (iqr) of 66.25 %, while the median value for those without tp53 mutations was 10 % with an iqr of 10.5 % (range: 0-30 %). when 42 tumors with missense mutations were considered, the median labeling index was calculated as 77.5 % (mean 64.24 %), and the median labeling index for cases with frameshift mutations was 12.5 % (mean 21 %). however, there was a wide range of labeling indices for missense (1-95 %) mutations. of note, out of 11 tumors with absent p53 staining, 6 had tp53 mutations (two frameshift, two missense, one splice site mutation, and one structural variant). figure 2. labeling index of p53 is significantly higher in tp53-mutated group as shown in the violin plot (a). radar chart for tp53 mutation types in tumor with high p53 li (>40 %) and low p53 (<1 %) li (b). roc analysis demonstrated 40 % as an optimal cutoff point for tp53 mutation based on ucsf500 ngs confirmation (c and d). (loh: loss of heterozygosity, cutpoint: mean value of the optimal cutoff, density: distribution of cutoff values). when the cut-off values for p53 immunohistochemistry were considered, the positive predictive value of 10 % labeling index was calculated as 60.4 %, while the negative predictive value at the same cut-off point was 70.6 %. an roc analysis based on the 99 patients demonstrated that strong p53 staining in more than 40 % of tumor cells provided the most accurate prediction of mutation based on youden index (figure 2). using this cutoff value, positive predictive value (ppv), negative predictive value (npv), sensitivity, and specificity were 100 %, 73.6 %, 67 %, and 100 %, respectively. most of the inaccuracy could be attributed to tumors with frameshift mutations, which frequently demonstrated the null cell pattern with absent immunostaining (with p53 li of <1 %). after excluding the tumor with frameshift mutations, an roc analysis redemonstrated that the best cutoff for p53 li as still 40 % with ppv, npv, sensitivity, and specificity calculated as 100 %, 81 %, 88 %, and 100 %, respectively. these findings suggest, as previously demonstrated by many, that p53 immunostaining cannot be taken as prima facia evidence of tp53 mutations. furthermore, our data suggest that the cut-off value recommended by the cns who classification to predict tp53 mutations may be inaccurate and should be reevaluated. this issue underscores the problem of accepting results of single studies without appropriate validation and confirmation by more than one independent scientific observation for the purposes of standardized classification systems that would be used worldwide. we believe that previously suggested p53 labeling index of 10 % as predictive of tp53 mutation significantly overestimates mutations, which could potentially lead to erroneous conclusions in the absence of molecular confirmation. considering possible variations when performed in different laboratories and using different antibodies, this cut-off value becomes even less predictive. for practical purposes, we recommend the use of >40 % as a more robust and predictive cut-off value for p53 immunohistochemistry, while still acknowledging that there will always be outliers regardless of the cut-off number utilized. finally, the absence of staining does not rule out a tp53 mutation, and thus should be interpreted with caution. references 1. appin cl, brat dj. biomarker-driven diagnosis of diffuse gliomas. mol aspects med [internet] 2015 [cited 2023 mar 4];45:87–96. https://doi.org/10.1016/j.mam.2015.05.002 2. dj b, rg v, kd a, et al. comprehensive, integrative genomic analysis of diffuse lower-grade gliomas. n engl j med [internet] 2015 [cited 2023 mar 4];372(26):2481–98. https://doi.org/10.1056/nejmoa1402121 3. eckel-passow je, lachance dh, molinaro am, et al. glioma groups based on 1p/19q, idh, and tert promoter mutations in tumors. new england journal of medicine [internet] 2015 [cited 2023 mar 4];372(26):2499–508. https://doi.org/10.1056/nejmoa1407279 4. brat dj, reuss de, von deimling a, et al. who classification of tumours editorial board. central nervous system tumours. 5th ed. 2021. 5. takano s, ishikawa e, sakamoto n, et al. immunohistochemistry on idh 1/2, atrx, p53 and ki-67 substitute molecular genetic testing and predict patient prognosis in grade iii adult diffuse gliomas. brain tumor pathol [internet] 2016 [cited 2023 mar 4];33(2):107–16. https://doi.org/10.1007/s10014-016-0260-x 6. ogura r, tsukamoto y, natsumeda m, et al. immunohistochemical profiles of idh1, mgmt and p53: practical significance for prognostication of patients with diffuse gliomas. neuropathology [internet] 2015 [cited 2023 mar 4];35(4):324–35. https://doi.org/10.1111/neup.12196 7. shao lw, pan y, qi xl, et al. atrx loss in adult supratentorial diffuse astrocytomas correlates with p53 over expression and idh1 mutation and predicts better outcome in p53 accumulated patients. histol histopathol [internet] 2016 [cited 2023 mar 4];31(1):103–14. https://doi.org/10.14670/hh-11-664 8. antonelli m, buttarelli fr, arcella a, et al. prognostic significance of histological grading, p53 status, ykl-40 expression, and idh1 mutations in pediatric high-grade gliomas. j neurooncol [internet] 2010 [cited 2023 mar 4];99(2):209–15. https://doi.org/10.1007/s11060-010-0129-5 9. newcomb ew, madonia wj, pisharody s, lang ff, koslow m, miller dc. a correlative study of p53 protein alteration and p53 gene mutation in glioblastoma multiforme. brain pathol [internet] 1993 [cited 2023 mar 4];3(3):229–35. https://doi.org/10.1111/j.1750-3639.1993.tb00749.x 10. faria mhg, neves filho ehc, alves mks, burbano rmr, de moraes filho mo, rabenhorst shb. tp53 mutations in astrocytic gliomas: an association with histological grade, tp53 codon 72 polymorphism and p53 expression. apmis [internet] 2012 [cited 2023 mar 4];120(11):882–9. https://doi.org/10.1111/j.1600-0463.2012.02918.x 11. gillet e, alentorn a, doukouré b, et al. tp53 and p53 statuses and their clinical impact in diffuse low grade gliomas. j neurooncol [internet] 2014 [cited 2023 mar 4];118(1):131–9. https://doi.org/10.1007/s11060-014-1407-4 12. takami h, yoshida a, fukushima s, et al. revisiting tp53 mutations and immunohistochemistry-a comparative study in 157 diffuse gliomas. brain pathol [internet] 2015 [cited 2023 mar 4];25(3):256–65. https://doi.org/10.1111/bpa.12173 13. oka h, utsuki s, tanizaki y, et al. clinicopathological features of human brainstem gliomas. brain tumor pathol [internet] 2013 [cited 2023 mar 4];30(1):1–7. https://doi.org/10.1007/s10014-012-0099-8 14. lucas chg, solomon da, perry a. a review of recently described genetic alterations in central nervous system tumors. hum pathol [internet] 2020 [cited 2023 mar 4];96:56–66. https://doi.org/10.1016/j.humpath.2019.10.009 15. chenn a, parker s, degeare sp, et al. path-05. implementation of a targeted next-generation sequencing panel for the diagnosis and precision medicine treatment of adult patients with who grade iv diffuse gliomas. neuro oncol [internet] 2018 [cited 2023 mar 4];20(suppl_6):vi158–9. https://doi.org/10.1093/neuonc/noy148.661 16. kline cn, joseph nm, grenert jp, et al. targeted next-generation sequencing of pediatric neuro-oncology patients improves diagnosis, identifies pathogenic germline mutations, and directs targeted therapy. neuro oncol [internet] 2017 [cited 2023 mar 4];19(5):699–709. https://doi.org/10.1093/neuonc/now254 copyright: © 2024 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. the presence of shrunken neurons with pyknotic nuclei in the dentate nucleus is a common postmortem change associated with autolysis of the cerebellar granular cell layer feel free to add comments by clicking these icons on the sidebar free neuropathology 5:13 (2024) letter the presence of shrunken neurons with pyknotic nuclei in the dentate nucleus is a common postmortem change associated with autolysis of the cerebellar granular cell layer bilge dundar1, busranur agac2, eyas alzayadneh3, randy tashjian4, kyle s. conway4 mayo clinic, department of pathology, rochester, mn, usa washington university in st. louis, department of neurology, st. louis, mo, usa university of iowa, department of pathology, iowa city, ia, usa university of michigan, department of pathology, ann arbor, mi, usa corresponding author: kyle s. conway · neuropathology section · university of michigan · 2800 plymouth rd., building 35 · ann arbor · mi 48109 · usa kyconway@med.umich.edu submitted: 29 february 2024 accepted: 03 may 2024 copyedited by: lauren walker published: 24 may 2024 https://doi.org/10.17879/freeneuropathology-2024-5398 keywords: cerebellum, autolysis, dentate nucleus, post-mortem, granular cell layer in 1937, g.e. smyth observed that “isolated pyknotic cells and even small groups of three or four pyknotic shrunken cells are quite commonly seen in the normal dentate nucleus”.1 although this change is commonly encountered in postmortem material, it is not well-described in the literature, possibly due to a presumption it is an artifactual change. while the cerebellum is well-known to be exquisitely sensitive to postmortem autolytic change, this change is described almost exclusively in the granular cell layer and not in the dentate nucleus (dn).2-8 to examine whether the presence of shrunken neurons with pyknotic nuclei in the dn represents a postmortem change or a true antemortem/perimortem injury, we retrospectively examined formalin-fixed, paraffin-embedded sections of cerebellum containing both granular cell layer and dn from randomly selected adult autopsies performed at the university of iowa. we qualitatively examined the dn for eosinophilic and pyknotic neurons, then photographed five fields at 200 x magnification (equal to 0.42 mm2 each), and manually measured soma diameter of all dentate neurons in these fields. blinded to the findings in the dn, we qualitatively assessed the cerebellar granular cell layer autolysis (gca) as absent, mild, moderate, or severe according to the scheme described by sheedy et al. (2012).7 for decedents who underwent full autopsy and had available clinical information, we obtained a limited selection of clinical and pathologic data from autopsy reports and patient records to assess for clinicopathologic associations with this change. pathologic data obtained included the small vessel arteriolosclerosis in the cerebellar dn, the presence or absence of neurodegenerative pathology, and the presence or absence of global hypoxic-ischemic injury, as assessed in three locations: anterior cerebral artery / middle cerebral artery (aca / mca) watershed region, hippocampus, and cerebellar purkinje cell layer (as either ischemic purkinje cells, purkinje cell loss, or bergmann gliosis). our cohort consisted of 81 brains, including 28 brain-only autopsies (primarily obtained for neurodegenerative evaluation) and 53 full autopsies of hospital inpatient decedents. the mean age was 66.5 years (+/- 15.0 years), and 54 of the patients (67 %) were male. in 32 cases (40 %), the dentate neurons were identified as having hypereosinophilic cytoplasm and pyknotic nuclei. the mean neuronal diameter (mnd) of dentate neurons across all cases was 19.3 microns (+/- 3.8 microns), and the presence of hypereosinophilia and pyknosis was associated with mnd (student’s t-test, p < 0.01). therefore, we chose mnd as an endpoint for further analysis using univariate linear regression with mnd as the dependent variable. mnd was inversely associated with the severity of gca (beta = -1.65 microns per qualitative level of autolysis, p < 0.01) (figure 1f). no significant relationship was identified between mnd and hypoxic-ischemic injury, either globally or in specific sampled regions: aca/mca watershed region (p = 0.21, sampled in 76/81 cases, 93 %), hippocampus (p = 0.35, sampled in 80/81 cases, 99 %), or cerebellar purkinje cell layer (p = 0.43, sampled in all cases). age and the presence of a neurodegenerative disease were associated with an increased mnd, and both acute liver failure and a history of diabetes were associated with a decreased mnd (table 1). given these results, we evaluated laboratory markers associated with liver injury, including aspartate transaminase (ast), alanine transaminase (alt), ammonia, and bilirubin. no single marker showed a significant association with mnd, although these data were available only for a limited number of cases. for 26 patients, arterial blood gas (abg) measurements were available, and ph was obtained as the most recent value in the last 48 hours of life. this value was not associated with mnd (p = 0.27). by univariate logistic regression, at least moderate gca was associated with acute liver failure (odds ratio = 6.34, p = 0.04) and most-recent ph (odds ratio = 1.55, p = 0.04) and was suggestive of an association with diabetes (odds ratio = 3.12, p = 0.06). figure 1. (a) moderate gca showing significant loss of viable granular-cell neurons. (b) severe gca showing significant loss of viable granular-cell neurons and extensive vacuolation. (c) normal, unaffected dentate nucleus is composed of large neurons (arrow) with abundant cytoplasm and large nuclei with prominent nucleoli. (d) a case with moderate gca showing shrunken, pyknotic neurons (arrow) with some relative preservation of smaller, intermediate neurons (arrowhead). (e) a case with severe gca showing prominent neuronal change and the presence of scattered swollen astrocytes (arrow). (f) mnd decreases in a linear fashion from mild through severe gca (bars show 95 % confidence interval). these findings help to better characterize the nature of this common postmortem finding. the strong association between decreased mnd and increasing severity of gca suggests that shrunken neurons with pyknotic nuclei in the dn are a postmortem change. the relative preservation of dentate neurons in cases of mild gca suggests that gca will typically precede changes to the dn neurons. the absence of an association with global hypoxic-ischemic injury argues against an ischemic component to the change. the positive association between age and the presence of a neurodegenerative disease may reflect that these brains were typically obtained from outpatient settings with fewer metabolic disturbances. table 1: clinical and pathologic associations with mnd beta p-value demographics (n = 81) age 0.97 < 0.01 male 1.20 0.18 neuropathologic features (n = 81) granular cell layer autolysis (severity; 0 - 3) -1.65 < 0.01 postmortem interval (24 hours) -0.65 0.12 cerebellar arteriolosclerosis (severity; 0 - 3) -0.03 0.94 hypoxic-ischemic injury (present/absent) -0.65 0.52 neurodegenerative disease (present/absent) 2.91 < 0.01 clinicopathologic features (n = 53) acute liver failure (present/absent) -4.39 < 0.01 chronic liver disease (present/absent) -1.93 0.15 acute kidney injury (present/absent) -1.15 0.26 chronic kidney disease (present/absent) 1.44 0.22 diffuse alveolar damage (present/absent) -2.05 0.85 history of smoking (present/absent) -1.03 0.31 history of hypertension (present/absent) -1.54 0.19 heart weight (per 100 grams) 0.00 0.30 history of diabetes (present/absent) -2.29 0.03 the fact that both significant clinicopathologic relationships – acute liver injury and a history of diabetes – appear to be also associated with gca suggests that these clinical states predispose decedents to cerebellar autolytic change. gca has been associated with acid-base status,2, 8 and a case of rapid granular-cell autolysis in the setting of acute metabolic insult has been described,8 suggesting that acid-base disturbance may be the predisposing factor. the fact that ph was associated with gca in our data but not clearly associated with mnd raises the possibility of another contributing component of premortem toxic/metabolic injury, but our conclusions are limited by a small number of cases with laboratory data regarding potential sources of such injury. qualitatively, swollen astrocytes consistent with alzheimer’s type 2 astrocytes were seen in severe cases, but we did not formally document this finding given the subjectivity of identifying these cells in the setting of autolytic change. while our data overall support the postmortem nature of this change, the presence of gliosis suggests a possible antemortem component in some cases. assessing the presence of alzheimer’s type 2 astrocytes with markers such as p6210 could help better establish whether their presence is associated with injury to dentate neurons. similarly, assessing the cerebellum with markers of ischemic injury such as map2 could help further support the conclusion that this change is not ischemic in nature.11 finally, we did not specifically examine changes in neuronal morphology in the thalamus and inferior olivary nucleus, which were sampled in a limited number of cases. given the significant projections between the dn and these structures, a further examination into whether they undergo similar morphologic changes may be warranted. our study is also limited by the heterogeneous and complex nature of dentate neurons, of which four morphologically distinct subtypes have been described. our data do not establish whether this neuronal change is due to a global reduction in neuronal diameter or preferentially affects a particularly neuronal subtype.9 nonetheless, while single-dimension morphometric analysis of neuronal cell bodies is inherently limited, our data show that mnd is a meaningful marker that reflects the practical setting in which the dn is evaluated in the routine practice of autopsy neuropathology. recognition of this postmortem change to neurons of the dn is important for several reasons. care should be taken not to overinterpret this change as a manifestation of acute ischemic injury, particularly if it is an isolated finding. in cases without significant gca or a poorly sampled granular cell layer, changes to the dn may imply an autolytic change that should be considered when determining tissue viability for additional studies. the diameter of dn neuronal cell bodies may provide a measurable proxy for cerebellar autolysis to assist in semi-quantitative assessment of gca. further investigation into a potential mechanistic relationship between acid-base status, metabolic injury, changes to the dn, and cerebellar autolysis is warranted. references 1. smyth, ge. the significance of lesions in the dentate nuclei apparently consecutive to disease of the frontal lobes. brain. 1941;64(1):63-72. 2. agarwal an, gilbert ar, mais dd. correlation of cerebellar granular layer autolysis with ante-mortem systemic acid-base status. cell tissue bank. 2021;22(3):505-509. https://doi.org/10.1007/s10561-021-09900-4 3. albrechtsen r. the pathogenesis of acute selective necrosis of the human cerebellar cortex. acta neuropathol. 1977;37:31-34. https://doi.org/10.1007/bf00684537 4. finnie jw, blumbergs pc, manavis j. temporal sequence of autolysis in the cerebellar cortex of the mouse. j comp pathol. 2016;154(4):323-8. https://doi.org/10.1016/j.jcpa.2016.03.005 5. krassner mm, kauffman j, sowa a et al. postmortem changes in brain cell structure: a review. free neuropathol. 2023;4. https://doi.org/10.17879/freeneuropathology-2023-4790 6. j. o, yutani c, imakita m et al. autolysis of the granular layer of the cerebellar cortex in brain death. acta neuropathol. 1986;70:75-78. https://doi.org/10.1007/bf00689517 7. sheedy d, harding a, say m, stevens j, kril jj. histological assessment of cerebellar granule cell layer in postmortem brain; a useful marker of tissue integrity? cell tissue bank. 2012;13(4):521-7. https://doi.org/10.1007/s10561-011-9265-1 8. suarez-pinilla m, fernandez-vega i. an acute metabolic insult highly increased postmortem cerebellar autolysis: an autopsy case. clin neuropathol. 2015;34(3):166-8. https://doi.org/10.5414/np300809 9. ristanovic d, milosevic nt, maric dl. on the classification of normally distributed neurons: an application to human dentate nucleus. biol cybern. 2011;104(3):175-83. https://doi.org/10.1007/s00422-011-0426-x 10. gelpi e, rahimi j, klotz s et al. the autophagic marker p62 highlights alzheimer type ii astrocytes in metabolic/hepatic encephalopathy. neuropathology. 2020;40(4):358-366. https://doi.org/10.1111/neup.12660 11. barranco r, bonsignore a, ventura f. immunohistochemistry in postmortem diagnosis of acute cerebral hypoxia and ischemia. medicine (baltimore). 2021; 100(25). https://doi.org/10.1097/md.0000000000026486 copyright: © 2024 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. from amaurotic idiocy to biochemically defined lipid storage diseases: the first identification of gm1-gangliosidosis feel free to add comments by clicking these icons on the sidebar free neuropathology 4:12 (2023) flashback from amaurotic idiocy to biochemically defined lipid storage diseases: the first identification of gm1-gangliosidosis burkhard s. kasper1, christian thomas2, anne albers2, ekkehard m. kasper3, konrad sandhoff4 friedrich-alexander-universität erlangen-nuremberg, university hospital, dept. neurology; erlangen, germany institute of neuropathology, university hospital münster, münster, germany division of neurosurgery, hamilton health sciences, mcmaster university faculty of health sciences, hamilton, on, canada limes institute, membrane biology & biochemistry unit, bonn university, bonn, germany corresponding author: burkhard s. kasper · epilepsy center · department of neurology · friedrich-alexander university erlangen-nuremberg · schwabachanlage 6 · 91054 erlangen · germany burkhard.kasper@uk-erlangen.de additional resources and electronic supplementary material: supplementary material submitted: 22 may 2023 accepted: 15 july 2023 copyedited by: jeffrey nirschl published: 08 august 2023 https://doi.org/10.17879/freeneuropathology-2023-4845 keywords: lipid storage disease, gm1-gangliosidosis, amaurotic idiocy, tay-sachs-disease, beta-galactosidase original paper: on a biochemically special form of infantile amaurotic idiocy. jatzkewitz h., sandhoff k., biochim. biophys. acta 1963; 70; 354-356. see supplement 1. abstract on february 23rd 1936, a boy-child (“kn”) died in an asylum near munich after years of severe congenital disease, which had profoundly impaired his development leading to inability to walk, talk and see as well as to severe epilepsy. while a diagnosis of “little’s disease” was made during life, his postmortem brain investigation at munich neuropathology (“deutsche forschungsanstalt für psychiatrie”) revealed the diagnosis of “amaurotic idiocy” (ai). ai, as exemplified by tay-sachs-disease (tsd), back then was not yet understood as a specific inborn error of metabolism encompassing several disease entities. many neuropathological studies were performed on ai, but the underlying processes could only be revealed by new scientific techniques such as biochemical analysis of nervous tissue, deciphering ai as nervous system lipid storage diseases, e.g. gm2-gangliosidosis. in 1963, sandhoff & jatzkewitz published an article on a “biochemically special form of ai” reporting striking differences when comparing their biochemical observations of hallmark features of tsd to tissue composition in a single case: the boy kn. this was the first description of “gm1-gangliosidosis”, later understood as resulting from genetically determined deficiency in beta-galactosidase. here we present illustrative materials from this historic patient, including selected diagnostic slides from the case “kn” in virtual microscopy, original records and other illustrative material available. finally, we present results from genetic analysis performed on archived tissue proving beta-galactosidase-gene mutation, verifying the 1963 interpretation as correct. this synopsis shall give a first-hand impression of this milestone finding in neuropathology. introduction in history of science, there has always been and will be discussion, to whom to ascribe to the first description of a disease. addressing a specific publication as the first description can turn out quite differently, depending on whether or not one is focusing on a disease’s clinical manifestations, its tissue pathology, pathophysiological mechanism or underlying genetic cause. further complicating matters, similar findings are sometimes reported independently or even simultaneously by separate researchers at different places. as thinking and knowledge about human disease evolve over time, which include terminology and classifications, it could well be that we end up with more than one author credited for the first description of a particular disease. individual patients and the details behind groundbreaking findings, however, are rarely appreciated in detail, albeit some of them have become linked to (neuro-)history at least by their initials, such as “h.m.” [1]. concerning the “lipidoses”, exemplified by tay-sachs disease (tsd), syndromal appearance of certain symptoms in children had been well observed and reported as early as at the end of the 19th century by warren tay [55] and bernard sachs [36] establishing “tay-sachs-disease” as an eponym [6], as well as “amaurotic family idiocy” (afi) as another common term [35]. the rather dramatic clinical presentation in tsd/afi and comparable diseases, such as niemann-pick’s, was accompanied by striking pathologic findings post-mortem, especially in the cns, where neurons were found enlarged and ballooned, obviously filled by aberrant material [25]. the first pathoanatomic description of tsd appears to have been given by sachs himself in 1887 [36]. in the 20th century tsd/afi brains were studied extensively both histopathologically and later also ultrastructurally (e.g. [8], [9], [10], [38], [55], [56]) laying the foundation for a concept of lysosomal lipid storage disease (review see [11], [25], [27], [58]). cns tissue pathology in tsd and other storage diseases appeared rather uniform, resulting from accumulation of some storage material strikingly altering neuronal morphology, a finding early appreciated in cns histopathology ([12], [57], [46-48]). afi subtypes for quite a long time were separated on clinical grounds only by predominating manifestation age. specific techniques allowing a more precise analysis, deciphering the underlying molecular pathobiology and establishing a clear understanding of different disease forms, were not available. this changed in the mid-to-late 1930s, when klenk coined the term “gangliosides” to describe characteristic acid glycolipid components of neuronal cells isolated from tsd brains [19-21]. it took more than 20 years from then to identify the first ganglioside structure g1 [24], now called gm1, and to apply better techniques such as thin-layer chromatography allowing for separation and characterization of the different ganglioside components in diseased versus normal brain tissues in the 1960s. further important breakthrough discoveries were achieved in the understanding of nervous system within a few years ([14], [15], [24], [34], [43], [53]) deciphering the “gangliosidoses”, including classic tsd as gm2-gangliosidosis. research activities and knowledge gain then exploded, leading to one of the most astonishing advances in understanding of lipid storage-related cns-disease (for review see: [2], [16]), which for decades before had remained enigmatic and summed up under different terms of afi, “amaurotic idiocy” (ai) or “infantile amaurotic idiocy” (iai), a hot topic in early neuropathology. up to today, many aspects of lipid storage related disease mechanisms have been elucidated. this includes many of the enzymes involved, their functional dynamics in health and disease, their structure(s), co-factors and substrates including cellular pathways & regulation, the role of the lysosomal compartment, the regulating genes and their mutational spectrum (for review see [2], [16], [22], [28], [42]). after decades characterized by restriction to symptom-oriented diagnosis and, at best, post mortem validation in a few selected cases, we are now able to recognize these diseases intra vitam by surrogate biomarkers and/or molecular testing. it is a fascinating journey to follow the historic lines of these developments. here we present and re-appreciate a key patient, “kn”, including his post mortem tissues as well as the understanding derived from analyzing his tissue probes biochemically: this boy died 1936 in munich from a fatal course of iai at the age of 7¾. by diligent biochemical analysis of this patient’s brain tissue, performed after more than 25 years of formalin-fixation, jatzkewitz & sandhoff revealed a “biochemically special form of infantile amaurotic idiocy” [14] later to be named “gm1-gangliosidosis” [51]. this can be acknowledged as the first definite characterization of this disease [16, 28], eliciting that tsd/ai/iai was a spectrum of different diseases rather than a uniform entity. what exactly did the authors describe in this study? jatzkewitz & sandhoff presented a surprising result of a thin-layer-chromatographic lipid compound analysis performed on brain tissue from an “ai”-patient, for this single case (patient “kn”) out of an ai-series clearly displayed an unusual “aberrant” pattern on chromatography when compared to chromatography bands of other “ai-tsd”-materials and normal brain (see figure 1). kn’s analysis did not show the accumulation of “tay-sachs-ganglioside” (=gm2 ganglioside) but showed accumulation of the major normal “ganglioside g1” according to kuhn & wiegandt [24], i.e. ganglioside a according to klenk and gm1-ganglioside according to svennerholm. why is this a milestone paper? the paper’s key finding summarized on less than three pages convincingly demonstrates that a special and biologically different variant of tsd-like disease had been found, which turned out to be correct. in contrast to classic tsd showing accumulation of gm2-ganglioside and its sialic acid free residue ga2, here a different metabolic block had to be suggested since other substances were stored, identified as “ganglioside gm1” and its sialic acid free residue [14] [41], which turned out to be correct [2]. the distinct identity of these storage compounds was proven by chemical, biochemical and enzymatic procedures [43]. while several papers and textbooks refer to a different study as the one having identified gm1 as the storage compound in this type of disease [30], this clearly came later ([16], [28]). at this stage, all the knowledge to be revealed later about metabolic chains and enzymes involved had not yet been known [44]. figure 1. original chromatography analysis by sandhoff: “kn” = lane 7. formalin-fixed control = lane 5 (photography by k. sandhoff, gangliosides labelled; original legend). see full article by jatzkewitz & sandhoff 1963 [14] in supplement 1 (illustrating the metabolic path).thin-layer chromatogram of lipid extracts of brain tissue from two usual infantile cases and one from one special form of late-infantile amaurotic idiocy (jatzkewitz and sandhoff 1963) adsorbent, 400-µm-thick layer of kieselgel g, merck; solvent system, propanol-conc. ammonia-water (6:2:1); height of the solvent front, 15cm; detection, anisaldehyde, sulfuric acid in acetic acid (reagent of kägi-miescher). 1, 20µg of neuraminic acid-free residue of ganglioside tay-sachs (b’, now named ga2, rf 0.33); 2, 250µg of total lipid extract of the brain cortex of a case of iai (fresh tissue); 3, 20µg of ganglioside tay-sachs (a’, gm2, rf 0.37); 4, 250µg of total lipid extract of the brain cortex of a case of iai, preserved for formalin for 26 years; 5, 250µg of total lipid extract of normal brain cortex, preserved in formalin for 26 years; 6, 20µg of ganglioside a (gm1, rf 0.30); 7, 250µg of total lipid extract of the brain cortex of a special form of late iai, preserved in formalin for 26 years; 8, 20µg of neuraminic acid-free residue of ganglioside a (ga1, rf 0.26). [author’s addendum: rf means “retention factor”]. what exactly was new based on the knowledge back then? up to this 1963 study’s result, ai and tsd was perceived as an entity with varying clinical courses and was classified according to manifestation age. classic microscopic histomorphological analysis was not able to clearly differentiate between various entities, and therefore could not resolve the ai-enigma, maybe except separation of the “neuronal ceroid-lipofuscinoses” (ncl) as a separate group in ai-like disease ([46-48] [57]). of note, ncl as a term was coined later ([50],[62]). the biology and biochemical nature of these diseases were largely unknown. what turned out as being correct or incorrect since then? some authorities were convinced that essentially everything was already known about tsd up to 1960. as late as 1957, seitelberger had stated that “…it has been…proven, that the cellular metabolic disturbance constituting the individual forms [of tsd] are mainly the same” [49]. when k. sandhoff following his analysis of a randomly selected first ai-case searched for further brain specimens in munich neuropathology archives, he was confronted by the institute’s head g. peters, who told him boldly: “…if you aim to know about tay-sachs-disease, read my book and you will know everything” (sandhoff, personal memory). this was the starting point. newly developed biochemical analytic methods opened new windows: it turned out that diverse lipid compounds represent essential building blocks of nervous tissue, that their metabolism is highly sophisticated biochemically and can be altered causing various specific diseases (for review see [45], [2]). concerning the “biochemically special form” of ai as identified in 1963, the postulated metabolic block in ganglioside a degradation later was identified and characterized as impairment of specific enzyme activities, mostly beta-galactosidase ([2], [16], [44]). at what stage of the author´s career was the paper published? k. sandhoff was a young student at the very beginnings of his career as a researcher (figure 2). figure 2: konrad sandhoff 1962, during the time of his thesis (photo shared by k sandhoff). this study was his debut publication. having finished studying chemistry in 1962, he turned towards lipid compound analysis from nervous tissue by joining the department of neurochemistry headed by horst jatzkewitz at munich max-planck institute for psychiatry, encouraged and supported by 1964 nobel laureate prof. fedor lynen. after failed attempts to isolate presumed sialic-acid-free gangliosides (“asialo-gangliosides”) from cattle brain, his attention turned towards new targets for investigation: rather by chance, in 1962 he decided to choose a first human brain sample in the lab labelled “ai” [44]. in this very first analysis, using chromatographic techniques, he was able to reveal two bands, which he named “a” and “b” (later identified as gm2-ganglioside and its corresponding asialo-residue ga2). in order to validate his finding, he widened his analysis to include tissue probes from other 12 brains with ai/tsd picked from the archives at munich neuropathology, “kn” being one of them ([43], [44]). how was the paper received over time? issues were raised whether the results presented were rather a methodological artifact caused by long-term formalin fixation, although this was largely ruled out by the experiments presented, since matching long-term fixed control samples had been included ([14], see figure 1). in the realm of anglo-american work this type of lipidosis became rather known as norman-landing-disease or landing’s disease for after an early case reported by norman [29] landing published a clinical report in 1964 ([26], [37], which was followed by identification of gm1-storage [30] and galactosidase deficiency [31]. in one early review, o’brien questioned, whether the patient from the jatzkewitz-sandhoff-paper had the disease at all [30]. the jatzkewitz-sandhoff paper [14] prompted further group-activities supported by national and international colleagues, who agreed to provide selected tissues probes incl. fresh and fresh-frozen samples from “ai” patients for further analysis by sandhoff. several researchers knew each other personally and also cooperated, such as sandhoff and o’brien or sandhoff and h. moser/boston (sandhoff, personal communication, [39]). such cooperation rapidly expanded the experience and led to description of further lipid storage disease variants, especially with respect to the gm2-gangliosidoses, including the hexosaminidase-0-variant later known as “sandhoff’s disease” ([23], [33], [40]). beyond this, further variants were described, including cases with storage but normal enzyme levels, indicating the presence of co-factors [44]. what happened to the original histological slides and patient documentation until today? kn’s original slides were found within the historical archive of the max planck institute for psychiatry in munich by registrar c. dücker after intense search by bs kasper had revealed enough detail in order to clearly identify them correctly, for there was no specific finding aid. the crucial informations were patient initials (“kn”) and age (“7¾“), as well as the institutional archive case number (55/36) listed in the sandhoff doctoral thesis only ([43], see supplement 2). an earlier paper on ai from munich [8], which had recruited its cases from the munich neuropathology collection also, served as cross reference, since it happened to list respective overlapping information including patient initials and age (see supplement 3). thus, tissue samples of “case 55/36” could be identified in two case boxes labelled “ai” at mpi archives (see supplement 4). luckily, the mpi archive stored one single additional document relating to “kn”, a clinical report giving some more details about the patient child, including full name, date of birth, date of death and the name of the caring medical institution prior to his death (see supplement 5). this finally allowed to identify further clinical records preserved within another munich archive, i.e. the archbishop of munich & freising’s archive (see supplement 6); for reasons of clerical law and tradition, this archive keeps documents much longer than done by average public hospitals. the munich psychiatric hospital’s archive did no longer store any documents (explored by p. rauh, see acknowledgement). what is known about the patient behind the paper? the patient designated “kn” in the respective publications ([8], [14], [15]) was a boy named johann knott, born april 11th 1928. early on, he lived with a foster mother in munich, but soon was put under guardianship by munich city administration. this way he came into an asylum in schönbrunn near munich early due to his severe disability labelled “little’s disease”. asylum notes describe the kid as unable to talk, unable to walk, suffering from epilepsy and being “very poor”. he was referred to the munich university hospital (“nervenklinik”) in june 1932, where he stayed until april 1933. at admission he was described as apathic and without reaction to external stimuli. he was noted to show impaired swallowing and an aberrant breathing pattern. physical and neurological examination was reported with brisk reflexes, abnormal muscular tone, but ocular fundi were reported as normal. the boy had multiple epileptic seizures every day described as tonic without further detail. affected by pneumonia, he died at schönbrunn february 23rd 1936 (see supplements 5-7). it is important to note, that kn’s death and postmortem investigation in no way is related to the nazi euthanasia program (confirmation with support by p. rauh; see acknowledgement, please read supplement 10). the role of german neuropathology as well as clinical medicine during the ns-regime is at the focus of ongoing research (see supplement 10). what can be seen on the original slides? at the historical archive of the munich max-planck-institute for psychiatry, 194 microscopic slides were found stored together with the original diagnostic report (see supplements 4 & 9) and the clinical note mentioned above (supplement 5). the slides encompass a systematic selection of cns sites including specimens of the spinal cord, selected brain stem levels such as medulla and pons, cerebellum and various cortical samples quite differently stained. stains according to the original report and (inconstant) slide labelling included bielschowsky-, nissl-, holzer-, mallory-, heidenhain-, “fat-” and “pikrofuchsin”-stains. while a significant number of slides were no longer useable due to severe aging and bleaching, several slides were preserved quite well. the reader is invited to explore a selection of well-preserved slides here (see figure 3 and slide-links). it is interesting to compare these examples to the original drawings drawn by van gieson, provided in bernard sachs’s 1887 publication [35] on “amaurotic idiocy” (see figure 4, and supplement 8). figure 3. original historical slides from selected cns sites of patient “kn”. specimen were well preserved stained mallory, an additional neocortical specimen underwent some “fat-stain”, not otherwise specified (scanned with zeiss axioscan.z1). the cerebral slides (lower row: neocortex and hippocampal region) illustrate the severe cortical pathology characterized by shrinking of the cortical band with profound loss of neurons. residual neurons show the typical swollen, ballooned morphology of a cns lipidosis. when going into detail on large magnifications, the neuronal pathology is seen well in brainstem also, esp. in the medulla/olive specimen (upper row, middle). within the hippocampus proper there seems to be profound loss of pyramidal neurons, which could represent hippocampal sclerosis (this specimen was not available in other stains). clicking into the picture will lead you to the full virtual slide. figure 4. original drawings by i. van gieson (1866-1913), from sachs’s publication 1887 [36] (courtesy of the national library of medicine). see supplement 8. have the original materials been used for modern molecular approaches and what was the result? to the best of our knowledge, the original materials of the patient johann knott presented here so far were not used for any modern molecular analysis later on, although materials participated in one subsequent ganglioside quantification analysis [15]. for this re-visitation, we performed a genetic analysis using a selected fraction of kn’s archived material using only sections no longer usable for microscopy due to aging effects. this procedure was covered by a positive vote of the ethics commission at munich university/lmu (nr. 22-1021): materials & methods: formalin-fixed paraffin-embedded (ffpe) cerebellar sections from the historical archive of the max planck institute for psychiatry in munich were used as follows. genomic dna was extracted from three stained sections. the slides were incubated in xylene (bergchemie j. c. bröcking) for 14 days to enable lifting of the cover slip. tissue was overlayed with incubation buffer containing proteinase k (maxwell ffpe plus dna kit, promega), scraped from the surface using a scalpel and resuspended for transfer into a 1.5 ml reaction tube to perform dna extraction using the maxwell ffpe plus dna kit (promega). dna was quantified using the quantifluor one dsdna system (promega) and 20 ng were subjected to the infinium hd ffpe restore protocol (illumina, zymo). finally, 1 µl of the restored gdna was inspected by genomic dna screentape assay (agilent) to determine dna integrity number (din) and concentration (figure 5a). although dna integrity was low (1.0), the exome 2.0 kit (twist bioscience) could be successfully applied for library preparation and sequencing was performed on a novaseq 6000 device. after base calling and adapter trimming, alignment to the human reference genome (homo_sapiens.grch37.75_complete) and variant calling were performed using dragen v07.021.624.3.10.10. variants were annotated using the variant effect predictor (v100) for functional annotations, pathogenicity scores, population allele frequencies and to determine the effect of called variants on genes, transcripts, and protein sequence. variants were manually inspected with the integrative genomics viewer (igv). results are illustrated in figure 5. figure 5. (a) dna fragment analysis. fragment analysis of gdna isolated from formalin-fixed paraffin-embedded tissue shows high dna degradation. the figure shows dna fragment sizing and quantification using genomic dna screentape assay. (b) igv screenshots showing representative images of the two heterozygous mutations: glb1 (nm_000404.4): c.400g>a; p.g134r (top) and glb1 (nm_000404.4): c.931g>a; p.g311r (bottom). both mutations have been described in patients with gm1 gangliosidosis [28]. epilogue amaurotic idiocy (ai), as exemplified by tay-sachs-disease (tsd), had remained a fatal, enigmatic condition for a long period of time. ai’s impressive cellular pathology, affecting neuronal cells throughout the nervous system quite specifically, was recognized as early as 1887 ([36] supplement 8). after separating the tissue pathology later to be known as ceroid-lipofuscinosis [62; 46-48] classic neuromorphology for decades had not much to contribute to the differentiation of ai, for its cns pathology appeared rather uniform [25]. significant advances in this field were largely driven by biochemical insight into composition and distribution of central nervous compounds and by applying newly emerging techniques between 1920 and 1960, exemplified by the works of klenk [19-21], kuhn & wiegandt [24], svennerholm [53] and sandhoff ([42], [45]). sandhoff’s original observation [14] revisited here opened the door to the realization that tsd-like ai was not a homogenous entity, but a spectrum of neurometabolic diseases [2]. triggered by detailed analysis of johann knott (kn) and subsequent findings the field expanded and evolved rapidly ([2], [16], [44], [45]) leading to in depth characterization of the different forms of lipid storage disease, including enzymes & genes involved. thus, gm1-gangliosidosis today can be summarized as a rare autosomally inherited lipid catabolism defect caused by deficiency of beta-galactosidase, encoded by the gene glb1 on chromosome 3, leading to lysosomal storage of ganglioside gm1 and its sialic-free residue ga1 ([2], [16], [28]] encompassing various clinical forms ([3], [16], [52]). the term “ai” has long been abandoned and is now replaced by more specific terms representing our refined understanding. acknowledgements we thank: c. dücker / historical archive registrar at mpi psychiatry munich for generous cooperation e. binder / head of mpi psychiatry munich for kindly supporting this project p. rauh / institute of history and ethics of medicine, tu munich, for fruitful discussion, sharing his knowledge about munich archives, and help with clarifying the historical context of the patient dr rauh is co-head of the research project entitled "brain research at institutes of the kaiser wilhelm society in the context of nazi injustices: brain specimens in institutes of the max planck society and identification of victims" (tu munich, university vienna, oxford brookes university, leopoldina) r. egensperger, h. leithäuser / lmu neuropathology munich for excellent support with digital scanning d. heuss / neurology at erlangen university, for support with his “flying microscope” g. treffler / munich from the munich-freising archbishop‘s archive c. döbereiner / erlangen university library for great help with many remote references supplementary material suppl. 1 jatzkewitz & sandhoff (1963) [14]; 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pediatrics 1969; 44: 570-583. pmid: 5346636. copyright: © 2023 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. striking intraneuronal neurofilament inclusions restricted to the locus coeruleus in a patient with creutzfeldt-jakob disease feel free to add comments by clicking these icons on the sidebar free neuropathology 5:22 (2024) letter striking intraneuronal neurofilament inclusions restricted to the locus coeruleus in a patient with creutzfeldt-jakob disease felix leitner1,2, sigrid klotz1,2, karoline ornig3, serge weis3, ellen gelpi1,2 division of neuropathology and neurochemistry, department of neurology, medical university of vienna, 1090 vienna, austria austrian reference center for human prion diseases (oerpe) division of neuropathology, department of pathology and molecular pathology, neuromed campus, kepler university hospital, linz, austria corresponding author: ellen gelpi · division of neuropathology and neurochemistry · department of neurology · medical university of vienna · 1090 vienna · austria ellen.gelpi@meduniwien.ac.at submitted: 29 july 2024 accepted: 14 september 2024 copyedited by: georg haase published: 02 october 2024 https://doi.org/10.17879/freeneuropathology-2024-5721 keywords: neurofilament inclusions, locus coeruleus, neurofilament, neuronal cytoplasmic inclusion, creutzfeldt-jakob disease introduction creutzfeldt-jakob disease (cjd) is the most frequent human prion disease, but it is overall a very rare, rapidly progressive neurodegenerative disease.1 physiopathologically, prion diseases have set the basis for the concept of protein misfolding as a common mechanism for the most prevalent protein-related neurodegenerative conditions. in addition, interactions between misfolded proteins have been an intense topic of basic research in the field of neurodegeneration, as has been the analysis of concomitant “proteinopathies” in neuropathology. several cases of cjd featuring concomitant pathologies such as alzheimer’s disease 1,2,3 or lewy body dementia 4,5,6 have been reported over the last years. most of the concomitant pathologies have been attributed to age rather than to a direct effect of the prion protein7,8, although this is still controversial. here we report the neuropathological findings of a cjd patient with striking intraneuronal inclusions that are restricted to the locus coeruleus and that display a unique immunohistochemical profile not seen in other neurodegenerative diseases. we propose that these unique intraneuronal inclusions might be associated with subtle and slowly progressing neurological symptoms like rapid-eye movement behavior disorder (rbd) or reduced cognitive flexibility. case presentation the patient was an 84-year old female patient who developed a rapidly progressive dementia accompanied by rigidity, dysarthria and dysphagia within a few months. brain mri revealed a restricted-diffusion pattern of the left basal ganglia, and the eeg showed partially steeply configured signals. the cerebrospinal fluid tests revealed an increased 14-3-3 protein concentration of over 80 000 au/ml (reference values < 10 000 au/ml) and a positive real-time quaking-induced conversion (rtquic) of the prion protein (prp). the patient fulfilled criteria for “probable” creutzfeldt-jakob disease (cjd)9 and died within less than 4 months. the brain weight was 1275 gram. on gross examination, there were no signs of brain atrophy. the circulus of willis showed only a few non-stenosing plaques in the arterial wall. there were no signs of brain swelling with tonsillar notch. the ventricular system was not enlarged. the substantia nigra and locus coeruleus appeared to be regularly pigmented. microscopic examination revealed the spongiform neuropil change characteristic of cjd10 as well as nerve cell loss and gliosis in the deeper cortical layers of the frontal and parietal lobes as well as in the basal ganglia, thalamus, brainstem and cerebellum. pathological prion protein deposits (anti-prp, bertin bioreagent, 12f10) were immunohistochemically detected in these regions. in cortical regions, there was a predominantly deep-laminar perineuronal pattern while the basal ganglia, the cerebellum and the nuclei of the brainstem, including the locus coeruleus and the solitary tract showed a dense synaptic deposition pattern. in addition, there were some plaque-like deposits in the neighbouring white matter and also deposits along axons and perivascularly in basal ganglia and thalamus. the morphological and immunohistochemical distribution pattern was overall well compatible with the vv2 histotype according to the 2009 parchi classification of cjd (figure 1).11 figure 1. characteristic features of cjd neuropathology consistent with the vv2 histotype a, b: immunohistochemistry for prp shows a deep laminar, perineuronal deposition pattern. c, d: in the basal ganglia, spongiform change is relatively prominent (c) and anti-prp immunohistochemistry shows a strong synaptic deposition pattern (d). e-h: in the cerebellum, spongiform change is prominent in the molecular layer and there is a moderate reduction of granule cells (e). single torpedoes are also observed (g). immunohistochemistry for prp reveals pathological synaptic deposits in the cerebellar cortex and coarse as well as plaque-like and periaxonal aggregates in the cerebellar white matter (h). synaptic pathological prp deposits were also detected in the brainstem including the locus coeruleus and solitary nucleus (not shown). in addition to the characteristic features of a spongiform encephalopathy, several fibrillar neuronal inclusions restricted to the locus coeruleus (lc) were identified in this patient. the locus coeruleus showed a moderate reduction of pigmented neurons and an accompanying gliosis. the inclusions were relatively large, partly eosinophilic and partly pale basophilic on h & e-stained sections and partly argyrophilic on bielschowskyand gallyas silver impregnation methods (figure 2). in contrast, by fluorescence microscopy, only a small fraction of the inclusions was weakly and irregularly stained by thioflavin t, while the majority of inclusions was not stained by thioflavin t. figure 2. microscopic features of intraneuronal cytoplasmic inclusions in the locus coeruleus a-c: frequent filamentous intraneuronal cytoplasmic inclusions in the locus coeruleus after hematoxylin-eosin staining. note the large size and the variable morphology of the inclusions that range from pale basophilic and fibrillar to dense hyaline and eosinophilic. d-e: p62 highlights the compact and filamentous nature of the large neuronal inclusions. f: intraneuronal inclusions were negative for alpha-synuclein, tau, phosphorylated tdp-43 and fus (arrows denote negative inclusion). g-i: in contrast, neuronal inclusions were positive for phosphorylated neurofilaments (smi31, g, arrow points to a non-stained segment of a large fibrillar inclusion) and non-phosphorylated neurofilaments (smi32, h, i). j-l: the inclusions were partly argyrophilic (j, k) and strongly positive for alpha-internexin (l). immunohistochemically, the neuronal inclusions in the lc were immunoreactive for p62, an indicator of autophagic degradative activity and they were strongly immunoreactive for phosphorylated neurofilaments (biolegend, clone smi 31), non-phosphorylated neurofilaments (biolegend, clone smi 32) and alpha-internexin (invitrogen, clone 2e3), a class iv neuronal intermediate filament. although the histological and immunohistochemical profile of these neuronal inclusions strongly resembled that of neurofibrillary tangles, immunohistochemistry for phospho-tau (thermo scientific, at8) remained negative. immunostaining against alpha-synuclein (ajroboscreen, clone 5g4), ptdp43 (cosmobio, clone 11/9) and fus (sigma life science, clone g75241) were also negative. interestingly, these fibrillar inclusions were not found in any other areas of the brain and were exclusively identified in the lc. discussion we present a unique combination of sporadic cjd with striking neurofilamentous inclusions restricted to the locus coeruleus. this combination has not been described so far, and particularly, this type of neurofilament-bearing inclusions restricted to the locus coeruleus seems to be a singular finding. the significance of the inclusion bodies in this region is unclear and no obvious clinical correlate could be retrospectively extrapolated from the history of the patient, as symptoms were dominated by the rapidly progressive dementia as well as the brainstem and cerebellar symptoms of cjd. of note, some important functions have been attributed to the locus coeruleus. the pigmented neurons in the pontine tegmentum have highly branched axon terminals that innervate many different areas of the cns, including the neocortex, hippocampus, amygdala, thalamus, cerebellum and spinal cord and plays a decisive role in arousal, concentration and stress response as it is the most important source of norepinephrine in the cns.12 the locus coeruleus seems to be prone to the development of certain types of intraneuronal inclusions, particularly neurofibrillary tangles and lewy bodies, and its cells are affected in various neurodegenerative conditions.13 in parkinson's disease or lewy body dementia, alpha-synuclein aggregates in form of lewy bodies and lewy neurites occur in the locus coeruleus prior to the substantia nigra.14 the neuronal dysfunction of the locus coeruleus/subcoeruleus also plays a role, together with the gigantocellular nucleus of the formatio reticularis, in the development of rapid-eye movement behavior disorder (rbd), a disorder in which atonia lacks during rem sleep, leading to dream-enacting behavior with flapping, kicking and biting.15,16,17 rbd most often represents an early or even prodromal symptom of parkinson's disease18 or lewy body dementia and is less often encountered in multisystem atrophy. rbd is most frequently associated with alpha-synuclein pathology. synuclein aggregates were however not observed in our patient. while we have no definite data on whether the patient had rbd or not, the large neurofilament inclusions in the locus coeruleus could theoretically be an additional non-lb related cause of rbd. moreover, reduced cognitive flexibility in pd has been related to reduced neuromodulation of the prefrontal cortex from subcortical structures such as the locus coeruleus.19 these symptoms are however very difficult to assess retrospectively and separate from the development of cjd in our patient. concerning prion diseases, experimental studies in mice have shown that the locus coeruleus, the solitary tract and the pre-boetzinger complex are the first brainstem target areas of early prion deposition.20 interestingly, the vv2 subtype of cjd also involves these brain regions, which all showed pathological prp deposits in our case. neurofilament inclusions are the hallmark of neuronal intermediate filament inclusion disease (nifid), as described by cairns et al.21 in patients with frontotemporal dementia. in these cases however, the morphology and particularly the extent of the inclusions are very different and affect also cortical areas, in particular fronto-temporal regions, basal ganglia, amygdala, hippocampus, brainstem including the substantia nigra, and cerebellum. the neurofilament inclusions were originally described as alpha-internexin positive and are currently considered as a subtype of fus/fet proteinopathy.22 in our case, inclusions were positive for alpha-internexin, negative for fus and in the majority negative for thioflavin t. while thioflavin t is a dye that binds to β-sheet rich structures, it may show variable affinity for different amyloid fibres. whether the pale and irregular thioflavin t staining of some intraneuronal neurofilament inclusions and the negative thioflavin t staining in other inclusions reflect different fibrillation states and/or affinities of the aggregated protein to the dye is currently unclear. in summary, we report on a patient who, in addition to cjd, presented an additional and likely independent neuropathological alteration. the strict limitation of the large neurofilamentous inclusions to the locus coeruleus and their immunohistochemical profile do not match to any specific neurodegenerative condition but may be associated with subtle and slowly progressing neurological symptoms such as rbd or reduced cognitive flexibility. declarations conflicts of interest statement the authors declare that they have no conflicts of interest directly related to the topic of this article. funding statement the authors declare that they have not received any specific funding for this study. the austrian reference center for human prion disease (örpe) is supported by the austrian ministry of health (federal ministry republic of austria for social affairs, health, care and consumer protection). author contributions ok and sw performed the autopsy; fl, ok, sw and eg conducted the neuropathological studies; sk and cs collected the clinical information, mri and csf data; fl and eg drafted the manuscript. all authors reviewed the manuscript. acknowledgements we would like to thank the laboratory technicians at the division of neuropathology and neurochemistry, medical university vienna for their assistance. we deeply thank patients and their families. references 1. hainfellner, j. a. et al. coexistence of alzheimer-type neuropathology in creutzfeldt-jakob disease. acta neuropathol 96, 116–122 (1998). doi: https://doi.org/10.1007/s004010050870 2. tousseyn, t. a. et al. coexistence of alzheimer-type neuropathology in creutzfeldt-jakob disease. alzheimer’s & dementia 4, t432–t433 (2008). doi: https://doi.org/10.1016/j.jalz.2008.05.1285 3. grau-rivera, o. et al. clinicopathological correlations and concomitant pathologies in rapidly progressive dementia: a brain bank series. neurodegenerative diseases 15, 350–360 (2015). doi: https://doi.org/10.1159/000439251 4. iida, t. et al. an atypical case of sporadic creutzfeldt-jakob disease with parkinson’s disease. neuropathology 21, 294–297 (2001). doi: https://doi.org/10.1046/j.1440-1789.2001.00407.x 5. haraguchi, t. et al. coexistence of creutzfeldt-jakob disease, lewy body disease, and alzheimer’s disease pathology: an autopsy case showing typical clinical features of creutzfeldt-jakob disease. neuropathology 29, 454–459 (2009). doi: https://doi.org/10.1111/j.1440-1789.2008.00964.x 6. fernández-vega, i. et al. coexistence of mixed phenotype creutzfeldt-jakob disease, lewy body disease and argyrophilic grain disease plus histological features of possible alzheimer’s disease: a multi-protein disorder in an autopsy case. neuropathology 35, 56–63 (2015). doi: https://doi.org/10.1111/neup.12150 7. rossi, m. et al. the characterization of ad/part co-pathology in cjd suggests independent pathogenic mechanisms and no cross-seeding between misfolded aβ and prion proteins. acta neuropathol commun 7, 53 (2019). doi: https://doi.org/10.1186/s40478-019-0706-6 8. kovacs, g. g. et al. non-alzheimer neurodegenerative pathologies and their combinations are more frequent than commonly believed in the elderly brain: a community-based autopsy series. acta neuropathol 126, 365–384 (2013). doi: https://doi.org/10.1007/s00401-013-1157-y 9. diagnostic criteria | creutzfeldt-jakob disease, classic (cjd) | prion disease | cdc. 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dauvilliers, y. et al. rem sleep behaviour disorder. nat rev dis primers 4, 19 (2018). doi: https://doi.org/10.1038/s41572-018-0016-5 16. boeve, b. f. et al. insights into rem sleep behavior disorder pathophysiology in brainstem-predominant lewy body disease. sleep medicine 8, 60–64 (2007). doi: https://doi.org/10.1016/j.sleep.2006.08.017 17. arnulf, i. rem sleep behavior disorder: motor manifestations and pathophysiology. movement disorders 27, 677–689 (2012). doi: https://doi.org/10.1002/mds.24957 18. iranzo, a. et al. neurodegenerative disorder risk in idiopathic rem sleep behavior disorder: study in 174 patients. plos one 9, e89741 (2014). doi: https://doi.org/10.1371/journal.pone.0089741 19. vazey, e. m. & aston-jones, g. the emerging role of norepinephrine in cognitive dysfunctions of parkinson’s disease. front behav neurosci 6, 48 (2012). doi: https://doi.org/10.3389/fnbeh.2012.00048 20. mirabile, i. et al. identification of clinical target areas in the brainstem of prion-infected mice. neuropathol. appl. neurobiol. 41, 613–630 (2015). doi: https://doi.org/10.1111/nan.12189 21. cairns, n. j. et al. patients with a novel neurofilamentopathy: dementia with neurofilament inclusions. neuroscience letters 341, 177–180 (2003). doi: https://doi.org/10.1016/s0304-3940(03)00100-9 22. gelpi, e. et al. phenotypic variability within the inclusion body spectrum of basophilic inclusion body disease and neuronal intermediate filament inclusion disease in frontotemporal lobar degenerations with fus-positive inclusions. j neuropathol exp neurol 71, 795–805 (2012). doi: https://doi.org/10.1097/nen.0b013e318266efb1 copyright: © 2024 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. brainstem inflammation in sudden unexpected death in infancy and childhood (sids/sudc) free neuropathology 5:12 (2024) letter brainstem inflammation in sudden unexpected death in infancy and childhood (sids/sudc) herbert budka1, daniele u. risser2, fabio c. monticelli3 division of neuropathology and neurochemistry (obersteiner institute), department of neurology, medical university of vienna, austria center for forensic medicine, medical university of vienna, austria department of legal medicine and forensic psychiatry, paris lodron university salzburg, austria corresponding author: herbert budka · division of neuropathology and neurochemistry (obersteiner institute) · department of neurology · medical university of vienna · 1140 wien · utendorfgasse 2/7 · austria herbert.budka@meduniwien.ac.at submitted: 2 april 2024 accepted: 29 april 2024 copyedited by: bert m. verheijen published: 3 may 2024 https://doi.org/10.17879/freeneuropathology-2024-5479 keywords: sids, sudc, brain inflammation, brainstem, neuroinflammation, microglia sudden unexpected infant death (suid) is the leading cause of death in the first year of age in developed countries [4] and a mystery in medicine still to be clarified. its major component is sudden infant death syndrome (sids) [4], defined as “sudden death of an infant under one year of age that remains unexplained after a thorough case investigation, including performance of a complete autopsy, examination of the death scene and review of the clinical history” [3]. identification of risk factors in the infant sleep environment resulted in sids being perceived as a sleep accident, with some decrease in prevalence by providing appropriate warnings [4]. however, renewed scientific efforts to identify biologic causes were recently urged [4]. most research suggests heterogeneity in the etiology of sids and its analogue after the first year of life, sudden unexpected death in childhood (sudc). there is evidence that slight infection and an activated immune system are involved, summarized in [9]. most recently a “multiomics” study renewed interest in brain inflammation being responsible for some sids cases, demonstrating increased neopterin, a biomarker for activated macrophages considered to indicate neuroinflammation, in csf of 6 among 71 sids deaths; one case was studied in more detail, but histology was suboptimal for interpretation [10]. so, what is the pathological substrate in such cases? prompted by that paper [10], one of us (hb) reviewed his personal forensic neuropathological archive for brains with sids or sudc, excluding trauma, epilepsy or pre‑existing pathology. since 2010, when immunocytochemistry for microglial activation markers has been regularly employed in the forensic setting, 6 cases with a clinical diagnosis or suspicion of sids (n = 3, ages 3.5, 5 and 7 months) or sudc (n = 3, ages 15, 35 and 36 months) were revisited. these children were found dead in bed 40 minutes, or 3, 3, 5, 8 hours after last seen alive, respectively; became suddenly blue and had cardiac arrest. general autopsy did not reveal any definite cause of death. neuropathologically, two cases showed multiple microglial nodules in the brainstem, two cases more diffuse and widespread microglial activation of basal brain regions including the brainstem, one a mixture of these, and another none of these. intensity ranged from few small nodules to abundant (fig. 1a), including confluent nodules (fig. 1b), whereas lymphocytic infiltration was absent. we believe that this type of neuropathology perfectly fits as substrate of the reported neopterin increase in csf [10]. it indicates early pathology as to be expected in sudden death, and is observational but plausible as a cause for sudden cardio-respiratory dysfunction due to affection of vital brainstem centers, possibly by local excess of proinflammatory mediators produced by activated microglia, as a neuro‑cytokine connection [9]. figure 1. brain sections of a 35-month-old, found dead 3 hours after last seen alive. a. many labeled microglial nodules (tiny brown/dark patches) in a transverse section of the pons. b. single and larger confluent nodules composed by large-bodied activated microglia in the tegmentum of the medulla oblongata. a and b immunohistochemistry with anti-hla-dr (mhc ii, clone cr3/43 dako, diluted 1:400), performed in a dako autostainer with dako envision kit and diaminobenzidine (dab) as chromogen, with hematoxylin as counterstain. a x 2, b x 200. the selective mode of our case recruitment (see declarations below) precludes any sound conclusion on the frequency of these changes. regardless, the phenomenon does occur, is frequent in our series, and substantiates the heterogeneity of sids/sudc. however, neuroinflammation is not specifically listed among the - mainly unspecific neuropathological abnormalities described in sids [3]. possible reasons for underrating might be that, first, such a finding does no longer formally comply with the definition of sids that requires absence of pathology. second, and in our view more importantly, routine autopsy work-up may not have focused on visualization of activated microglia that requires appropriate markers, since routine stains such as h&e, luxol fast blue or immunohistochemistry for gfap may fail to reveal such pathology. for such visualization, iba1 has been considered as a general marker of microglia by ambrose et al. [1], but their study did not equate hypertrophic iba1+ microglia with activation. moreover, in addition to hla-dr (mhc-ii), cd68 has been used for visualization of activated microglia [1, 10]. however, it is not our experience that cd68 is equally suited as hla‑dr to show early microglial activation; instead, in our hands cd68 is particularly valuable when microglia convert to macrophages, i.e., as a marker of phagocytosis. clearly there is more work to be done in this area. regardless of how valuable the visualization of microglia is, it is necessary to emphasize that their activation just indicates a quick, highly sensitive, but primarily unspecific reaction to pathogenic stimuli, ranging from hypoxia and vascular impairment to trauma, infections and inflammation, neurodegeneration and even tumors. in a detailed study of infants, microglial heterogeneity in regional distribution and activation across the brain was described as characteristic, including a series of sids/sudc cases with immunohistochemistry for iba1, cd68 and hla-dr; however, the brainstem had the “smallest” activation, and hla-dr was most infrequently observed across brain regions; in the context of sids, the dentate gyrus was found as region of interest for microglial activation [1], rather than the brainstem. in earlier publications, microglia in infant brains including sids and sudc have been studied in various regions by various methods with variable results (supplementary table 1 in [1]). the nucleus tractus solitarii in sids was found to harbor a significant increase of reactive astrocytes, a significantly higher percentage of necrotic cells, and an increase of microglial cells [2]. in contrast to these previous publications, our small series demonstrated multiple classical microglial nodules and/or more diffuse microglial activation, with focus on the brainstem but not other brain regions. in absence of other pathogenic clues, we believe that such neuropathology suggests an early inflammatory process that has not yet progressed to elicit lymphocytic infiltration and astrogliosis. it would be well in accordance with a viral infection as early brainstem encephalitis, although the virus type is not evident. indeed, 4 of our 6 cases were reported to have had a mild cold, what has been considered as a risk factor for sids; a range of viruses has been reported in sids [9]. in our case illustrated in fig. 1, a tracheal swab was positive for parainfluenza 2 virus, and human parechovirus 3 was found by others in one case [10]. previously, meningeal inflammation in sids/suds was described as lymphocytic infiltration, interpreted as unspecific [5], and lymphocytic pleocytosis in postmortem csf [7]. massive pathology of brain edema and lymphocytic meningitis was present in 2 sudc cases positive for influenza a or adenovirus [6]. sars-cov-2 infection in infants (although possibly coincidental) may present as suid with subtle and unspecific neuropathology, and one case was described to have prominent groups of microglia and mononuclear cells in the brainstem [11]. in any case, a thorough investigation of sids/sudc should include a complete brain autopsy with immunocytochemistry for activated microglia. research for additional signs of neuroinflammation and deep sequencing for pathogen discovery as recommended [8] will hopefully improve our understanding of the nature of these tragedies. declarations competing interests hb is a member of the editorial board of free neuropathology. all three authors are providing (neuro)pathological examinations in forensic cases as sworn‑in experts, ordered by district attorneys or courts in austria. brains are examined on suggestion by departments of forensic medicine performing complete legal autopsies. hb has no influence on the decision to submit such brains or not, and there are other neuropathological experts available as well. financial disclosure our expert forensic assessments are paid for by the contracting authority, according to legally fixed rates that are divided between the examiner and the university. we have no other financial disclosure to make. no funds, grants, or other support was received. ethics approval this is a retrospective analysis of anonymized patient data obtained in routine care. the paris lodron university of salzburg research ethics committee raised no objections to the publication of the results. acknowledgements we are grateful to dr. sigrid klotz and prof. romana höftberger for help with photomicrographs, to mrs. carmen haider and susanne schmid for archival help, and to the staff of the histology lab for excellent technical work-up, all at the division of neuropathology and neurochemistry (obersteiner institute), department of neurology, medical university of vienna, austria. references 1. ambrose n, rodriguez m, waters ka, machaalani r (2020) microglia in the human infant brain and factors that affect expression. brain behav immun – health 7: 100117. 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non-functioning pituitary neuroendocrine tumours kristin astrid øystese1, nicoleta cristina olarescu1,2, cecilia lindskog3, fabjola xheka4,5, jon berg-johnsen2,6, jens petter berg2,7, jens bollerslev1,2, olivera casar-borota4,8 department of endocrinology, morbid obesity and preventive medicine, oslo university hospital, oslo, norway institute of clinical medicine, faculty of medicine, university of oslo, oslo, norway cancer precision medicine, department of immunology, genetics and pathology, uppsala university, uppsala, sweden department of clinical pathology, uppsala university hospital, uppsala, sweden department of clinical genetics, karolinska university hospital, solna, stockholm, sweden department of neurosurgery, oslo university hospital, oslo, norway department of medical biochemistry, oslo university hospital, oslo, norway department of immunology, genetics and pathology, uppsala university, uppsala, sweden corresponding author: kristin astrid øystese · oslo university hospital · section for specialised endocrinology · postboks 4950 nydalen · 0424 oslo · norway k.a.b.oystese@ous-research.no additional resources and electronic supplementary material: supplementary material submitted: 29 february 2024 accepted: 06 may 2024 copyedited by: georg haase published: 06 june 2024 https://doi.org/10.17879/freeneuropathology-2024-5396 keywords: pituitary neoplasm, non-functioning pituitary adenoma, neoplastic stem cells, stem cells abstract background: cells with stem cell features have been described in pituitary neuroendocrine tumours (pitnets). transcription factors sox2 and sox9 are stem cell-associated markers while the pituitary progenitor marker prop1 is involved in anterior pituitary development. we characterised the presence of these markers known to be present in the human pituitary in non-functioning (nf) pitnets. methods: we investigated the pituitary transcription factors sox2, sox9 and prop1 by immunohistochemistry (ihc) (n = 125) and rt-qpcr (n = 78) in a retrospective cohort of clinically nf-pitnets. the markers were scored based on the percentage of immunolabeled cells. ihc staining scores were compared to reintervention rates for the whole cohort, and to expression of fsh, lh or er in gonadotroph nf-pitnets. results: most tumours showed no or few cells positive for sox2, sox9 and prop1. more patients with sox2-negative tumours went through reintervention (40 % vs 19 %, p = 0.03). sox2, sox9 and prop1 staining correlated positively to each other (sox2 and sox9 rs = 0.666, sox2 and prop1 rs = 0.704, sox9 and prop1 rs = 0.570, and p < 0.001 for all). in gonadotroph nf-pitnets, staining for sox2 and prop1 was positively associated to fshβ staining (p < 0.001 for both). staining for sox2, sox9 and prop1 was positively associated with gene expression of estrogen receptor 1 (esr1) (p < 0.001, p = 0.004 and p < 0.001) and ihc staining for erα (p = 0.001, p = 0.03 and p = 0.05, respectively). conclusion: sox2, sox9 and prop1 were present at low levels in nf-pitnets. absence of sox2 staining was associated with a higher reintervention rate. the stem cell markers correlated positively with markers of gonadotroph differentiation in gonadotroph nf-pitnets. sox2 and sox9 were frequently coexpressed and showed positivity in intratumoural cells with epithelial features, however without coexpression of pituitary transcription factors. introduction the pituitary gland is the main regulator of the endocrine systems, and has a complex development and maturation during life. the pituitary orchestrates growth, metabolism and reproduction in humans, and adapts to major physiological life changes such as puberty, pregnancy and menopause. the different cell lineages of the anterior pituitary lobe are derived from rathke's pouch during embryogenesis [1]. rathke's pouch contains stem cells and progenitor cells expressing transcription factors, e.g. sry-box transcription factor 2 (sox2), sry-box transcription factor 9 (sox9) and prophet of pit1 (prop1) [2]. single cell sequencing shows that sox2, sox9 and prop1 are expressed in stem cell populations in the human fetal pituitary [3]. adult stem cells have been identified in several tissues including the pituitary, and have the capability of self-renewal, and the ability to differentiate in specific cell types that are typically restricted to the tissue of origin [3, 4]. the pituitary adaptation to different life events might be facilitated by the presence of stem cells [5]. prop1 is a transcription factor found in the early development of the pituitary gland and is known to be expressed in pituitary stem cells [3]. prop1 is necessary for the development of the pit1 and sf1 lineages. moreover, prop1 has a role in the migration of developing pituitary cells from rathke's pouch in early pituitary development [6–8]. for simplicity, prop1 will be addressed as a stem cell-associated marker throughout the manuscript together with sox2 and sox9. pituitary neuroendocrine tumours (pitnets) are common intracranial neoplasms of epithelial origin. they are classified immunohistochemically based on the staining for the different adenohypophysial hormones fsh, lh, acth, gh, prolactin and tsh and the pituitary transcription factors sf1, tpit and pit1 [9]. further, the tumours are clinically characterised by their functionality. approximately half of the pitnets are clinically non-functioning, lacking clinical features and symptoms related to hormone overproduction [10, 11]. the majority of non-functioning pitnets (nf-pitnets) are gonadotroph (sf1) in origin, followed by corticotroph (tpit) and rarely by pit1 cell lineage tumours [12, 13]. the nf-pitnets are usually detected due to compression symptoms from the surrounding structures, mainly yielding symptoms from the visual apparatus or from hypopituitarism. however, an increasing number of tumours are detected incidentally by imaging procedures taken for unrelated reasons [14]. to date there is no medical treatment available for the nf-pitnets. tumour-directed treatment involves pituitary surgery. a substantial proportion of patients presents with postoperative tumour remnants that may grow, and there are no reliable markers differentiating the tumours needing adjuvant or repeated intervention. the tumourigenesis of pitnets is poorly understood. most tumours are sporadic, and only a small proportion is associated with gene mutations e.g. in aip or men [15]. in a rat experimental pituitary tumour model, the level of sox9 was decreased at the initial stage of tumourigenesis; however both sox2 and sox9 expression levels were similar to those observed in normal pituitary gland at a more advanced stage of tumourigenesis [16]. cells with stem cell-like features have been found in histological specimens from human adenohypophysial tumours [17] and single cell mrna-sequencing has shown clustering of pituitary stem cell markers in both normal and tumourous pituitary stem cells [18]. sox2 has been found in approximately 20 % of human nf-pitnets [19]. furthermore, nuclear coexpression of prop1 and sox2 was found in tumour stem cells isolated from nf-pitnets [20]. it has been hypothesised that pituitary stem cells play a role in the development of pitnets by paracrine effects, while tumour models have shown that the stem cells themselves do not give rise to tumour cells [21, 22]. yet there is still a substantial knowledge gap concerning the role of tumour stem cells at different stages of tumourigenesis. in this study, we characterised the presence of stem cell-associated transcription factors known to be present in the adult human pituitary in nf-pitnets and correlated them to clinical and molecular markers. methods patients the study retrospectively included patients operated for clinical nf-pitnet between 1998 and 2009, where tumour tissue from primary pituitary surgery was available for immunohistochemical (ihc) subclassification (n = 158). all tumours were clinically classified as non-functioning at the time of surgery. seventeen patients lacked tumour tissue available for ihc characterisation or frozen tumour tissue available for rt-qpcr (9 sf1, 4 null-cell, 3 tpit and 1 pit1 tumour), leaving 141 patients (129 tumour samples available for ihc analyses and 81 for qrt-pcr) for analysis of the stem cell-associated markers. additionally four null-cell nf-pitnets (not staining for anterior pituitary hormones or transcription factors) were excluded from the analyses due to lack of consistent ihc characterisation. all patients were operated at the same tertiary referral centre. data from magnetic resonance imaging (mri) were available for 60 patients (46 with gonadotroph, 9 with corticotroph and 5 with pit1 tumours). tumour volume was measured by the cavalieri method, as previously described [23], and invasion was defined by the knosp criteria. a tumour with a knosp grade ≥ 3 on either side was defined as an invasive tumour [24, 25]. repeated surgery or radiation therapy less than 12 months after primary surgery was defined as adjuvant treatment to the primary surgery. hence, reintervention was defined as surgery or radiation therapy more than 12 months after primary surgery. informed consent was obtained from all living patients. the regional ethics committee (rek 2014/635) and the hospital authority approved the study. histopathology and immunohistochemistry the original hematoxylin and eosin stained sections from all tumours were reviewed by a pathologist (ocb) to confirm the presence of representative tumour tissue. tissue microarrays (tmas) were constructed containing two replicate 1 mm cores from formalin-fixed paraffin-embedded (ffpe) tissue samples of representative areas [26]. tma blocks were sectioned, mounted on adhesive slides and dried at room temperature overnight, followed by baking at 50 °c for 12–24 h. the sections were deparaffinised in xylene, hydrated in graded alcohols and blocked for endogenous peroxidase in 0.3 % hydrogen peroxide diluted in 95 % ethanol. for antigen retrieval, slides were boiled for 4 min at 125 °c in citrate buffer ph6 (dako target retrieval solution (agilent technologies, santa clara, ca) using a decloaking chamber (biocare medical, walnut creek, ca). automated immunohistochemistry for sox2, sox9 and prop1 was performed essentially as previously described [27, 28] using an autostainer 480 instrument (thermo fischer scientific, waltham, ma). primary antibodies against sox2 (371r-15, cell marque, rocklin, ca), sox9 (amab90795, atlas antibodies, bromma, sweden) and prop1 (hpa049839, atlas antibodies) were diluted 1 : 50, 1 : 150 or 1 : 1500, respectively, in ultraab diluent (thermo fisher scientific) followed by incubation for 30 min at room temperature. the slides were further incubated with the secondary reagent anti-rabbit/mouse horseradish peroxidase-conjugated ultravision (thermo fischer scientific) for 30 min at room temperature, and developed for 10 min using diaminobenzidine (dab) quanto (thermo fisher scientific) as chromogen. all incubations were followed by rinse in wash buffer (thermo fisher scientific) twice for 5 min. slides were counterstained in mayer's hematoxylin (histolab, gothenburg, sweden) and coverslipped using pertex (histolab) as mounting medium. the slides stained for sox2, sox9 and prop1 were scanned in hamamatsu nanozoomer s60 at 40 x magnification. the microphotographs illustrating the immunohistochemical results were taken from the scanned images. multiplex immunofluorescence (mif) slides were baked overnight at 50 °c, deparaffinised in xylene and rehydrated in graded alcohols (99.9 %, 95 %, and 80 %) down to deionised water. endogenous peroxidase was blocked using 0.3 % hydrogen peroxide in 95 % alcohol, and heat-induced epitope retrieval (hier) was performed in a decloaking chamber (biocare medical) at 125 °c for 4 min while the slides were immersed in 1x target retrieval solution ph 6.0 (agilent technologies inc., santa clara, ca, usa). slides were then cooled to approximately 90 °c before rinsing with deionised water. after hier the slides were treated with a led-light bleaching process immersed in a bleaching solution consisting of 0.2m glycine, 1.5 % hydrogen peroxide, and 1x tbs+tween (ta-999-tt, thermo fisher scientific) for 1 h in rt. slides were incubated using a multiplex panel of six antibodies in a 6-cycle antibody staining process with intermediary elution steps after each cycle. antibody stripping/elution was performed by boiling the slides in a decloaking chamber (biocare medical) at 90 °c for 20 min and slides immersed in 1x target retrieval solution, ph 6.0 (agilent technologies). full staining cycle information (panel antibody markers, dilutions, reagents, incubation times, opal fluorophores) are available in supplementary table 1. one cycle of staining included blocking, primary antibody incubation, anti-rabbit igg (h+l) with horseradish peroxidase (hrp) polymer, and an opal fluorophore (akoya biosciences, marlborough, ma, usa). all cycles were performed at room temperature (rt) using the austostainer 480s (thermo fischer scientific) and hrp-kit from epredia (epredia ultravision lp hrp-kit, breda, netherlands). after the last cycle, slides were incubated with the opal 780 fluorophore-conjugated anti-dig antibody and 4',6-diamidino-2-phenylindole (dapi) (invitrogen™, d1306, thermo fisher scientific). slides were then mounted using prolong™ glass antifade mounting media and left overnight at rt after which they were digitalised at 40 x magnification using phenoimager (akoya biosciences). spectral unmixing and export of images were performed using the built-in spectral library of the inform software (akoya biosciences). multiplex ihc was performed on one tma including cores from 50 nf-pitnets of different types. classification of immunohistochemically stained samples the immunohistochemical classification was based on the expression of the anterior pituitary lobe hormones fshβ, lhβ, acth, gh, prl, tsh and alpha subunit and the transcription factors sf1, tpit and pit1 as previously described [13, 29]. the tumours were classified into three groups based on their cell line of origin: gonadotroph nf-pitnets (sf1), corticotroph nf-pitnets (tpit) and pit1 nf-pitnets. in the pit1 group, 4 tumours stained for prl, 1 for prl and gh, 2 stained for alpha subunit alone, and 1 tumour was plurihormonal expressing tsh in a proportion of cells and fshβ and/or lhβ as well as alpha subunit in scattered cells. the staining for fshβ and lhβ was graded from 0–4 based on the percentage of positive cells (0 = no positive cells, 1 = 0–10 % positive cells, 2 = 10–50 % positive cells, 3 = 50–80 % positive cells and 4 = > 80 % positive cells). the staining for estrogen receptor α (erα) was based on the immunoreactive score (irs), being the product of the percentage of positive staining cells (0 = 0 %; 1 = 1–10 %; 2 = 10–50 %; 3 = 50–80 %; and 4 = > 80 %) and the predominant staining intensity (0: no staining; 1: weak staining; 2: moderate staining; 3: strong staining). the stem cell-associated markers sox2, sox9 and prop1 were scored based on the proportion of positively stained cells (staining score 0 = no positive cells, 1 = staining in scattered cells, 2 = staining in 1–5 % of cells, 3 = staining in 5–10 % of cells, 4 = staining in 10–30 % of cells, 5 = staining in 30–50 % of cells and 6 = staining in > 50 % of cells). all positive cells showed a distinct nuclear immunolabeling. rt-qpcr reverse transcription (rt) was performed using a high-capacity cdna reverse transcription kit (applied biosystems, foster city, ca, usa) by a labnet multigene gradient thermal cycler (labnet international inc., edison, nj, usa) according to the manufacturer's protocol. after the reaction, the cdna was diluted to a ratio of 1 : 10. quantitative real time polymerase chain reaction (rt-qpcr) was performed in an abi 7900 apparatus (applied biosystems, foster city, ca, usa) using power sybr green master mix (applied biosystems, foster city, ca, usa). samples were dispensed in the corresponding wells by an automated pipetting system (epmotion® 5070 cb, hamburg, germany). rt-qpcr was performed using a previously described protocol [30]. the primer sequences are available upon request. all rt-qpcr experiments were in accordance with the minimum information for publication of quantitative real-time pcr experiments (miqe) guidelines [31]. gapdh or geometric mean of gapdh and alas were used as housekeeping genes [30, 32]. frozen tumour tissue was available for rt-qpcr analyses of sox2, sox9 and prop1 in 66, 69 and 70 gonadotroph, three, three and four tpit and three, three and four pit1 samples, respectively. statistics chi square and fisher's exact tests were used for group comparison of nominal data. mannwhitney u and kruskall wallis tests were used to compare continuous data between different groups. spearman's correlation coefficient (rs) were analysed for correlation analyses. cox regression and kaplan-meier analyses were used to analyse the association between reintervention and presence of sox2. the cox regression analysis was performed only for the gonadotroph tumours to prevent bias from the other groups with a small number of tumours. group differences were considered statistically significant at the 5 % significance level. all statistical tests were two-sided. statistical analyses were performed using spss version 28, and figures 4 and 6 were made using graphpad prism 9.3.1. results clinical data and stem cell-associated markers tumour tissues for rt-qpcr and/or for ihc from 137 patients with nf-pitnets (table 1) were eligible for analysis. six patients (5 sf1 and 1 tpit) had less than 12 months follow-up and were excluded from reintervention analysis. six patients went through radiation therapy less than 12 months after primary surgery (4 sf1 and 2 tpit); one of these went through additional reintervention. sf1 tpit pit1 p-value total (n = 137) 112 16 9   available ihc 101 16 8   female/male 39/73 7/9 6/3   age mean (sd) 60 (51 - 72) 57 (52 - 71) 38 (26 - 58)* 0.01 follow-up (months) 128 (99 - 160) 110 (95 - 171) 125 (117 - 168) 0.45 tumour volume (mm3) 6480 (4055 - 10795) 6351 (2104 - 17697) 2670 (1962 - 4950)* 0.05 table 1: clinical data of patients *there was a significant difference in age between the pit1 and tpit group and between the pit1 and sf1 group (p = 0.03 and p = 0.002, respectively), and also in tumour volume between the sf1 and pit1 group (p = 0.007). however, there were only nine patients in the pit1 group, five of them having available preoperative mri. the patients in the pit1 group were younger than the patients in the sf1 and tpit groups. the tumours in the pit1 group were smaller than in the sf1 group; however preoperative mri was only available for five of the pit1 tumours. the subgroups were otherwise equal concerning age, gender and follow-up (table 1). stem cell associated-markers are expressed at a low level in nf-pitnets the majority of nf-pitnets were negative for sox2, sox9 and prop1 (staining score 0) or positive only in scattered cells (staining score 1) (fig 1). the staining score was concordant between replicate tma punches from the same tumour. no tumours presented staining in more than 50 % of tumour cells (staining score 6). we found a moderate to strong correlation between the relative mrna levels for sox2, sox9 and prop1 and the ihc staining for the corresponding proteins (table 2). due to the large number of tumours negative (staining score 0) for the three markers, we also analysed the correlations when excluding these tumours. the correlations between mrna and ihc levels remained significant for sox2 and sox9 despite a lower number of tumours (sox2 n = 18, rs = 0.51, p = 0.03; sox9 n = 51, rs = 0.58, p < 0.001). there was a borderline significance for prop1, though the correlation coefficient remained at approximately the same level (prop1 n = 15, rs = 0.51, p = 0.054). sox2, sox9 and prop1 showed a moderate to strong correlation between themselves, both at the gene and the protein expression levels (table 2). figure 1: two gonadotroph tumours (upper and middle row) demonstrating immunolabeling for sox2, sox9 and prop1 with score 4 (a, b), score 5 (c) and score 2 (d-f). a silent corticotroph tumour (bottom row) demonstrating immunolabeling for sox2, sox9 and prop1 with score 1 (g), score 5 (h) and score 1 (i). prop1-positive cells in i are pointed with arrows to distinguish them from erythrocytes that show unspecific staining by prop1 ihc analysis.   ihc sox9 ihc prop1 δct sox2 δct sox9 δct prop1 ihc sox2 rs p-value n 0.67 < 0.001 125 0.704 < 0.001 125 0.500 < 0.001 66 0.548 < 0.001 69 0.478 < 0.001 70 ihc sox9 rs p-value n   0.573 < 0.001 125 0.309 < 0.011 66 0.605 < 0.001 69 0.336 < 0.004 70 ihc prop1 rs p-value n   0.511 < 0.001 66 0.515 < 0.001 69 0.405 < 0.001 70 δct sox2 rs p-value n   0.680 < 0.001 69 0.800 < 0.001 67 δct sox9 rs p-value n   0.709 < 0.001 70 table 2: the spearman's correlation coefficient rs between the immunohistochemical (ihc) staining score and the relative gene expression (δct) of sox2, sox9 and prop1 is shown in bold characters. the correlation between the different markers sox2, sox9 and prop1 was moderate to strong both for ihc and gene expression. the stem cell-associated markers presented a distinct nuclear immunolabeling in positive cells. some of these cells had a morphology similar to the tumour cells, whereas some positive cells seemed to be endothelial cells by location and morphology. occasionally, positive cells aggregated in small groups surrounding luminal spaces. if identified within the tumour, epithelial cystic remnants demonstrated strong expression of sox2 and sox9 (fig. 2). all immunolabeled cells except the epithelial cells in the cystic remnants were taken into analysis. figure 2: immunolabeling of stem cell-associated markers in cells with a location and morphology corresponding to endothelial cells (a, sox9, x 200) and in cells aggregating in small groups (niches, arrows) around luminal structures (b, sox9, x 400). epithelial cells in the remnants of rathke's pouch within a gonadotroph tumour (c, he) demonstrating immunolabeling for sox2 (d) and sox9 (e), but not prop1 (f), (x 200). multiplex ihc was performed on a subset of the tumours and demonstrated frequent coexpression of sox2 and sox9. moreover, stem cell-associated markers were usually coexpressed with the epithelial marker. however, pituitary transcription factors, usually positive in all or the vast majority of the tumour cells, were not expressed in cells positive for stem cell-associated markers (fig. 3). figure 3: multiplex ihc on a section from a gonadotroph tumour. sf1 (in yellow) and cytokeratin (in green) are illustrated on all three microphotographs. coexpression of sox9 (in pink) and cytokeratin is shown in a; and coexpression of sox2 (in red) and cytokeratin is shown in b. in c (no filter for sox2 and sox9), a blue colour indicates nuclear staining in the cells labelled for sox2 and sox9 in a and b; however, no sf1 staining is seen in the nuclei of those cells. stem cell-associated markers between different subgroups of nf-pitnets the distribution of sox2, sox9 or prop1 staining in the different subgroups of nf-pitnets is presented in fig. 4. there was no difference in sox2, sox9 or prop1 staining between the gonadotroph (fig. 4a) and the corticotroph (fig. 4b) subgroups of nf-pitnets (p = 0.91, p = 0.37 and p = 0.74, respectively). none of the tumours in the pit1 group (fig. 4c) (n = 8) stained for sox2 or prop1, while four out of eight tumours stained for sox9, without statistical difference in comparison with the other two groups. figure 4: distribution of stem cell-associated markers in different nf-pitnets. most tumours presented staining scores of 0 or 1 for sox2 (n = 91 [64.5 %] and n = 15 [10.5 %] respectively), sox9 (n = 41 [29 %] and n = 51 [36 %] respectively) and for prop1 (n = 106 [75 %] and n = 4 [3 %] respectively). proportion of positively stained cells in subgroups of nf-pitnets. a total of 125 tumours were investigated by immunohistochemistry: 101 gonadotroph (sf1), 16 corticotroph (tpit) and 8 from the pit1 lineage. we did not find a significant difference in staining between the groups. stem cell-associated markers and reintervention the tumours with no staining for sox2 presented a higher rate of reintervention than the tumours with a sox2 staining score ≥ 1 (n = 34 [41 %] and n = 7 [19 %], respectively) for the whole cohort (p = 0.02), and for the gonadotroph cohort separately (n = 27 [42 %] and n = 6 [19 %], respectively, p = 0.03). there was no association between the gene expression of sox2 and reintervention (p = 0.69). in the gonadotroph tumours, survival analyses showed that younger age (exp(b) 0.95 [ci 0.92–0.98]) and absence of sox2 staining (exp(b) 0.35  [ci 0.14–0.89]), but not gender (exp(b) 1.2 [ci 0.61-2.71]) were associated with a higher risk of reintervention (fig. 5). we did not find an association between sox9 or prop1 and reintervention. figure 5: kaplan-meier curve showing the difference in reintervention rate between patients with gonadotroph tumours without sox2 staining and tumours with sox2 staining (p = 0.03). the number at risk at 25, 50, 100 and 150 months were 52, 40, 22 and eight, respectively, for the tumours not stained for sox2, and 26, 24, 16 and three, respectively, for the tumours with sox2 staining. stem cell-associated markers and regulators of the gonadotroph axis for the gonadotroph nf-pitnets, we found no difference in gender, age, tumour volume or invasiveness between tumours stained or not for the stem cell-associated markers sox2, sox9 or prop1 (suppl. table 2). the staining for sox2, sox9 and prop1 was associated with higher fshβ but not lhβ gene expression and staining (suppl. table 2). sox2-positive tumours showed higher levels of fshβ and estrogen receptor α (erα) staining as well as higher estrogen receptor 1 (esr1) and gnrhr gene expression (fig 6. a, c, d and suppl. table 2), whereas no difference was observed for lhβ (fig 6 b). similar patterns were observed for sox9and prop1-positive tumours. no correlation was found between sox2, sox9, prop1 and sf1 gene expression (suppl. table 2). figure 6: expression of sox2 and regulators of the gonadotroph axis. the presence of sox2 was positively associated with higher levels of fshβ, erα and gene expression of gnrhr in the gonadotroph subgroup of nf-pitnets (n = 101). sox2 staining is classified as positive (staining score ≥ 1) or negative (staining score 0). horizontal bars indicate median and interquartile values. statistical comparisons are calculated by mann-whitney u test for all associations. discussion the stem cell-associated markers sox2, sox9 and prop1 were distinctly present, usually in a small proportion of cells, only in a subset of nf-pitnets. the ihc staining and the gene expression levels correlated well for the same marker and between different markers. we did not find any association between the presence of the investigated markers, age at surgery, tumour volume or invasiveness. absent sox2 staining was associated with a higher rate of reintervention in the whole cohort of tumours and in the gonadotroph subgroup of tumours. the presence of the selected markers was positively associated with some of the regulators of the gonadotroph axis such as fshβ, gnrhr and erα in gonadotroph nf-pitnets. in accordance with previous experimental studies [33–35], we demonstrate that cells with stem cell features are present at a low number in a subset of pitnets. they tend to occur in small niches and have heterogeneous morphology corresponding to tumour cells and occasionally to endothelial cells. in the murine pituitary, sox2-positive cells are present in a small proportion of cells (3–5 %) and usually scattered throughout the gland, and more concentrated lining the pituitary cleft [36]. a previous ihc study found sox2 positivity in 20 % of human nf-pitnets, which is not far from our results [19]. interestingly, by using multiplex ihc, we demonstrate expression of the stem cell-associated markers sox2 and sox9 in intratumoural cells with epithelial features. however, in the cells positive for these markers, there was no immunolabeling for pituitary-specific transcription factors. it is not clear whether the cells coexpressing stem cell and epithelial markers may correspond to the previously described “follicular cells” of gonadotroph tumours, though these cells were found to express gonadotroph transcription factors in the study by delfin et al [37]. expression of stem cell-associated markers in a population of endothelial cells is in line with previous research, and may support speculation that transdifferentiation of stem cells into endothelial cells may contribute to tumour neovascularisation [33]. moreover, sox2 and sox9 were strongly expressed in the epithelial cells of the rathke's pouch remnants embedded in the tumour tissue, in accordance with previous studies demonstrating expression of stem cell markers in rathke's pouch epithelium [18, 36] as well as in craniopharyngiomas originating from the epithelial remnants of rathke's pouch [38]. the tumours lacking sox2 staining presented a higher rate of reintervention in our cohort, contradictory to what we assumed initially. surprisingly, zhang et al found that the well differentiated sf1-tumours showed a higher rate of recurrence than the poorly differentiated tumours [18]. we have also previously presented data on markers associated with tumours being more differentiated and having a higher rate of reintervention in nf-pitnets [30, 39]. the association between recurrence and differentiation in the sf1-tumours deviated from pituitary tumours of other cell lineages in the study from zhang et al, corresponding to previous investigations in functioning pituitary tumours [40, 41]. why the sf1-tumours deviate from the remaining pituitary tumours in this matter has not been explored. we found a moderate to strong correlation between the three markers sox2, sox9 and prop1. previous studies have shown that pituitary stem cells express both sox2 and sox9 [42], and cells with stem cell properties lining the borders of the marginal zone are also positive for prop1 [1]. in our study, the presence of the stem cell-associated markers was consistently associated with markers of the gonadotroph cell phenotype. as previously described, stem cells lose sox2 expression and generate committed progenitor cell lineages during embryonic development [43]. recently it was hypothesised that sox2-positive cells may give rise to a well organised structural and functional network that coordinates the responses to challenges, similar to the hormone secreting cells [5, 44]. our data suggest that the sox2-positive cell network is closely related to the gonadotroph cell network, or that both cell types respond to similar factors (e.g. nutrients, blood supply), and are located in similar areas in the parenchyma of the pituitary tumours. whether the cells with stem cell features have a role in the hormonal regulation of the gonadotroph neoplastic cells, or whether the correlation presented here mirrors the normal pituitary phenotype is currently unknown. limitations the study was of retrospective design, and tumour tissue was not available for all patients operated during the given time period, which may imply an inclusion bias. the ihc analyses were based on tmas and not whole section slides. this might underestimate the presence of the markers investigated, knowing that stem cells or cells with stem cell features are few and might reside in niches in the tumour. however, the strong correlation between gene expression data and immunohistochemistry, as well as the high concordance between staining score in replicate tma punches from the same tumour, as described in this study, supports the reliability of data acquired by tma assessment. the expression of the proteins analysed by ihc was quantified based on different scales, i.e. erα by immunoreactive score (irs) and sox2 by the percentage of distribution. this was due to a usually low number of positive cells and almost uniform strong staining, which makes irs score unapplicable. due to the low number of patients with radiological follow-up, we used the need for reintervention as the main end point concerning tumour aggressiveness. this might be influenced by the primary surgical result and by the operability of the patient. tumour volume and invasiveness were not included in the regression analysis for the same reason. these variables would have been of great interest to be included in the analysis if they were available for the whole cohort. we did not succeed in performing ihc with endothelial markers as a part of multiplex ihc. thus, we could not explore coexpression of endothelial and stem cell-associated markers in the endothelial-looking cells positive for stem cell markers. conclusion the stem cell-associated markers sox2, sox9 and prop1 are present at low levels in nf-pitnets. they are correlated with each other, and their expression seems to be associated with the regulation of gonadotropins in the gonadotroph subgroup of tumours. present or increased staining for the markers was not associated with tumour volume or invasiveness, though the absence of sox2 seems to characterise tumours with a higher rate of reintervention. the stem cell-associated markers sox2 and sox9 are frequently coexpressed and are usually positive in the intratumoural cells with epithelial features lacking however immunolabeling for pituitary transcription factors. declarations and statements competing interests olivera casar-borota is an associate editor of free neuropathology. the other authors declare no competing interests. funding olivera casar-borota was supported by the swedish cancer society (grant number 190157), the lions cancerforskningsfond and a grant from the swedish state under the agreement between the swedish government and the county councils (alf-agreement). for the remaining authors, no funding was received to assist with the preparation of this manuscript. ethics approval this study was performed in line with the principles of the declaration of helsinki. approval was granted by the regional ethics committee for the south-east part of norway (rek 2014/635), and by the hospital authorities. consent to participate informed consent was obtained from all living patients. data sharing statement the data underlying this article cannot be shared publicly due to the privacy of individuals that participated in the study. the data will be shared on reasonable request to the corresponding author. author contribution kristin astrid øystese, nicoleta cristina olarescu, jens petter berg, jens bollerslev and olivera casar-borota contributed to the study conception and design. material preparation, data collection and analysis were performed by kristin astrid øystese, nicoleta cristina olarescu, cecilia lindskog, fabjola xheka, jon berg-johnsen and olivera casar-borota. the first draft of the manuscript was written by kristin astrid øystese and all authors commented on previous versions of the manuscript. all authors read and approved the final manuscript. acknowledgment m. sc jonas gustavsson for performing multiplex ihc. references 1. cox b, roose h, vennekens a, vankelecom h. pituitary stem cell regulation: who is pulling the strings? j endocrinol. 2017;234(3):r135-r58. epub 20170614. https://doi.org/10.1530/joe-17-0083. pubmed pmid: 28615294. 2. garcia-lavandeira m, quereda v, flores i, saez c, diaz-rodriguez e, japon ma, et al. a grfa2/prop1/stem (gps) cell niche in the pituitary. plos one. 2009;4(3):e4815. epub 20090313. 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https://doi.org/10.1016/j.tem.2012.02.004. pubmed pmid: 22436593. copyright: © 2024 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. pituitary adenoma classification: tools to improve the current system feel free to add comments by clicking these icons on the sidebar free neuropathology 5:2 (2024) opinion piece pituitary adenoma classification: tools to improve the current system william charles mcdonald allina health laboratories – abbott northwestern hospital, minneapolis, mn 55407, usa corresponding author: william c. mcdonald · hospital pathology associates · 2800 10th avenue south · suite 2200 · minneapolis, mn 55407 · usa william.mcdonald@allina.com additional resources and electronic supplementary material: supplementary material submitted: 12 december 2023 accepted: 05 january 2024 copyedited by: shino magaki published: 10 january 2024 https://doi.org/10.17879/freeneuropathology-2024-5226 keywords: pituitary, classification, machine learning, statistical learning abstract the world health organization classification of pituitary tumors provides a framework for pathologists and researchers to classify pituitary adenomas. from the perspective of a practicing pathologist, this classification can be improved by pooling immunohistochemical data in a more standardized way, and by deliberately distinguishing features that assist in classification from those that do not. this article illustrates one general workflow to examine classification features consisting of immunohistochemical stains for anterior pituitary tumors, in order to promote debate and advance an evidence-based framework for classification. introduction: opportunities for improvement every pathologist spends their career building or adapting tools to better characterize the diseases encountered in their practice. for neoplasms, this often begins by becoming familiar with the microscopic spectrum of the disease, the demographics of commonly affected individuals, and common clinical presentations. consciously or unconsciously, we build a list of features that we find useful in classifying a disease, and then move on to refine the classification or provide additional prognostic or predictive information. to use personal experience alone in the diagnosis of disease is treacherous, since neoplasms overlap in appearance and human judgement is fraught with various cognitive biases (1). generalizations and simple heuristics can be helpful, but data are necessary to test our assumptions and to develop better tools for classification. ideally, the world health organization (who) 5th edition pituitary tumor classification (2) would provide pathologists with a solid evidence-based classification to aid in diagnosis. however, the current system has several shortcomings that hinder its ability to accurately and reproducibly classify pituitary adenomas, including: failure to carefully distinguish between features used for classification and other descriptive variables, some of which may have a role in estimating prognosis or predicting response to therapy. among the first tasks of the pathologist is to decide which ancillary tests to perform for accurate tumor classification. other testing may be employed, but our first goal is typically to classify the disease in question. fuzzy language as to what constitutes a positive test. statements offering clarification as to what constitutes "limited", "variable", or "focal" immunoreactivity are not provided. unproven claims about the distribution of presumably continuous variables, specifically cellular maturity, acidophilia, or cytoplasmic granularity. ask yourself "on what scale do we measure maturity, or acidophilia, or granularity?". these variables are so subjective as to be unhelpful in most circumstances. apart from sparsely granulated somatotroph adenomas—where fibrous bodies have been cataloged and are used as a proxy for granularity (3)—the spectrum of granularity in lactotroph or corticotroph adenomas has not been sufficiently delineated. while granularity might be essentially bimodal in somatotroph adenomas, other adenoma lineages or classes are not necessarily bimodal. literature lacking negative controls. negative control groups permit the calculation of specificity. without adequate negative controls, the resulting lack of context makes claims about ultrastructural findings, for instance, spheridia (4), impossible to validate. multilineage and plurihormonal tumors show fuzzy boundaries between and within tumor lineages, further underscoring the importance of negative controls. to wit, how useful is a marker like alpha subunit if it can appear—or not appear—in any lineage? we cannot currently answer this with any statistical rigor. unsubstantiated generalizations. for instance, for mature plurihormonal pit1 lineage adenoma, a blanket statement that they are "positive for er and gata3" is based on a single reference that includes only a handful of such tumors (5). conversely, we are forced to content ourselves with overly general statements that invite greater precision; for instance, "most" immature pit1 lineage adenomas are said to express alpha subunit, er, and gata3. surely data can be cited that would improve this broad generalization. small numbers. claims made about the role of gata3 immunohistochemistry (ihc) in pit1 lineage tumors are particularly concerning. only a handful of citations with primary gata3 data are included (5-7), with just mete et al. (5) showing gata3 immunoreactivity in 10 of 53 pit1 lineage tumors examined. furthermore, gata3 immunopositivity was defined in that series as greater than or equal to 5% of neoplastic cells, a very permissive cutoff. merely 3 pit1 lineage adenomas showed diffuse immunoreactivity in that series (5). poorly supported ultrastructural arguments. for instance, small mitochondria were encountered in 6 of 15 of the early series of acidophil stem cell adenoma by horvath et al. (8); given the wide spectrum of possible findings in acidophil stem cell adenoma, it is unclear, then, which additional features would prove useful in supporting this particular tumor type. confusion between "plurihormonal" and "multilineage". using the who 5th edition classification, it is currently possible to diagnose a plurihormonal tumor with only a single positive hormone stain. conversely, multiple synchronous tumors within the same lineage are occasionally encountered in clinical practice, but do not appear to fit within the group "multiple synchronous adenomas of distinct lineages". failure to address technical hurdles like marker co-expression, which is especially important in pit1 lineage tumors. is dual or multiplex ihc necessary to establish the diagnosis of mammosomatotroph tumors, for instance? must we use electron microscopy? if we must resort to electron microscopy, what is the sensitivity and specificity of the relevant ultrastructural findings? while who committees do not endorse particular tests, marker co-expression is a fundamental feature of pituitary adenoma classification that needs to be addressed. before i go further, let me address the unnecessarily prominent role in pituitary tumor conversations that nomenclature occupies these days. i agree with ho et al. (9) that a compelling case for "pituitary neuroendocrine tumor" (pitnet) has not been made, despite the prominence of its advocates. as it is still permissible to use the term "pituitary adenoma" according to the 5th edition of the who classification (2), i do so, and beg forgiveness from passionate adherents to the pitnet label. in any event, such academic debates are less substantial than deficits in the who classification of pituitary tumors, and distract from more important concerns. improving the who classification it would be a disservice to register these criticisms without offering some means of improvement. better methods are available, and are already employed, for instance by the architects of dna methylation profiling, which has now spread from its origins in germany to laboratories in the rest of the world, including north america. it’s not an overstatement to say that methylation profiling has radically revised our understanding of central nervous system tumor classification, and is poised to do so for tumors arising in other systems. yet before we abandon our current strategy for pituitary adenoma classification using ihc in favor of methylation profiling, i think that two key lessons can be extracted from the methylation profiling experience in order to improve our ihc-based approach. firstly, any attempt to establish a classification system requires the registration and comparison of large numbers of cases including all relevant classes of tumor. methylation profiling could not have been successful without assembling large numbers of cases. as in methylation profiling efforts, cases need to be assembled into repositories that include all relevant tumor classes; this is the opposite of the current common practice of publishing limited, highly biased series designed to illustrate but not to test a classification or classification variable. secondly, diagnostic features that are useful in classification must be chosen and applied uniformly throughout the registry. indeed, authors of the who pituitary tumor classification seem to sense this, and recommend performing the "complete panel of stains" (presumably, all possible ihc that might relate to pituitary adenoma diagnosis) during the routine evaluation of pituitary adenomas (10). nonetheless, data derived from the "complete panel of stains" are almost never provided in supporting literature, which tends to consist of reviews or biased collections of rare tumor types and upon individual classification variables, without documentation of other tests performed to corroborate the conclusions. claims by who authors pertaining to ihc for alpha subunit and gata3 are especially concerning in their lack of context and/or low numbers of published cases. one possible workflow to address the noted challenges, which incorporates these lessons from methylation profiling, is provided here as an illustration, using data from cases previously published. case selection, recording of pre-operative data, tissue microarray (tma) construction, ihc, and scoring were performed as previously described (11, 12). in addition to previously reported stains, we also performed ihc for gata3 (hg3-31 (sc-268), santa cruz biotech, 1:100 dilution) and ihc for estrogen receptors (alpha) (sp1, thermo scientific, 1:160 dilution). in brief, 157 pituitary adenomas from allina health (n = 136) and the university of pennsylvania (n = 21) were incorporated into tma’s and stained with the indicated ihc. ihc was scored in a blinded fashion using the allred method (13), and adenoma class was assigned based upon median ihc scores, using clinical and serological data to confirm the classification. a simplified managerial class was assigned to facilitate analysis, but this class did not supervise the machine learning process. estimating correlation between classification variables to start our thought experiment, classification variables should be distinguished from other descriptive variables and explicitly stated. this helps to establish limits on the classification problem, and allows more refined discussion of diagnostic algorithms. it is also an essential step in establishing relationships among these variables. in our work, the "complete panel of stains" (that is, the classification variables) consists of ihc for sf1, pit1, tpit, gata3, alpha subunit, luteinizing hormone (lh), follicle-stimulating hormone (fsh), prolactin, thyroid-stimulating hormone (tsh), growth hormone, estrogen receptor, cytokeratin cam5.2, and adrenocorticotropic hormone (acth). when one considers our series of pituitary adenomas (11, 14), correlation among variables is readily shown in a matrix (figure 1). figure 1. correlation matrix comparing immunohistochemical stains, ordered using hierarchical clustering. correlation coefficient is displayed. abbreviations: acth, adrenocorticotropic hormone; asu, alpha subunit; cam 5.2, cytokeratin cam 5.2; er, estrogen receptor; fsh, follicle-stimulating hormone; gh, growth hormone; lh, luteinizing hormone; sf1, steroidogenic factor 1; tpit, t-box transcription factor; tsh, thyroid-stimulating hormone. it's easy to see that the variables that one might use for classifying pituitary adenomas are variably correlated with one another, exactly as one should expect. practical problems arise for the pathologist, however, in considering which variables to use when trying to classify an adenoma. incorporating redundant and correlated classification variables with inferior test performance reduces the usefulness of a classification algorithm and falsely bolsters the confidence of the diagnostician (1). put another way, adding redundant and correlated variables that hope to resolve cases into groups leads to a classification system that produces overfitting and a classification process that generates expensive and distracting false positives and false negatives. this is a statistical truism, but seems to be forgotten by many pathologists. in concrete terms, when the authors of the who endocrine pituitary tumor classification recommend that "the complete panel of stains" be performed on all pituitary adenomas, they promote a classification system that overfits the data and reduces reproducibility while at the same time generating expensive, wasteful, and distracting false positives and false negatives in laboratories worldwide. establishing context: observing multi-variable relationships among classification variables it has never been easier to move beyond simple correlations to show broader, multi-variable relationships using widely available, free statistical software (15, 16). of the 157 pituitary adenomas in our series (11, 14), the relationships between classification variables, demographics, and tumor size can be readily illustrated in a so-called heatmap (figure 2). figure 2. the figure shows a heatmap of median ihc scores (allred scores) from 157 pituitary adenomas with annotation for managerial diagnoses (at top), tumor size, patient sex, and patient age (at bottom). alpha subunit staining and cam5.2 pattern are included in the annotation, but are not used for clustering. columns represent individual cases. rows of the heatmap represent allred scores for the ihc indicated to the right, and range from 0 (no staining) to 8 (strong staining in greater than 2/3 of the cells); gray boxes indicate missing data. abbreviations: acth, adrenocorticotropic hormone; cam 5.2, cytokeratin cam 5.2; er, estrogen receptor; fsh, follicle-stimulating hormone; gh, growth hormone; lh, luteinizing hormone; sf1, steroidogenic factor 1; tpit, t-box transcription factor; tsh, thyroid stimulating hormone. the advantage of such clustered displays is that they illustrate many relationships within the context of other potentially important variables. for instance, the extent to which gata3 immunoreactivity is restricted to one or another group of adenomas in this collection of tumors can be estimated at a glance. figure 2 shows, for example, that gata3 immunoreactivity is observed in a subset of corticotroph adenomas, as observed by ricklefs et al. (7); conversely, we do not find pit1 family members with strong gata3 immunoreactivity in our series, although mete et al. have reported this in limited numbers (5). likewise, a group of sf1-immunoreactive/pit1-immunoreactive tumors, possibly of the sort described by asa et al. (17), is readily found. unlike most of the works cited by the current who classification, however, the immunohistochemical and demographic context of these tumors is apparent. this context is vital to establishing an evidence-based classification as well as for developing sensible diagnostic algorithms in individual laboratories. only when classification variables are tabulated can the difficult work of estimating test characteristics occur. these test characteristics allow us to select a combination of tests that maximize accuracy and minimize wasteful complexity. estimating test performance: sensitivity and specificity using the data we tabulated for the classification variables, we may now estimate test characteristics. generally, we think in terms of sensitivity and specificity. these are surprisingly nuanced ideas, even for those who have worked in pathology for many years. sensitive to what? specific for what? these two questions are often left unstated, leading to much confusion. the answers to these questions determine the parameters of the calculations performed and constrain the uses of any test. the goal of a test can greatly alter its usefulness (that is, its positive predictive value and negative predictive value). to consider the sensitivity of a test, it is equivalent to ask "what is true positive (tp) and how well does the test capture all cases of the disease in question?" for specificity, the equivalent question is "what is true negative (tn) and how well does the test capture all cases that should not be classified as the disease in question?". definitions of what constitutes tp or tn can be made more narrowly (for instance, assuming that sf1 immunoreactivity is only tp when identifying gonadotroph adenomas) or more broadly (for instance, including sf1 immunoreactivity in multilineage pituitary tumors, as well as gonadotrophs). this mental exercise is applied to each classification variable. why go through the trouble of such mental gymnastics for each classification variable? in short, because it permits the estimation and comparison of test characteristics and, importantly, their confidence intervals (ci). figures 3-5 show the results when such a thought experiment is applied to the classification variables considered above. figure 3 might be considered when deciding what stains are the most appropriate for the classification of a gonadotroph adenoma. each stain is considered under narrow (tp = gonadotrophs) or broad (tp = gonadotrophs or plurihormonal tumors) assumptions, with positive immunoreactivity defined as median allred score greater than 4 for all immunostains except lh and fsh, where a lower cutoff of greater than 2 was selected in recognition of the frequently more limited proportion of tumor cells expressing lh or fsh in gonadotroph adenomas. figure 3. test characteristics and 95% ci of immunohistochemical stains pertinent to the sf1 family of tumors using restrictive (narrow) or inclusive (broad) definitions of true positivity. abbreviations: ihc, immunohistochemistry; ppv, positive predictive value; npv, negative predictive value. positive immunoreactivity was defined as a median allred score greater than 4 for all markers, except lh and fsh, where a median allred score greater than 2 was interpreted as positive. in our hands, sf1 ihc enjoys better sensitivity than other markers, while lh and fsh ihc have very high specificity, but low sensitivity, even when criteria for immunopositivity are relaxed. others have reported a similar pattern (18). in my laboratory, to perform ihc for lh and fsh in addition to ihc for sf1 is unnecessary for the majority of tumors, adding to complexity and cost, without providing additional value, at least within an initial panel of immunostains. for the corticotroph lineage, both silent and functional corticotroph adenomas were accepted as tp, and estimates are made without broad or narrow conditions. figure 4 shows that in our hands ihc for the transcription factor tpit outperforms acth, showing greater sensitivity and comparable specificity. figure 4 also shows that the negative predictive value of cam5.2 is quite high, suggesting that when one encounters an adenoma that lacks cam5.2 immunoreactivity, it is unlikely to be a corticotroph adenoma. personal experience suggests (data not shown), that tpit ihc is much easier to interpret than acth, which in our laboratory is never as clearly and darkly staining as tpit. figure 4. test characteristics and 95% ci of immunohistochemical stains pertinent to the tpit family of tumors. abbreviations: ihc, immunohistochemistry; ppv, positive predictive value; npv, negative predictive value. positive immunoreactivity was defined as a median allred score greater than 4 for all markers. finally, most pathologists would agree that the pit1 lineage represents the most complicated and diagnostically thorny part of the who pituitary adenoma classification. figure 5 shows estimated test characteristics for ihc that target pit1 lineage and offers several important insights. among the most obvious features is the dependence of a test on the number of data points available. tsh ihc sensitivity, for instance (figure 5, panel a), shows very wide confidence intervals largely due to the paucity of cases with positive results. given the heterogeneity of the pit1 family, and the relatively porous boundaries between pit1 family member ihc results, the mere exercise of deciding "what is true positive" is worthwhile. except for pit1 itself, where all pit1 classes are accepted as tp, i’ve undertaken to show broad and narrow definitions of tp and tn in figure 5, but acknowledge the inherent difficulty in such an exercise, especially given the paucity of prevalence estimates for some of the rarer adenoma classes, and the current ambiguity in classification guidelines. figure 5. test characteristics and 95% ci of immunohistochemical stains pertaining to pit1 family of tumors using restrictive (narrow) or inclusive (broad) definitions of true positivity. abbreviations: ihc, immunohistochemistry; ppv, positive predictive value; npv, negative predictive value. positive immunoreactivity was defined as a median allred score greater than 4 for all markers. armed with this these estimates, i am now able to make more informed choices about which stains to use in a diagnostic algorithm for my practice. the workup of a pituitary adenoma in my laboratory currently entails careful documentation of available clinical, radiological, and serological information. all pituitary adenomas are examined using a panel of ihc including sf1, pit1, and tpit. for nonfunctional adenomas, this panel resolves most macroadenomas into the gonadotroph adenoma class without the need for additional ihc. in the setting of cushing disease, or when a nonfunctional adenoma is found to be tpit immunoreactive, i add ihc for acth and cytokeratin cam5.2 to the initial panel. in the setting of acromegaly or significant hyperprolactinemia, in addition to sf1, pit1, and tpit ihc we also perform ihc for prolactin, growth hormone, tsh, estrogen receptor, and gata3 in the initial panel. finally, when clinical findings or initial ihc are ambiguous, a liberal approach is used, with the "complete panel of stains" being necessary in only a minority of cases. the approach related above resembles in some ways the efforts by neou et al. (19) to build a pituitary adenoma classification, but restricts itself to readily available ihc. although simple immunohistochemical stain scores are illustrated here, other potential classification variables, including molecular features or categorical variables like ultrastructural findings, would also profit from such interrogation. one day the use of methods such as multiple factor analysis (19) might allow incorporation of various disparate variables into a single model. we might finally be able to estimate the usefulness of molecular, serological, demographic, immunohistochemical, and ultrastructural variables within a common context. the strengths of our approach include the blinded review of ihc prior to interpretation by a pathologist and that our series includes tumors commonly encountered in the community. weaknesses include limited clinical follow-up following resection, paucity of rare tumor types, and underrepresentation of microadenomas (necessitated by study design, which employed tma’s with four cores per adenoma). the classification lifecycle: provisional models as the british statistician george box once noted, "all models are wrong, some are useful". tests and classifications come and go. in this sense, all classifications are provisional (20) and our collective task is simply to improve the current draft of the classification. i’m optimistic that clearly stated classification variables, attention to correlation, joint registry of a wide range of cases rather than subsets of rare adenoma types, and attention to test performance would generate a more useful, transparent, and reproducible classification of pituitary adenomas. for the practicing pathologist, this would be invaluable. an appendix of the immunohistochemical data and basic characteristics of our series are provided in supplement 1 in order that others may provide their own definitions of true positive and true negative in the calculation of test characteristics, to compare our series with their own, and to draw their own conclusions. references 1. tversky a, kahneman d. judgment under uncertainty: heuristics and biases. science. 1974;185(4157):1124-31. doi: https://doi.org/10.1126/science.185.4157.1124. 2. who classification of tumours editorial board. endocrine and neuroendocrine tumours [internet]. lyon (france): international agency for research on cancer; 2022 [cited 2022 aug 9]. 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bruce jn, otten m, khandji ag, flowers xe, siegelin m, et al. gonadotroph tumours with a low sf-1 labelling index are more likely to recur and are associated with enrichment of the pi3k-akt pathway. neuropathol appl neurobiol. 2021;47(3):415-27. doi: https://doi.org/10.1111/nan.12675. 7. ricklefs fl, fita kd, rotermund r, piffko a, schmid s, capper d, et al. genome-wide dna methylation profiles distinguish silent from non-silent acth adenomas. acta neuropathol. 2020;140(1):95-7. doi: https://doi.org/10.1007/s00401-020-02149-3. 8. horvath e, kovacs k, singer w, smyth hs, killinger dw, erzin c, et al. acidophil stem cell adenoma of the human pituitary: clinicopathologic analysis of 15 cases. cancer. 1981;47(4):761-71. doi: https://doi.org/10.1002/1097-0142(19810215)47:43.0.co;2-l. 9. ho kky, fleseriu m, wass j, van der lely a, barkan a, giustina a, et al. a tale of pituitary adenomas: to net or not to net: pituitary society position statement. pituitary. 2019;22(6):569-73. doi: 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2020;37(1):123-34 e5. doi: https://doi.org/10.1016/j.ccell.2019.11.002. 20. louis dn. a vade mecum for crossing the second translational "valley of death" in brain tumor classification. brain pathol. 2023:e13183. doi: https://doi.org/10.1111/bpa.13183. copyright: © 2024 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. hypothesis: entrapment of lipoprotein particles in the brain causes alzheimer’s disease feel free to add comments by clicking these icons on the sidebar free neuropathology 2:30 (2021) opinion piece hypothesis: entrapment of lipoprotein particles in the brain causes alzheimer’s disease delphine boche1, james ar nicoll1,2 1 clinical neurosciences, clinical and experimental science, faculty of medicine, university of southampton, southampton, united kingdom 2 department of cellular pathology, university hospital southampton nhs foundation trust, southampton, united kingdom corresponding author: delphine boche · clinical neurosciences, clinical and experimental sciences academic unit · faculty of medicine · university of southampton · southampton general hospital · mailpoint 806 · southampton so16 6yd · united kingdom d.boche@soton.ac.uk submitted: 05 august 2021 accepted: 21 october 2021 copyedited by: cinthya agüero published: 02 november 2021 https://doi.org/10.17879/freeneuropathology-2021-3459 keywords: lipoprotein particles, extracellular matrix, cholesterol transport, apolipoprotein e, alzheimer’s disease abstract we present for consideration a hypothesis that impaired movement of lipoprotein particles in the extracellular space in the brain in ageing is central to and causes all the key pathophysiological features of alzheimer’s disease (ad). the role of lipoprotein particles is to transport cholesterol from glial cells, where it is synthesised, to neurons, which require cholesterol for synaptic plasticity. the lipoprotein particles have a cholesterol-containing hydrophobic core, in which amyloid-β (aβ) can be solubilised. the core is surrounded by a hydrophilic surface containing apolipoprotein e (apoe) which, as neurons bear receptors for apoe, determines the destination of the particles. the problem arises because the extracellular space is a narrow cleft, barely wider than the lipoprotein particles themselves, which they have to navigate in order to perform their crucial cholesterol-transporting function. we explain how lipoprotein particles could become trapped in the ageing extracellular matrix and that this primary abnormality results in reduced delivery of cholesterol to neurons leading to impaired synaptic plasticity, crucial for learning and memory. it can also explain extracellular aβ accumulation, to which a microglial response generates a neurotoxic reaction, and intraneuronal tau aggregation, each of which exacerbate the problem. all these players have been known for many years to be important in alzheimer’s pathogenesis but a single unifying mechanism to explain how they are linked has been lacking. this proposed mechanism, with entrapment of lipoproteins particles as key to the development of ad, can explain the failure of so many clinical trials and points out new directions to be taken. genetics shows the overriding importance of apolipoprotein e in a complex multifaceted disease process, such as alzheimer’s disease (ad), genetic factors are important in highlighting key elements relevant to the onset of the disease. by far the major genetic risk factor for ad, in terms of scale of effect, is the apolipoprotein e genotype (gene: apoe, protein: apoe). there are 3 common apoe gene alleles (ε2, ε3 and ε4); as each person inherits one allele from either parent there is a total of 6 apoe genotypes (ε2/ε2, ε2/ε3, ε3/ε3, ε3/ε4 and ε4/ε4). the apoe ε4 allele carriage rate (i.e. the proportion of people in a population who possess one or two ε4 alleles) is typically about 25% of people of european ancestry [1], but varies considerably around the world among different populations [2]. a single copy of the apoe ε4 allele confers a two to fourfold increased risk of developing ad, with ε4 homozygotes at fourteen times increased risk of developing ad, whereas the less common ε2 allele confers relative protection, approximately halving the risk [3]. the effect is so substantial that a person who is ε4 homozygous and lives to 85 years of age has a lifetime risk of ad of more than 50%, comparable to the risk associated with brca1 mutation in breast cancer [4]. on the other hand, the protective effect of apoe ε2 is so substantial that ε2 homozygotes have a very low likelihood of developing ad; ε2 homozygotes have a 87% lower odds ratio than ε3 homozygotes and a 99.6% lower odds ratio than ε4 homozygotes [5]. the apoe gene polymorphism is lacking from non-human primates [6, 7] although, interestingly, rhesus monkeys which have an ε4-like apoe sequence develop aβ plaques as they age [8]. the fact that there are more than 200 animal models of ad highlights the difficulty in mimicking the complexity of the human disease in experimental animals. a complete understanding of the pathophysiology of ad ideally would explain all aspects of the human disease, and in particular incorporate the role of the apoe protein; how it causes and interacts with the accumulation of amyloid-β (aβ) and tau proteins, the glial cell activity and most importantly, the neuronal and synaptic dysfunction and loss. the function of apoe is to deliver cholesterol, packaged in lipoprotein particles, to neurons apoe is the principal cholesterol carrier in the brain, acting as a detergent with hydrophobic and hydrophilic moieties. its main role is to solubilise cholesterol and other lipids and lipid-soluble substances to enable them to be transported in the aqueous extracellular environment of the brain. the importance of cholesterol to the brain is highlighted by the fact that 25% of the body’s cholesterol is contained within the brain, despite the brain representing only 2% of the body weight [9]. cholesterol forms about 30% of the lipid bilayer of the membrane of all cells and is important in maintaining membrane fluidity. in the brain, the cell membrane is essential for conducting the action potential and for communication between neurons at synapses. however, despite its importance to neuronal function, neurons do not synthesise their own cholesterol, but rely on cholesterol which is synthesised by glial cells and then transported to neurons [10]. delivery of cholesterol to neurons is in particular demand when the neuron changes, as in synaptic plasticity which underpins learning and memory which are particularly affected in ad [11]. of note, genome-wide association studies have highlighted polymorphisms in genes in addition to apoe that are involved in cholesterol handling (clu, picalm, bin1 and abca7) as risk factors for ad [12, 13], supporting an important role for cholesterol handling in the disease mechanism [14, 15]. apoe is located in the shell of lipoprotein particles, with hydrophobic substances including cholesterol, which is loaded onto the lipoprotein particle by the enzyme abca1, being transported in the core [16]. the destination of the lipoprotein particles is determined by the receptors for the proteins on the surface of the lipoprotein shell. apoe, on the outer surface of the lipoprotein particles, binds to receptors of the low-density lipoprotein (ldl) receptor family (principally lrp1/apoe receptor) present on neuronal cell membranes and, by this mechanism, the cholesterol is internalised within the neurons. some cns lipoprotein particles also bear apoj (clusterin) on their surface; ependymal cells, but not neurons or other glia, bear apoj receptors so it is unlikely to be relevant for cholesterol delivery to neurons [17]. however, it is notable that in genome-wide association studies, polymorphism of apoj/clusterin gene has also been shown to influence risk for ad [18], raising the possible importance of clearance of lipoprotein particles and cholesterol to the csf. outside the brain, in the rest of the body, there are several other lipoproteins that can fulfil the function of cholesterol transport in addition to apoe, including apoa which is synthesised in the liver [19]. apoa-containing lipoprotein particles are detectable in the csf but appear to be excluded from the brain parenchyma by the blood-brain barrier. consequently, the cholesterol required by neurons is synthesised within the brain and delivered to neurons by apoe-containing lipoprotein particles [20] [9, 21]. lipoprotein particles become entrapped in the ageing extracellular matrix the lipoprotein particles, which resemble the high-density lipoproteins (hdl) present in the bloodstream, are in the region of 11-20 nm in diameter [22]. in order to transport cholesterol to neurons, they have to travel in the extracellular space in the brain which itself measures only about 40 nm between adjacent cells [23], below the resolution of light microscopy. small molecules can pass readily, by diffusion and potentially by active flow, through the extracellular space but diffusion even of conventional macromolecules much smaller that lipoprotein particles is hindered, particularly in pathological processes such as gliosis (activation of glial cells) [24], which occurs in association with ageing and the neurodegeneration in ad. an additional complexity is that the extracellular space resembles a sponge, containing the extracellular matrix (ecm). in the brain, the ecm is a complex multimolecular three-dimensional structure consisting of proteoglycans/glycosaminoglycans, proteins, proteinases, and cytokines [25]. expression of collagen iv, laminin and fibronectin is upregulated in the cerebral cortex in early ad. with ageing-related changes to the extracellular matrix, exacerbated by other risk factors for ad (hypertension, diabetes, obesity, inflammation and physical inactivity), the narrow extracellular space likely becomes compromised (‘fibrosed’), impeding the movement of, and trapping, lipoprotein particles in between cells. interestingly, oxidative modification of plasma lipoproteins is one of the earliest steps in the development of atherosclerosis, involving accumulation of lipoprotein particles in the vessel wall provoking inflammatory reaction [26]. in ad patients, there is evidence of greater oxidation of plasma and csf lipoproteins [26] and this could conceivably further impair the passage of lipoprotein particles through the extracellular space in the brain. consequently, the apoe-mediated system for transporting cholesterol and lipids from glial cells to neurons in the brain is unique, crucial for neuronal function and plasticity and vulnerable to age-related failure (figure 1). it is proposed that this results in a number of consequences leading to the key features of ad pathophysiology as follows. figure 1. entrapment of lipoprotein particles in the brain causes alzheimer’s disease. lipoprotein particles transport cholesterol from glial cells, where it is synthesised, to neurons which require cholesterol for synaptic plasticity. the lipoprotein particles have a cholesterol-containing hydrophobic core, in which aβ is solubilised, and a hydrophilic surface containing apolipoprotein e. receptors for apoe on the neuronal cell membrane determine the destination of the particles. the lipoprotein particles must navigate the extracellular space to reach the neurons, a narrow cleft barely wider than the particles themselves. as the extracellular matrix ages, lipoprotein particles become trapped between cells and this primary abnormality results in reduced delivery of cholesterol to neurons leading to impaired synaptic plasticity, crucial for learning and memory. impaired movement of lipoprotein particles in the extracellular space in the brain in ageing is central to and causes all the key pathophysiological features of alzheimer’s disease: degeneration of entrapped lipoprotein particles releases aβ which aggregates in the aqueous environment of the extracellular space; a microglial reaction to the aβ results in secretion of neurotoxic substances; neuronal cholesterol deficiency causes intraneuronal tau accumulation. [artwork by dr jennifer m dewing] entrapment of lipoprotein particles can explain the key features of alzheimer’s disease aβ deposition in the parenchyma as plaques: aβ peptide is notoriously insoluble in an aqueous environment, but it is lipid soluble and is transported in the hydrophobic core of lipoprotein particles [27-30]. therefore, trapping lipoprotein particles would immobilise aβ in the extracellular space. as the trapped particles degrade and rupture, we propose they release aβ peptide into the surrounding aqueous environment where it aggregates, initiating the formation of extracellular aβ plaques. co-localisation of apoe with aβ plaques is consistent with the origin of the aβ as being from apoe-containing lipoprotein particles [31]. immunohistochemistry for aβ on semi-thin sections (1μm) shows that diffuse plaques, usually interpreted as the earliest stage of aβ deposition, are formed from a cluster of dot-like structures [32], and electron microscopy shows small vesicles associated with amyloid fibrils in the extracellular space [32]. in the context of the current hypothesis, it is intriguing to speculate that these might be entrapped lipoprotein particles from which the amyloid has originated. app transgenic mice, over-expressing the v717f human amyloid precursor protein, develop aβ plaques as they age. interestingly, when crossed with apoe knock out mice so that they lack apoe, these mice do not accumulate plaques [33]. this finding indicates that apoe is essential for the deposition of aβ plaques, supporting the mechanism we propose. with ageing, the primary risk factor for ad, the human brain decreases in weight and size with an overall decline in cortical cholesterol that accelerates from the age of 80 [34, 35]. this is consistent with age-associated entrapment of the lipoprotein particles impairing delivery of cholesterol to neurons and resulting in neuronal cholesterol deficiency [36]. this has further effects on aβ as cholesterol deficiency leads to thinning of the cell membrane, shifting the site for secretase cleavage of the amyloid protein precursor (app) from producing predominantly the shorter form of aβ (aβ40) to the longer form (aβ42) which is relatively even more insoluble and prone to aggregation [37, 38]. this exacerbates the effect of trapped lipoprotein particles as the aβ42 then coalesces onto the initial plaque seeds facilitating their growth. in physiological conditions, aβ is suggested to act as a regulator of cholesterol homeostasis as observed in experimental models [39]. in humans, the familial forms of alzheimer’s disease are due to an imbalance in aβ production and are associated with increased cholesterol levels [40]. the binding of apoe to lipoprotein receptor-related protein (lrp) [41], the major apoe receptor on neurons, is also involved in the cellular uptake of cholesterol and aβ, consistent with apoe, cholesterol and aβ all being components of the lipoprotein particle. in the blood vessel walls as cerebral amyloid angiopathy (caa): aβ also colocalises with apoe in the walls of cerebral blood vessels suggesting that lipoprotein particles become trapped in the extracellular space of the vessel wall basement membrane as a consequence of age-related changes. the blood vessel dysfunction caused by the presence of caa further compromises brain function by haemorrhage, ischaemia and paralysing autoregulation of cerebral blood flow [42]. impaired neuronal and synaptic function deficiency of neuronal cholesterol results in impaired communication between neurons at the synapse, and particularly it interferes with the alterations in neurons which underpin learning and memory (i.e. synaptic plasticity) [43-46]. synaptic plasticity requires neuronal cell membrane to be synthesised to form and re-form synapses as they are remodelled. in neuronal cultures, the presence of glial cells enhances the formation and function of synapses and the essential factor mediating this effect has been identified as glia-derived cholesterol, delivered to the neurons by apoe-containing lipoprotein particles binding to the neuronal ldl receptors [11, 47, 48]. interestingly, in a number of clinical studies of traumatic brain injury, the possession of apoe ε4 is associated with a worse outcome and severe neurologic deficits [49-51]. this seems particularly so in young people in whom neuronal plasticity after injury might otherwise be more pronounced [50]. experimental studies in apoe ε4 transgenic mice confirmed impaired neuronal plasticity after brain injury [52-56]. further evidence comes from experimental models of global cerebral ischaemia in which apoe-deficient mice have increased neuronal damage which is ameliorated by intraventricular infusion of lipoprotein particles [57]. in addition, agonists of liver x receptors (lxr), which act as cholesterol sensors and promote lipidation of apoe by atp-binding cassette transporter a1 (abca1), ameliorate neuronal injury in experimental models of trauma [58] and reverse deficits in mouse models of ad [59]. the effects of neuronal and synaptic dysfunction due to lack of cholesterol [60] would be expected to be most pronounced in neuroanatomical locations in which plasticity is greatest (i.e. hippocampus involved in memory, association cortex involved in interpretation of sensations) and less severe or absent where plasticity is least (i.e. primary motor and sensory cortex, cerebellum, spinal cord). this is consistent with the clinical observations that the hierarchical sequence of loss of functions over time in ad follows the distribution of neuroplasticity [61]. tau protein accumulation we propose that neuronal cholesterol deficiency, resulting from the entrapment of lipoprotein particles, leads to the intracellular aggregation of tau. tau is involved in maintaining the cytoskeletal structure of neurons and, in particular, the transport of proteins from the cell body along axons to the synapses. accumulation of hyperphosphorylated tau occurs early in regions where the cholesterol is most in demand, that is where the rate of plasticity and synaptic remodelling is greatest (i.e. hippocampus, association cortex) and later or not at all where plasticity is least (i.e. primary motor and sensory cortex, cerebellum, spinal cord). a difficulty in trying to explain a direct link between aβ and tau pathology in ad has been that they appear to arise in different neuroanatomical locations; tau accumulation occurs earlier in the hippocampus and associated structures, only spreading later to the cerebral neocortex, whereas aβ accumulation occurs early in the cerebral neocortex. the hypothesis proposed here that there is not a direct link between aβ and tau pathology, but rather that they are each driven by cholesterol deficiency, resolves this conundrum. direct experimental support comes from cholesterol depletion in neuronal cultures which induces tau hyperphosphorylation that can be prevented by treatment with lipoproteins and cholesterol [62]. the link between neuronal cholesterol deficiency and tau accumulation is further highlighted by the occurrence of tangles at a young age in niemann-pick type disease type c, a rare progressive genetic disorder characterised by the inability of the body to transport cholesterol and lipids [63-64]. additional circumstantial evidence supporting a link between cholesterol deficiency and tangle formation potentially comes from the study of chronic traumatic encephalopathy (cte). cte is a condition caused by repeated blows to the head, typically in boxing and other sports, and is associated with the development of dementia with the formation of tangles as the key pathological feature [65]. it has been found that after a head injury, levels of apoe and cholesterol-containing lipoproteins in the cerebrospinal fluid plummet, just at the time when there is an increased demand for cholesterol for neuronal repair [66-68]. this relative deficiency of cholesterol, when recurrent over time with repeated blows to the head, could explain the development of tangles in cte [51-56] [65] studies using cerebral organoids derived from ad patients highlight an association of tau pathology with apoe ε4 carriage [69]. glial cell dysfunction astrocytes and microglia have a major role in supporting neurons and activation of glial cells is a hallmark of ad. in particular, they play an important role in the cycling of lipoprotein particles, scavenging lipid debris from degenerating neuron/synapses, lipidation the particles and releasing them for transport and uptake by neurons [70]. in apoe ε4 carriers, the brain is relatively deficient in apoe and the lipoprotein particles are smaller [71], more prone to aggregate and carry less cholesterol, rendering them particularly vulnerable to the effects of age-related cholesterol deficiency. human induced pluripotent cell (ipsc)-derived astrocytes from ε4 homozygotes produce lipoprotein particles that are smaller, carry less cholesterol and support neurons less well in terms of viability and expression of synaptic proteins compared with those from ε3 carriers [72]. microglia are the immune cells of the brain and are markedly activated in ad [73, 74]. many of the genes identified in genome-wide association studies are expressed by microglia, indicating that they play an important role in the development and/or progression of the disease [75]. the presence of extracellular aβ expelled by trapped and degraded lipoprotein particles is putatively recognised by the microglial pattern recognition receptors (pprs) evolved to detect molecular patterns associated with the bacterial cell walls. the consequent pro-inflammatory state provoked by this response causes release of cytotoxic substance evolved to destroy invading micro-organisms, which inadvertently has a harmful effect on neurons, compounding the effects of cholesterol deficiency [73, 76]. weaknesses/limitations of the hypothesis the ideas presented here form a hypothesis to explain the development of ad which is coherent and explains many facets of the disease. it is important to emphasise that a hypothesis is also an extrapolation of what is currently known and so some of the statements above are not firmly established but are potentially controversial and remain to be explored in the testing of the hypothesis. potential thorns in the side of this hypothesis include: localisation of aβ within apoe-containing lipoprotein particles has not been directly demonstrated in the human brain. this needs to be explored and represents an important gap in our knowledge. available technology is a limitation because the lipoprotein particles are below the resolution of light/confocal microscopy but are potentially amenable to study by novel 3d electron microscopy methods combined with multilabel immunostaining. direct evidence that lipoprotein particles become trapped in the extracellular space, disintegrate and release aβ is currently lacking. studies of immunohistochemistry for aβ and apoe identify apoe in some, but not all plaques. apoe seems to be present particularly in cored plaques, which are interpreted as later stage plaques, rather than early diffuse plaques as might be predicted from the hypothesis. the presence of apoe in later stage plaques could possibly reflect involvement of apoe-containing lipoprotein particles in attempted removal of aβ, in addition to a role in plaque formation. evidence that high peripheral cholesterol levels are associated with increased risk of ad and that statins, which reduce circulating cholesterol by inhibiting the cholesterol-synthesising enzyme hmg-coa reductase, may reduce risk for ad [77] might seem to contradict the hypothesis presented here. it seems that statins do reduce cholesterol levels in the brain [78]. however, it may be that the relative amounts of cholesterol, aβ and apoe are important i.e., that there is sufficient cholesterol as required by neurons, sufficient apoe to solubilise the cholesterol and a sufficient volume of lipophilic core within the lipoprotein particles to transport aβ and prevent its escape into the aqueous environment of the extracellular space where it is prone to aggregate. therapeutic consequences over the past decades, human clinical trials of new therapies for ad have been unrelentingly disappointing. this is despite there being no shortage of endeavour and expenditure, and no lack of successful therapeutic studies in animal models of specific aspects of ad. why is this? we suggest it is because the wrong targets have been addressed. whereas each of the major suspects listed above (aβ, tau, glial cells) may exacerbate the neurodegeneration and set up self-perpetuating vicious cycles, we propose they are not the initiating factor in sporadic ad, either singly or in combination. the pathophysiological scheme outlined above indicates that, according to this hypothesis, aβ and tau are not the cause of 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(https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. 63rd annual meeting of the canadian association of neuropathologists association canadienne des neuropathologistes (canp-acnp) feel free to add comments by clicking these icons on the sidebar free neuropathology 4:20 (2023) meeting abstracts 63rd annual meeting of the canadian association of neuropathologists association canadienne des neuropathologistes (canp-acnp) meeting abstracts october 18th–21st, 2023 montreal, qc submitted: 14 november 2023 accepted: 15 november 2023 published: 20 november 2023   the canadian association of neuropathologists – association canadienne des neuropathologistes (canp-acnp) held their 63rd annual meeting at the mcgill faculty club and the montreal neurological institute in montreal, qc, from october 18th to 21st, 2023, under the leadership of dr. robert hammond, president of the canp-acnp, dr. peter schutz, secretary treasurer of the canp-acnp, and with technical support from canp administrator colleen fifield. the academic program comprised 18 scientific abstracts, 14 unknown cases, a mini-symposium on molecular testing of brain tumours in canada, and the presidential symposium on pediatric neuropathology. digital pathology images from the 14 unknown cases are available for viewing online (www.canp.ca). the unknown case sessions were moderated by dr. peter schutz. the presidential symposium 2023 on pediatric neuropathology featured the gordon mathieson lecture given by dr. m. del bigio on in-utero infections and their effects on the developing brain, and the david robertson lecture given by dr. d. van essen entitled models and mechanisms of cerebral cortical expansion and folding. the program was completed by three invited presentations with dr. m. oskoui presenting on the epidemiology of cerebral palsy in canada, dr. p. ballabh presenting on cerebral gray matter injuries in infants with intraventricular hemorrhage, and dr. w. foulkes presenting on cns manifestations of dicer-1 tumour predisposition syndrome. the mary tom award for best clinical science presentation by a trainee went to dr. k. c. martin (supervisor dr. s. yip), and the morrison h. finlayson award for best basic science presentation by a trainee was won by dr. r. cotau (supervisor dr. m. richer). the following abstracts were presented at the 63rd annual meeting of the canadian association of neuropathologists – association candienne des neuropathologistes (canp-acnp) in october 2023.   https://doi.org/10.17879/freeneuropathology-2023-5195 keywords: canadian association of neuropathologists, canp, meeting abstracts, 63rd meeting oct. 2023 contents abstract 1: aqueductal stenosis and mesencephalosynapsis in fetal brains, part 1: classification, and morphology abstract 2: aqueductal stenosis and mesencephalosynapsis in fetal brains, part 2: associations abstract 3: a novel association: distal arthrogryposis, kif21a and fetal neuroaxonal dystrophy abstract 4: polymicrogyria through a tensegrity lens abstract 5: imaging axonal pathology in the retina as a potential non-invasive biomarker for amyotrophic lateral sclerosis abstract 6: progressive supranuclear palsy: a novel phenotype of germline sqstm1 (p62) mutation abstract 7: a 108 year old brain with near normal cognition abstract 8: cellular activation patterns of cd10+ fibro-adipogenic progenitors across acquired disease states in human skeletal muscle biopsies abstract 9: assessment challenges and headaches in the implementation of competence by design (cbd): the experience in a diagnostic and molecular pathology residency program abstract 10: isolated small vessel inflammation in temporal artery biopsies: small red flags abstract 11: the international spinal cord injury biobank: neuropathological contributions to global translational research in traumatic spinal cord injury abstract 12: death and the maid at the calgary brain bank abstract 13: pathology of the vertebral artery in medicolegal autopsies abstract 14: study of gamma-aminobutyric acid and glutamate signaling pathways in glioblastoma abstract 15: using molecular and immunohistochemical features to predict outcomes in grade 3 meningiomas abstract 16: pediatric-type gliomas in adults: pathologic and molecular features abstract 17: extra-pineal papillary tumor of the pineal region (ptpr) masquarading as a subependymal giant cell tumor (sega) abstract 18: ethical considerations in the use of dna methylation profiling for tumor diagnostics     abstract 1 free neuropathol 4:20:4 aqueductal stenosis and mesencephalosynapsis in fetal brains, part 1: classification, and morphology yael fisher1, orli greenberg1, patrick shannon1 1 pathology and laboratory medicine, mount sinai, toronto, on, canada systematic studies of the histology of fetal aqueductal stenosis (as) are uncommon. mesencecphalosynapsis, (midline fusion of the midbrain colliculi with absence of the dorsal medial septum), is infrequently described in the literature in association with brain malformations. we conducted a 12 year review of our institutional experience with fetal obstructive hydrocephalus and as using text word searches for aqueductal stenosis, aqueductal atresia, ventriculomegaly, hydrocephalus and rhombencephalosynapsis. we obtained 274 cases. we excluded chiari malformations, skeletal dysplasias, ex-vacuo ventriculomegaly and cases with unsatisfactory representation of the midbrain. 118 cases of obstructive ventriculomegaly with aqueductal pathology remained. clinical and histological features of each case were reviewed. the median gestational age was 23 weeks, interquartile range = 21 to 25 weeks. we identified 5 major morphological patterns of as. 1) aqueductal atresia (14 of 118, 11.9%); 2) severe stenosis (22 of 118, 18.6%); 3) mild stenosis (51 of 118, 43.2%); 4) borderline stenosis (10 of 118, 8.5%); 5) slit-like aqueduct (13 of 118, 11%). 8 cases had a seemingly patent aqueduct (6.8%), but 7 of these had a clear obstructive lesion (2 had isolated glial webbing of the aqueduct, 5 had other outflow obstructions). mesencephalosynapsis was seen in 42 of 118 cases (35.6%), more commonly in cases of atresia and severe stenosis. hemosiderin laden macrophages were present in the aqueduct 39 of 118 cases (33.1%). atresia and severe as showed a stronger association with multiple congenital anomalies and other central nervous system malformations when compared with the milder forms of as.   abstract 2 free neuropathol 4:20:5 aqueductal stenosis and mesencephalosynapsis in fetal brains, part 2: associations yael fisher1, orli greenberg1, patrick shannon1 1 pathology and laboratory medicine, mount sinai, toronto, on, canada as part of our review of fetal aqueductal histopathology, we reviewed the brain and autopsy findings accompanying the morphologies of aqueductal stenosis (as) and mesencephalosynapsis (mes), the absence of the median dorsal glial septum, in 118 cases of fetal hydrocephalus. we encountered some novel associations. 1. rhombencephalosynapsis: 21 of 24 (87.5%) cases of rhombencephalosynapsis demonstrated as with mes and 6 cases (25%) demonstrated a mass of neurons resembling heterotopic purkinje cells obstructing the rostral 4th ventricle/caudal aqueduct. 2. hemifacial hypoplasia: we encountered 7 cases of hemifacial hypoplasia (hh) with as and mes. on review of central nervous system pathology in hh, of the 21 cases of hh in our archives 7 had hydrocephalus, and all 7 had as and mes, including two rhombencephalosynapses. as with mes seems to be a principal pathology causing hydrocephalus in hh. 3. tegmental injury: we identified 3 cases of symmetrical tegmental injury with calcifications and microglial aggregates, in a pattern suggestive of fetal hypoxic ischemic injury. two cases had as with atresia, and one a slit like aqueduct, none had mes. tegmental injury in a hypoxic ischemic pattern is an infrequent but distinct pattern producing as and hydrocephalus. other distinctive pathologies associated with as are the vacterl association (2 with mes, 5 without), holoprosencephaly (n=8) and amniotic rupture sequence (n=3, all with mes).   abstract 3 free neuropathol 4:20:6 a novel association: distal arthrogryposis, kif21a and fetal neuroaxonal dystrophy leslie e. hamilton1, priya t. bhola2, radha mishra3, gail e. graham2, care4rare canada consortium4, kym m. boycott2,4, damien d’amours3, kristin d. kernohan4,5 1 department of pathology and laboratory medicine, children’s hospital of eastern ontario and university of ottawa, ottawa, canada 2 department of genetics, children’s hospital of eastern ontario, ottawa, canada 3 department of cellular and molecular medicine, ottawa institute of systems biology, university of ottawa, ottawa, canada 4 children’s hospital of eastern ontario research institute, university of ottawa, ottawa, canada 5 newborn screening ontario (nso), ottawa, canada although fetal neuroaxonal dystrophy is an established etiology for fetal akinesia, the underlying genetics were not known. kif21a is a kinesin motor protein that plays an important role in microtubule dynamics, including regulation of axonal growth. recently, falk et al. (2023) linked bi-allelic loss-of-function variants in kif21a to severe fetal akinesia with arthrogryposis. we present the case of an young infant diagnosed in utero with distal arthrogryposis, who died at 3 weeks of age, that represents an independent discovery of this autosomal recessive kif21a-related condition. her course was complicated by respiratory issues, hypotonia, seizures and neurological deterioration. brain mri was significant for polymicrogyria, diffuse brain volume loss and mild thinning of optic nerves. autopsy findings confirmed the bilateral extensive polymicrogyria and reduced brain volume, with probable thinning of the corpus callosum and descending corticospinal tracts. histologically, axonal spheroids were seen throughout multiple regions of the central and peripheral nervous system. other findings included cerebellar heterotopia, simplification of the inferior olives and a congenital neurogenic myopathy. clinical exome sequencing revealed biallelic compound heterozygous variants in kif21a. our case represents an independent discovery and second report of the autosomal recessive condition related to kif21a and revealed fetal neuroaxonal dystrophy as the underlying neuropathology, which has not been previously described in the literature. our findings expand the spectrum of kif21a-related disorders, and highlight the importance of a multidisciplinary approach, exome sequencing, and multi-site collaboration in the investigation of rare genetic conditions.   abstract 4 free neuropathol 4:20:7 polymicrogyria through a tensegrity lens robert hammond1, christopher dunham2 1 department of pathology and laboratory medicine, western university, london, ontario, canada 2 department of pathology and laboratory medicine, university of british columbia, vancouver, british columbia, canada polymicrogyria is an anomaly of cortical development. it may be pure or associated with other lesions. polymicrogyria is etiologically diverse and associated with a spectrum of neurological consequences proportional to its extent and associated abnormalities. the literature has mediated a debate on the timing and significance of particular etiologies, inviting a variety of theories to account for its stereotypic architecture. congenital cytomegalovirus (cmv) infection is a common cause of polymicrogyria. in a collection of such cases, ranging from 16 to 39 weeks gestational age, several findings were almost invariable: subventricular zone necrosis, subventricular germinal cell infection, radial glial disruption and injuries to the pial-glial border. subcortical neuronal heterotopias and leptomeningeal heterotopias were also common. clinical details and pathological findings indicate that the responsible insults occurred before neuronal migration was complete, lasted several weeks and led to a variety of structural perturbations to the developing brain. these cases add to the evidence that polymicrogyria is not solely post-migrational and that a variety of injuries (and timings) can replicate its relatively stereotyped morphology. a reductionist focus on timing and etiology has distracted from a more fundamental and versatile construct built on the principles of tensegrity, whereby the brain’s unique morphology (and disruptions of the same) can be explained on the basis of forces that exist at the tissue, cellular and subcellular levels. the natural experiment of polymicrogyria in congenital cmv infection lends support for tensegrity as a basis for normal and abnormal morphologies while embracing the variables of timing and etiology.   abstract 5 free neuropathol 4:20:8 imaging axonal pathology in the retina as a potential non-invasive biomarker for amyotrophic lateral sclerosis farbod khorrami1,2*, maryam amin mohammed amin1*, xun zhou1, you liang4, neeru gupta1,2,3,5, yeni h. yucel1,2,3,5,6 1 keenan research centre for biomedical science, unity health toronto. st. michael's hospital, toronto, on, canada 2 department of laboratory medicine & pathobiology, temerty faculty of medicine, university of toronto, toronto, on, canada 3 department of ophthalmology & vision sciences, temerty faculty of medicine, university of toronto, toronto, on, canada 4 department of mathematics, toronto metropolitan university, toronto, on, canada 5 department of ophthalmology and visual sciences, university of british columbia, vancouver, bc, canada 6 institute of biomedical engineering, science and technology (ibest), st. michael's hospital, toronto metropolitan university, toronto, on, canada imaging biomarkers of axonal pathology in amyotrophic lateral sclerosis (als) remain limited. we identified axonal spheroids, a pathological hallmark of als, in the post-mortem retina of als patients. we aim to establish the imaging of axonal pathology in the retina as a non-invasive als biomarker. following ethics approval, 10 post-mortem als and 8 age-matched control eyes were imaged using a clinical eye imaging device (spectralis, heidelberg engineering) combining confocal scanning laser ophthalmoscope and optical coherence tomography (oct). confocal microscopy imaging of phosphorylated neurofilament (p-nf) immunostained retinas was performed for histological validation. sequential in vivo retinal imaging was performed in als mice (n=28) and age-matched controls (n=28) with spectralis. statistical analysis was performed using generalized linear mixed modeling (rstudio). ex vivo human retinal fundus imaging (blue reflectance) detected more hyperreflective puncta in als patients compared to controls (19.04±22.5 vs. 2.7±1.6 /mm2, p<0.05). oct confirmed puncta were localized in the retinal nerve fiber layer (rnfl). in vivo mouse fundus imaging (infrared reflectance) showed significantly more puncta in the retina of als mice, compared to controls (7.2±5.2 vs. 1.8±0.9, p<0.05), with a progressive increase over time in als mice (p<0.001). immunofluorescence studies showed the presence of p-nf-positive axonal spheroids in the rnfl in human and mouse als retinas. axonal pathology in als is detectable with a widely available clinical eye imaging device. this study suggests imaging of the axonal pathology in the eye is a promising biomarker for assessing ocular phenotype, progression, and treatment response in clinical trials in als.   abstract 6 free neuropathol 4:20:9 progressive supranuclear palsy: a novel phenotype of germline sqstm1 (p62) mutation madison t. gray1, elizabeth finger2, lee cyn ang1,2 1 department of pathology and laboratory medicine, schulich school of medicine, western university, london, on, canada 2 department of clinical neurological sciences, schulich school of medicine, western university, london, on, canada a woman presented at age 61 with anhedonia and emotional lability with an mmse of 28/30. two years after presentation, she developed disinhibition ― notably inappropriate giggling ― as well as a wide-based gait. five years after initial presentation, there was the development of ocular abnormalities including saccadic intrusions and upgaze limitation. fine motor movements were slower and less elegant on the left. the patient passed away in a nursing home seven years after presentation. family history was notable for a father and paternal grandmother who were prone to giggle and have angry outbursts. neither carried a formal neurological diagnosis. antemortem genetic testing in our patient revealed a c.1211t>c(p. met404thr) mutation in sqstm1(p62). at autopsy, there was moderate frontotemporal atrophy. histological sections showed atrophy and gliosis of the subthalamic nucleus. grumose degeneration of the dentate nucleus was also present. p62 and phosphorylated-tau(at8) immunostaining demonstrated globose tangles most prominent in substantia nigra, pontine nuclei, and inferior olive. neurofibrillary tangles were rare in cortex and basal ganglia where tufted astrocytes and coiled bodies predominated. no abnormal beta-amyloid, tdp-43, or alpha-synuclein staining was present. this was consistent with a pathological diagnosis of progressive supranuclear palsy (psp). mutations of sqstm1(p62) have been associated with ftld/als, and inclusion body myositis-like myopathy, and familial paget disease of bone. here, we report (to our knowledge) the first case of sqstm1 mutation presenting with pathologically-confirmed psp. the mutation in this case (p.met404thr) has been reported to impair ubiquitin binding and has not previously been associated with neurodegenerative disease.   abstract 7 free neuropathol 4:20:10 a 108 year old brain with near normal cognition david g. munoz1,2,4, julia keith3,4 1 division of pathology, unity health, toronto, on, canada 2 keenan research centre for biomedical research, the li ka shing knowledge institute, st. michael’s hospital, toronto, on, canada 3 division of pathology, sunnybrook health sciences centre, toronto, on, canada 4 department of laboratory medicine and pathobiology, university of toronto, on, canada a 108 year old male veteran lived in a nursing home, and participated in regular cognitive testing. despite being legally blind and hearing impaired, his last mmse, 3 years prior to death at age 105 was 25/30. the brain weighed 1365 gr and showed severe bilateral hippocampal atrophy. h&e sections showed complete neuronal loss of ca1 bilaterally, and severe loss of vermal purkinje cell, but the neuronal populations of the neocortex and entorhinal cortex were mostly preserved. reactive gliosis was prominent throughout the cingulate cortex, mostly restricted to grey-white junction in the frontal, parietal and temporal cortices, and absent in the occipital cortex. the main protein deposit was tdp-43, present in the frontotemporal neocortex as neuronal cytoplasmic inclusions and thick short process in the superficial layers (type a), and in the hippocampus as a dense network of thin process in ca1. tau labeled neurofibrillary tangles in the entorhinal cortex and the locus ceruleus, and plaque-like structures in the absence of amyloid in the hippocampus. beta amyloid plaques were moderately dense in the neocortex, and accompanied by vascular deposits in the occipital cortex only. alpha-synuclein lewy bodies were present in the locus ceruleus, but not the substantia nigra. the surprising findings were the extent of hippocampal lesions compatible with near normal function, and the severe cortical astrocytosis out of proportion to abnormal protein deposits or neuronal loss. we propose that astrocytosis may develop in response to neuronal tdp-43, made conspicuous by ageing, and play a neuroprotective role, as postulated in als.   abstract 8 free neuropathol 4:20:11 cellular activation patterns of cd10+ fibro-adipogenic progenitors across acquired disease states in human skeletal muscle biopsies peter w. schutz1, simon cheung1, lin yi2, fabio m.v. rossi2 1 department of pathology, university of british columbia and vancouver general hospital, vancouver, bc, v6t 2b5, canada 2 biomedical research centre, university of british columbia, vancouver, bc, v6t 1z3, canada background: fibro-adipogenic progenitors (fap) are resident mesenchymal stem cells of skeletal muscle with defined roles in muscle fiber repair and fibrosis. experimental studies have shown their involvement in fibrofatty degeneration, denervation atrophy, and their susceptibility for pharmacological intervention. fap reaction patterns in human muscle biopsies are largely unknown, but have potential to inform translational approaches. method: 32 muscle biopsies from the archives of vancouver general hospital were selected from 8 groups (normal, dermatomyositis, ibm, anti-synthetase syndrome, imnm, denervation, type 2 atrophy, rhabdomyolysis). fap reaction patterns were analysed on routine cd10 immunohistochemical staining and compared between groups. double staining with mxa was performed on a subset. groups were compared histologically and by semi-quantitative scoring. results: activated endomysial cd10+ faps showed thickening and expansion of their normally delicate cell processes surrounding muscle fibers, and endomysial cell clusters evidencing proliferation. comparison across groups confirms fap activation in association with fiber degeneration/regeneration, foci of inflammation, and denervation, in keeping with experimental results. unexpectedly, dermatomyositis and anti-synthetase biopsies show diffuse activation. fap activation in dermatomyositis coincided with sarcoplasmic mxa expression. conclusion: assessment of cd10+ fap activation is routinely possible using cd10 immunohistochemistry and demonstrates cellular fap reaction patterns often in keeping with preclinical results. prominent expansion of fap processes surrounding myofibers suggests enhanced interaction between myofiber/basement membranes and faps during activation. the presence of diffuse fap activation in dermatomyositis and anti-synthetase biopsies raises the possibility of fap activation as part of the autoimmune process. future diagnostic applications, clinical significance and therapeutic potential remain open questions.   abstract 9 free neuropathol 4:20:12 assessment challenges and headaches in the implementation of competence by design (cbd): the experience in a diagnostic and molecular pathology residency program hao li1, sarah j. aziz1, lindsay ninivirta1, louise mui1, david k. driman1, christopher j. watling2, emily a. goebel1 1 pathology and laboratory medicine, schulich school of medicine & dentistry, western university, london, on, canada 2 centre for education research & innovation, western university, london, on, canada background/objectives: the recent implementation of competence by design (cbd) in neuropathology residency creates changes in both education philosophy and practical administration. this study analyzes perceptions of specific assessment criteria in cbd at one diagnostic and molecular pathology (formerly anatomical pathology) residency program, and the lessons learned. methodology: we focused on assessment interpretation of transition to discipline (ttd) entrustable professional activities (epas), which overlap with those in neuropathology – basic specimen handling, basic microscopy, and summarizing clinical information for clinicopathologic correlation. eight pathology faculty were interviewed. they were first shown the epa titles only (“gestalt” impression), then the full descriptions, and expressed their perceived expectations of the epas during each phase. interviews were recorded, transcribed, and subjected to thematic analysis. results: the gestalt impression of specimen handling and microscopy epas varied widely in the expected level of performance at a “basic” stage. while the descriptions clarified expectations, participants disagreed on whether they were valid and complete, due to contrasts with their own training experience, daily work, and subspecialty niche. it was also felt that all epa descriptions contained internal inconsistencies that were challenging to interpret. consequently, faculty described varying approaches to resident assessment despite identical criteria. conclusions: our findings demonstrate significant subjectivity in the interpretation of cbd epas, even with detailed assessment criteria. this is due to both inadequate clarity of descriptions as well as faculty attitudes and biases. therefore, further refinement of epa descriptions may be necessary, along with education of faculty on the underlying purpose of each assessment item.   abstract 10 free neuropathol 4:20:13 isolated small vessel inflammation in temporal artery biopsies: small red flags shervin pejhan1, lillian barra2, pari basharat2, larry allen3, lulu l.c.d. bursztyn3,4 alain proulx3, ruo yan chen2, morgan smith1, montana hackett5, robert hammond1,4 1 department of pathology & lab medicine, london health sciences centre, schulich medicine & dentistry, university of western ontario, london, ontario, canada 2 department of medicine, division of rheumatology, london health sciences centre, schulich medicine & dentistry, university of western ontario, london, ontario, canada 3 ivey eye institute, st. joseph's hospital, schulich medicine & dentistry, university of western ontario, london, ontario, canada 4 department of clinical neurological sciences, london health sciences centre, schulich medicine & dentistry, university of western ontario, london, ontario, canada 5 schulich medicine & dentistry, university of western ontario, london, ontario, canada temporal arteritis (ta) is the most common vasculitis over age 50. untreated, many patients will suffer blindness or stroke. gold standard diagnosis is achieved by temporal artery biopsy. the aim of this research is to investigate the relevance of small vessel inflammation in such biopsies. our dataset is comprised of 72 temporal artery biopsies subjected to a uniform re-examination paired with clinical data including demographics, history, physical examination, and laboratory findings. documented pathology variables include the presence or absence of ta, angiitis of vasa vasorum (avv), and inflammation of small peri-adventitial vessels consistent with small vessel vasculitis (svv). clinical and pathological variables were subjected to multivariate analysis. in brief, 25% of cases were identified as ta, 21% as isolated avv, 7% as isolated svv, and 5% as mixed avv/svv, while 42% showed no inflammation. all cases of ta were accompanied by small vessel inflammation: 94.4% exhibiting avv with or without svv, and 5.6% exhibiting svv, demonstrating a strong association between ta and small vessel inflammation. of the 24 cases with isolated avv/svv, 25% progressed to a clinical diagnosis of ta within one year whereas only 10% of cases with no identifiable inflammation showed such progression. furthermore, isolated avv/svv was identified in 25% of patients with a high clinical probability for ta, 60% of whom acquired a diagnosis of ta on clinical grounds within one year of follow up. our findings suggest that isolated avv/svv identifies a subgroup of patients with a higher risk of harboring or developing ta.   abstract 11 free neuropathol 4:20:14 the international spinal cord injury biobank: neuropathological contributions to global translational research in traumatic spinal cord injury veronica hirsch-reinshagen1,2, adam velenosi2,3, sarah r. morris2,4, kevin dong2,3, pushwant mattu1, zahra samadi-bahrami2,3, sureyah nassimbwa2,3, eslam abdelaziz 2,3, piotr kozlowski2,5, g. r. wayne moore1,2, cornelia laule1,2,4,5, brian k. kwon2,6 1 department of pathology and laboratory medicine, university of british columbia, british columbia, canada 2 international collaboration on repair discoveries, university of british columbia, british columbia, canada 3 praxis spinal cord institute, british columbia, canada 4 department of physics and astronomy, university of british columbia, british columbia, canada 5 department of radiology, university of british columbia, british columbia, canada 6 department of orthopedics, university of british columbia, british columbia, canada much of the scientific research dedicated to traumatic spinal cord injury (tsci) has focused on animal models of tsci. our understanding of human tsci is hampered in part by lack of biospecimens from patients. this gap in knowledge represents an important void in tsci translational research, as biological differences between animal models and the human condition need to be considered in the development of therapeutic approaches. herein we introduce the international spinal cord injury biobank (iscib; www.sci-biobank.org), a vancouver-based, multi-user biorepository with global range and the mission of accelerating therapeutic development in sci, including tsci, through improved biological understanding of human sci. certified by the canadian tissue repository network, iscib will be introduced to the community of canadian neuropathologists as a possible resource for research or post-mortem spinal cord donation. we describe the translational research gap that iscib is helping to fill; its structure, governance, and certification; how data and samples are accrued, processed and stored; the process through which samples and data are shared with global researchers; and finally, an example of novel clinicopathological insights we are gathering in human tsci. by expanding awareness of the existence of iscib within the community of canadian neuropathologists, we hope to further the mission of this pioneering world-class sci biorepository.   abstract 12 free neuropathol 4:20:15 death and the maid at the calgary brain bank jeffrey t. joseph1 1 department of pathology and laboratory medicine, university of calgary, calgary, ab, canada objective: determine tissue quality parameters in medical assistance in dying (maid) brain donations. background: in canada, medical assistance in dying (maid) was decriminalized in 2015 and was legalized in canada and alberta in june 2016. methodology: review 18 brain donations from maid patients to the cbb and compare them to non-maid donations. four quality parameters were compared to non-maid patients in the cbb: post-mortem interval (pmi), neuronal acidophilia (surrogate marker for hypoxia-ischemia), type 2 astrocytes in the globus pallidus externus (surrogate marker for systemic metabolic derangements), and cerebellar internal granular neuron autolysis (reflection of patient's pre-mortem medical state, including systemic acidosis). results: two procedural changes have brought the pmi from an average of 44 hours to 6 hours in maid patients. maid patients have a significantly shorter pmi than non-maid patients. unlike many brain bank patients, who die from non-neurological terminal diseases, most maid patients die without significant systemic diseases; they die without undergoing prolonged dying. maid patient brains have no or only early acidophilia in sensitive sites. maid patient brains have no type 2 astrocytes. maid patient cerebellums lack internal granular neuron autolysis. conclusions: maid patients represent an important source of autopsy tissue, which circumvents degradation of brain tissue during the dying process that affects many brain banks.   abstract 13 free neuropathol 4:20:16 pathology of the vertebral artery in medicolegal autopsies fabio a. tironi1, sarathchandra kodikara2, michael s. pollanen1 1 department of laboratory medicine and pathobiology, university of toronto, and ontario forensic pathology service, toronto, on, canada 2 department of forensic medicine, university of peradeniya, kandy, sri lanka objective: describe the clinicopathologic features of the vertebral artery (va) in medicolegal autopsies. methodology: cases with fatal or significant va pathology from 1996 to 2011 in the provincial forensic pathology unit, toronto, were studied. reports and histologic slides were reviewed. va examination by segment and pathological findings were analyzed. results: twenty-six cases were included, 2.7 male to female ratio, 15 to 76 years. fourteen cases (14/26, 54%) were trauma related, 11 cases (11/26, 42%) were non-traumatic, and 1 overlap (1/26, 4%). the 14 traumatic cases had mild to moderate external injuries. thirteen of the 14 trauma cases (93%) had a basal subarachnoid hemorrhage (sah) and showed transmural tears in the intracranial (v4) segment. the other trauma case had dissection between v1 and v2, associated with cerebellar, brainstem, and upper spinal cord (cbsusc) infarctions. col3a1 mutations and segmental mediolytic arteriopathy (sma) were found among the cases. the eleven (42%) non-traumatic cases included basal sah, cbsusc and thalamic infarctions. va thrombosis, atherosclerosis, dissection, fibromuscular dysplasia (fmd), sma and col3a1 mutations were present among cases. the overlap case presented upper posterior neck trauma and basal sah due to a v3 dissection. in this case, there was also fmd, sma, multifocal intramural hemorrhages and dissections involving craniocervical and visceral arteries and a col3a1 mutation was found. conclusion: traumatic rupture of v4 resulted frequently in sah. non-traumatic pathology predominately occurs in extracranial va. we recommend that postmortem examination of these cases includes examination or the entire vertebral artery.   abstract 14 free neuropathol 4:20:17 study of gamma-aminobutyric acid and glutamate signaling pathways in glioblastoma raul cotau1,2,3,4, étienne audet-walsh1,4,5, maxime richer3,6 1 department of molecular medicine, université laval, québec, canada 2 department of medicine, université laval, québec, canada 3 neuroscience axis, centre de recherche du chu de québec-université laval, québec, canada 4 cancer research center, université laval, québec, canada 5 endocrinology and nephrology axis, centre de recherche du chu de québec-université laval, québec, canada 6 department of molecular biology, medical biochemistry, and pathology, université laval, québec, canada glioblastomas (gbm) represent the majority of malignant primary brain tumors, and cannot be cured. the identification of their survivaland proliferation-associated mechanisms may help introduce new therapeutic targets. several lines of evidence suggest that neurotransmitters gamma-aminobutyric acid (gaba) and glutamate are involved in gliomagenesis. their signaling pathways within the tumor microenvironment (tme) remain nonetheless poorly defined. we hypothesize that the modulation of gaba and glutamate genes may control tumoral aggressivity. we (1) identify vulnerable biomarker genes for gbm (stage iv glioma), and (2) assess their validity in in vitro and clinical studies. a gaba-treated u87 gbm cell line was bulk rna sequenced, and single-cell rna-seq data of 9 resected new-diagnostic, and 5 recurrent idh-wildtype gbm allowed for differential and enrichment analysis. data suggested that gaba regulates cancer-associated pathways, such as survival, and metabolism. using a novel algorithmic cell type identification approach, gbm cells, along with neuroimmune proand anti-inflammatory populations were identified. potential gaba, glutamate, and calcium neurotransmitter-associated biomarkers previously identified in the laboratory were found to be differentially expressed between cell-group clusters. further enrichment aims to identify cell types, and genes associated with protumoral activity. a cohort comprising 50 resected gbm samples was utilized to associate those new genes, as well as previous markers c5ar1, vgat, gad-1, and gaba-b to histologic characteristics including necrosis, angiogenesis, and infiltration. immunohistochemical alignment between our, and clinical markers (ex. mib1 for proliferation) was performed. gaba was significantly overexpressed in mib1-rich zones. this study introduces gabaand glutamate-associated biomarkers as potential gbm therapeutic vulnerabilities.   abstract 15 free neuropathol 4:20:18 using molecular and immunohistochemical features to predict outcomes in grade 3 meningiomas karina chornenka martin, md1, kira tosefsky, bsc2, alexander d. rebchuk, md msc2, justin z. wang, md3,4,5, gelareh zadeh, md phd3,4,5, serge makarenko, md2, stephen yip, md phd1 1 department of pathology & laboratory medicine, faculty of medicine, university of british columbia, vancouver, british columbia, canada 2 division of neurosurgery, university of british columbia, vancouver, canada 3 division of neurosurgery, university of toronto, toronto, canada 4 macfeeters hamilton neuro-oncology program, princess margaret cancer centre, university health network and university of toronto, toronto, ontario, canada 5 princess margaret cancer centre, university health network, toronto, ontario, canada objective: the who 2021 classification introduces molecular alterations in meningiomas that are associated with increased risk of recurrence and/or shortened overall survival (os). these include tert promoter (tertp) mutations, cdkn2a/b homozygous deletion, h3 k27me3 loss, and bap1 loss. with precise prognostic implications pending further characterization, we explored these molecular alterations and associated clinical outcomes in a single-center cohort of grade 3 meningiomas. furthermore, we examined whether mtap and p16 immunohistochemistry can predict cdkn2a/b status. methods: clinical and histopathological information were obtained from the electronic medical records of grade 3 meningiomas resected at a tertiary center between 2007-2020. molecular testing for tertp mutations and cdkn2a/b status, methylation profiling, and immunohistochemistry for h3 k27me3, bap1, p16, and mtap were performed. predictors of survival were identified by cox regression. mtap and p16 expression were assessed and correlated with cdkn2a/b status. results: out of 15 cases included in the study, 8/15 were classified as anaplastic, 6/15 as rhabdoid and 1/15 as papillary. one rhabdoid tumour exhibited bap1 loss, four tumours harboured tertp mutations and three demonstrated cdkn2a/b homozygous deletion. tertp mutations and cdkn2a status were associated with significant reductions in os, which was independent of simpson resection grade. meningiomas with cdkn2a/b homozygous deletion showed consistent loss of p16 and mtap immunoreactivity. conclusion: tertp mutations and cdkn2a/b homozygous deletion are significantly associated with reduced os independent of resection extent. our findings support using these molecular markers for prognostication in meningiomas. additionally, this small series demonstrates that p16 and mtap immunohistochemistry can predict cdkn2a/b status.   abstract 16 free neuropathol 4:20:19 pediatric-type gliomas in adults: pathologic and molecular features andrew f. gao1, josé-mario capo-chichi2, adam smith2, gelareh zadeh3, lili-naz hazrati4, cynthia hawkins4, phedias diamandis1 1 division of anatomic pathology, laboratory medicine program, university health network, toronto, on 2 division of clinical laboratory genetics, laboratory medicine program, university health network, toronto, on 3 division of neurosurgery, krembil neuroscience program, university health network, toronto, on 4 dept. of pediatric laboratory medicine, hospital for sick children, toronto, on the 2021 who classification of cns tumours recognizes adultand pediatric-type gliomas, which have important clinical and biological differences. while pediatric-type gliomas occur most commonly in children, their frequency and pathologic and molecular features in the general adult population have not been well studied. we prospectively characterized a single-institution cohort of 498 consecutive gliomas (diffuse gliomas, circumscribed gliomas, and glioneuronal tumours) using immunohistochemistry, sanger sequencing, panel-based ngs, fish, snp array, and/or dna methylation profiling. of 490 cases that could be evaluated (median age 58, range 18-91), pediatric-type alterations were identified in 61 (12%, median age 34, range 18-73). excluding patients >55yo with glioblastoma (for whom no molecular testing was performed; n=227, 46%), pediatric-type alterations comprised 23% of the remaining cohort (61/263), compared to 45% for idh mutations, and 32% for adult-type gbm. the frequency of pediatric-type alterations exceeded that of non-canonical idh mutations in histologic gbms ≤55yo (8% vs 3%, n=96), and exceeded both non-canonical idh mutations and adult-type gbm in tumours with diffuse lower-grade histology (20% vs 8% and 17%, respectively, n=138). h3-3a, braf, nf1, and mismatch repair were the most commonly altered genes in pediatric-type diffuse high-grade gliomas, while braf, fgfr1, and fgfr2 alterations prevailed in diffuse low-grade gliomas, circumscribed gliomas, and glioneuronal tumours. in our cohort of adult patients, pediatric-type alterations are relatively common, especially in younger adults and lower-grade tumours. pediatric-type alterations should be routinely included in the diagnostic evaluation and molecular testing of gliomas in adults.   abstract 17 free neuropathol 4:20:20 extra-pineal papillary tumor of the pineal region (ptpr) masquarading as a subependymal giant cell tumor (sega) kenrick wysong md1, matija snuderl md phd2, john c. dewitt md phd1 1 department of pathology and laboratory medicine, university of vermont, medical center, burlington, vt, usa 2 department of pathology, nyu langone medical center, new york, ny, usa papillary tumor of the pineal region (ptpr) is a rare neuro-epithelial tumor arising in the pineal region characterized typically by papillary and solid architecture with epithelial-like cells and reactivity for cytokeratins, while subependymal giant cell tumor (sega) is a periventricular tumor composed of large ganglion-like astrocytes. here we describe the case of 75-year-old man with a slowly enlarging superior cerebral aqueduct mass first discovered and followed over six years prior to biopsy. given its increasing size a small biopsy was performed which was characterized by large pleomorphic ganglion-like giant cells with abundant cytoplasm and prominent nucleoli. immunohistochemistry showed that tumor cells were strongly positive for s100, but negative for gfap. despite the patient not carrying a diagnosis of tuberous sclerosis, given the ventricular location, histologic appearance, and immunohistochemical results a diagnosis of a low grade tumor consistent with subependymal giant cell astrocytoma (sega) was issued, with outside expert consultation in agreement with this interpretation. subsequently, despite surgery the mass continued to slowly grow over the next few years and the decision was made to perform next generation and methylation sequencing 4 years following the initial biopsy. next generation sequencing returned only the presence of an nf2 mutation, while methylation sequencing was a match for papillary tumor of the pineal region. subsequent immunohistochemistry for keratin ae1/3 was strongly positive on the original biopsy, consistent with this new diagnosis. this case demonstrates the utility of methylation sequencing, particularly when biologic behavior and sequencing results do not correlate with histologic diagnosis.   abstract 18 free neuropathol 4:20:21 ethical considerations in the use of dna methylation profiling for tumor diagnostics kyle s. conway1, pouya jamshidi2, lauren b. smith1 1 department of pathology, university of michigan, ann arbor, mi, usa 2 department of pathology, northwestern university, chicago, il, usa dna methylation profiling has become essential for cns tumor classification. unlike traditional molecular testing, methylation profiling does not interrogate specific genetic alterations. rather, it evaluates a genome-wide methylation profile, typically in conjunction with a machine-learning classifier that assigns a diagnostic category. patients benefit from an unprecedented ability to accurately diagnose and classify tumors, but there is little formal evaluation of the unique ethical considerations raised by this modality. our objective is to apply principles of medical ethics to the use of methylation profiling, with a focus on three principles: (1) informed consent: the diagnostic paradigm of methylation is complex for patients and clinicians to understand and risks confusion regarding the diagnostic significance of a methylation profile. patients may be unaware of what data is stored and how it will be stored, transferred, and re-analyzed for clinical and research purposes. (2) harm to patients: methylation profiling generates epigenetic data that exists indefinitely and can be analyzed in a dynamic fashion. as new classifiers are implemented, a previously unclassifiable tumor may become classifiable without re-sequencing or a clinically significant change in methylation class may occur. (3) justice and resource utilization: the extensive granularity of diagnostic categories defined by methylation profiling creates a risk of over-testing that may not generate clinically actionable results. the use of machine learning carries specific considerations of justice, including inherent biases in the populations used for classifier generation. neuropathologists should keep these ethical considerations in mind when implementing methylation as a clinical test or in clinical practice. copyright: © 2023 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. how and why i fell into neuropathology feel free to add comments by clicking these icons on the sidebar free neuropathology 4:21 (2023) reflections how and why i fell into neuropathology jean-jacques hauw address for correspondence: jean-jacques hauw · 6, rue du collège de l’arc · 39100 dole · france jean-jacqueshauw@laposte.net additional resources and electronic supplementary material: supplementary material submitted: 07 september 2023 accepted: 08 september 2023 copyedited by: couger jimenez jaramillo published: 23 november 2023 https://doi.org/10.17879/freeneuropathology-2023-5112 keywords: neuropathology, reflections, autobiography, pitié-salpêtrière paris i'm going to explain how and why i fell into the department of neuropathology at the pitié-salpêtrière hospital of paris. i'd also like to sketch the history of french neuropathology in the years 1960-2010, as seen by a naive young student, and then by a practicing neuropathologist (often still very naive). as a matter of fact, although the history of neurosciences [1-2] and the pitié-salpêtrière hospital in paris [3-4] have been the subject of numerous publications, the history of neuropathology in this hospital has been rarely documented [5-6]. i spent more than forty years strolling along the alleys of la salpêtrière, among its old pavilions, the saint louis chapel, the “pavillon des folles”, the courtyard of manon lescaut and the guard room. i worked full-time between the escourolle laboratory, the “amphithéâtre des morts” and the university. it has been a real pleasure to be part of this world. i would also like to offer young doctors in training and future neuropathologists some advice that might help them in the choice and development of their future careers. how did i turn to medicine? i was raised by devoted parents, teachers of english and economics in secondary school and technical education, who had managed to move close to paris (from boulogne-sur-mer in the north of france, to dreux, then to versailles) so that their children could study there. actually, most of my career followed a succession of accidents. before i chose my future profession, i hesitated between sciences politiques school (humanities and social sciences) and medicine. as my first stroke of luck, i missed the registration to sciences politiques, so i registered to medicine. in fact, i registered to a pre-training year which included practical work (microscope examination, dissection, etc.). as chance would have it, i was seated next to fellow student claude-françois degos, whose father was a "grand patron" and a professor of medicine at paris university. i heard him talking with one of his friends about his future career: “to access a bright future, one would have to prepare for competitive examinations to become an "extern" [7] and then an "intern" (resident) (paid functions i had never heard of).” the best way to do so would be to follow the “conferences for externship and then those for internship (training courses given by young pedagogical doctors).” that is what i did. it paid. i passed the externship competition. and then, i was lucky enough to be accepted into one of dr. jean-claude hesse’s internships, one of the best conferences. thanks to him, i successfully passed the boarding school competition. how did a young student turn to neurology? every morning except sunday, “externs” examined selected patients of their department and drew up their files under the supervision of “interns” and other doctors. each 6-month “externship” was chosen on the basis of the year in which the student passed the competition and his admission rank. i started my externship at the raymond poincaré hospital in garches, a suburb of paris (near versailles, where i lived with my parents), in doctor jean benassy's neuro-rehabilitation department. more specifically, it turned out to be a ward for tetraplegic patients. it was an emotional shock and perhaps the origin of my vocation. another stroke of luck: a year later, when i was ending another externship in medical consultation at boucicaut hospital in paris, dr. pierre thiroloix, who oversaw this sector, kindly asked me: “what specialty would you like to do?” i answered, “neurology.” he recommended me to professor pascal brunet (fig 1) at la salpêtrière hospital, who proved to be an excellent mentor throughout my whole career. following his advice, i did one externship in neurology in professor paul castaigne’s and an additional one in professor françois lhermitte’s department at la salpêtrière hospital. i began my residency in internal medicine and then transitioned to neurosurgery (a specialty for which i obviously had no talent!) in professor marcel david’s department at la pitié hospital [8]. at the same time, i began studying psychology at the universities of nanterre and paris. figure 1: from left to right: dean pascal brunet, alex novikoff, jeanne-marie boutry, françoise cathala in her office at la salpêtrière, and miss besse, influential secretary of dean paul castaigne. how did a young student in neurology fall into research (nerve tissue culture)? in the neurosurgery department of professor marcel david, a neurologist, dr. elie bernard-weil told me one day (yet another stroke of luck!): “i've invented a treatment that allows damaged nerve tissue to regenerate. i'm looking for someone to study it. would you like to do it on nerve tissue cultures in a research laboratory: the service de physiopathologie cellulaire (department of cellular physiopathology) of saint-antoine hospital in paris?” of course! it was a turning point. in doctor roger robineaux's laboratory (part of the new research association “association claude bernard pour la recherche médicale”), i was given an exceptionally warm welcome by all the members of the teams, including a researcher from the centre national de la recherche scientifique (cnrs), pierre goube de laforest. i learned from them a wide range of tissue culture techniques (of cells: "histiotypic", of organs with migration of certain cells around the explant: "organized", of organs without this cell migration: "organotypic", now called "organoids”) and first applied them to nerve tissue from chicken embryos (one of the least expensive to obtain). i also learned how to study them "in vitro" by phase contrast (or nomarski) differential interference contrast (dic) and microcinema or by light and electron microscopy [9]. this made it possible to study both nerve growth factors and the toxicity of certain molecules (e.g., lipidosis induced by some drugs) [10]. i used to go there every afternoon, except for those devoted to internship boarding duty, to the psychology courses i followed, and to the histology tutorials i was giving to young students at the paris faculty of medicine under the supervision of professor christian da lage for financial reasons (which turned out to be another stroke of luck!). with the help of two women researchers, who were culturing kidneys and livers from human stem cells derived from aborted fetuses, i was able to grow human cerebellar tissue [11]. ethical issues were quite different in the 1960s! when i asked pierre goube de laforest about human fetal tissue culture, he replied: "i don't see any problem", and, the following week: "i asked around: should we not baptize the fetus before harvesting it, and culturing its nervous tissue?" among the doctor roger robineaux's laboratory's researchers, two americans on sabbatical became interested in my nerve tissue cultures. they were alex novikoff (fig 1) and his wife, phyllis. they used electron microscopy to study lysosomes, recently described by christian de duve. they studied the relationship between the neuronal golgi apparatus and lysosomes. they were helped by an exceptional technician, jeanne-marie boutry (fig 1), whom they later brought to new york for a year [12-13]. a few years later, they invited me to new york, where i met the american neuropathologist robert terry [14] at the albert einstein college of medicine, solidifying my orientation towards neuropathology. how did a young student in neurology fall into neuropathology? a few months later, i met another researcher in roger robineaux's laboratory, françoise cathala (another stroke of luck!) (fig 1). she was an exceptional woman an unconventional neurologist and virologist, very passionate, brilliant, and enthusiastic, but often little understood (even despised) by her colleagues. clearly interested in my nerve tissue cultures, she whispered to me: "did you know that an internship position in neuropathology has just been created at la salpêtrière hospital in professor raymond escourolle’s new neuropathology department? it is alongside professor castaigne’s department, just above my virology laboratory. you would be able to perform your nerve tissue cultures there. as no one is yet aware of this new position, you have a great chance of getting the job." françoise cathala was right: i realized my dream of becoming an intern at la salpêtrière. and that's where i met the world of neuropathology with which i was completely unfamiliar, as my training had only included internal medicine, neurology, psychiatry, surgery, and neurosurgery. neuropathology at la salpêtrière and la pitié hospitals (paris) in the late 1960s the situation of neuropathology wasn’t simple. the adjacent hospitals la pitié and la salpêtrière were still distinct. initially, there were four different small laboratories ("laboratoires de service") attached to clinical departments [6]. two were part of the neurology departments. the charles foix laboratory was headed by raymond escourolle (fig 2, 4 and 6) and belonged to the “clinique du système nerveux” headed by the dean paul castaigne (fig 2) and his very influential secretary, miss besse (fig 1) [4]. his numerous assistants included pascal brunet (another lucky break: miss besse appreciated pascal brunet and helped me in any way she could). when i joined as a resident, this laboratory also carried out neuropathology procedures for the neurological clinics run by professors andré buge and françois lhermitte. the other neuropathology laboratory of the neurology department was located at the “clinique neurologique” of la salpêtrière and was headed by raymond garcin, followed by georges boudin (managed by michel fardeau, jean lapresle, jacqueline mikol, and others) [5,15]. the two other neuropathology laboratories were part of the neurosurgery department. one was located at la salpêtrière (in the department of professor jacques lebeau, successor to jean-marie guillaume who established it in 1942, with the neuropathologist jean-françois foncin, successor to jean-emmanuel grüner). the other was located at la pitié hospital in the neurosurgery department of professors marcel david (who was followed by bernard pertuiset and jacques philippon). the neuropathologists of this department were histologists roger messimy, christian da lage, jean racadot, jacques poirier and michèle kujas [5-6]. in addition to these neuropathology laboratories, there was also a general pathology department (headed by guy chomette, then yves le charpentier and isabelle brocheriou). this last one used to carry out all the general pathology activity, including autopsies, for patients from non-neurological departments. figure 2: from left to right: dean paul castaigne, raymond escourolle and jean-claude gautier. then, gradually, the various neuropathology "laboratoires de service" were grouped together in the charles foix (currently known as raymond escourolle) neuropathology department. this one progressively took over all the autopsies of the pitié-salpêtrière hospital, and then those of the parisian region (whether or not they concerned patients with neurological or neurosurgical conditions). this was not without some adventures. the most amusing was the retirement of jean-françois foncin, which led to the closure of his laboratory. this quirky neuropathologist had decided to take home (in the paris suburbs) a number of brain samples! but the many formalin-fixed brains that remained had to be moved from the cellar below his laboratory. together with the administrative director of the pitié-salpêtrière hospital group, we decided to organize their transfer to the raymond escourolle neuropathology department. to ensure that the operation did not shock hospital staff and patients, the director staged "renovation work" on j-f foncin's laboratory. then "monsieur grimi" (abdelmalek grimi, technician specializing in autopsies) and i discreetly transported these brains and the related files in my car over several trips. today, the remaining facilities are the general pathology department and the raymond escourolle department of neuropathology with one exception. certain muscle biopsies are carried out at the "institute of myology", set up in 1996 at the instigation of an association of patients with muscular diseases and their parents, the afm-téléthon. michel fardeau and his students launched this activity. charles foix department of neuropathology in the late 1960s the charles foix neuropathology department carried out cerebrospinal fluid (csf) cytology examination and muscle and nerve biopsies (with a dedicated sampling room, where we performed them). above all, autopsies were by far the most important activity for the intern. i used to perform 4 to 10 per week, first under the guidance of professor raymond escourolle with the help of “monsieur grimi”, then alone with him. later, when i was an intern in clinical neurology at la salpêtrière, i was sometimes asked by the manager of the neuropathology laboratory to help her intern carry out autopsies. these led to brain-cutting sessions and clinicopathological correlations for multiple departments at la salpêtrière, but also for other hospitals, such as the hospice d'ivry sur seine (renamed hôpital charles-foix in 1976) or saint-germain-en-laye hospital. raymond escourolle (1924-1984), who directed the charles foix neuropathology department, was a pupil of paul castaigne (fig 2) initially trained in clinical neurology and then mentored in neuropathology by serge brion. he succeeded yvan bertrand whose predecessors had been jean-martin charcot, pierre marie, and charles foix. when i arrived in this laboratory, raymond escourolle was surrounded by numerous collaborators, all former neurologists: brigitte berger, an electron microscopy specialist, gérard percheron (fig 3), an anatomist who had described a variant of the human posterior cerebral circulation (a solitary arterial trunk supplying blood to the paramedian thalami and the rostral midbrain bilaterally) and jacques poirier (fig 3), a histologist and specialist of medicine history. there were also a number of clinicians who performed specialized clinico-pathological research, such as christian derouesné, jean-claude gautier (fig 2), jean-louis ribadeau-dumas and jean-louis signoret (fig 3). when raymond escourolle died [16], i became head of the neuropathology department (which took his name), and was followed by charles duyckaerts (fig 3 and 7), and danielle seilhean (fig 7). figure 3: from left to right. above: nicole baumann with gérard percheron; jacques poirier; jean-louis signoret. below: general louis court; dominique dormont; charles duyckaerts and annick alpérovitch. an internship at la salpêtrière: the guardroom the residents (“internes”) had some advantages, the most significant of which was the use of the guardroom (“salle de garde”). they slept there when they were on night duty and, most importantly, had lunch there whenever they wanted. “frescoes" decorated the guardroom. these were murals, mostly erotic cartoons, depicting the hospital doctors (fig 4-6) [17]. after a few introductory rites, the “salle de garde” was an opportunity to meet the other interns, the foreign trainees and also the so-called "fossils" that became “dinosaurs” when older. these were the former residents who continued to frequent the guardroom and performed the rituals there ("welcoming newcomers", "bawdy guardroom songs”, etc.). one of the most addicted to the salle de garde was the neuropathologist jean-françois foncin, whom i met there before becoming a fossil myself. he was a remarkable singer, a great specialist in "chansons de salle de garde" (guardroom songs), which are usually erotic and divisive. figure 4: raymond escourolle at the salpêtrière guardroom with mural “frescoes” circa 1960. figure 5: mural “frescoes” at the salpêtrière guardroom circa 1980. bottom right: "the whole mystery of jacques' life is made of consent". figure 6: the 1980 “frescoes” of the salpêtrière guardroom with the names of the people caricatured. the arrival of prion diseases in france françoise cathala, who had just returned from the laboratory of future nobel prize winner carleton gajdusek's in bethesda, was unique among researchers in france in that she was in love with latent viruses and prion diseases. "i was fascinated by the notion of a latent virus in the nervous system after a primary infection with occasional manifestations appearing subsequently, a phenomenon well known with herpes simplex virus and the herpes virus of varicella–zoster, the subject of my thesis. i wanted to research slow virus diseases with the intention of eventually studying multiple sclerosis, then without any knowledge regarding kuru or the transmission of creutzfeldt–jakob disease to chimpanzees!" [18] this caused her some career difficulties (notably at the french research institute inserm), as these unconventional infectious agents were far from unanimously accepted by the infectiologists [19]. actually, the transmission of bovine spongiform encephalopathy from cattle to man had not yet been recognized at that time. it was thanks to her that i met the french pioneers in this field, the researcher dominique dormont (fig 3) and the army medical officer general louis court (fig 3). together, we carried out the first french autopsy on a chimpanzee inoculated with creutzfeldt-jakob disease at the percy armed forces training hospital. i took part in numerous activities, including the "comité sur les encéphalopathies subaiguës spongiformes transmissibles et les prions” (committee on transmissible spongiform encephalopathies and prions) headed by dominique dormont and known as the "dormont committee", and in research and meetings on the subject in collaboration with jean-philippe brandel, jean-philippe deslys, stéphane haïk and corinne lasmézas. these activities resulted in the establishment of the neuropathological diagnostic criteria for prion diseases [20] and the discovery of the phenomenon of "transconformation", or the spread of protein misfolding from cell to cell. this was a new paradigm in the field of neurodegenerative disorders, brain aging and several other disorders [21]. a passion: research after being introduced to research through nerve tissue culture, and being made aware of prions, i aimed to perform and encourage research practical for the field of neuropathology and, more broadly, for clinical, neurological or neurosurgical disciplines. i carried out this project under the direction and with the help of the inserm neurochemistry researcher, nicole baumann (fig 1), and then with the epidemiology researcher annick alpérovitch (fig 3). one of my main themes was the neuropathology of dementia (particularly degenerative diseases: alzheimer's, parkinson's, steele-richardson-olszewski, fronto-temporal, vascular and 'mixed' dementias as well as the infectious diseases aids and prions). i initiated prospective clinical, genetic, biochemical and pathological studies related to this theme with several clinical teams from the pitié-salpêtrière (notably the departments of professors yves agid and olivier lyon-caen, françois bricaire and marc gentilini), charles foix hospital and other long-stay centers. for instance, we investigated statistical links between the density and distribution of cerebral lesions and the presence, type and intensity of cognitive disorders. other studies aimed to provide new insights into the neuropathology of alzheimer's disease, mixed dementia and aids. development of research techniques in the neuropathology department as soon as i was appointed “assistant” at the neuropathology laboratory of the pitié-salpêtrière hospital, i was determined to develop research techniques of the nervous system. in addition to electron microscopy and histochemistry, we introduced tissue cultures, morphometry, immunohistochemistry and molecular biology (in situ hybridization, pcr, nested pcr, in situ pcr). a confocal microscope was installed (institut fédératif de neurosciences de la salpêtrière). a bsl3 laboratory (p3) was built on the premises of the pitié-salpêtrière faculty of medicine. it enabled the study of diseases that may be caused by prions and other infectious agents under safety conditions. neuropathology and autopsy four main varieties of autopsies are distinguished in france: the forensic autopsy, medical scientific autopsies, the “gift of corpse to the university” for teaching, and the sanitary autopsy. the neuropathology department at la pitié-salpêtrière carried out the largest number of medical-scientific autopsies in france. unfortunately, the number of autopsies requested has fallen steadily since diagnostic techniques, particularly radiological, have improved. this decline is of concern for three reasons. for one, the predictive value of clinical diagnosis (even after using the most modern diagnostic methods) is still poor in some cases, especially when several conditions co-exist. the proportion of unexpected findings that may have modified the patient therapy (20-25 %) has remained unchanged for many years. second, the autopsy is an important piece of the health watch, i.e., of public welfare. third, modern post-genomic research needs tissue samples that often cannot be obtained by other means for ethical reasons. i have also carried out neuropathological examinations of forensic autopsies performed by dominique lecomte at the paris institute of forensic medicine. numerous patients’ associations campaign for making gifts of organs for research purposes easier. we stress the importance of autopsy among medical doctors and caregivers, the prerequisites for restarting autopsy activity and the modifications of regulations and practices that are required [22-23]. neuropathology of stroke at the charles foix laboratory, autopsies were carried out according to a very strict protocol. in addition to the classic autopsy and the removal of the brain and the spinal cord, a special technique was used to allow a complete examination of the cerebral circulation. brought back from england by professor jean-claude gautier, it consisted of whole block removal of the aorta and vessels of the neck, cervical spine, and skull base, including the courses of the carotid and vertebral arteries and the cerebral venous circuits. the block was dissected after formalin fixation, and the results of the macroscopic (and, if necessary, microscopic) examination were recorded on pre-printed diagrams. this allowed very detailed studies of the lesions of the vessels destined for the brain, giving at times surprising results [24-25]. a young neuropathologist's career after my residency, i was successively “assistant”, “chef de travaux-assistant”, “professeur agrégé”, professor of pathology and laboratory head”, then “consultant” and “professor emeritus” at la salpêtrière. this enabled me to become a member of the "club français de neuropathologie”, which became the “société française de neuropathologie." i will always remember the meetings of this society, particularly the one held in marseille at the invitation of dean maurice toga, when i went in the car of raymond escourolle. i was surprised and delighted to discover, in addition to the interesting scientific papers, the famous marseilles creeks from a sailboat maurice toga made available to his guests! i also remember my (unplanned) participation in the 6th international congress of neuropathology which was held in paris (1970), where i discovered with admiration magdeleine bérard-badier, serge brion, edith farkas, jean-emmanuel gruner, gilles lyon, ludo van bogaert, julio oscar trelles and many other international leading neuropathologists. i then became a member (and for a time vice-president) of the european confederation of neuropathological societies [26]. the charles foix, then raymond escourolle laboratory: a second family the daily stays in the laboratory, the professional and often friendly contacts with all the members of the laboratory (head of department, colleagues, assistants, interns, secretaries, technicians, nurses (fig 7)), the biopsy, cytology and autopsy procedures ... quickly led to what might be considered an addiction! working in the laboratory wasn't just a duty or a routine. it was often a pleasure. all the more so because some of the events that took place were puzzling (and sometimes very amusing!). figure 7: group photo of members of the raymond escourolle laboratory in 1994.   from left to right: josiane (nurse for nerve and muscle biopsies), mrs. houet (care assistant for nerve and muscle biopsies), marguerite (polish visitor), hassan husseini (resident), toshiki uchihara (japanese visitor), mireille (hospital agent), pia delaere (belgian visitor), jean-pierre brion (belgian visitor), christelle py (technician), odile russaouen (technician), sylviane (care assistant), marianne candau (technician), serge duckett (australian visitor), jean-jacques hauw (neuropathologist), marie-christine (secretary), yi he (chinese visitor), charles duyckaerts (neuropathologist), karima mokhtari (neuropathologist), eléonore tang, (secretary), danielle seilhean (neuropathologist), pascale lacor (technician), thierry maisonobe (neurophysiologist), sophie camillieri (pathologist), christiane raiton (technical manager), annie laquerrière (neuropathologist from rouen). an exceptional story: ravachol’s head françois-claudius koënigstein (more commonly known as ravachol) was a french anarchist militant, born on october 14, 1859, in saint-chamond (between saint-etienne and lyon in france). having committed several offenses, murders, and attacks, he was guillotined in montbrison, near saint-chamond, on july 11, 1892. as was usual at the time, half his head was preserved in a glass jar containing formalin, and given to “science”, i.e. in practice, to jean-martin charcot’s laboratory. when i was resident, ravachol's head was kept in professor escourolle's office, in a locked cupboard with a glass door, allowing it to be admired (fig 8). a few years later, when i arrived in the neuropathology department in april 1975, police special forces occupied the site as the head of ravachol had been stolen! indeed, raymond escourolle's wardrobe had been broken into and the head had disappeared. within a few days, i understood what had happened from the gossip in the on-call room. some inebriated residents (one of whom worked in andré buge’s department) had stolen the head as a gift for one of their girlfriends (granddaughter of karl marx, who cried out in horror at the sight of this gift!). not knowing what to do, the friends came up with a fantastic idea to make a splash. and this was implemented a few days later: ravachol’s head was found in front of the pantheon, a mausoleum for the remains of distinguished french citizens in the 5th arrondissement of paris, modelled on the pantheon in rome. it was accompanied by a missive: « ceci est la tête de ravachol. elle devrait être au panthéon, et non pas chez escourolle. » (“this is ravachol’s head. it should be in the pantheon, not at escourolle’s”). and the story was confirmed by the concerned intern jean-christophe rufin, who gave up medicine, became a humanitarian (notably in ethiopia), then french ambassador, then novelist and member of the renowned académie française who later recounted it in his book: “un léopard sur le garrot” [27]. figure 8: ravachol’s head (sorbonne university collection, under the inventory number: su.pm.t.2023.0.1.). administrative work and learned societies other strokes of luck also befell me! very early on, i was appointed director of the “experimental and clinical neuropathology” associated training course, then of the “neurology, neuropathology” center of the claude bernard association. i was elected as a member of the board of directors of this association (later renamed the robert debré association for medical research). i was appointed treasurer, a post i still hold. i was then elected a member of the medical committee of pitié-salpêtrière hospital, then representative of the pathologists on the medical committee of assistance publique des hôpitaux de paris. i was then appointed a member of the pathological anatomy section of the french conseil supérieur des universités. professor (now dean) pascal brunet asked me to become a member of the board of directors of the pitié-salpêtrière faculty (i have also been vice-dean). i was also appointed a member of the institut fédératif de neurosciences de la salpêtrière and vice chairman of the specialized scientific commission 6, neurosciences b of the french national medical research institute (inserm). then, for obscure reasons (probably to avoid the election of someone else), i was asked by jean cambier and jean-claude gautier to apply for membership of the french national academy of medicine, which had never had a neuropathologist member before. and for the same obscure reasons, with the help of pathologist louis orcel, i was elected (by just one vote!). this gave me the opportunity to rub shoulders with the leading french and foreign specialists in all areas of human and animal medicine and pharmaceuticals and to help draft numerous press releases and reports for the french authorities. i was later elected treasurer for four years (fig 9) and remain chairman of the "mental health, neuroscience and addiction" commission. i was also given the opportunity to develop, with professor jean-paul tillement, the french academy of medicine's franco-québec relations. figure 9: dressed as an academician, when i was treasurer with professor jean cambier. a large number of countries visited and host of colleagues and friends from all over the world i was fortunate enough to be invited to give conferences in most french regions (including guadeloupe) and many other countries (including argentina, australia, austria, belgium, canada, germany, hungary, israel, italy, japan, peru, spain, sweden, tunisia, the united kingdom and the united states of america (usa)), which gave me the opportunity to make some very interesting visits. i have always been warmly welcomed, and have also had the pleasure of welcoming and working with a large number of foreign colleagues, some of whom came to live in france (in particular from algeria, armenia, australia, belgium, brazil, canada, china, colombia, germany, hungary, india, italy, iran, lebanon, madagascar, mexico, morocco, japan, peru, poland, portugal, romania, russia, spain, switzerland, tunisia, united kingdom and the usa (fig 7)). i remember the friendly welcomes i received in many countries, notably germany, italy, and spain. the relationships that gave me the best memories are those developed with five countries: peru, india, usa, japan, and the united kingdom. in peru, thanks to the invitation and warm welcome of julio oscar trelles [28], a former student of jean lhermitte in paris (and his son luis, who entered the charles foix laboratory), it was a delight to discover one of the wonders of the world: machu pichu. it was also a pleasure to welcome t. vasudev rao, a pathologist from bangalore. during our holidays we were able to lend him the paris flat we were renting, overlooking the seine at the foot of the eiffel tower, which enabled him to spend a few days there with his family. in gratitude, he gave us a very pretty hindu statue, which still hangs in our flat in dole. umberto de girolami, a neuropathologist at brighman and women’s hospital in boston (usa), stayed several times at la salpêtrière and returned regularly to paris as a foreign member of the french académie de médecine. as far as japan is concerned, i had the pleasure of discovering kyoto, nara and tokyo with toshiki uchihara. in the united kingdom, it was queen’s square national hospital and the institute of neurology in london. professors leo wilfried duchen and francisco scaravilli invited me, and came to paris in return, enabling us to make fruitful contacts and even enjoy pleasant trips several years later. it also gave me the opportunity to meet my second wife, chantal hausser, a canadian neurologist, and neurophysiologist who had been a trainee for 3 months at the charles foix laboratory and whom i met again 12 years later in montreal. i had the pleasure of welcoming some of these foreign colleagues (leo duchen, umberto de girolami and toshiki uchihara) in our house in dole or in our country house at the abbey of baume les messieurs, in the french jura, and taking them on a tour of the landscapes and little-known beauties of this area (dole and arbois, the cities of louis pasteur, the cirque de baume-les-messieurs and chateau-chalon, the royal saltworks of arc et senans, lake chalain, the medieval city of nozeroy etc.). a difficult (and risky) diagnosis thanks to my english friends! one of my colleagues and friends at la salpêtrière told me, "i've just admitted to neurosurgery one of my patients, the wife of a very important person. she has a brain tumor. can you let me know what the diagnosis is?” i couldn't forget his request. a few days later, i received a message from a pathologist in the united states asking me, on behalf of the vip, for some slides from this biopsy. i sent them with the agreement of the attending physician. the diagnosis seemed obvious to me but, consulting recent publications on the subject, i also decided to send some white slides to francisco scaravilli. what a surprise it was to receive, a few days later, an invitation to meet a north american delegation in paris! in addition to the american pathologist, the meeting included a quebec pathologist who was supposed to translate english into french, and a series of secretaries and other people whose role was unclear to me. it was decided to hold two "multidisciplinary meetings" first with microscopes, at the raymond escourolle laboratory, then at the department of my colleague, the patient’s attending physician. thanks to the notoriety of francisco scaravilli, i could impose the diagnosis, and only after the opinion of another colleague, a hematologist and oncologist at la salpêtrière, could we start the treatment best suited to the patient’s illness! the new developments in neuropathology whatever the procedure brain biopsy, now performed very often by stereotaxy [29], including very challenging area such as the brain stem [30], muscle and nerve biopsies, skin biopsies for quantifying small nerve fibers, cytological examination of cerebrospinal fluid or autopsy neuropathology techniques have recently exploded and are continuing to develop. for example, stimulated raman scattering microscopy, a label-free optical imaging method performed on smear or frozen-sections, is developing to quickly generate digital hematoxylin-and-eosin-stained-like images for histopathological tissue analysis [31]. it can be used in the operating room to provide fast answers for brain biopsies. of course, artificial intelligence also enters the world of neuropathology. curtis langlotz’s, director of the center for artificial intelligence in medicine and imaging of sanford university, formula about radiologists [32] can be stated as: “artificial intelligence won't replace neuropathologists, but neuropathologists who use artificial intelligence will replace those who don't!” why fall into neuropathology? firstly, it is sometimes a matter of chance, which i would not hesitate to describe as fortunate. secondly, it has many advantages. some of these are very material: for example, it is an exception for a neuropathologist to work on weekends (apart from writing scientific articles or preparing lectures or presentations for scientific conferences) or to be summoned in a hurry! other advantages are more intellectual in nature. they involve, for example, puzzle solving: the cause(s) of any nervous system illnesses the patient may have or have had, or establishing the precise nature of a tumor and its prognosis, using increasingly sophisticated techniques, to which we must constantly adapt. to continue the comparison with games, it's the equivalent of a chess game or bridge, played with increasingly strong partners. artificial intelligence, far from being able to replace the experience and knowledge of the neuropathologist and his numerous coworkers, must now be used as an additional tool. and this requires a great deal of companionship, which is always important in medicine, but even more so in neuropathology, where getting things wrong can have serious consequences. so, neuropathology requires the development of multiple skills in a wide range of clinical and biological disciplines, from pathology and neurology to genetic and molecular biology. it therefore imposes a wide range of complementary training, which often proves fascinating. in addition, this discipline, more than most other medical ones, offers a wide variety of diagnostic, teaching, and research activities. what's more, this rare specialty brings us into contact with many colleagues from different regions and countries, leading to numerous trips and the discovery of other points of view and even civilisations. last but not least, to sum up: it's a fascinating job! by way of conclusion, taking inspiration from rudyard kipling: « if you can dream – and not make dreams your master; if you can think – and not make thoughts your aim; or walk with kings – nor lose the common touch, if neither foes nor loving friends can hurt you, if all men count with you, but none too much; and – which is more – you’ll be a neuropathologist, my son! » acknowledgements i would like to thank danielle seilhean, who provided figures 7 and 8, and chantal hausser-hauw, who reviewed 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stimulated raman scattering (srs) microscopy. plos one 12(5): e0178750. https://doi.org/10.1371/journal.pone.0178750 32. langlost cb (2019) will artificial intelligence replace radiologists? radiol artif intell 1(3):e190058. https://doi.org/10.1148/ryai.2019190058 copyright: © 2023 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. 66th annual meeting of the german society of neuropathology and neuroanatomy (dgnn) meeting abstracts feel free to add comments by clicking these icons on the sidebar free neuropathology 3:20 (2022) meeting abstracts 66th annual meeting of the german society of neuropathology and neuroanatomy (dgnn) meeting abstracts berlin, november 1–5, 2022 submitted: 09 august 2022 accepted: 10 august 2022 published: 10 august 2022   liebe kolleginnen und kollegen, zur 66. jahrestagung der deutschen gesellschaft für neuropathologie und neuroanatomie im rahmen der neurowoche vom 1. bis zum 5. november 2022 begrüße ich sie herzlich in berlin. die letzten jahre haben eine enorme erweiterung der analytischen methodik mit schwerpunkt auf molekularen untersuchungen gebracht. ein großer teil dieser untersuchungen wurde in unseren einrichtungen entwickelt und wird dort erbracht. in der tat hat sich die neuropathologie zu einem motor der neuroonkologischen und neurowissenschaftlichen forschung entwickelt und deutschsprachige neuropathologische institutionen haben wesentlich dazu beigetragen. ganz neue therapien bauen auf diese erkenntnisse auf. dadurch sind wir für die versorgung unserer patienten wichtiger denn jeher. deswegen sehe ich einen großen und zunehmenden bedarf dem wir neuropathologen nachkommen müssen. alle schwerpunkte unseres faches sind hiervon betroffen, die gehirntumordiagnostik, die neurodegenerativen erkrankungen, entzündung und erkrankungen der muskeln und der nerven. wir arbeiten eng mit unseren kollegen aus der neuroonkologie, neuropädiatrie, neurologie neurochirurgie und neuroradiologie zusammen. der interdisziplinäre austausch hat einen hohen stellenwert und wir freuen uns deshalb besonders, dass unsere jahrestagung in diesem jahr wieder im rahmen der neurowoche stattfindet, was die kommunikation und den wissenstransfer über die fächergrenzen beflügelt. dieses jahr wollen wir besonders die jungen neuropathologen und neuropathologinnen in den vordergrund stellen. sie sollen unser fach als lebendig und besonders zukunftsfähig erleben. von ihnen erwarten wir die dynamik, den einsatz und den ideenreichtum, der die neuropathologie in den nächsten jahren noch weiter zu einer zentralen querschnittsplattform für die neurofächer machen wird. wir haben einen kongressstrang mit wissenschaftlichen sitzungen am donnerstag, freitag und samstag ausgerichtet. sie dürfen vorträge mit jungen neuropathologischen expertinnen und experten sowie von jungen nachwuchswissenschaftlerinnen und nachwuchswissenschaftlern erwarten. ich freue mich auf lebhafte diskussionen und spannende interdisziplinäre debatten! ihr prof. dr. andreas von deimling universitätsklinikum heidelberg neuropathologie   https://doi.org/10.17879/freeneuropathology-2022-4366 keywords: german society of neuropathology and neuroanatomy, dgnn, meeting abstracts contents 1. new diagnostic methods 1.01 methylthioadenosine phosphorylase immunostaining as a surrogate marker for cdkn2a/b homozygous deletion in gliomas 1.02 artificial intelligence in morphomolecular analysis of glioblastoma 1.03 histological and molecular correlates of tspo labelling in human brain tissue 1.04 towards swift and accessible precision cns tumour diagnostics using third generation sequencing and deep transfer learning 1.05 retinal pathology as potential biomarker of symptom severity and impairment in patients with stiff person syndrome 1.06 deep learning based cerebrospinal fluid diagnostics 1.07 proteomic profiling of idh-mutant gliomas identifies hip1r/vimentin as surrogate markers for 1p/19q codeletion and enables prediction of chromosomal copy number variations 1.08 cellular digital neuropathology 2. neurooncology 2.01 using spatial transciptomics for diagnostic analysis of glioma 2.02 single cell dna amplicon sequencing reveals order of mutational acquisition in traf7 and klf4 or akt1 co-mutated meningiomas 2.03 alterations in ptpn11 and other noonan syndrome associated map-kinase signaling pathway genes accumulate in histopathologically atypical ganglioglioma with adverse postsurgical outcome 2.04 molecular refinement of pilocytic astrocytoma in adult patients 2.05 exploration of cellular origins and therapeutic targets by modeling high grade pediatric glioma of the mycn subclass in mice 2.06 the genomic and transcriptional landscape of primary central nervous system lymphoma 2.07 pmolecular mechanisms of therapy resistance in malignant melanoma brain metastasis 2.08 a peripheral nerve sheath tumor syndrome caused by postzygotic erbb2 mutations 2.09 cns-tumor patients within the impress-norway trial: first year experiences 3. neurodegeneration 3.01 cnn-supported quantification of fat compartments at abdominal mri applied to als patients 3.02 neurodegenerative iron storage disease (neuroferritinopathy) caused by a novel frameshift mutation in the ferritin heavy chain gene (fth1 c.341-342del) 3.03 the contribution of late-nc to neuron loss, granulovacuolar degeneration and dementia in alzheimer’s disease 3.04 the role of c3 inhibition in an ipsc nmj model of neuroinflammation 3.05 fast-track procedure for the neuropathological assessment of neurodegenerative diseases 3.06 neurodegeneration in hsan1 due to atl1 (gly66gln) mutation is associated with defective erprotein quality control and compromised autophagy 3.07 single-nucleus chromatin accessibility profiling in four-repeat tauopathies 3.08 application of a human stem cell transplantation model of alzheimer’s disease to examine disease-associated changes at a single cell level in vivo 4. neuroinflammation 4.01 pathological and genetic characterization of jc virus encephalopathy with an eleven-year-long disease course 4.02 reduction of oligodendrocyte populations in patients with late-onset multiple sclerosis 4.03 schwann cell remyelination is a salient feature of spinal nmo with neuroprotective potential 5. muscle / nerve 5.01 molecular profiling of skeletal muscle in infantile, juvenile and adult patients with pompe disease 5.02 expression of immune regulating proteins in skeletal muscle of different idiopathic inflammatory myopathies (iim) subtypes 5.03 long term safety and efficacy outcomes for x-linked myotubular myopathy (xlmtm) with gene replacement therapy, resamirigene bilparvovec (aspiro): preliminary results from cohort 1 in aspiro, a phase 1/2/3 study 5.04 lymphotoxin-driven chronic mucle inflammation interdepends with impaired autophagy, self-perpetuates and models inclusion body myositis in mice 5.05 novel form of congenital myopathy caused by bi-allelic mutations in uncoordinated mutant number-45 myosin chaperone b 6. free topics 6.01 deep genotype-phenotype analysis of focal cortical dysplasia type 2 differentiates between a gator-positive autophagy altered subtype 2a and mtor-positive migration deficit subtype 2b 6.02 age-dependent increase of perineuronal nets in the human hippocampus of patients with and without temporal lobe epilepsy 6.03 vakuolisierung der dura als nicht-lymphassoziierte veränderung 6.04 moghe with or without slc35a2 brain somatic mutations reveal a common phenotype of oligodendroglial regeneration and remyelination     1. new diagnostic methods 1.01 free neuropathol 3:20:5 methylthioadenosine phosphorylase immunostaining as a surrogate marker for cdkn2a/b homozygous deletion in gliomas theoni maragkou1, ekkehard hewer1,2, erik vassella1, baptiste pasquier1, stefan reinhard1, maja neuenschwander1, philippe schucht3 1 university of bern, institute of pathology, institute of pathology, bern, switzerland 2 lausanne university hospital, institute of pathology, institute of pathology, lausanne, switzerland 3 inselspital, bern university hospital, dept. of neurosurgery, dept. of neurosurgery, bern, switzerland background: homozygous deletion (hd) of the cdkn2a/b locus has emerged as an unfavorable prognostic marker in diffuse gliomas, both idh-mutant and idh-wildtype. testing for cdkn2a/b deletions can be performed by a variety of approaches, including copy number variation (cnv) analysis based on genome-wide dna methylation data, next generation sequencing (ngs) or fluorescence in-situ hybridization (fish), but questions remain regarding the accuracy of and correlation between different testing modalities. aims: in this study, we assessed the utility of s-methyl-5'-thioadenosine phosphorylase (mtap) and cellular tumor suppressor protein pl61nk4a (p16) immunostaining as surrogate markers for cdkn2a/b hd in gliomas, across different histological tumor grades and idh mutation status. question: are mtap and p16 accurate surrogate markers for cdkn2a/b hd in gliomas? methods: idh1 r132h, atrx and mtap immunohistochemistry was performed on tissue microarrays (tmas) of 301 diffuse gliomas. survival analysis was performed to assess the prognostic value of mtap. furthermore, 100 consecutive cases of gliomas were collected, in order to correlate mtap and p16 expression with the cdkn2a/b status in cnv plot of each tumor. results: mtap deficiency was associated with shortened survival in idh-mutant astrocytomas (n=75; median survival 61 vs. 137 months; p<0.0001), idh-mutant oligodendrogliomas (n=59; median survival 41 vs. 147 months; p<0.0001) and idh-wildtype gliomas (n=117; median survival 13 vs. 16 months; p=0.011). in a cohort of 100 gliomas, complete loss of mtap and p16 by immunohistochemistry was 100 % and 90 % sensitive as well as 97 % and 89 % specific for cdkn2a/b hd, respectively, as identified on cnv plot derived from genome-wide dna methylation analysis. two cases with mtap and p16 loss of expression did not demonstrate cdkn2a/b hd in cnv plot, however fish analysis confirmed the hd for cdkn2a/b. conclusions: mtap immunostaining is an important complement for diagnostic work-up of gliomas, because of its excellent correlation with cdkn2a/b status, robustness, rapid turnaround time and low-costs, while p16 immunostaining represents a good alternative for detecting cdkn2a/b hd. discovering cdkn2a/b hd through mtap and/or p16 immunohistochemistry seems to be a more accurate method than the cnv analysis derived from genome-wide dna methylation data.   1.02 free neuropathol 3:20:6 artificial intelligence in morphomolecular analysis of glioblastoma stephan balogh1, karen brengmann1, jannik sehring1, thomas kauer1, gudrun schmidt1, till acker1, daniel amsel1, hildegard dohmen1 1 institute of neuropathology, justus liebig university giessen, giessen, deutschland background: glioblastoma is the most common primary malignant brain tumor and has a poor prognosis despite existing treatment options. it is characterized by its inhomogeneous appearance and molecular heterogeneity. a detailed diagnosis is desirable, especially with regard to the emerging personalized medicine to guide treatment decisions. the increasing application of whole slide image scanners enables the digitalization of histopathological slides, collected in clinical routine diagnostics, into high-quality images that offer new possibilities for computer-aided precision diagnostics. objective: the goal of our work-in-progress project is to curate a high-resolution dataset with annotations to train an artificial intelligence to independently recognize characteristic structures of glioblastoma tissue sections, such as tumor area, vascular proliferation and necrosis. using these ai-based algorithms, we aim to gain new insights that could help refine the characterization of glioblastomas by correlating morphological information with available clinical and molecular data. methods: initially, clinical and molecular information on 200 glioblastoma patients was gathered. the corresponding hematoxylin-eosin-stained histopathological slides were then digitized using a high-throughput whole slide image scanner (hamamatsu nanozoomer s360). the annotation of key features in the images was divided into two distinct phases. prior to the actual annotation phase, two md students completed a training phase with a small batch of images (n=10) in order to get hands-on experience with the annotation software and difficult issues (qupath version 0.3.0). the ground truth was determined by an experienced neuropathologist. evaluation: the interand intraobserver variability of the two students will be evaluated with regard to the learning progress (e.g. accuracy, classification of a tissue area) and differences between the annotations of the two students, derived from subjective assessment. in the second part, correlations between morphological information (vascular proliferation and necrosis with pseudopalisading) and clinical and molecular information will be examined. these annotated slides will serve as training and test sets for in-house ai-based predictions. perspective: our data will be used as a resource for an in-house developed app that will serve as a learning solution for medical students, but also as a crowdsourcing platform for the annotation of features on small patches of whole slide images. in addition, the curated high-resolution data set will serve as input for further internally developed ai algorithms to support tumor diagnosis and therapy decisions.   1.03 free neuropathol 3:20:7 histological and molecular correlates of tspo labelling in human brain tissue lorraine weidner1,2, franziska dekorsy3, stefanie quach4, viktoria ruf5, julia lorenz1,2, peter hau2,6, jörg-christian tonn4,5, peter bartenstein3,7, matthias brendel3,7, nathalie l. albert3,7, markus j. riemenschneider1,2 1 regensburg university hospital, department of neuropathology, regensburg, deutschland 2 regensburg university hospital, wilhelm sander neuro-oncology unit, regensburg, deutschland 3 university hospital of munich, lmu munich, department of nuclear medicine, münchen, deutschland 4 university hospital of munich, lmu munich, department of neurosurgery, münchen, deutschland 5 lmu munich, center for neuropathology and prion research, münchen, deutschland 6 regensburg university hospital, department of neurology, regensburg, deutschland 7 german cancer research center (dkfz), german cancer consortium (dktk), partner site munich, heidelberg, deutschland background: tspo is frequently upregulated in neoplastically transformed tissues, including glioblastomas. this may be of use for pet imaging of brain tumors. however, due to the heterogeneity of cell populations that could contribute as tspo-pet signal source in gliomas, the imaging biomarker interpretation may be challenging. aim: we therefore dissect tspo labelling in connection with the underlying histopathological and molecular features in biopsy samples from glioma patients. question: to decipher the underlying histopathological and molecular features of tspo-pet enrichment. methods: we aim to collect a total of 75 glioma patients all characterized by mri, tspoand fet-pet. tspo protein expression and expression of cell differentiation markers are assessed immunohistochemically on consecutive sections and by multiplex stains. rna isolation has been optimized to perform rna-seq on biopsy samples and to compare regions of high and low tspo-pet signal/protein expression. to identify relevant hallmarks and go terms we use deseq2 followed by fuma and reactome as well as gsea with normalized counts. furthermore, exceeding the biopsy study we stain tissue microarrays for tspo that cover a broader spectrum of human brain pathologies as well as a spectrum of non-neoplastic tissues from different brain regions. to better understand tspo regulation, we consult data of large patient cohorts from the tcga database, perform in vitro epigenetic investigations on azaor tsa-treated patient-derived glioblastoma cell lines and analyze the tspo promoter in gliomas by direct bisulfite sequencing. results: we report the interim analysis of the glioma patients that have been included and fully histologically characterized in the biopsy study so far. our results suggest that (apart from microglia and macrophages) the glial tumor cells relevantly contribute to the overall tspo signal in these patients. rna-seq analyses comparing tspo high and low regions (both by pet and protein expression) indicate three tspo-dependent functional clusters, i.e apoptosis/dna repair, extracellular matrix organization and immune system. furthermore the tissue microrarrays show heterogeneity of tspo expression between different brain pathologies and non-neoplastic brain regions. bringing this information together with tspo-pets from respective patients/brain regions will generate a map of tspo expression in healthy and diseased brain for clinical use. finally, our epigenetic investigations suggest that a loss of tspo methylation in high-grade neoplasms may mechanistically contribute to the tspo overexpression observed in these tumors. conclusion: taken together, our approach of integrating histological, molecular and imaging data will provide unique insights into tspo-pet enrichment patterns and will help to better understand and to comprehensively describe the clinical relevance of this novel imaging biomarker.   1.04 free neuropathol 3:20:9 towards swift and accessible precision cns tumour diagnostics using third generation sequencing and deep transfer learning areeba patel1,2, helin dogan1,2, alexander wolfgang jung3, zaira seferbekova4, alexander payne5, natalie schoebe1,2, elena krause1,2, michael ritter1,2, daniel schrimpf1,2, damian stichel1,2, stefan hammelmann1,2, christina blume1,2, philipp euskirchen6, violaine goidts7, martin sill8,9, stefan pfister8,9, matthew loose5, wolfgang wick10,11, andreas von deimling1,2, david jones8,12, matthias schlesner13, moritz gerstung3,4, felix sahm1,2 1 german cancer research center, clinical cooperation unit neuropathology, heidelberg, deutschland 2 university hospital heidelberg, neuropathology, heidelberg, deutschland 3 european molecular biology laboratory, european bioinformatics institute embl-ebi, hinxton, united kingdom 4 german cancer research center, division of artificial intelligence in oncology, heidelberg, united kingdom 5 university of nottingham, deepseq, school of life sciences, nottingham, united kingdom 6 charité-universitätsmedizin berlin, neurology, berlin, deutschland 7 german cancer research center, brain tumor translational targets, heidelberg, deutschland 8 hopp children's cancer center (kitz), heidelberg, deutschland 9 german cancer research center, division of pediatric neurooncology, heidelberg, deutschland 10 german cancer research center, clinical cooperation unit neurooncology, heidelberg, deutschland 11 national center for tumor diseases, department of neurology and neurooncology program, heidelberg, deutschland 12 german cancer research center, pediatric glioma research group, heidelberg, deutschland 13 augsburg university, biomedical informatics, data mining and data analytics, augsburg, deutschland background: molecular markers are now unequivocally a requirement for integrative brain tumour diagnostics. the 2021 who classification of central nervous system (cns) tumours substantially increases the set of genes required in routine evaluation, and significantly increases the relevance of dna methylation analysis in the diagnostic process. owing to extensive setup costs and batch requirements, smaller labs and clinics might not be able to deliver molecular results for prompt clinical decisions. deep neural network architectures have been shown to predict whole genome duplications, driver gene mutations, transcriptomic associations, immune cell localisation and prognostic effects from h&e slides. third generation sequencing has enabled sequencing longer reads, shorter library preparation protocols, ability to call base modifications natively, real time analysis, and low-cost, portable devices. aims: to make precision diagnostics accessible, we introduce an integrated computational histopathology and third generation sequencing workflow for real-time cns tumour molecular diagnostics. methods: we present cns-chipa multi-task lightweight deep transfer learning model to predict key molecular alterations, methylation classification and survival from h&e stained cns tumour slides. the model provided basic information regarding the tumor type instantly. for further detail (e.g. variant of idh alteration) and subtyping, we subsequently used the predictions to formulate a custom panel for each patient. targeted sequencing and analyses were performed using rapid-cns2a custom neurooncology third generation sequencing pipeline for parallel copy-number profiling, mutational and methylation analysis that is highly flexible in target selection, requires no additional library preparation for targeting, runs efficiently on single samples, and can be initiated upon receipt of frozen sections. rapid-cns2 leverages adaptive sampling through readfish and was run using a portable minion or gridion device. results: we show that cns-chip can predict a multitude of key pathognomonic alterations (eg. idh mutation, 7 gain/10 loss, etc.) using a single model with reasonable accuracy. using a personalised panel for targeted sequencing of each sample enabled smaller target sizes, thus reducing sequencing time to an average of 24 hours. cns-chip predictions were compared to their respective rapid-cns2 results and corresponding conventional data (ngs panel sequencing and epic array analysis). we demonstrate our workflow on prospective diagnostic samples received by the department of neuropathology, university hospital heidelberg. the average turnaround time per sample was 48h. conclusions: our workflow harnessing histology-based molecular predictions to instruct targeted sequencing can be set up with low initial investment, reduces hands-on time and has the potential to facilitate reporting of integrated molecular diagnostic results in less than 48h. cns-chip combined with rapid-cns2 thus aims to make cns molecular diagnostics affordable and accessible to smaller hospitals and labs especially in lowand middle-income countries.   1.05 free neuropathol 3:20:11 retinal pathology as potential biomarker of symptom severity and impairment in patients with stiff person syndrome sabine seefried1, claudia sommer1 1 ukw, würzburg, deutschland background: stiff-person syndrome (sps) is a rare chronic autoimmune disease characterized by painful spasms and rigidity, predominantly of the axial and lower extremity muscles. autoantibodies have been reported in 80% of cases, disrupting the function of the inhibitory neurotransmitters gaba and glycine, and leading to overexcitability of the neuromuscular system and psychological comorbidities. since the retina is highly enriched with gabaergic neurons, retinal pathology may occur in sps, especially in the ganglion cell and inner plexiform layer (gcipl) of the retina, which contains most of the retinal gabaergic neurons. study aims: we aimed to detect potential abnormalities in the retina in sps patients and correlate these with symptom severity and impairment. hypotheses: we hypothesized a lower retinal thickness in sps patients in comparison to normal healthy controls and patients with diabetes and furthermore correlations between gad antibody levels and findings in retina layer thickness and correlations with severity of the disease. methods: 24 gad positive sps patients (17 female; 7 male; aged 53 ±7; 9 with diabetes) received a clinical examination and optical coherence tomography (oct) for retina layer thickness. blood was drawn for autoantibody detection. data were compared to a matched healthy cohort and a matched patients group with diabetes but no diabetic polyneuropathy. the severity of sps symptoms and impairment was assessed on the basis of the ability to walk: free walking, use of a crutch, use of two crutches, walking on the rollator or sitting in a wheelchair. results: oct showed lower retinal thickness in gcipl and lower average macular thickness (amt) in the sps patients (gcipl: 73.34±5,3; amt: 302.98 ±12,1) in comparison to healthy controls (gcipl: 76.01 ± 4,2; amt: 311.76±13,4). comparison of the 15 sps patients without diabetes with the total number of 25 sps patients showed no difference between these results. the control patients with only diabetes had no abnormalities in the oct results compared to the healthy controls, indicating that the atrophy of gcipl and aml is likely to be caused exclusively by the sps and not by diabetes. there was a positive correlation between walking ability and gcipl thickness in sps patients; higher gcipl thickness was associated with better walking ability (r² = 0,92). conclusion: this study indicates retinal involvement in sps. oct might be useful as a complementary diagnostic tool in sps, and retinal layer thickness measurements might be developed as a non-invasive biomarker for disease progression.   1.06 free neuropathol 3:20:12 deep learning based cerebrospinal fluid diagnostics leonille schweizer1,2, philipp seegerer3, hee-yeong kim4, rené saitenmacher3, amos münch1, liane barnick1, anja osterloh1, carsten dittmayer1, ruben jödicke1, debora pehl1, annekathrin reinhardt5, klemens ruprecht6, annika k wefers7, patrick harter8, ulrich schüller7, frank l heppner1, maximilian alber3, klaus-robert müller3, frederick klauschen9 1 institute of neuropathology, charité – universitätsmedizin berlin, corporate member of freie universität berlin, humboldt-universität zu berlin and berlin institute of health, berlin, berlin, deutschland 2 german cancer consortium (dktk), partner site berlin, and german cancer research center (dkfz), berlin, deutschland 3 machine-learning group, department of software engineering and theoretical computer science, technical university of berlin, berlin, deutschland 4 systems medicine of infectious disease, robert koch institute, berlin, deutschland 5 department of neuropathology, university hospital heidelberg, heidelberg, deutschland 6 department of neurology, charité university medicine berlin, berlin, deutschland 7 institute of neuropathology, university medical center hamburg-eppendorf, hamburg, deutschland 8 neurological institute (edinger institute), goethe university, frankfurt, deutschland 9 institute of pathology, ludwig-maximilians-universität münchen, münchen, deutschland background: the analysis of cerebrospinal fluid (csf) specimens is essential for the diagnostic workup and clinical management of neurological patients and relies on differential cell typing. because blood cell cytometers are unable to identify diagnostically relevant cell types in csf samples, the current gold-standard is based on microscopic examination by specialized technicians and neuropathologists. manual differential cell count is time-consuming, labor-intensive and subjective. we therefore set out to compile a real-world csf dataset including all diagnostically relevant cell types to train a robust algorithm for cell type differentiation with the potential to solve complex diagnostic tasks. methods: we therefore developed an image analysis approach based on expert annotations of 127.455 digitized csf objects from 78 patients corresponding to 15 clinically relevant categories and trained a multiclass convolutional neural network (cnn). we applied explainable artificial intelligence (xai) methods to elucidate the most relevant image pixels for cnn predictions and compare pattern recognition to humans. we further developed a new data partitioning strategy for further machine learning projects. to assess the realistic usefulness in diagnostic practice, we validated the cnn-based approach by comparing its performance to that of seven board certified neuropathologist from different academic institutions. results: the cnn classified the 15 categories with high accuracy (mean auc 97.3%). by using xai, we could demonstrate that the cnn identified meaningful substructures in csf cells recapitulating human pattern recognition. we validated the diagnostic performance of the cnn by comparing the predictions of 511 cells selected from 12 different csf samples to seven board-certified neuropathologists blinded for clinical information. inter-rater agreement between the cnn and the ground truth was non-inferior (krippendorff’s alpha 0.79) compared to the agreement of seven human raters and the ground truth (mean krippendorff’s alpha 0.72, range 0.56-0.81). the cnn assigned the correct diagnostic label (inflammatory, hemorrhagic or neoplastic) in 10 out of 11 clinical samples compared to 7-11 out of 11 correctly labeled csfs by human raters. similar to four human raters, the cnn misclassified single highly activated b-cells as cancer cells in two samples, but indicated reduced confidence by low predicted probability vectors for the difficult cases. conclusions: our approach not only provides the basis to overcome current limitations in automated cell classification for routine diagnostics, but also demonstrates how a visual explanation framework can connect machine decision-making with cell properties and thus provide a novel versatile and quantitative method for investigating csf manifestations of various neurological diseases.   1.07 free neuropathol 3:20:14 proteomic profiling of idh-mutant gliomas identifies hip1r/vimentin as surrogate markers for 1p/19q codeletion and enables prediction of chromosomal copy number variations marius felix1, dennis friedel1, ashok kumar jayavelu2, katharina filipski3, anne-kathrin reinhard1, uwe warnken4, damian stichel1, daniel schrimpf1, andrey korshunov1, yueting wang1, tobias kessler4, nima etminan5, andreas unterberg6, christel herolod-mende6, laura heikaus7, felix sahm1, wolfgang wick4, patrick n. harter3, andreas von deimling1, david e. reuss1 1 department of neuropathology, institute of pathology, heidelberg, deutschland 2 clinical cooperation unit pediatric leukemia, german cancer research center (dkfz), heidelberg, deutschland 3 institute of neurology, edinger institute, heidelberg, deutschland 4 clinical cooperation unit neurooncology, german consortium for translational cancer research (dktk), german cancer research center (dkfz), heidelberg, deutschland 5 department of neurosurgery, mannheim, deutschland 6 department of neurosurgery, heidelberg, deutschland 7 bruker gmbh, bremen, deutschland background: idh-mutant gliomas are a common but heterogenous group of diffuse gliomas. chromosomal copy number variations (cnv) are a hallmark of many different types of cancer and 1p/19q codeletion is mandatory to differentiate “astrocytoma, idh mutant” from “oligodendroglioma, idh-mutant and 1p/19q-codeleted”. currently, loss of nuclear atrx is the only surrogate marker for an 1p/19q-wildtype status accepted by who. unmet diagnostic needs are improved surrogate markers for 1p/19q codeletion in atrx retaining gliomas and a rapid determination of cnvs in general. aims: we aimed at the identification of protein-level surrogate markers for 1p/19q codeletion suitable for immunohistochemical assays as well as proteomic signatures associated with chromosomal alterations in general. methods: we used mass-spectrometry (ms) based proteomics to analyze idh-mutant gliomas pre-characterized by dna methylation profiling. a discovery series containing 35 fresh frozen (ff) and 72 formalin fixed and paraffin embedded (ffpe) tumors were analyzed and potential biomarkers for 1p/19q codeletion were identified. a subsequent validation series consisting of 50 oligodendrogliomas and 50 astrocytomas was evaluated using immunohistochemistry to confirm potential biomarker identifications based on proteomic discoveries. furthermore, an additional validation cohort of 69 idh-mutant gliomas was stained and evaluated in a separate institution. results: proteomic data from ff and ffpe tissues were comparable. highly specific protein patters were identified, which could distinguish between oligodendroglioma and astrocytoma. oligodendrogliomas showed high hip1r and low vimentin (vim) staining intensities and astrocytomas low hip1r and high vim staining intensities. blinded evaluation of the validation cohort revealed a specificity of 100% and sensitivity 90-94% between two observers for 1p/19q prediction. combined evaluation with atrx increased sensitivity to 96%. an additional verification cohort stained and evaluated in a separate institution revealed similar prediction performances. further analyses revealed that a high proportion of the differentially regulated proteins between astrocytoma and oligodendroglioma are coded on the 1p and 19q chromosome arms. by generating virtual protein abundance means from unregulated chromosome arms, chromosomal protein ratios (cprs) were calculated, which helped predict copy number variations, showing high correlation with cnv plots from genome wide dna methylation profiles. conclusions: ms based analysis of ffpe tissue highly correlates with ff tissue, allowing in depth differential proteomic profiling. proteomics enables the discovery of new biomarkers and has great potential for the future of brain tumor diagnostics. immunohistochemistry for hip1r, vim and atrx can predict 1p/19q status with high specificity and sensitivity. cprs are a promising tool for the rapid proteome-based determination of chromosomal copy number variations.   1.08 free neuropathol 3:20:16 cellular digital neuropathology jonas franz1, christine stadelmann1 1 institut für neuropathologie, universitätsmedizin göttingen, göttingen, deutschland background: neuropathology is traditionally based on histological analysis of tissue. aside molecular pathology also the introduction of digital microscopy is changing the working environment of neuropathologists. digital pathology comes with modern algorithms for image quantification. mostly these algorithms are capable of solving sophisticated classification or segmentation problems. while classification is often applied to whole disease entities and segmentation is used to find, e.g., immunpositive areas we tried to adopt the algorithms to the traditional concept of „cellularpathologie“ as defined by r. virchow. aims: we aimed at optimizing existing image analysis algorithms and concepts specifically to the need of classification of cells on whole slide images. question: the main question was to determine a workflow which integrates on the one hand the needs of neuropathologists to steer the analysis and on the other hand to implement even advanced computer technology, e.g., with deep learning-based analyses. method: our main method focused on supervised deep learning to classify single cells in immunfluorescence stainings based on existing nuclear segmentation algorithms. result: as a result we deployed locally an image classification server for cell annotation by neuropathological experts without background in computer science in combination with an open microscopy (omero) server. manually annotated images were used to train various classification algorithms in combination with data augmentation and other techniques to optimize performance. after model selection we could reach almost human performance (>98% accuracy with <2-3% false positive rate) in an exemplary project of microglial cell classification stained by iba1. conclusion: we conclude that this concept of image analysis generalizes to various sorts of immunfluorescence stainings and might thus help to elevate the single cellular analysis to a whole tissue-based analysis with millions of cells.   2. neuroonocology 2.01 free neuropathol 3:20:17 using spatial transciptomics for diagnostic analysis of glioma michael ritter1,2, christina blume1,2, areeba patel1,2, philipp sievers1,2, helin dogan1,2, christel herold-mende3, wolfgang wick4,5,6, andreas von deimling1,2, felix sahm1,2 1 university hospital heidelberg, department of neuropathology, heidelberg, deutschland 2 german cancer research center (dkfz), german consortium for translational cancer research (dktk), clinical cooperation unit neuropathology, heidelberg, deutschland 3 university hospital heidelberg, department of neurosurgery, heidelberg, deutschland 4 german cancer research center (dkfz), german consortium for translational cancer research (dktk), clinical cooperation unit neurooncology, heidelberg, deutschland 5 university hospital heidelberg, neurology clinic, heidelberg, deutschland 6 german cancer research center (dkfz), national center for tumor diseases (nct), department of neurology and neurooncology program, heidelberg, deutschland background: the lack of specific in-situ markers of idh-wildtype glioblastoma makes it hard to distinguish between infiltrating tumour cells and reactive tissue. determination of copy number variations (cnv) of chromosomes may assist in this diagnostic challenge, but often requires a large amount of tissue, which is not always available. especially for stereotactic biopsies the available material is often quite limited. aims: in our study we wanted to test the feasibility of using spatial transcriptomics to distinguish between invading tumour cells and reactive/adjacent tissue and if it is possible to generate useful data like cnvs from small tissue fragments. methods: we applied spatial transcriptomics on 12 ffpe gbm samples indicating reactive tissue or an infiltration zone and 4 stereotactic biopsies. we determined the cnv for all samples and used a single cell dataset of infiltrating tumour cells (darmanis et al., 2017) to map the different cell types onto the tissue. results: the mapping of the tumour cells onto the sections clearly distinguished the tumour from the adjacent tissue and also allowed to further distinguish between majorly reactive tissue and tissue with a high number of infiltrating tumour cells. nevertheless, mapping on single cell level resolution requires higher resolution methods. both mapping by chr. 7 gain and chr. 10 loss and expression-based mapping produced similar results. using spatial transcriptomics on stereotactic biopsies we were able to determine the major cnvs from a 5 µm thick tissue section with a 1 mm diameter. conclusion: in summary we prove the feasibility to identify the infiltration zone and distinguish from reactive tissue using spatial transcriptomics, and we were able to derive cnv from very small tissue fractions. this can be leveraged especially if immunohistochemical stainings are not informative or too little tissue is left for the determination of cnv profiles.   2.02 free neuropathol 3:20:18 single cell dna amplicon sequencing reveals order of mutational acquisition in traf7 and klf4 or akt1 co-mutated meningiomas helin dogan1, christina blume1, areeba patel1, gerhard jungwirth2, miriam ratliff3, ralf ketter4, wolfgang wick5, christel herold-mende2, david reuss1, andreas von deimling1, felix sahm1 1 clinical cooperation unit neuropathology, german cancer consortium (dktk), german cancer research center and dept. of neuropathology, university hospital heidelberg, heidelberg, deutschland 2 dept. of neurosurgery, university hospital heidelberg, heidelberg, deutschland 3 dept. of neurosurgery, university hospital mannheim, mannheim, deutschland 4 dept. of neurosurgery, university hospital saarland, homburg, deutschland 5 dept. of neurology and neurooncology program, national center for tumor diseases, university hospital heidelberg, heidelberg, deutschland background: most meningiomas carry mutations in the tumor suppressor neurofibromatosis gene 2 (nf2) on chromosome 22q, while nf2-wildtype meningiomas account for about one third of all. in non-nf2-mutated cases, smo, polr2a, pik3ca, akt1 and klf4 mutations, the latter both typically with traf7 mutations, have been described. the combination of akt1 and klf4, respectively, with traf7 is intriguing: traf7/akt1 co-mutations are associated with meningothelial histology and basal localization, while traf7/klf4 co-mutations are highly specific for secretory meningioma without any predominant localization. since bulk molecular profiling indicates a step-wise mutational acquisition, the mutational sequence, whether the alteration in traf7 or in akt1/klf4 occurs first, has remained elusive. methods: single-cell sequencing technologies have allowed direct insight into the clonal architecture and complexity of thousands of individual cells. after evaluation of a patient with two independent meningiomas having identical somatic traf7 mutation but separate akt1/klf4 hotspot mutation, variant allele frequencies (vafs) of 62 retrospectively collected meningiomas carrying either coor single-mutations in traf7 and/or akt1 or klf4 were compared using bulk hybrid-capture panel sequencing data. additionally, a custom tumor panel comprising 392 amplicons covering 28 genes as well as the tert promoter was used along with the amplicon-based tapestri technology for single cell dna sequencing. genotype clustering analysis was finally performed to reveal the order of mutational acquisition in our cohort of traf7mut/akt1mut and traf7mut/klf4mut meningiomas (n=7). results: looking at mutational co-occurrence in bulk data, mutations assigned with higher vafs, unless explained by copy number changes, are thought to be acquired earlier than those with lower vafs. our bulk data of 28 co-mutated cases showed no significant difference in bulk-measured vafs, suggesting there was no major gap between the two time points of mutational acquisition. however, the majority of single-mutated cases (21/36) harbored mutations in traf7, while the others were either only akt1 (n=12) or klf4 (n=3) mutant. while it remains impossible to delineate clonal architecture from bulk data, our single cell data allowed grouping of cells into clonal populations. a total of 875,000 cells from 7 samples were prepared resulting in a median throughput of 2315 cells per sample and a median sequencing coverage of 105 reads per cell per amplicon. our data revealed three subclones in each sample: one wildtype clone (potentially stroma cells), one clone carrying a single mutation in traf7 (detected for 6/7 samples) and another clone harboring the co-mutations in traf7 and klf4 or akt1. conclusions: our findings strengthen the hypothesis that in traf7mut/akt1mut and traf7mut/klf4mut meningiomas, the mutation in traf7, which can occur throughout the wd40 domain of the protein, is acquired in an earlier stage than the hotspot mutation in akt1 or klf4. this study shows, that single-cell technologies on dna are useful in elucidating clonal architecture and phylogenetic trees. although single-cell dna sequencing in particular is associated with technical challenges such as false positive variant calling and allelic dropouts, high numbers of recovered cells as well as high-quality sequencing allow conclusive information on cellular zygosity and a robust analysis of mutational acquisition.   2.03 free neuropathol 3:20:20 alterations in ptpn11 and other noonan syndrome associated map-kinase signaling pathway genes accumulate in histopathologically atypical ganglioglioma with adverse postsurgical outcome lucas hoffmann1, roland coras1, katja kobow1, javier lopez-riviera2,3,4, costin leu3,4,5,6, dennis lal3,4,5,6, peter nürnberg6, christian g. bien7, thilo kalbhenn7, markus müller7, hajo hamer8, sebastian brandner9, karl rössler9,10, samir jabari1, ingmar blümcke1 1 department of neuropathology, universitätsklinikum erlangen, fau erlangen-nürnberg, partner of the european reference network (ern) epicare , erlangen, deutschland 2 department of molecular medicine, cleveland clinic lerner college of medicine, case western reserve university, cleveland, united states 3 genomic medicine institute, lerner research institute, cleveland clinic, cleveland, oh 44195, united states 4 charles shor epilepsy center, neurological institute, cleveland clinic, cleveland, united states; 5 stanley center for psychiatric research, broad institute of harvard and m.i.t, cambridge, ma 02142, united states 6 cologne center for genomics (ccg), medical faculty of the university of cologne, university hospital of cologne, cologne, germany 7 department of epileptology (krankenhaus mara), medical school, bielefeld university, bielefeld, germany 8 epilepsy center, universitätsklinikum erlangen, fau erlangen-nürnberg, erlangen, germany, and epicare partner, erlangen, germany 9 department of neurosurgery, universitätsklinikum erlangen, fau erlangen-nürnberg, erlangen, germany, and epicare partner, erlangen, germany 10 department of neurosurgery, medical university of vienna, vienna general hospital, vienna, austria, vienna, austria background: the ptpn11 gene is a tyrosine phosphatase non-receptor type protein linked to the map kinase signaling pathway. it was recently discovered as novel lesional epilepsy gene by large exome-wide sequencing studies. ptpn11 germline mutations have been associated with noonan syndrome, a multisystem disorder characterized by facial features, developmental delay and other organ diseases. sporadically, low-grade epilepsy-associated brain tumors (leat) also occur in noonan patients. herein, we performed a first deep phenotype-genotype analysis of low-grade developmental brain tumours with brain somatic alterations of the ptpn11 gene as compared to commonly observed leat with or without map kinase signaling pathway alterations. methods: we selected 87 leat cases recently submitted to whole exome sequencing and genotyping including 17 dysembryoplastic neuroepithelial tumours (dnt) and 70 ganglioglioma (gg). clinical data were retrieved from hospital files including postsurgical outcome (engel outcome, seizure onset, age at surgery, mri findings, location). available histopathology slides were fully digitalized for systematic microscopy analysis, including h&e and immunohistochemistry for cd34, p16, map2, neun, ki67, idh1 and p53. results: we identified a series of eight gg with ptpn11 alterations, i.e. gains in copy number variations (cnv) of the locus 12q, which showed a systematic pattern of additional cnv gains in fgfr4, rheb, nf1, kras as well as braf alterations (figure 1). histopathology pattern analysis revealed an atypical and complex glio-neuronal phenotype with subpial tumour spread and large, pleomorphic and multinuclear cellular features (figure 2). only three out of eight gg with ptpn11 alterations were free of disabling-seizures two years after surgery (engel ia outcome, 38%). this was remarkably different from our series of gg with braf alterations (n=35), gg without any genetic alteration detectable by our study paradigm (n=27) and dnt with fgfr1 alterations (n=6) with engel ia rates of 85%, 76% and 83%, respectively. conclusions: we identified a subgroup of ganglioglioma characterized by ptpn11 alterations in association with other noonan syndrome related alterations of the map kinase signaling pathway, i.e., kras, rheb, braf, and fgfr4. these tumours were further characterized by histopathological features of cellular atypia in glial and neuronal cell components as well as adverse postsurgical outcome. these features were strikingly different from other leat with defined genetic alterations in braf, e.g., v600e mutation, and fgfr1. notwithstanding, these findings need further validation as they argue for a three-tiered who grading system also for developmental, glio-neuronal tumors associated with early-onset focal epilepsy. genetic similarities to noonan syndrome and noonan syndrome associated disorders may also suggest the use of targeted treatment options against the map kinase and mtor signaling pathway. figure 1: oncoplot of ptpn11 altered leat compared to a braf-v600e mutated gg (arrow on left) and a fgfr1 altered dnt (arrow on right) figure 2: histopathology findings in a ptpn11 altered atypical ganglioglioma. a: subpial growth (arrow) with large, pleomorphic and glio-neuronal phenotype shown in b. c: abundant cd34 immunoreactivity (sp – subpial region). d: the arrow points to a bi-nucleated neuron (map2 immunohistochemistry) confirming the diagnosis of ganglioglioma.   2.04 free neuropathol 3:20:23 molecular refinement of pilocytic astrocytoma in adult patients helena bode1,2, catena kresbach1,2,3, dörthe holdhof1,2, mario m. dorostkar4,5, patrick n. harter6, jürgen hench7, stephan frank7, alicia eckhardt1,2,8, annika k. wefers3, sina neyazi1,2, david capper9,10, michael bockmayr1,2,11, ulrich schüller1,2,3 1 department of pediatric hematology and oncology, university medical center hamburg-eppendorf, hamburg, deutschland 2 research institute children’s cancer center hamburg, hamburg, deutschland 3 institute of neuropathology, university medical center hamburg-eppendorf, hamburg, deutschland 4 center for neuropathology, ludwig-maximilians-university, munich, deutschland 5 german center for neurodegenerative diseases, munich, deutschland 6 institute of neurology (edinger institut), university hospital frankfurt, frankfurt, deutschland 7 division of neuropathology, institute of medical genetics and pathology, university of basel, basel, switzerland 8 lab of radiobiology & experimental radiation oncology, hubertus wald tumorzentrum – university cancer center hamburg, university medical center hamburg-eppendorf, hamburg, deutschland 9 german cancer consortium (dktk), partner site berlin, and german cancer research center (dkfz), heidelberg, deutschland 10 department of neuropathology, corporate member of freie universität berlin, charité, universitätsmedizin berlin and humboldt-universität zu berlin, berlin, deutschland 11 institute of pathology, corporate member of freie universität berlin, charité, universitätsmedizin berlin and humboldt-universität zu berlin, berlin, deutschland background: pilocytic astrocytomas (pa) are the most common primary central nervous system neoplasms in children. the vast majority of cases harbor kiaa1549-braf fusions and usually go along with an excellent prognosis. in contrast, pa in adult patients are rare, lack kiaa1549-braf fusions in many cases, and demonstrate a more aggressive clinical course. purpose: this project aims at characterizing adult pa regarding their molecular profile and clinical course. methods: we identified 55 cases with a histological diagnosis of pa in adulthood (≥18 years). molecular analyses of these cases included dna methylation analysis, copy number profiling, and dna sequencing for the most common mutations in the mapk-pathway. results: the mean age of our cohort was 35 years. tumors were located infratentorially (41%), supratentorially (41%), and spinally (18%). after performing global dna methylation analyses and applying the dkfz brain tumor classifier (v12.5), only 25% of these cases received a significant match to one of the reference methylation classes of pa (score ≥ 0.9). 20% matched to different entities, and 55% did not match to any brain tumor class. furthermore, only 23% of the tumors exhibited the kiaa1549-braf fusion. further analyses of tumors with a significant match to one of the three pa reference classes showed that adult patients mostly had supratentorial pa (lgg_pa_gg_st, mean age: 20 years, n=45), while children had pa in midline structures (lgg_pa_mid, mean age: 9 years, n=51) or in the posterior fossa (lgg_pa_pf, mean age: 11 years, n=159, p<0.005). among these tumors defined by dna methylation, the typical kiaa1549-braf fusion was found in 94 % of pediatric tumors and only in 45 % of tumors occurring in adults. conclusions: in summary, according to dna methylation profiling, a particularly high fraction of tumors histologically appearing as pa in adult patients do not match known reference cohorts of pa. many tumors are even reflecting other tumor entities, indicating ambiguous histological features. furthermore, even in cases that significantly match to pa regarding dna methylation, the distribution of genetic drivers differs from their pediatric counterparts.   2.05 free neuropathol 3:20:25 exploration of cellular origins and therapeutic targets by modeling high grade pediatric glioma of the mycn subclass in mice melanie schoof1,2, shweta godbole3, carolin walter4,5, matthias dottermusch3,6, thomas albert5, annika ballast5, carolin göbel1,2, sina neyazi1,2, dörthe holdhof1,2, catena kresbach1,6, gefion dorothea epplen1, mirjam blattner-johnson7,8, franziska modemann9,10, ann-kristin afflerbach1,2, alicia eckhardt1,11, vanessa thaden1, nina struve11,12, david t. w. jones7,8, kornelius kerl5, julia neumann3,6, ulrich schüller1,2,6 1 research institute children’s cancer center hamburg, hamburg, germany 2 department of pediatric hematology and oncology, university medical center hamburg-eppendorf, hamburg, deutschland 3 center for molecular neurobiology (zmnh), university medical center hamburg-eppendorf, hamburg, deutschland 4 institute of medical informatics, university of muenster, muenster, deutschland 5 department of pediatric hematology and oncology, university children’s hospital münster, muenster, deutschland 6 institute of neuropathology, university medical center hamburg-eppendorf, hamburg, deutschland 7 hopp children's cancer center (kitz), heidelberg, deutschland 8 pediatric glioma research group, german cancer research center (dkfz), heidelberg, deutschland 9 department of oncology, hematology and bone marrow transplantation with division of pneumology, university medical center hamburg-eppendorf, hamburg, deutschland 10 mildred scheel cancer career center, university cancer center hamburg, university medical center hamburg-eppendorf, hamburg, deutschland 11 department of radiotherapy, university medical center hamburg-eppendorf, hamburg, deutschland 12 mildred scheel cancer career center hatrics4, university medical center hamburg-eppendorf, hamburg, deutschland pediatric gliomas of the mycn subclass, a recently described highly aggressive brain tumor entity, frequently carry amplifications of mycn and mutations in tp53. these tumors present with a median age of 8 years and a median overall survival of only 14 months. better treatment options are urgently needed, as the current treatment is ineffective and causes severe side effects. here, we describe the generation of a novel mouse model, which can be used for preclinical research. we bred hgfap-cre::tp53fl/fl::lsl-mycn mice, which develop large forebrain tumors with 100 % penetrance within the first 80 days of life. the murine tumors show a high similarity with human tumors in histology, gene expression, and global dna methylation pattern. single-cell gene expression analyzes of these tumors revealed a large intratumoral cell heterogeneity and, due to the similarity of the tumor cells with oligodendrocytes in different developmental stages, suggests a glial origin of these tumors. additionally, we tested the preclinical potential of our mouse model by showing sensitivity of mouse and human tumor cells to aurka inhibition in vitro. we believe that further characterization and utilization of the model will pave the way to improved treatment strategies for patients with these highly aggressive tumors.   2.06 free neuropathol 3:20:27 the genomic and transcriptional landscape of primary central nervous system lymphoma josefine radke1, naveed ishaque2, reiner siebert3, stefan wiemann4, frank heppner5 1 universität greifswald, pathologie, greifswald, deutschland 2 berlin institute of health (bih), digital health center, berlin, deutschland 3 ulm university & ulm university medical center, human genetics, ulm, deutschland 4 deutsches krebsforschungszentrum (dkfz), heidelberg, deutschland 5 charité, neuropathology, berlin, deutschland background: primary lymphomas of the central nervous system (pcnsl) are mainly diffuse large b-cell lymphomas (dlbcls) confined to the central nervous system (cns). despite extensive research, the molecular alterations leading to pcnsl have not been fully elucidated. aims: in order to provide a comprehensive description of the genomic and transcriptional landscape of pcnsl, we here performed whole-genome and transcriptome sequencing and integrative analysis of 51 lymphomas presenting in the cns, including 42 ebv-negative pcnsl, 6 secondary cns lymphomas (scnsl) and 3 ebv+ cnsl and matched controls. the results were compared to an independent validation cohort of 31 ffpe cnsl specimens (pcnsl, n = 19; scnsl, n = 9; ebv+ cnsl, n = 3) as well as 39 fl and 36 systemic dlbcl cases outside the cns. results: somatic genomic alterations in pcnsl mainly affect the jak-stat, nfkb, and b-cell receptor signaling pathways, with hallmark recurrent mutations including myd88 l265p (67%) and cd79b (63%), cdkn2a deletions (83%) and also non-coding rna genes such as malat1 (70%), neat (60%), and mir142 (80%). kataegis events, which affected 15 of 50 identified driver genes and 21 of the top 50 mutated ncrnas, played a decisive role in shaping the mutational repertoire of pcnsl. compared to systemic dlbcl, pcnsls exhibited significantly more focal deletions in 6p21 targeting the hla-d locus that encodes for mhc class ii molecules as a potential mechanism of immune evasion. mutational signatures correlating with dna replication and mitosis (sbs1, id1 and id2) were significantly enriched in pcnsl (sbs1: p = 0.0027, id1/id2: p < 1x10-4). furthermore, tert gene expression was significantly higher in pcnsl compared to abc-dlbcl (p = 0.027). although pcnsl share many genetic alterations with systemic abc-dlbcl in the same signaling pathways, transcriptome analysis clearly distinguished both into distinct molecular subtypes. ebv+ cnsl cases may be distinguished by lack of recurrent mutational hotspots apart from ig and hla-drb loci. conclusion: we show that pcnsl can be clearly distinguished from dlbcl, having distinct expression profiles, ig expression and translocation patterns, as well as specific combinations of genetic alterations.   2.07 free neuropathol 3:20:28 molecular mechanisms of therapy resistance in malignant melanoma brain metastasis elisa schumann1, randi koll2, julia onken2, karsten jürchott2, torben redmer3, josefine radke4 1 charité universitätsmedizin berlin, institut für neuropathologie, berlin, deutschland 2 charité universitätsmedizin berlin, berlin, deutschland 3 veterinärmedizinische universität wien, wien, austria 4 universität greifswald, greifswald, deutschland background: malignant melanoma (mm) is among the tumor entities with the highest potential to spread to the cns. about 45% of mm patients suffer from brain metastasis likely proceeding continuously during the course of disease. genetically and molecularly distinct subclones lead to tumor heterogeneity which is followed by therapy resistance and poor prognosis. previous studies suggested increased metastatic potential to and within the brain under braf inhibitor (brafi) therapy, which is caused by upregulation of a subset of molecular drivers controlling migratory and invasion such as the nerve growth factor receptor cd271/ngfr. aims: to gain insight into the molecular features of migration and invasion of patient derived cell lines from mm brain metastases (bm) that were therapy-responsive or therapy-resistant to brafi, radiotherapy and immune checkpoint inhibitors. methods: we generated patient derived cell lines from mm bm (n = 9) and performed dna-sequencing (n = 5) and transcriptome analyses (n = 2) of cell lines and concordant tumors (n = 2). furthermore, we used the incucyte® live-cell analysis to perform high throughput scratch wound assays with patient derived cell lines, which were genetically modified leading to overexpression or downregulation of ngfr. results: transcriptome profiling of brafi resistant mm bm revealed that the invasive potential increased during disease progression. this process was accompanied by upregulation of ngfr expression. moreover, it was preserved in patient derived cells lines, which demonstrated significantly higher potential of two-dimensional in vitro migration (90% vs. 76% after 100 hours). furthermore, cd271 knockdown was associated with loss-of-expression of several genes involved in migration and invasion. conclusions: brain metastases are the major cause of death in metastasized mm. probably, bm emerge and progress by the concerted interaction of several molecular programs that are triggered by cells of the tumor microenvironment and/or in response to therapeutic interventions. our study provides a longitudinal perspective on the progression of brain metastasis and their mechanisms leading to therapy resistance.   2.08 free neuropathol 3:20:29 a peripheral nerve sheath tumor syndrome caused by postzygotic erbb2 mutations michael ronellenfitsch1,2,3,4, isabel gugel5, dusica babovic-vuksanovic6, maximilian rauch2,7, jens schittenhelm5, martin u. schuhmann5, silvia hofer8, martina kirchner9, gerhard marquardt10, rouzbeh banan911, benedikt sauer1,3, ulrich schüller12,13,14, werner paulus15, matthias meinhardt16, tareq juratli16, albrecht stenzinger9, stefan fröhling17,18, eric legius19, andreas von deimling9,11, felix sahm9,11, joachim p. steinbach1,2,3,4, patrick harter2,3,10, victor-felix mautner12, david reuss11,20 1 dr. senckenberg institute of neurooncology, frankfurt, deutschland 2 university cancer center (uct) frankfurt, frankfurt, deutschland 3 german cancer consortium (dktk), frankfurt, deutschland 4 frankfurt cancer institute, frankfurt, deutschland 5 university hospital tübingen, tübingen, deutschland 6 mayo clinic college of medicine, rochester, united states 7 goethe university hospital, frankfurt, deutschland 8 university hospital and university of zurich, zürich, switzerland 9 heidelberg university hospital, heidelberg, deutschland 10 university hospital frankfurt, frankfurt, deutschland 11 dkfz, heidelberg, deutschland 12 university hospital hamburg-eppendorf, hamburg, deutschland 13 university medical center hamburg-eppendorf, hamburg, deutschland 14 research institute children's cancer center hamburg, hamburg, deutschland 15 university hospital münster, münster, deutschland 16 university hospital carl gustav carus, dresden, deutschland 17 nct heidelberg and dkfz, heidelberg, deutschland 18 dktk, heidelberg, deutschland 19 ku leuven and university hospital, leuven, belgium 20 universitätsklinikum heidelberg, neuropathologie, heidelberg, deutschland introduction: peripheral nerve sheath tumors (nst) are common manifestations of different tumor syndromes within the neurofibromatosis spectrum, comprised of nf1, nf2 and schwannomatosis. these are caused by inactivating germline mutations in the nf1, nf2, smarcb1 or lztr1 tumor suppressor genes respectively. neurofibromas are closely associated with nf1 and schwannomas occur in both nf2 and schwannomatosis. occurrence of neurofibroma/schwannoma hybrid tumors is reported in all these syndromes. we recently described erbb2 mutations in a significant portion of neurofibroma/schwannoma hybrid nerve sheath tumors. based on clinical criteria, these cases resembled schwannomatosis. however, the somatic genetic profile of the tumors was distinct. additionally, features untypical of schwannomatosis were present, resembling previously published descriptions of four patients with distinctive but unclassifiable clinical and pathological findings. objectives: the aim of the study was the clinicopathological and molecular characterization of erbb2-mutant peripheral nerve sheath tumors. methods: tumors were evaluated by histology. next generation sequencing (hd-panel or whole exome sequencing) was used to determine the presence of an erbb2 mutation in at least one tumor of every patient. pyro-sequencing was used to verify erbb2 mutations and to determine their presence in additional tumors from a given patient. 850k methylation profiling was used for additional characterizations. results: we identified 13 non-related patients with erbb2 mutant nst, including all 4 previously published patients with an unclassified syndrome. all but two patients were females and tumors developed slowly during adulthood. all patients had multiple nst, which were restricted to a specific anatomic region in several patients while a more widespread distribution of tumors was present in others. the histology showed quite distinctive features within the spectrum of hybrid nerve sheath tumors. strikingly, in all patients, tumors from distinct anatomic locations harbored the very same activating erbb2 mutation. the median mutant allele frequency of 12% was comparatively low (range 4%-23%) suggesting that only a subpopulation of cells harbored the mutation. no other candidate driver alteration was found by ngs panel or wes and rna-sequencing. dna methylation profiling provided evidence for a distinctive epigenetic profile of erbb2-mutant nsts. no chromosomal copy number alterations were detectable. ongoing molecular analyses will provide additional insight in the pathogenesis of erbb2-mutant nsts. conclusion: erbb2 mutations in nst do not occur as isolated somatic events in sporadic tumorigenesis or in the setting of nf1, nf2 or schwannomatosis but represent manifestations of a distinct tumor syndrome most likely caused by postzygotic erbb2-mosaicism. diagnosis of erbb2-mutant nst is of high clinical relevance due to the availability of specific erbb2 inhibitors and preliminary evidence of their effectiveness. histology is sufficiently specific for screening purposes but molecular analyses with highly sensitive methods like deep coverage ngs are mandatory for the definitive diagnosis.   2.09 free neuropathol 3:20:31 cns-tumor patients within the impress-norway trial: first year experiences pitt niehusmann1,2, hege g russnes1,3,4, katarina puco2, åsmund flobak5,6, eli sihn s. steinskog7, åse haug7, sigmund brabrand2,8, egil s. blix9,10, anne j skjulsvik5,11, ragnhild m wold12, henning leske1, hrvoje miletic13,14, petter brandal8,15, gro l. fagereng16, kjetil taskén3,17, åslaug helland3,4,8 1 oslo university hospital, department of pathology, oslo, norway 2 oslo university hospital, division for cancer medicine, oslo, norway 3 university of oslo, institute of clinical medicine, oslo, norway 4 oslo university hospital, department of cancer genetics, institute for cancer research, oslo, norway 5 norwegian university of science and technology, department of clinical and molecular medicine, trondheim, norway 6 st. olav university hospital, the cancer clinic, trondheim, norway 7 haukeland university hospital, department of oncology, bergen, norway 8 oslo university hospital, department of oncology, oslo, norway 9 uit the arctic university of norway, institute of clinical medicine, tromsø, norway 10 university hospital of north norway, department of oncology, tromsø, norway 11 st. olav university hospital, department of pathology, trondheim, norway 12 university hospital of north norway, department of pathology, tromsø, norway 13 university of bergen, department of biomedicine, bergen, norway 14 haukeland university hospital, department of pathology, bergen, norway 15 oslo university hospital, section for cancer cytogenetics, institute for cancer genetics and informatics, oslo, norway 16 oslo university hospital, institute for cancer research, oslo, norway 17 oslo university hospital, department of cancer immunology, institute for cancer research, oslo, norway background: impress-norway is a nation-wide precision medicine trial for cancer patients in norway that launched april 1st 2021. in this investigator-initiated, prospective, open-label, non-randomized combined basketand umbrella-trial, patients are enrolled into multiple parallel treatment cohorts. patients with progressive cancer disease, including primary cns-neoplasms, with no further standard therapy to offer, are eligible. all drugs available in impress-norway are regulatory approved. currently, five different pharmaceutical companies provide 16 drugs, and we are in process to acquire eight additional drugs for patients in this study. methods: comprehensive genomic profiling (gene-panel analysis of >500 genes) is performed as part of the norwegian public health care system. patients consenting to the impress-norway profiling phase contribute their clinical and molecular data for research and are screened for cell-free circulating tumor dna. patients with identified biomarkers matching available drugs are referred by the national molecular tumor board for inclusion in the impress-norway treatment phase. these patients will have extensive biobanking as well as whole genome molecular profiling of their tumors before and during treatment. in the impress-norway treatment phase, each cohort is defined by the patients’ tumor type, molecular profile of the tumor, and study drug. treatment outcome in each cohort is monitored using a simon two-stage-like ‘admissible’ monitoring plan to identify evidence of clinical activity. the primary objective in the study is clinical benefit of treatment at 16 weeks of treatment; defined as complete response, partial response, or stable disease. here, we report on patients with cns-neoplasms included in the impress-norway profiling and treatment phases. results: as of april 30th, 2022, twenty-four patients with cns-neoplasms had been included in the molecular profiling phase of impress-norway and 22 had completed evaluation at the molecular tumor board (see table 1). tumor mutation burden (tmb) in cns-tumor tissue samples ranged from 1.6-250 somatic mutations per megabase (mut/mb; median=4.7, n=23). in liquid biopsies, blood tmb ranged from 0-6 mut/mb (median=0; n=21), indicating a limited efficacy of this analysis in cns-tumor patients. in five of the 22 patients with completed evaluation, we identified biomarker, which allowed allocation to an impress-norway treatment-cohort (ratio of cns-patients with targetable biomarker was similar to the overall inclusion of patients into treatment cohorts, 67/295). one glioblastoma patient showed complete response according to rano at 39 weeks.   table 1 diagnosis treatment cohort glioblastoma, idh-wildtype (n=13) n=3 astrocytoma, idh-mutant (n=4) n=0 anaplastic meningioma (n=1) n=0 atypical meningioma (n=1) n=0 diffuse midline glioma, h3 k27-altered (n=1) n=1 high-grade astrocytoma with piloid features (n=1) n=1 myxopapillary ependymoma (n=2) n=0 supratentorial ependymoma (n=1) n=0   due to increasing test capacity, we anticipate to double the number of included cns-tumor patients within the next 6 months. whole genome sequencing data from patients included into the treatment cohorts are obtained successively. conclusion: patients with advanced cancer progressing on standard treatment are referred to treatment in impress-norway after advanced molecular diagnostics. molecular alterations indicating benefit of drugs currently available in the study are detected in a reasonable number of patients with cns-neoplasms.   3. neurodegeneration 3.01 free neuropathol 3:20:33 cnn-supported quantification of fat compartments at abdominal mri applied to als patients ina vernikouskaya1, hans-peter müller2, dominik felbel1, francesco roselli2, albert christian ludolph2, volker rasche1, jan kassubek2 1 ulm university medical center, internal medicine ii, ulm, deutschland 2 university of ulm, neurology, ulm, deutschland background: amyotrophic lateral sclerosis (als) is the most frequent adult onset neurodegenerative motor neuron disease characterized by catabolism1, and patients begin to lose weight more than 10 years before the onset of motor symptoms2. als patients have been shown to display an expanded ratio between visceral adipose tissue (vat) and subcutaneous adipose tissue (sat)3. accurate segmentation of body fat compartments from mri is, however, a challenging task due to the limited reproducibility of semi-manual delineations and artifacts. concerning organ segmentation, learning-based algorithms and especially convolutional neural networks (cnn) have been proven to outperform traditional methods in speed and reproducibility. aims: the aim of this study was to automate the discrimination of abdominal body fat compartments into sat and vat from t1-weighted mri using deep cnn and to quantify the fat ratio in patients with als as compared to the control cohort. question: may cnn-supported segmentation of body fat compartments serve for unbiased analysis of the vat/sat ratio parameter as a potential biological marker? methods: 74 als patients (age 60 ± 12, m/f 50/24) and 81 healthy subjects (56 ± 15, 42/39) underwent mri examination with multi-slice t1-weighted spin-echo sequence. all available data were split in training (50 %), validation (6 %), and test (44 %) data, based on age and bmi strata. semi-automatic segmentation of subcutaneous and visceral fat was performed with an established reference method using software package atlas4. the obtained sat/vat masks were used for training of the cnn of u-net like architecture. performance of the segmentation using cnn was evaluated in terms of dice coefficients. volumetric computation of segmented sat and vat for all test objects was performed with reference and cnn-based methods and compared by pearson correlation. vat/sat ratio was assessed. results: the dice coefficients between the cnn-supported and reference segmentations comprised 0.87 ± 0.04 for sat and 0.64 ± 0.17 for vat in the control group and 0.87 ± 0.08 for sat and 0.68 ± 0.15 for vat in the als group. a significant linear correlation between the cnn predicted and reference method with pearson coefficients 0.992 in controls and 0.977 in als patients was observed for sat, whereas lower pearson coefficients 0.653 in controls and 0.814 in als patients were obtained for vat. significant difference for the vat/sat ratio was observed when comparing als patients versus healthy subjects with the p-value of 0.002. figure 1 shows the sat and vat segmentation results in healthy controls and als patients. conclusions: the obtained results in the t1-weighted mri data in the als patient cohort could reproduce the results of a reference technique in a user-independent manner with high accuracy. cnn-supported quantification of vat/sat ratio might serve as a biological marker in als body composition assessment, potentially as a secondary read-out for clinical trials. references: 1. dupuis et al, lancet neurol. 2011 2. peter et al, eur j epidemiol. 2017 3. lindauer et al, plos one. 2013 4. müller et al, nmr biomed. 2011 figure 1: comparison of ccn-based segmentation vs. reference segmentation and vat/sat ratio plot in healthy controls and als patients.   3.02 free neuropathol 3:20:35 neurodegenerative iron storage disease (neuroferritinopathy) caused by a novel frameshift mutation in the ferritin heavy chain gene (fth1 c.341-342del) vincent umathum1,2, daniel amsel1, christina becker3, corinne kasan3, andrea may4, klaus nehmer4, andreas günther4,5, carmen selignow1, anna nishimura1, ioannis alexopoulos6, attila németh1, nadja ritschel1, axel weber7, till acker1, anne schänzer1 1 institut für neuropathologie, justus-liebig-universität, gießen, deutschland 2 institut für pathologie und molekularpathologie, bundeswehrkrankenhaus ulm, ulm, deutschland 3 institut für pathologie, zytologie und molekularpathologie mvz, wetzlar, deutschland 4 pneumologische klinik, agaplesion evangelisches krankenhaus, gießen, deutschland 5 zentrum für interstitielle und seltene lungenerkrankungen, justus-liebig-universität, gießen, deutschland 6 institute for lung health, justus-liebig-universität, gießen, deutschland 7 institut für humangentik, justus-liebig-universität, gießen, deutschland introduction: neuroferritinopathy (nf) is a rare hereditary neurodegenerative disorder associated with increased iron deposition in the brain and extracerebral organs such as the kidney, liver, skin and skeletal muscle. the ferritin complex consists of 24 subunits of ferritin light and heavy chains and converts free iron into a non-redox active storage form. clinically, the focus is on chorea-huntington-like movement disorders. so far, only mutations in the ferritin light chain gene (ftl) have been described in nf. material and methods: a 78-year-old female patient died of covid-19-associated pneumonia and was autopsied. standard staining (he, prussian blue), immunohistochemical and immunofluorescence staining as well as electron microscopic analyses were performed on paraffin-embedded formalin-fixed (ffpe) tissue of the patient from different brain regions as well as heart, lung, liver and kidney. whole slide images scans were done by hamamatsu nanozoomer s360 and evaluated morphometrically with qpath. whole-exome sequencing (wes) was done from ffpe material of the basal ganglia. results: macroscopically, no pathology was found in the brain. microscopically, numerous inclusion bodies (ib) were seen in the brain and sporadically in the liver and kidney. the ib were homogeneously sharply defined on he stains and up to 13 µm in size (normal nuclear diameter: approx. 5-6 µm), with strong fe3+ deposits in the prussian blue staining. ultrastructurally, the ib showed intranuclear, fine granular aggregates with lateralisation of the chromatin to the inner side of the membrane. immunohistochemistry for ftl and ferritin heavy chain protein (fth) showed clear nuclear expression in the ib. in contrast, the cells in the control tissue had predominantly perinuclear, cytoplasmic expression. quantitative evaluation showed increased fth expression in the patient: frontal cortex (cf): 0.2%, occipital cortex (co): 0.3%, hippocampus: 0.08%, basal ganglia (bg): 0.7%, dentate nucleus (dn): 1.7% compared to control tissue (cf: 0.06% co: 0.04% hippocampus: 0.04% bg: 0.08% dn: 0.4%. the ratio of fth expression (compared to control tissue) was highest in the bg (1:8.4), followed by the co (1:8.3) and lowest in the hippocampus (1:2.1). wes revealed a previously undescribed variant (double deletion) in the ferritin heavy chain gene (fth1 c.341-342del). wild-type sequence in ftl. discussion: the present study describes for the first time a patient with nf caused by a previously undescribed mutation in the fth1 gene with presentation of numerous ib in the brain and sporadically in extracerebral tissue. these results suggest that the function of the ferritin complex can be disturbed not only by an ftlbut also by an fth1-mutation, leading to pathological deposition of ferritin complexes. with extended analyses, it could be shown that the ib correspond to enlarged cell nuclei with intranuclear ferritin accumulations. furthermore, a high variability in distribution of the ib in different brain regions was found. summary: mutation in fth1 (c.341-342del) is associated with a rare neurodegenerative disease with increased intranuclear iron deposition mainly in the bg and dn, possibly showing a similar pathomechanism to the known ftl-mutation. if nf is clinically suspected, mutations should therefore be investigated not only in the ftl-gene but also in the fth1-gene.   3.03 free neuropathol 3:20:37 the contribution of late-nc to neuron loss, granulovacuolar degeneration and dementia in alzheimer’s disease dietmar thal1,2,3, klara gawor1,3, evelien van schoor1,3,4, sebastiaan moonen1,3,4, jolien schaeverbeke1,3,4, rik vandenberghe3,4,5, mathieu vandenbulcke3,4,6, christine a. f. von arnim7,8, marta koper1,3,4, sandra tomé1,3 1 ku-leuven, department of imaging and pathology, laboratory for neuropathology, leuven, belgium 2 uz leuven, department of pathology, leuven, belgium 3 leuven brain institute, leuven, belgium 4 ku leuven, department of neuroscience, leuven, belgium 5 uz leuven, department of neurology, leuven, belgium 6 uz leuven, department of psychiatry, leuven, belgium 7 göttingen university, department of geriatrics, göttingen, deutschland 8 ulm university, neurology, ulm, deutschland background: tdp-43 pathology in alzheimer’s disease (ad) is currently considered as a co-pathology belonging to the spectrum of limbic-predominant, age-associated tdp-43 encephalopathy (late). ad cases with tdp-43 pathology have greater medial temporal lobe atrophy and cognitive decline compared to ad cases without tdp-43. recently, we showed that the accumulation of the necrosome (executer complex of necroptosis which is a programmed from of necrosis) in granulovacuolar degeneration (gvd) in ad is associated with neuron loss. aims: here, we aim to clarify whether necroptosis in ad is related to tdp-43 pathology, i.e., late neuropathological changes (late-nc) and indicates a contribution of late-nc to the degeneration/ death of neurons in ad. by doing so, we will determine the impact of late-nc on ad-nc, neuron loss, especially necroptosis, and cognitive decline. research question: does tdp-43 pathology in late contribute to neurodegeneration via necrosome accumulation and necroptosis in ad and, if so, is this related to tau pathology. methods: we used 234 human post-mortem brains from 89 non-ad controls, 82 p-pread and 63 ad cases. we determined amyloid-β (aβ) (mtl = medial temporal lobe-based) phases, braak neurofibrillary tangle (nft) stages, late-nc stages and gvd stages, which described the anatomical spread of gvd bodies, and the frequency of gvd-affected neurons in a given region. from these, 66 cases were assessed for neuronal density in the ca1 subfield of the hippocampal formation and 186 were retrospectively assessed for clinical dementia rating (cdr) scores. in a subset of 27 cases covering 9 non-ad controls (without tdp-43 pathology), 8 symptomatic ad cases without tdp-43 pathology (adtdp-) and 10 symptomatic ad cases with tdp-43 proteinopathy (i.e.: late-nc; adtdp+), we quantified neuronal density, severity of ptdp-43, pmlkl (a component of the activated necrosome) and ptau pathology. results: we found that late-nc and braak nft stages contribute independently to gvd severity when being covariates the same model term (p < 0.001). late-nc and braak nft stages were associated with neuronal density in the hippocampus (p = 0.022 and p = 0.004, respectively). braak nft stage alone showed also an association with neuronal density (p < 0.001). partial correlation analysis corroborated these data and revealed that braak nft and late-nc stage are significantly correlated with gvd stage and cdr score. in the 27 cases used for quantitative assessments, the absence of ptdp-43 was associated with less neuronal loss in adtdpcases compared to adtdp+ (p = 0.0204). adtdp+ cases exhibited significantly more pmlkl-positive neurons, when compared to adtdpcases (p = 0.025) and non-diseased controls (p < 0.0001). consistently, adtdp+ cases also showed enhanced numbers of ptau exhibiting neurons compared to adtdp(p = 0.045) and controls (p < 0.0001). conclusions: thus, late-nc contributes independently to neuronal loss and gvd severity. further, these data highlight the impact of cytoplasmic tdp-43 aggregation on ptau pathology as well as on necroptosis activation in ad. support: fwo.   3.04 free neuropathol 3:20:39 the role of c3 inhibition in an ipsc nmj model of neuroinflam-mation scott baver1, virginia smith2, yan li1, david eyerman1, ashley robertson2, leticia lenkiu2, heather cannon2, daisy martinez2, hannah hanson2, james hesperos2 1 apellis, waltham, united states 2 hesperos, orlando, united states background: the complement cascade is a critical component of the immune system, and dysfunction of complement has been implicated in als. components of the complement cascade, including c3, are reported to be deposited on neuromuscular junctions (nmjs) of muscle biopsies in people with als. objectives: evaluate the effect of complement c3 inhibition on nmj function in response to inflammatory stimulations in a human ipsc-derived nmj model. question: while the therapeutic potential of complement system modulation has been explored via use of embryonic knockout animals in als models, its effects in clinically relevant human cell-based models are unknown. methods: nmj systems were established by plating human ipsc-derived motoneurons, skeletal myoblasts, schwann cells, microglia, and activated or inactivated thp monocytes in a compartmentalized co-culture system. activated and inactivated monocytes were plated at ratios of 4:1-1:50 (vs. skeletal myoblasts). als model systems were created using sod-1 (e100g) or tdp-43 ipsc-derived cells compared to control nmjs established from wild-type ipsc-derived cells. to determine the role of c3 on nmj function, the c3 inhibitor apl-2 (50 µg/ml) and human complement serum (0.05%) were acutely dosed for 3 hours. complement c3 expression was assessed by immunocytochemistry, and nmj number and fidelity were calculated by assessing the number of functional myotubes under indirect stimulation and the ratio of number of successful contractions to number of pulses at a given frequency, respectively. all experiments were replicated twice in triplicate. results: functional nmj systems were assessed with the addition of various ratios of activated or inactivated monocytes to skeletal muscle-side of the culture chamber. addition of activated monocytes resulted in reduced nmj number and function. in addition, while c3 expression was observed with thp-monocytes, activation of m1 macrophages increased c3 activity. human complement serum potentiated the effects of m1 macrophages; further decreasing nmj numbers and reducing nmj fidelity. acute treatment with apl-2 attenuated these effects. compared to wild-type, sod-1 and tdp-43 nmj systems reduced nmj number and fidelity. the reduction in sod-1 nmj number and fidelity was greater than that of tdp-43 nmj system. conclusions: these data demonstrate that modulating c3 with apl-2 in the presence of an inflammatory nmj environment could improve overall function of the nmj in related disease states.   3.05 free neuropathol 3:20:40 fast-track procedure for the neuropathological assessment of neurodegenerative diseases benjamin englert1,2,3, viktoria ruf1, jochen herms1,2,3 1 ludwig-maximilians university, center for neuropathology and prion research, munich, deutschland 2 german center for neurodegenerative diseases (dzne), munich, deutschland 3 munich cluster for systems neurology (synergy), munich, deutschland background: in order to exclude creutzfeldt-jakob disease we have established a fast-track assessment of all brains that we receive at the neurobiobank munich. aims and questions: we aim to evaluate the concordance between fast-track working diagnosis and the full histological work-up of the brain of patients clinically diagnosed with a neurodegenerative disease. methods: two predefined, easily accessible brain regions (frontal superior gyrus and cerebellar hemisphere) were sampled, fixed in formalin, treated with formic acid and embedded together in one paraffin block. h&e stain and six immunostains (antibodies against prion protein, α-synuclein, β-amyloid, phospho-tau, phospho-tdp43, p62) were performed. 133 cases were analysed. diseases in which a diagnosis cannot be made by analysing these two brain regions like pure amyotrophic lateral sclerosis and huntington disease have been excluded from this analysis. results: in 96.2% of cases the fast-track neuropathological diagnosis was confirmed by the conventional pathological work-up of the entire brain. only in four cases evaluation of additional brain regions was necessary to make a conclusive histopathological diagnosis. only in one case in our study (of an unusual tauopathy) the fast-track diagnosis needed to be corrected. the suspected clinical diagnosis was neuropathologically confirmed by fast-track histology in only 60.2 % of cases. in 39.8 % the clinical diagnosis was either different or ambiguous. conclusion: our study shows that histological and immunohistochemical screening of two selected brain regions (superior frontal gyrus and cerebellum) is indeed sufficient for establishing a reliable working diagnosis of patients with a neurodegenerative disease in over 96 % of cases. given the rapid workflow, a prompt response from the neuropathology to clinicians may improve the accuracy of clinical diagnosis. this procedure does not allow precise neuropathological disease staging and certain co-pathology cannot be fully appraised. diseases like pure als or hd cannot be neuropathologically verified on these two brain regions and the contribution of vascular pathology to the clinical picture cannot be precisely assessed.   3.06 free neuropathol 3:20:41 neurodegeneration in hsan1 due to atl1 (gly66gln) mutation is associated with defective erprotein quality control and compromised autophagy istvan katona1, hülya-sevcan daimagüler2, haihong guo1, priyanka tripathi1, antonio sechi3, alfred yamoah1, shelisa tey1, michael schröder1, jürgen klingelhöfer4, joachim weis1, anand goswami1 1 uniklinik rwth aachen, institut für neuropathologie, aachen, deutschland 2 uniklinik köln, kinderklinik, köln, deutschland 3 uniklinik rwth aachen, institut für zellund tumorbiologie, aachen, deutschland 4 klinikum chemnitz , klinik für neurologie, chemnitz, deutschland background: atlastin-1 (atl1) functions as a gtpase and is crucial for endoplasmic reticulum (er) shaping and er-microtubule interactions. mutations in atl1 have been reported to cause hereditary sensory and autonomic neuropathy type 1d (hsan1d) as well as hereditary spastic paraplegia 3a (spg3a). atl1 mutation have been linked to abnormal er morphology; still the molecular pathomechanism of defective er structures and their pathological consequences contributing to hsan1d and spg3a have not been investigated in detail so far. question: we asked if/how atl1 participates in autophagy process. methods: we used biochemical and immunocytochemistry approaches followed by live cell imaging in cell culture models overexpressing normal and mutant atl1 proteins. we corroborated our findings with comparative ultrastructural analysis on cell culture models and on biopsy samples. results: we observed that over-expression of mutant atl1 (gly66gln) forms protease resistant large, globular er associated aggregates in cell culture models, which further leads to er stress and structural abnormalities of er and associated compartments. interestingly while endogenous atl1 protein is degraded by ubiquitin proteasome system (ups), mutant atl1 impairs ups, induces proteotoxicity and cell death. autophagy, which activates as a compensatory mechanism, also compromises at multiple steps, probably due to deformities of er and persistent proteotoxic stress. extensive workup of skin, sural nerve and muscle biopsy material of a rare gly66gln hsan1 patient revealed prominent loss of myelinated and unmyelinated sural nerve fibres, but only minor neurogenic muscular atrophy. ultrastructural analysis on this biopsy revealed signs of altered autophagy in axons as well as prominent alterations of schwann cell nuclei/nuclear envelope. in line with this, hsan1 patient's fibroblasts showed similar defects. conclusions: overall, our results support the notion that neurodegeneration in hsan1 due to atl1 mutation is closely linked with the deformed er and associated functions including autophagy. neurons and distal axons are particularly vulnerable to such pathomechanism, thus explaining the degenerative phenotype in hsan 1 and related diseases.   3.07 free neuropathol 3:20:42 single-nucleus chromatin accessibility profiling in four-repeat tauopathies viktoria ruf1, nils briel1,2, sigrun roeber1, janina mielke1, mario m dorostkar1, otto windl1,2, thomas arzberger1,2,3, felix l. strübing1,2, jochen herms1,2,4 1 zentrum für neuropathologie und prionforschung, münchen, deutschland 2 deutsches zentrum für neurodegenerative erkrankungen, münchen, deutschland 3 klinik für psychiatrie und psychotherapie, münchen, deutschland 4 munich cluster of systems neurology (synergy), münchen, deutschland background: progressive supranuclear palsy (psp) and corticobasal degeneration (cbd) are sporadic neurodegenerative diseases characterized by aggregates of hyperphosphorylated four-repeat tau (4r-tau) in neurons, oligodendrocytes and astrocytes, where tufted astrocytes (ta) are a typical hallmark of psp, whereas astrocytic plaques (ap) are pathognomonic for cbd. the molecular mechanisms underlying tau aggregation and neurodegeneration are largely unclear. aims and questions: to characterize chromatin accessibility profiles of psp and cbd using atac-seq (assay for transposase-accessible chromatin using sequencing) to contribute to a better understanding of the underlying pathomechanisms of psp and cbd. methods: atac-seq was performed on 45,000 isolated single nuclei from the frontal cortex of 4 psp and 4 cbd patients and 5 healthy controls. after preprocessing and quality control, gene accessibility (ga), gene ontology (go) and transcription factor motif enrichment (tfme) analysis were conducted using snapatac. gchromvar was applied to map genetic risk variants to peaks and cicero/tradeseq was used for pseudotime analysis and repeated tfme analysis. results: we found that ga of tauopathy-associated genes was substantially altered in the brains of psp and cbd patients and could demonstrate that pspand ftd-associated genetic risk variants are particularly linked to astrocytic chromatin accessibility profiles. go enrichment analysis of significantly different transcription factor motifs (tfm) in astrocytes identified numerous motifs belonging to the immediate early response (ier; e.g. fos and jun family) or intracellular homeostasis and protein degradation (e.g. maf family and nfe2). moreover, pathway analysis highlighted immunological terms related to both innate and acquired immunity. pseudotime analysis of transcription factor motif enrichment (tfme) revealed for cbd a trajectory terminating in a population of cbd-derived astrocytes, whereas for psp no such disease-defined cluster was apparent. accordingly, a decrease was observed for tfs associated with early state astrocytic differentiation in cbd, while tfme of ier-related tfs considerably increased along the pseudotime axis. conclusions: our study revealed genetic dysregulation affecting neurodegenerative, neuroinflammatory and degradation processes and particularly points to a major role of astrocytes in the pathogenesis of psp and cbd. however, to understand the downstream mechanisms, further independent validation especially at the transcriptome and protein level will be necessary. github ressource: https://github.com/nes-b/snatac-seq_psp_cbd   3.08 free neuropathol 3:20:43 application of a human stem cell transplantation model of alzheimer’s disease to examine disease-associated changes at a single cell level in vivo wenhui qu1, matti lam2, aayushi mahajan3, nelson humala3, osama al dalahmah1, jason mares2, trang nguyen1, ismael santa-maria1, andrew sproul1, markus siegelin1, james goldman1, peter canoll1, vilas menon2, gunnar hargus1 1 columbia university medical center, department of pathology and cell biology, new york, united states 2 columbia university medical center, department of neurology, new york, united states 3 columbia university medical center, department of neurosurgery, new york, united states background: alzheimer’s disease (ad) is the most common type of dementia and is characterized by widespread degeneration of the central nervous system with amyloid and tau pathology leading to severe impairment of learning and memory. despite the high number of patients with ad, the molecular mechanisms leading to neurodegeneration are only partially understood and effective treatment options still do not exist. aims/questions: here, we applied a human stem cell model of ad to study disease-modifying roles of neural cells at a single cell level in vivo. methods: to this end, we differentiated induced pluripotent stem cells (ipscs) with the familial ad-associated appv717i mutation in the amyloid precursor protein as well as isogenic control cells into neural progenitor cells (npcs) and neurons. results: appv717i neurons showed reduced neurite outgrowth and demonstrated an increased susceptibility towards oxidative stress with changes in metabolic programs. we injected appv717i and control npcs into the brains of immunocompromised nsg mice resulting in neuronal grafts in both groups two months after injection with activation of microglial cells and presence of reactive astrocytes within and around the grafts. we then performed single nucleus rna sequencing (snrna-seq) on microdissected grafts to characterize gene expression and dysregulated pathways in appv717i versus control neurons as well as in astroglial cells and microglia in response to appv717i and control neurons at a single cell level. conclusions: our findings show that ipscs represent a powerful cell source to study mechanisms of disease development in vitro and in vivo. this stem cell model of ad could also be used as a cellular platform for high-throughput drug screening purposes to identify potential therapeutic targets in ad.   4. neuroinflammation 4.01 free neuropathol 3:20:44 pathological and genetic characterization of jc virus encephalopathy with an eleven-year-long disease course marco mlynek1, adriane kuttlovci1, marek jauß2, cornelia tennstedt-schenk3, lidia stork1, christine stadelmann1, imke metz1 1 institut für neuropathologie universitätsmedizin göttingen, göttingen, deutschland 2 ökumenisches hainich klinikum ggmbh, mühlhausen/thüringen, deutschland 3 institut für pathologie, mühlhausen/thüringen, deutschland the jc polyomavirus (jcpyv) typically causes progressive multifocal leukoencephalopathy (pml) in immunocompromised humans. the pathology shows demyelinated white matter lesions with infection of glial cells, but very few infected cortical pyramidal neurons. in contrast, another jcpyv-associated disease is jc virus encephalopathy (jcve), which is characterized by numerous infected neurons. while the non-coding control region of jcpyv in pml shows genetic rearrangements (so-called prototype), in jcve the archetype virus is found, which is also present in healthy individuals. we provide a detailed clinical, (histo)pathological and genetic description of a patient with jcve. we analyzed different lesion areas for viral infection and replication of glial and neuronal cells. in addition, demyelination, cell loss and axonal damage were investigated. we present a 54-year-old male patient with an exceptionally long disease course over eleven years who was diagnosed with jcve. he developed slowly progressing myoclonia, numbness of the upper and lower extremities, pharmacoresistant seizures and a general cognitive decline. mri showed a progressive atrophy of the cortex with cortical and subcortical parenchymal lesions. there was no clear evidence of immunosuppression. a variant of unclear significance in the interferon-induced with helicase c domain 1 (ifih1) gene was found. he died due to status epilepticus and pneumonia. macroscopy showed extensive cystic white matter lesions with preservation of the cortical ribbon (figure 1a). histology revealed in addition lesions with ongoing viral infection. here, the number of infected neuronal and glial cells outnumbered clearly the number of replicating cells, suggesting a limited viral replication (figure 1b). nevertheless, a pronounced decrease in oligodendrocytes was observed, while no major loss of astrocytes and neurons was found. juxtacortical lesions showed prominent axonal damage and loss. late lesions were characterized by a necrotic-cystic tissue defect located primarily at the gray-white junction and later spreading throughout the white matter. only few publications describe jcve as a new entity of jc virus pathologies characterized by a prominent infection of neurons. what distinguishes our case from others is the extraordinarily long disease course without clear evidence of immunodeficiency. a variant of unclear significance in the ifih1 gene was found. ifih1 encodes the mda5 protein (pattern-recognition-receptor), which is part of the innate immune system and is mainly responsible for the activation of the antiviral immune response such as the induction of interferon-1/-β/-α. this mutation potentially predisposed to the jcpyv infection. the slow disease progression in our patient is possibly due to relatively well-preserved immune function. the presence of a stable, archetype variant could also indicate a less productive viral variant. our detailed histological description shows that infected cells clearly outnumbered replicating cells, leading to production of virus in only single cells. despite numerous infected neurons, in the long run white matter lesions with a pronounced tissue destruction prevailed. thus, destructive white matter lesions seem to be a pivotal histopathological characteristic of jcve. figure 1: (a) cortex with juxtacortical lesion (arrow) and massive cystic changes within the white matter (wm). (b) multiple infected cortical pyramidal neurons (t-ag = early viral protein, indicating infection, neun = neuronal marker).   4.02 free neuropathol 3:20:46 reduction of oligodendrocyte populations in patients with late-onset multiple sclerosis schirin stephan1, lidia stork1, wolfgang brück1, christine stadelmann-nessler1, imke metz1 1 university medical center göttingen, institute of neuropathology, göttingen, deutschland background: over 2.8 million people worldwide suffer from multiple sclerosis (ms) a chronic inflammatory demyelinating disease of the cns. typical or normal-onset ms (noms) manifests in young adults (20-40 years old). in 3-12% of cases, the disease begins in elderly patients >50 years of age (late-onset ms or loms). such patients more often suffer from primary progressive ms, faster disease progression, and worse recovery from relapses, as well as age-related comorbidities. animal studies suggest a less efficient remyelination in elderly animals. marked remyelination, which is observed in 23-50 % of ms lesions in young adult-onset patients, could be slower and less successful in loms. a recent study showed that cells expressing bcas1 represent a subpopulation of actively myelinating oligodendrocytes, and can serve as a reliable marker of ongoing remyelination. questions: we addressed the question, if myelin regeneration in loms patients is less effective. methods: we performed histological analyses of three oligodendrocyte markers that correspond to different oligodendrocyte maturation stages (olig2 positive cells with a strong nuclear signal for oligodendrocyte precursor cells, bcas1 for myelinating oligodendrocytes, and nogoa for mature oligodendrocytes) in biopsy specimens from loms patients (n=30), and compared them with noms patients (n=25). results: the number of mature oligodendrocytes was significantly lower in the normal appearing (non-demyelinated) (p=0.02) and perilesional white matter (p<0.0001) in patients with loms compared to noms. moreover, mature oligodendrocytes in these regions showed a negative correlation with the age of patients (r=-0.5, p=0.01). there were no differences in the number of oligodendrocyte precursor cells between the two groups. also, the population of active myelinating bcas1+ oligodendrocytes was significantly lower in loms compared to noms patients in the normal-appearing white matter (p=0.03). again, the number of myelinating cells correlated negatively with the age of patients (r=-0.5, p=0.01). in both groups, the highest number of active myelinating oligodendrocytes was found in early active demyelinating lesions and here at the lesion edge. although no significant differences in the number of active myelinating cells was found in lesion areas comparing loms and noms, a trend for higher numbers of myelinating cells was observed in the center of late active lesions in noms. in general, older lesions (late active demyelinating and inactive lesions) showed lower numbers of active myelinating cells. loms patients had a significantly higher edss score (median 3.5, p=0.003) at last follow-up as compared to noms patients (median 2.0). a higher edss score was associated with a lower number of both mature and oligodendrocyte precursor cells in active ms lesions (r=-0.42, p=0.01 and r=-0.54, p=0.002 respectively). conclusions: we observed a significant reduction of active myelinating and mature oligodendrocytes in the periplaque and normal appearing white matter of loms patients. this may result in a lower remyelination within lesions. our results suggest a more efficient remyelination in noms compared to loms. importantly, we show that a higher edss at last follow-up in loms patients negatively correlates with the number of oligodendrocytes within lesions, emphasizing the importance of a loss of oligodendrocytes for clinical disability. study supported by sanofi genzyme.   4.03 free neuropathol 3:20:47 schwann cell remyelination is a salient feature of spinal nmo with neuroprotective potential carolina thomas1,2, josephine sophia owens1, parinaz yavarzadeh1, anne winkler1, christine stadelmann1 1 institut für neuropathologie universitätsmedizin göttingen, göttingen, deutschland 2 max-planck-institut für multidisziplinäre naturwissenschaften, city campus abteilung molekulare neurobiologie, göttingen, deutschland background: neuromyelitis optica (nmo) is a severe autoimmune demyelinating disorder characterized by the presence of pathogenic autoantibodies against the water channel aquaporin-4, and clinically by extensive myelitis with optic neuritis. intriguingly, despite the complete loss of astrocytes with consecutive damage and reduction of oligodendrocytes, a partial remyelination of nmo lesions has been previously described. aims: here we aimed to decipher the cellular components present in remyelinated spinal nmo lesions and to detect potential protective effects of remyelination on axonal integrity in these lesions. methods: we performed post-mortem histopathological and immunohistochemical examination of spinal lesions from a cohort of 8 patients. results: we demonstrate that schwann cells (sc) are present and are in part responsible for remyelination of nmo lesions. furthermore, we show a greater abundance of remyelination in the lesions localized along peripheral nerve entry-exit zones as well as perivascularly. in addition, no sc remyelination was observed in spinal lesions of patients suffering from multiple sclerosis. finally, axonal density was partially preserved in sc remyelinated areas. conclusions: taken together, our results indicate that scr is an exclusive feature of nmo that requires a breaching of the glia limitans with potential neuroprotective effects.   5. muscle / nerve 5.01 free neuropathol 3:20:48 molecular profiling of skeletal muscle in infantile, juvenile and adult patients with pompe disease alexander schaiter1, andreas roos2,3,4, andreas hentschel5, andreas hahn6, marek bartkuhn7,8, anne schänzer1 1 institute of neuropathology, justus liebig university, giessen, deutschland 2 department of pediatric neurology, centre for neuromuscular disorders, centre for translational neuroand behavioral sciences, university duisburg-essen, essen, deutschland 3 children's hospital of eastern ontario research institute; division of neurology, department of medicine, the ottawa hospital; and brain and mind research institute, university of ottawa, ottawa, canada 4 department of neurology, heimer institute for muscle research, university hospital bergmannsheil, ruhr-university bochum, bochum, deutschland 5 leibniz-institute für analytische wissenschaften – isas – e.v., dortmund, deutschland 6 department of child neurology, justus liebig university, giessen, deutschland 7 institute of biomedical informatics and systems medicine science unit for basic and clinical medicine, justus liebig university, giessen, deutschland 8 institute for lung health (ilh) platform for genomics and bioinformatics, justus liebig university, giessen, deutschland introduction: pompe disease is a lysosomal metabolic disease caused by mutations in the alpha 1,4-glucosidase (gaa). the gaa enzyme defect leads to accumulation of glycogen in striated muscles and a reduced muscle function. pompe disease is categorized in infantile onset (iopd) and late onset (lopd) based on gaa residual activity, genetic profile and clinical manifestation. furthermore, a juvenile onset is described, which is classified as lopd. the underlying mechanisms of pompe disease are still unknown. molecular signatures of iopd, lopd and juvenile patients are rare. the aim of this study is to identify specific differences between the subgroups of pompe disease using proteomic analysis. the first objective is to find novel protein expressions associated with the subgroups of pompe disease. the second objective is to compare the proteomic profiles of the subgroups with each other to prove the categorization by a proteomic approach. material and methods: skeletal muscle biopsies from 35 patients with pompe disease before start of enzyme replacement therapy (ert) (lopd n=22, iopd n=11, juvenile n=2) and 21 age matched controls (adult n=11, infantile n=10) were analysed using mass-spectrometry in an lfq (label-free-quantification) experiment. to identify significantly up or down regulated proteins between the groups, the raw proteomic data was further processed using maxquant, perseus and the r-programming language. principal component analysis (pca) and hierarchical clustering was used to show statistical difference between the sample groups. further, a pathway analysis of the significantly associated proteins using the kegg and the gene ontology (go) library was conducted. to validate associated pathways on another approach a gene set enrichment analysis (gsea) was calculated. to compare the proteomic profile of lopd, iopd and juvenile, the most significant proteins of each group were defined as gene-sets in gsea. results: in the iopd 13 proteins were up-regulated and 11 downregulated compared to controls. analyses of juvenile pompe showed 7 up-regulated, 37 downregulated and in lopd 19 up-regulated, 47 down-regulated proteins. the samples of lopd and juvenile shared certain protein expression e.g. fbn1, col1a2 and col3a1 were down-regulated in both groups. however, iopd and lopd showed divergent expression e.g. hk1 (phosphorylation of various hexoses and involved in innate immunity) was up-regulated in lopd and down-regulated in iopd whereas usmg5 (upregulated during skeletal muscle growth) was upregulated only in iopd. the gsea further shows a positive correlation between lopd and juvenile pompe and a negative correlation between iopd and lopd/juvenile pompe. discussion: mass-spectrometry analysis showed a significant altered regulation in skeletal muscle samples from patients with pompe disease compared to controls. interestingly most of the dysregulated proteins varied in iopd and lopd subgroup, whereas the expression profiling was similar in the juvenile patients compared to lopd. these data underline the heterogeneity in pompe disease and may indicate a divergent underlying muscle pathology in infantile patients with more immature skeletal muscle fibers compared to juvenile and adult patients.   5.02 free neuropathol 3:20:50 expression of immune regulating proteins in skeletal muscle of different idiopathic inflammatory myopathies (iim) subtypes anna nishimura1, rebecca hasseli2,3, heidrun h. krämer4, angela roth1, eva neuen-jacob5, tobias ruck6, anne schänzer1 1 institute of neuropathology, justus-liebig-university, giessen, deutschland 2 department of rheumatology and clinical immunology, campus kerckhoff, justus-liebig-university, giessen, deutschland 3 department of internal medicine ii, justus-liebig-university, giessen, deutschland 4 department of neurology, justus-liebig-university, giessen, deutschland 5 institute of neuropathology, heinrich-heine-university, düsseldorf, deutschland 6 department of neurology, medical faculty, heinrich-heine-university, düsseldorf, deutschland background: idiopathic inflammatory myopathies (iims) are autoimmune diseases classified as polymyositis (pm), dermatomyositis (dm), immune-mediated necrotizing myopathy (imnm), anti-synthetase syndrome (asys) and sporadic inclusion body myositis (sibm). adaptive and innate immune responses play a role in the pathogenesis of iim and immunomodulatory treatment is the recommended therapy. the individual regulatory mechanisms in iim subtypes might differ and a better understanding of the pathophysiology would improve the individual therapeutic approaches. with whole muscle section morphometry, we want to analyze the immune regulating proteins in certain subtypes of iim. methods: muscle biopsies from 24 adult patients with iim (average age at biopsy 54.6 years; 65.2 % female; dm n=5; imnm n=5; asys n=7; sibm n=7), neurogenic atrophy (na; n=4) and controls (hc; n=6) were included in the study. all biopsies were re-evaluated according to the common classification. the degree of pathology severity was estimated using a semiquantitative pathology score (p-score from 0 to 10). double immunofluorescence staining was performed with antibodies against mhc-1 (major histocompatibility complex 1), mhc-2, icam-1 (intercellular adhesion molecule 1) and vcam-1 (vascular cell adhesion molecule 1) and antibodies against spectrin or desmin. the sections were subsequently digitalized using a zeiss axio scan.z1 slide scanner. the coexpression was analyzed on entire sections using imagej software and quantified using the manders' coefficient (m). results: mhc-1 expression was significantly upregulated on muscle fibers of patients with asys (m=0.559), dm (m=0.609), sibm (m=0.557) and na (m=0.208) compared to hc (m=0.009). for imnm (m=0.079), the expression was significantly lower compared to asys and dm. significant upregulations of mhc-2 were found for asys (m=0.263), dm (m=0.504), sibm (m=0.336) and na (m=0.416) compared to hc (m=0.036), as well as a significant upregulation for dm compared to imnm (m=0.055). only asys samples showed a significant upregulation of icam-1 compared to hc (m=0.060), whereas in general, the expression varied strongly (asys m=0.263±0.182; dm m=0.270±0.256; sibm m=0.337±0.249; imnm m=0.335±0.295) with no significant difference between the iim subtypes. vcam-1 expression showed no significant difference between the iim subtypes. however, in comparison to hc (m=0.036) we found vcam-1 significantly upregulated in asys (m=0.200), dm (m=0.377) and sibm (m=0.371). correlating our results with the p-score, a positive correlation (r=0.770) for icam-1 expression in sibm was found. conclusion: we analyzed protein expression levels on muscle fibers in whole section analysis, which provides accurate data on larger sections. different expression patterns of mhc-1/2, icam-1 and vcam-1 were found in iim subtypes with lower expressions in imnm for mhc-1/2 and vcam-1. interestingly, in sibm icam-1 expression correlated with the pathology score. these insights might help to improve morphological diagnosis in iim subtypes and identify individual immune response patterns, which may improve the accuracy of future diagnoses.   5.03 free neuropathol 3:20:52 long term safety and efficacy outcomes for x-linked myotubular myopathy (xlmtm) with gene replacement therapy, resamirigene bilparvovec (aspiro): preliminary results from cohort 1 in aspiro, a phase 1/2/3 study astrid blaschek1, perry shieh2, nancy kuntz3, james dowling4, carsten bonnemann5, a. reghan foley5, dimah saade6, andreea seferian7, laurent servais8, neema lakshman9, cong han10, suyash prasad11, salvador rico11, westin miller9 1 klinikum der universität münchen, münchen, deutschland 2 university of california, los angeles, united states 3 ann & robert h. lurie children’s hospital of chicago, chicago, united states 4 the hospital for sick children, toronto, canada 5 national institutes of health, bethesda, united states 6 university of iowa hospitals and clinics medicine specialty clinics, iowa city, united states 7 institut de myologie, paris, france 8 mduk oxford neuromuscular centre, oxford, united kingdom 9 astellas gene therapies, california , united states 10 astellas pharma global development, northbrook, united states 11 formerly astellas gene therapies, california, united states background: xlmtm is a rare, life-threatening congenital myopathy caused by mutations in the mtm1 gene. there is no approved treatment for xlmtm. objectives: aspiro (nct03199469), a phase 1/2/3 randomized, open-label study is investigating the safety and efficacy of at132 (resamirigene bilparvovec), a single-dose gene replacement therapy for ventilatory-dependent xlmtm. question: what is the primary safety and efficacy data from asprio? methods: participants were young boys with genetically confirmed xlmtm. the primary efficacy outcome was the change in hours of daily ventilator support from baseline through week 48. the key secondary efficacy outcome was percentage of participants who achieve functionally independent sitting by week 48. we report long-term safety and key efficacy outcomes (up to 42 months [m]) for the first 6 participants dosed in aspiro, all receiving the lower-dose of 1.3 x 1014 vg/kg and compared with 15 untreated controls (including 12 participants from inceptus), as of 29jan2021. results: the mean age at dosing was 20·4m (range: 9·5-49·7m) and 19·6m (5·9-39·3m) at enrollment among dosed participants and controls, respectively. major developmental milestones achieved by all dosed participants over time is shown in figure 1. all dosed participants were ventilator dependent at first assessment; 5 (83.33%) requiring transtracheal invasive ventilation >22 hours/day and 1 (16.67%) used non-invasive ventilation of 12 hours/day. all dosed participants achieved ventilator independence, 5 remain so (mean durability 25.6m; range 18.3-36.6m) of which 4 have been decannulated. no control participants became ventilator independent or were decannulated. at baseline, 1/6 dosed participant was able to sit independently without support for 30 seconds; 5/6 participants did not have full head control and were unable to sit independently. major motor milestones were achieved in all dosed participants (figure 1); 5/6 remain independently ambulatory without assistive device (achieved mean [sd] time 21.92 [5.57]m); 4 of whom have achieved the ability to ascend stairs. 5/15 (33.3%) control participants achieved independent sitting; none achieved higher milestones. among 6 dosed in cohort 1, 4 (67%) participants experienced treatment-emergent severe adverse events; infections in 4 (67%) and respiratory/thoracic/mediastinal disorders in 1(17%). all dosed participants currently have stable liver function test values. as of january 2021, three deaths in the higher-dose cohort occurred following severe decompensated liver disease, and three deaths in the control cohort (aspiration pneumonia; cardiopulmonary failure; hepatic hemorrhage with peliosis) were observed. as of september 2021, a newly dosed participant in the lower-dose cohort experienced severe liver function test abnormalities and has died. conclusions: a rapid improvement in respiratory and motor outcomes was observed among 6 xlmtm participants dosed with at132 at 1.3 x 1014 vg/kg vs control participants; these improvements have been maintained and expanded upon over time, indicating improved strength, function, and quality of life for these dosed participants. these substantial improvements must be weighed against the occurrences of fatal serious adverse events, for which the aspiro program is on clinical hold while relevant clinical information is being gathered and reviewed. figure 1: developmental motor and respiratory milestones achieved in xlmtm patients dosed at at132 at 1.3 x 1014 vg/kg.   5.04 free neuropathol 3:20:54 lymphotoxin-driven chronic mucle inflammation interdepends with impaired autophagy, self-perpetuates and models inclusion body myositis in mice juliane bremer1, judith bauer2, jana zschüntzsch3, thomas blank4, kamil zajt1, laura anna fischer3, anna sensmeyer3, lara wiechers3, josef reichelt3, kai hofmann5, monika wolf6, corinna leuchtenberger5, priyanka tripathi1, claudia einer7, hans zischka7, adriano aguzzi6, regina reimann6, veronika kana6,8, elisabeth rushing6, marco prinz4, david liebetanz3, francesca odoardi3, joachim weis1, jens schmidt3,9, mathias heikenwälder5 1 uniklinik rwth aachen, institut für neuropathologie, aachen, deutschland 2 technische universität münchen, institut für toxikologie und umwelthygiene, münchen, deutschland 3 universitätsmedizin göttingen, klinik für neurologie, göttingen, deutschland 4 universitätsklinikum freiburg, institut für neuropathologie, freiburg, deutschland 5 dkfz heidelberg, heidelberg, deutschland 6 universitätsspital zürich, institut für neuropathologie, zürich, switzerland 7 helmholtz münchen, institut für molekulare toxikologie und pharmakologie , münchen, deutschland 8 universitätsspital zürich, klinik für neurologie, zürich, switzerland 9 universitätsklinik der medizinischen hochschule brandenburg, abteilung neurologie und schmerztherapie, rüdersdorf, deutschland background, goals and research question: inclusion body myositis (ibm) is a progressive muscular disorder characterized by muscle inflammation and degeneration including protein aggregates and altered autophagic activity. the combination of inflammatory and degenerative features has led to the assumption that this interrelationship may serve as a major driver of the disease pathology. in view of the lack of effective treatment there is an urgent need for useful model systems that reflect the presumed pathomechanisms. a reliable animal model for chronic inflammatory and degenerative features as in ibm has not been available. methods/results: here, we established mouse models with lymphotoxin expression-driven chronic inflammation of skeletal muscle and with impaired autophagy due to muscle cell specific atg5-knockout. both conditions alone induced weakness and muscular atrophy. gene expression analysis showed that, while chronic inflammation alone drives endoplasmic reticulum stress and alters autophagy/ proteostasis, autophagy disruption alone induces a pro-inflammatory state. this suggests that both, inflammation and autophagy disruption are interdependent in ibm pathogenesis. only when we genetically combined transgene-driven inflammation with autophagy impairment in mice, skeletal muscle fibers displayed characteristic molecular and neuropathological features of ibm, including protein aggregates with typical ultrastructural morphology. given that human ibm is refractory to established drugs, we aimed to mirror recent treatment failures and identify the underlying mechanisms by subjecting four months-old myositis mice to corticosteroids, anti-cd52 or anti-thy1.2 antibodies to deplete lymphocytes, or by blocking lymphotoxin beta-receptor signaling. none of these treatments was able to significantly improve muscular performance of the mice or expression profiles of molecular indicators of muscle pathology. this suggests that, once established, ibm-like pathology cannot be reverted or prevented from progression, but is a self-perpetuating condition. conclusions: in summary, the data provide unique evidence that inflammation and autophagy disruption are intertwined in ibm-like muscular pathology. it can be expected that this novel mouse model will substantially further our effort to identify better treatment modalities for ibm in the future.   5.05 free neuropathol 3:20:56 novel form of congenital myopathy caused by bi-allelic mutations in uncoordinated mutant number-45 myosin chaperone b sebahattin cirak1 1 uniklinik ulm, klinik für kinder-und jugendmedizin, sozialpädiatrisches zentrum und pädiatrische neurologie mit stoffwechsel, ulm, deutschland background: congenital myopathies (cm) form a genetically heterogeneous group of disorders, only 60% can be genetically solved. aims: discovery of novel genetically defined myopathies. question: deciphering the genomic landscape of congenital myopathies. methods: we recruited an 11-year old male of consanguineous parents, presenting with proximal weakness, gower's sign, without cardiomyopathy with a stable disease course. we performed exome sequencing and data analysis was performed with our in-house software varbank2 according to an autosomal recessive inheritance. we investigated the effect of the missense mutation by complementation assay on the zebrafish steif mutant, an unc-45b loss-of-function model. results: we have discovered and published a novel genetically defined form of cm due to a novel homozygous missense mutation in unc45b (nm_173167.2: c.2261g>a, p.arg754gln) also co-segregating in the family with three healthy siblings (dafsari et al., 2019). in our patient's muscle biopsy, core-like structures were detected mainly in the center of muscle fibers in nadh histochemistry. electron microscopy showed numerous focal core-like alterations of myofibrillar architecture with z-bands streaming. conclusions: three isoforms of unc45b are highly expressed in skeletal muscle, only one also in cardiac muscle. due to its high evolutionary conservation throughout species, a loss of unc45 results in different pathological conditions in various species: a knockdown of unc-45 resulted in dilated cardiomyopathy and a reduced muscle contractility in d. melanogaster. similarly, in unc-45b knockdown zebrafish and also in steif mutants, disrupted myofibrillogenesis associated cardiac dysfunction and paralysis was observed. injection of mutant unc-45b mrna did not rescue the steif mutant in contrast to wt mrna confirming the pathogenicity of the missense mutation.   6. free topics 6.01 free neuropathol 3:20:57 deep genotype-phenotype analysis of focal cortical dysplasia type 2 differentiates between a gator-positive autophagy altered subtype 2a and mtor-positive migration deficit subtype 2b jonas honke1, lucas hoffmann1, roland coras1, javier a. lópez-rivera2,3,4, costin leu3,4,5,6, dennis lal3,4,5,6, peter nürnberg6, tom pieper7, till hartlieb7, manfred kudernatsch7, christian g. bien8, friedrich woermann8, thomas cloppenborg8, hajo hamer9, sebastian brandner10, karl rössler11, arnd dörfler12, stéphanie baulac13, sara baldassari13, ingmar blümcke1,4 1 department of neuropathology, universitätsklinikum erlangen, fau erlangen-nürnberg, and partner of the european reference network (ern) epicare , erlangen, deutschland 1 department of molecular medicine, cleveland clinic lerner college of medicine, case western reserve university, cleveland, united states 1 genomic medicine institute, lerner research institute, cleveland clinic, cleveland, oh 44195, united states 1 charles shor epilepsy center, neurological institute, cleveland clinic, cleveland, united states 1 stanley center for psychiatric research, broad institute of harvard and m.i.t, cambridge, ma 02142, united states 1 cologne center for genomics (ccg), medical faculty of the university of cologne, university hospital of cologne, 50931 cologne, germany 1 center for pediatric neurology, neurorehabilitation, and epileptology, schoen-clinic, vogtareuth, germany 1 department of epileptology (krankenhaus mara), medical school, bielefeld university, bielefeld, germany 1 epilepsy center, universitätsklinikum erlangen, fau erlangen-nürnberg, and epicare partner, erlangen, germany 1 department of neurosurgery, universitätsklinikum erlangen, fau erlangen-nürnberg, erlangen, germany 1 department of neurosurgery, medical university of vienna, vienna general hospital, vienna, austria 1 department of neuroradiology, universitätsklinikum erlangen, fau erlangen-nürnberg, erlangen, germany 1 sorbonne université, institut du cerveau paris brain institute icm, inserm, cnrs, aphp, hôpital de la pitié salpêtrière, paris, france background: focal cortical dysplasia type 2 (fcd2) is the single most common cause of drug-resistant focal epilepsy in children. despite continuous progress in diagnostic methods, however, the recognition and treatment of fcd2 subtypes remain a challenging issue in clinical practice. herein, we performed a deep genotype-phenotype analysis to further elucidate the clinical-pathological and genetic presentation of fcd2. methods: patients with focal epilepsy submitted to neurosurgical treatment, histopathological confirmed diagnosis of fcd ilae type 2 and positive genetic testing were retrieved from the european epilepsy brain bank. clinical data were available from the contributing epilepsy centers. deep whole-exome sequencing with a coverage of >350x or mtor gene panel sequencing with a coverage of >1000x were obtained from fresh frozen tissue samples. histopathological analyses were performed from formalin-fixed, paraffin embedded tissue samples using he and immunohistochemistry for nf-smi32, neun, ps6, p62, and vimentin. all slides were digitalised and further analysed with qupath v.0.3.0. results: seventeen patients were identified by carrying pathogenetic variants in genes directly associated with the mtor pathway, i.e., loss-of-function in the gator1 complex (depdc5, n=7 and nprl3, n=3), or gain-of-function in the mtorc1 signalling pathway [mtor, n=7]. all patients were seizure-free after surgery with the exception of four patients carrying a depdc5 mutation. histopathological analysis revealed a fcd2a subtype in all cases with gator1 alteration, i.e., no balloon cells. in contrast, the fcd2b subtype was predominant in cases with gene variants affecting the mtorc1 signalling complex. specimens carrying mtor variants also had significantly larger dysmorphic neurons than gator1 variants [p=0.005]. in addition, five cases defined by gator1 variants showed a unique and predominantly vacuolizing phenotype (figure 1c). all cases with gator1 alterations were located in the frontal lobe and the majority was confined to the cortical ribbon not affecting the white matter (figure 1a). this pattern was reflected by subtle or negative mri findings in 9/10 patients with gator1 variants. discussion/conclusion: we describe a yet unrecognized genotype-phenotype correlation of gator1 variants with fcd2a in the frontal lobe. these lesions were histopathological further characterized by abnormally vacuolizing cells suggestive of an autophagy altered phenotype. from recently published evidence, we also hypothesize that this subtype will carry a second hit brain somatic variant in the depdc5 gene, which was, however, difficult to identify with techniques applied in our current study. in contrast, patients with fcd2b and brain somatic mtor variants showed larger lesions on mri including the white matter, suggesting compromised neural migration (figure 1b). these data may help to better understand difficult-to-diagnose and treat fcd subtypes, i.e., ilae type 2a and 2b. figure 1: genotype-phenotype analysis of fcd ilae type 2 a: 42-year-old male patient with frontal lobe epilepsy since age 5 years, histopathological confirmed fcd2a and a pathogenic depdc5 mosaicism. the arrow points to the sharp border between the cortical fcd and the normal appearing white matter (wm). ncx normal 6-layered neocortex. neurofilament smi32 immunohistochemistry. scale bar = 2.5mm (applies also to b). higher magnification in c reveals dysmorphic neurons with a vacuolizing predominant phenotype (red arrow) suggesting an autophagy altered phenotype. scale bar = 100µm (applies also to d). b: 19-year-old male patient with frontal lobe epilepsy since age 9 years, histopathological confirmed fcd2b at a bottom-of-sulcus (bos; higher magnification in d) and a pathogenic mtor mosaicism. dysmorphic neurons and balloon cells (latter not shown) were aggregating in the neocortex and white matter (arrow) suggesting a migration deficient phenotype.   6.02 free neuropathol 3:20:60 age-dependent increase of perineuronal nets in the human hippocampus of patients with and without temporal lobe epilepsy annika lehner1, lucas hoffmann1, friedrich paulsen2, hajo hamer3, katrin walther3, karl rössler4, sebastian brandner5, ingmar blümcke1 1 universitätsklinikum erlangen and fau erlangen-nürnberg, department of neuropathology, erlangen, deutschland 2 fau erlangen-nürnberg, institute of functional and clinical anatomy, erlangen, deutschland 3 universitätsklinikum erlangen and fau erlangen-nürnberg, epilepsy center, neurological institute, erlangen, deutschland 4 medical university of vienna, vienna general hospital, department of neurosurgery, vienna, austria 5 universitätsklinikum erlangen and fau erlangen-nürnberg, department of neurosurgery, erlangen, deutschland background: perineuronal nets (pnn) are a specialized extracellular matrix surrounding parvalbumin-positive gabaergic interneurons of the central nervous system, contain mainly aggrecan and lecticans, and play a role in the regulation of synaptic plasticity, brain maturation and cognitive impairment. in the human neocortex, pnn appear in their full expression at an age of eight, and alterations of pnn have been found in neurological disorders including alzheimer´s disease, schizophrenia or epilepsy. however, their role in functional maintenance of the cns as well as disease pathogenesis remains to be elucidated. aim: our aim was to histopathologically assess pnn in the hippocampus of patients with temporal lobe epilepsy (tle) compared to age-matched post-mortem control subjects. methods: formalin-fixed and paraffin-embedded neurosurgical tissue specimens of the human hippocampus were retrieved from the european epilepsy brain bank. twenty-nine patients had histopathologically confirmed hippocampal sclerosis (hs), and 11 patients suffered from tle with no hs. neuropsychology scores for cognitive tasks were retrieved from the files of the erlangen epilepsy center. hippocampus samples of 27 postmortem brains served as control (age range 3-84). pnn were visualized using antibodies directed against aggrecan (figure 1). pnn were manually recorded in the dentate gyrus, ca1, ca2, ca3, ca4 and subiculum. pnn density per mm² was then compared between different hs subtypes according to the ilae classification scheme and controls. selected cases were double immunofluorescence labeled with antibodies against parvalbumin and aggrecan (figure 1) and further analyzed on fully digitalized scans (hamamtsu nanozoomer s60) with qupath 0.3.0. results: the density of pnn increased with age in both, human controls and tle patients (figure 2). however, the density of pnn was significantly higher in all tle patients compared to age-matched controls with a non-significant increase in patients with hippocampal sclerosis compared to tle patients without hs. these alterations were also region-specific and most obvious in the subiculum, ca1 and dentate gyrus (figure 3). there was no significant correlation with cognitive impairment in our cohort of tle patients. discussion: we quantitatively described the normal distribution of pnn in the human hippocampal formation. we investigated changes with age in the number of perineuronal nets in older patients and found new evidence that epilepsy alters perineuronal nets. figure 1: triple immunofluorescence-staining at 40x magnification: a parvalbumin-positive neuron surrounded by aggrecan (left side). immunohistochemical staining of a pnn in the stratum plexiforme of the dentate gyrus (150 µm away from stratum granulosum), screenshot from qupath, 40x magnification (right side). figure 2: correlation between the age and the average number of pnn per mm² in patients with and without epilepsy figure 3: the normal distribution of pnn and alterations in epilepsy   6.03 free neuropathol 3:20:63 vakuolisierung der dura als nicht-lymphassoziierte veränderung ralf schober1, christian eisenlöffel1, max holzer2 1 klinikum st. georg, inst. f. pathologie u. tumordiagnostik, leipzig, deutschlandy 2 paul-flechsig-institut für hirnforschung, univ. leipzig, leipzig, deutschland die meningealen lymphgefäße stellen eine zwischenstation beim abtransport des liquors vom glymphatischen system des gehirns zu den zervikalen lymphknoten dar (louveau j ea 2015, aspelund a ea 2015). sie gelten als wichtiges immun-portal für das zentrale nervensystem (tavares ga u. louveau a 2021), und eine verlegung des glymphatischen systems wird pathogenetisch mit der demenz und insbesondere der alzheimer'schen erkrankung in verbindung gebracht (nedergaard m u. goldman sa 2020). histologische darstellungen der überwiegend sinus-nah in der dura gelegenen lymphkanäle beziehen sich bislang hauptsächlich auf die ratte, detailliertere untersuchungen beim menschen und insbesondere bei verschiedenen erkrankungen stehen noch aus. wir haben in 35 sektionsfällen die dura untersucht, meist im bereich des sinus sagittalis superior, teilweise zusätzlich des sinus transversus. das alter der 14 weiblichen und 21 männlichen patienten und patientinnen betrug 25-91 jahre, im durchschnitt 69 jahre. in 8 fällen war ein morbus alzheimer diagnostiziert worden, in 9 fällen alzheimer-assoziierte veränderungen, in je einem fall part und morbus parkinson. 6 fälle hatten meningitische veränderungen, 2 fälle eine meningeosis carcinomatosa, 2 fälle eine av-fistel, 3 fälle intra-/subdurale blutungen, und 3 fälle eine sinusthrombose. im histologischen bild vorherrschend und in allen außer 3 fällen nachweisbar waren jedoch nicht die lymphgefäße, sondern gruppen kleiner intraduraler vakuolen, 30-300 µ im durchmesser, meist glatt begrenzt und ohne zellbelag. bei immunhistochemischen untersuchungen in ausgewählten fällen zeigten sie keine positive reaktion auf podoplanin, cd31, cd34, s100, aβ und app. dagegen ließen sich lymphgefäße deutlich anhand ihrer positiven reaktion auf podoplanin identifizieren, sie erschienen durchweg streifenförmig-schmal und regelrecht strukturiert. die anzahl der vakuolen-gruppen und ihre dichte in der dura wurde semiquantitativ in 6 stufen von nicht vorhanden bis stark eingeteilt, und diese stufen wurden jeweils den falldaten gegenübergestellt. eine korrelation mit bestimmten erkrankungen ergab sich nicht, lediglich eine partielle korrelation mit dem alter. deutlich vermehrt waren vakuolen bei gleichzeitigem nachweis einer größeren anzahl fibrosierter pacchionischer granulationen vorhanden. zusammenfassend stellt eine vakuolisierung der dura im sinusdach-bereich eine relativ häufige veränderung dar, nicht im zusammenhang mit dem lymphsystem stehend sowie offenbar nicht krankheits-korreliert und am ehesten degenerativer natur. die befunde bestätigen und ergänzen kurze angaben der älteren literatur, wo zunächst von grüppchen großblasiger zellen gesprochen wird (schaffer j 1933), dann aber von altersveränderungen mit hohlraumbildungen durch aufquellung und zerfall von bindegewebsfasern (schaltenbrand g u. dorn e 1955).   6.04 free neuropathol 3:20:64 moghe with or without slc35a2 brain somatic mutations reveal a common phenotype of oligodendroglial regeneration and remyelination simon geffers1, lucas hoffmann1, roland coras1, katja kobow1, javier lopez-riviera2,3,4, costin leu3,4,5,6, dennis lal3,4,5,6, peter nürnberg6, stephanie bulac7, till hartlieb8,9, tom pieper8, manfred kudernatsch8,10, christian bien11, hajo hamer12, karl rössler13,14, sebastian brandtner14, christine stadelmann15, ingmar blümcke1 1 department of neuropathology, university hospital erlangen, erlangen, deutschland 2 department of molecular medicine, cleveland clinic lerner college of medicine, cleveland, united states 3 genomic medicine institute, lerner research center, cleveland clinic, cleveland, united states 4 charles shor epilepsy center, neurological institute, cleveland clinic, cleveland, united states 5 stanley center for psychiatric research, broad institute of harvard and m.i.t, cambridge, united states 6 cologne center for genomics (ccg), medical faculty of the university of cologne, university hospital of cologne, cologne, deutschland 7 sorbonne université, institut du cerveau , paris brain institute, paris, france 8 center for pediatric neurology, neurorehabilitation and epileptology, schoen klinik vogtareuth, vogtareuth, deutschland 9 research institute rehabilitation, transition, palliation, pmu salzburg, salzburg, austria 10 center for neurosurgery, epilepsy surgery, spine surgery and scoliosis, schoen klinik vogtareuth, vogtareuth, deutschland 11 department of epileptology (krankenhaus mara), bielefeld university, bielefeld, deutschland 12 epilepsy center, university hospital erlangen, fau erlangen nürnberg, erlangen, deutschland 13 department of neurosurgery, university hospital erlangen, fau erlangen nürnberg, erlangen, deutschland 14 department of neurosurgery, medical university of vienna, vienna general hospital, vienna, austria 15 institute of neuropathology, university medical center göttingen, göttingen, deutschland mild malformation of cortical development with oligodendroglial hyperplasia in epilepsy (moghe) is a recently identified disease entity associated with early onset frontal lobe epilepsy in the vast majority of reported cases. brain somatic mutations in the udp-galactose transporter slc35a2 gene were identified in about 50% of published moghe cases as underlying pathogenetic variant. herein, we performed a phenotype-genotype analysis of moghe with slc35a2 mutations compared to moghe without slc25a2 mutations in order to assess a potentially different phenotype-genotype relationship at the microscopy and clinical level. this may also help us to further untangle the yet unknown aetiological spectrum of the moghe disease. we retrieved 39 surgical brain specimens histopathologically characterized as moghe from the archives of the european epilepsy brain bank, 19 with and 20 without brain somatic slc35a2 variants and compared them with 20 age-matched controls, i.e., fcd 2a (n=10) and non-epileptic post-mortem specimens of the frontal neocortex (n=10). semi-quantitative analysis using a panel of immunohistochemical markers addressing oligodendroglial lineage (olig2), maturation (breast cancer amplifying sequence 1; bcas1), proliferation (ki67), and myelination (cnpase, nissl-lfb) were performed. the clinical presentation was not significantly different between both study cohorts (moghe with or without slc35a2 mutations), e.g., median seizure onset was at 1.2 and 1.8 years, respectively. patients were operated at 6.3 and 7.5 years, respectively, and 89% of lesions were localized in the frontal lobe. however, patients with slc35a2 mutated moghe presented with more infantile spasms (61% vs. 25%). patchy areas of increased oligodendroglial cellularity and decreased myelination were matching each other and were present in both groups (figure 1). in addition, regenerative oligodendrocytes immunoreactive for bcas1 were visible in both moghe groups and both groups had higher numbers of bcas-positive oligodendrocytes compared to our age-matched control cohort (figure 2). interestingly, moghe patients operated at an older age (>17 years; n=5) showed lower bcas and oligodendroglial cell numbers which were similar to those observed in our post-mortem control cohort. myelination deficits were also less visible in moghe patients operated at an older age (figure 3). in conclusion, our results revealed a similar genotype-phenotype correlation of moghe w/o slc35a2 brain somatic mutations. these results further suggest a similar oligodendroglial regeneration and remyelination capacity of early-onset moghe subtypes, and that the same udp-galactose transport pathway is likely to play a role in moghe with slc35a2 wildtype urging for a more systematic approach deciphering the pathogenic aetiology of the moghe disease spectrum. figure 1: patchy regions with increased oligodendroglial cell density (heatmap analysis in a, b, e) revealed a matching overlay with patchy areas of hypomyelination (f). moghe with slc35a2 mutations in a, c-f; b – moghe without slc35a2 mutation. figure 2: all moghe samples revealed a higher number of bcas1-immunoreactive oligodendrocytes at early ages, which normalize with further maturation. figure 3: all moghe samples revealed a higher degree of hypomyelination (nissl-luxol-fast-blue, lfb) at early ages, which normalize with further maturation. copyright: © 2022 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. parkinsonism associated with prolonged unresponsive wakefulness syndrome after blunt head injury: a clinico-pathological study feel free to add comments by clicking these icons on the sidebar free neuropathology 6:3 (2025) original paper parkinsonism associated with prolonged unresponsive wakefulness syndrome after blunt head injury: a clinico-pathological study kurt a. jellinger institute of clinical neurobiology, vienna, austria corresponding author: kurt a. jellinger · institute of clinical neurobiology · alberichgasse 5/13 · 1150 vienna · austria kurt.jellinger@univie.ac.at submitted: 23 october 2024 accepted: 11 november 2024 copyedited by: georg haase published: 29 january 2025 https://doi.org/10.17879/freeneuropathology-2025-5982 keywords: blunt traumatic brain injury, prolonged unresponsive wakefulness, chronic vegetative state, posttraumatic parkinson-like symptoms, brainstem lesions, neuropathology abstract objective: survival after traumatic brain injury (tbi) and posttraumatic parkinsonian-like symptoms is increasing, in particular in those patients developing during disease course an unresponsive wakefulness syndrome (uws) previously termed persistent vegetative state. material & methods: 100 patients with disorders of consciousness after a blunt tbi ranging from deep coma to defective states / minimal cognitive state survived between 12 and 900 days. 15 patients developed parkinsonian symptoms, which were correlated with their neuropathological changes. results: the patients, surviving either uws recovery (n = 10) or defective minimally conscious state (mcs) (n = 5), clinically presented with severe (n = 7), moderate (n = 5), or mild (n = 3) parkinsonian symptoms mainly comprising symmetrical rigidity, amimia, hypo- / akinesia and convergence disorder, which in six patients were associated with unilateral or bilateral resting tremor. following levodopa treatment, 11 patients showed mild to moderate improvement and four patients almost complete improvement of uws, parkinsonism or both. neuropathology revealed in most cases supratentorial traumatic lesions such as contusions, cerebral hemorrhages and diffuse white matter lesions. in addition to lesions in the basal ganglia and hippocampus, all cases displayed older lesions in the dorsolateral or lateral parts of the pons and in lower midbrain with various involvement of substantia nigra. the periaqueductal gray and upper midbrain tegmentum were however preserved. the pattern of brainstem lesions correlated with the sequelae of transtentorial shifting due to increased intracranial pressure. conclusions: these and other rare observations following blunt tbi confirm the importance of the pattern of secondary brainstem lesions for the development and prognosis of uws and rare parkinson-like symptoms. introduction survival rates after blunt traumatic brain injury (tbi) and chronic posttraumatic disorders of consciousness (doc) are increasing [1]. in view of recent advances in the prognosis of tbi and related docs and their impact on diagnostic accuracy, prognosis and treatment [2, 3], the european task force on disorders of consciousness proposed the term "unresponsive wakefulness syndrome" (uws) [4–8] for the challenging neurological conditions previously termed "apallic syndrome" [9, 10] or "persistent vegetative state" (pvs) [11, 12]. the functional outcomes following severe tbi are summarized in fig. 1. with increased survival rates after severe tbi, the incidence of posttraumatic parkinsonism (ptp) has increased [14]. ptp is commonly caused by injuries from accidents (mainly motor vehicle accidents and falls) with loss of consciousness and latency to symptom onset of one to six or more months, although up to 42 % of patients did not report loss of consciousness [14]. in the fully developed "apallic syndrome", akinesia, amimia and increased muscle tonus with cogwheel phenomenon are almost obligatory and associated with spasticity and disorders of optomotoric akin to decerebration rigidity, while tremor is usually absent [10]. on the other hand, some patients with doc following severe tbi have been reported to have parkinsonian symptoms [15]. both pvs and minimally conscious state (mcs) following severe tbi can include features of akinetic mutism and parkinsonism that may share midbrain network dysfunctions [16, 17]. extrapyramidal symptoms with bradykinesia, rigidity and resting tremor are the hallmark features of ptp, whereas postural instability was reported only in 15 % of cases [18]. figure 1. conceptual overview of functional outcomes following severe traumatic brain injuries (modified from [13]). functional communication refers to the most basic skills of communication that become better with improvement of the unresponsive wakefulness syndrome (uws) (horizontal arrow). the ellipse between uws and minimally conscious state reflects rare patients with fragments of behavior that arise spontaneously and not in response to stimulation. locked-in syndrome is a rare neurological disorder in which patients can think and reason. they cannot speak but they can move their eyes up and down and blink. neuroimaging shows either a subdural hematoma causing transient compression obstruction of the basal ganglia or hypointense lesions confined to the basal ganglia [14] or were negative [15]. in a prospective study of patients with posttraumatic movement disorders, basal ganglia were the most common sites affected (66.7 %), followed by thalamus and brainstem (16.7 % each), while diffuse white matter (wm) involvement was the most common radiological lesion in patients with tremor [19]. in a man aged 37 years, parkinsonian features following a blunt tbi were associated with hypodense computer tomography (ct) lesions in the substantia nigra (sn) region [20]. in three patients, a posttraumatic rigid-akinetic syndrome resembling parkinson disease appeared after a delay of one to five months after tbi. these patients displayed traumatic changes in the substantia nigra (sn) according to neuroimaging, and responded to levodopa [21]. according to a recent review, 53 % of ptp cases had a confirmed lesion; 53 % of the latter was located in the sn, 22 % more broadly in the midbrain [14]. neuropathological studies of ptp after blunt tbi emphasized the importance of brainstem lesions secondary to increased intracranial pressure (icp) [22–25] and extensive damage of the cerebral wm due to diffuse axonal injury [26–28], associated with hippocampal lesions and bilateral necrosis in thalamus and other basal ganglia [29]. among 35 patients who survived blunt tbi for one month and up to eight years, thalamic lesions were seen in up to 96 % of cases, while diffuse axonal injury and ischemic brain lesions were seen in 14 % to 71 % of cases [30]. brain-injured patients with doc showed lesions of thalamus and basal nuclei that were confirmed by mri [31] or proton magnetic spectroscopy [32]. thalamic lesions were associated with thalamo-cortical disconnectivity [33] and disordered brain networks [34, 35], whereas brainstem lesions after severe tbi were of prognostic value [36]. disruption of the ascending arousal network in the brainstem tegmentum causes doc after acute tbi [37]. neuroimaging studies suggested an important role for the default mode network to discriminate the uws from mcs [8, 38], while abnormalities in the brainstem were confined to the tbi group [39]. brainstem injuries detected by mri after severe tbi were shown to be of prognostic value [36]. most recent studies provided evidence for a key difference in the left frontoparietal connectivity when contrasting uws with mcs [40]. this paper presents the neuropathological findings in a series of 15 patients surviving after blunt tbi, showing either complete recovery or defective states of uws, presenting with severe to mild, mainly symmetrical rigid-akinetic parkinsonian symptoms. material and methods in a consecutive post-mortem series of 630 subjects who survived a blunt tbi, 100 subjects (59 males and 41 females, age from five to 85 years, mean age 41.5 years, 77 % traffic accidents, 23 % falls plus other causes) with prolonged doc and a survival between 12 and 900 days showed various states of consciousness ranging from deep coma or acute midbrain syndrome (n = 6, survival 12–35 days) to full uws state (n = 61, survival 12–257 days), partial recovery from uws (n = 22, survival 21–900 days) to remission state or partial clinical improvement comparable to mcs (n = 11, survival 21–293 days, mean 93.5 days). the neuropathological findings of this group have been described previously [25]. the 100 patients who survived blunt tbi for periods between 22 and 900 days (mean 564 days) included 15 patients (13 males, 2 females aged 21 to 56 years, mean 41.4 years) who showed not only posttraumatic motor and postural disorders but also flexor and / or extensor spasms and optomotor disorders (diplopia, convergence disorders) and who developed parkinson-like symptoms of moderate to severe intensity, mainly characterized by symmetrical hypo / akinesia, hypomimia / aminia and rigidity, with convergence disorders in 10 patients and unilateral or bilateral resting tremor in six patients. the clinical data of these 15 patients were evaluated retrospectively from their clinical and intensive care records. their state of consciousness was evaluated by intensive care specialists according to the glasgow scales and their extrapyramidal symptoms by movement disorder specialists. since a part of the patients died between 1966 and 1980, ct studies could only be performed in a few patients. in addition to supratentorial traumatic lesions, the patients showed recent unior bilateral necroses, hemorrhagic cysts, old necroses, and/or atrophy of striatum, globus pallidus and/or thalamus, unilateral or bilateral hippocampal lesions, extensive damage to the cerebral wm and unior bilateral lesions in the brainstem, usually in lateral or dorsolateral parts of the pontine tegmentum, cerebral peduncles and/or cerebellum. in some patients, cerebral blood flow measurements were performed with the 133xenon clearance method according to ingvar & lassen and gray matter hypoperfusion was correlated with the anatomical brainstem lesions [41]. most of the patients with parkinsonian symptoms received levodopa treatment, initially per gastric tube and later on orally (levodopa / benserazide carbidopa 100 / 25 mg three times per day). eleven patients showed partial improvement of the parkinsonian symptoms and less pvs, while four patients showed almost complete recovery of both syndromes. neuropathological examination was performed according to standard protocols, with macroscopic assessment of brain atrophy, focal supratentorial brain lesions, deep traumatic and posttraumatic brain lesions (residuals of subdural / epidural or subarachnoidal hemorrhages, cortical contusions / concussions or hematomas, etc.). pressure necroses / hemorrhages in the parahippocampal gyri were taken as evidence that icp had been increased. macroscopic changes in brainstem and cerebellum were described in detail. histological examination of multiple paraffin blocks was performed using routine stains (h&e, cresyl violett, kluver-barrera or heidenhain stain, bodian silver impregnation, holzer stain for fibrillary gliosis and immunohistochemistry for glial fibrillary acidic protein / gfap). amyloid-β, tau and synuclein pathologies were not specifically examined. results the type, location and extension of the essential morphological changes are presented in table 1. the key clinical and neuropathological data are presented in table 2. table 1: cerebral lesions in 15 patients with blunt traumatic brain injury showing distinct recovery states from unresponsive wakefulness syndrome (uws) and parkinsonian symptoms uws partial recovery uws defective state/mcs total number of patients 10 5 15 survival (days) 25–900 22–293   deep coma (days) 3–12 3–10   intracranial, epi-subdural hemorrhage 4 2 6 no superficial brain lesions 2 0 2 cortical contusions/lacerations 8 2 10 vascular/ischemic cortical lesions 1 1 2 white matter hemorrhages 1 0 1 diffuse white matter lesions 6 4 10 lesions corpus callosum 1 0 1 hippocampal/parahippocampal lesions 9 4 13 necroses/hemorrhages striatum 3 2 5 necroses/hemorrhages globus pallidus (total) 5 1 6 necroses thalamus 2 0 2 lesions globus pallidus + bilateral sn 2 1 3 lesions globus pallidus + unilateral sn 1 1 2 lesions floor 3rd ventricle, periaqueductal gray 0 0 0 brainstem lesions (total) 10 5 15 lesions midbrain tegmentum + sn 1 0 1 lesions lateral/dorsolateral pons tegmentum 9 5 14 diffuse gliosis pons 1 1 2 cerebellar lesions 1 1 2 uws: unresponsive wakefulness syndrome; mcs: minimal cognitive state; sn: substantia nigra table 2: key clinical and neuropathological data uws stage major secondary lesions age sex accident coma duration (days) survival (days) primary traumatic lesion partial recovery defect state parkinsonian symptoms hippocampus others brainstem lesion 48 f car accident 8 111 subdural h., concussions + – rigor, akinesia ++ thalamus dorsolat. pontine tegmentum 74 f car accident 10 112 subdural h., concussions + – " " ++ cerebellum " ", sn 32 m motorcyclist, blow to forehead 10 160 epidural h. + – " " ++ thalamus rostral pontine tegmentum, sn 21 m motorcyclist, hit by car 8 184 concussions + – " " ++ pallidum pontine tegmentum, sn 39 m drunken fall on occiput 8 228 wm lesions, frontal concussion + – " " ++ ? dorsal pontine tegmentum 47 f fall from ladder 4 230 concussions + – " " ++ pallidum rostral pontine tegmentum, sn 32 f skiing accident 10 527 epidural h. + – " " ++ pallidum " " 20 m motorcyclist, hit by car 10 301 concussions + – " " ++ pallidum " " 36 m motorcyclist 30 301 concussions + – " ", oculomotor palsy ++ thalamus, pulvinar caudal midbrain + pontine tegmentum 59 m scooter ride 4 360 skull fracture, concussions + – " " ++ pallidum " " 39 m drunken fall 4 456 none + – " " ++ thalamus midbrain, pontine tegmentum 30 m motorcyclist, hit by tram 5 236 none – + " " ++ cerebellum, gliosis caudal midbrain tegmentum, sn, gliosis pontine brain 23 m motorcyclist, hit by car 6 260 wm lesions – + +/– ++ – old hemorrhages, caudal midbrain tegmentum 72 m knocked over by car 8 290 depressed skull fracture, concussions – + + ++ – dorsolat. pontine tegmentum, sn 30 m motorcyclist, hit by car 4 301 subdural h., concussions – + + ++ striatum, pallidum dorsolat. pontine tegmentum, sn, demyelination pontine basis uws: unresponsive wakefulness syndrome, h.: hematoma; wm: white matter; sn: substantia nigra the 10 patients who survived for periods between 25 and 900 days with a partial recovery from uws showed residuals of intracranial epior subdural hemorrhages (n = 4), cortical contusions / lacerations (n = 8), vascular / ischemic cortical necroses or wm hemorrhages (n = 1 each), diffuse wm changes (n = 6), lesions of the corpus callosum (n = 1), old hemorrhagic / ischemic lesions of the hippocampus usually with unilateral predominance (n = 9) or old hemorrhages and / or (cystic) necroses of the globus pallidus (n = 5) or less frequently of the striatum and / or the thalamus (n = 3/2). among the latter patients, one case each also showed cystic necrosis of the globus pallidus with unilateral or bilateral lesions (small cysts, old hemorrhages, neuronal depletion) in the sn. the lesions mainly involved the lateral and dorsal parts of the sn (supply areas of small circumflexious rami) or oral and medial parts of the sn (supply areas of paramadian arteries). no definite lesions were seen around the walls of the third ventricle and in the periventricular gray matter. in one case, small cystic necroses and hemorrhagic residuals were detected in the dorsolateral midbrain tegmentum together with bilateral sn lesions. all the other cases showed superficial defects or hemorrhagic residuals in the lateral and dorsolateral pontine tegmentum. in one case each, myelin pallor and diffuse gliosis of the pontine basis and cerebellar lesions were present. a 32-year-old woman victim of a skiing accident was treated by excavation of an epidural hematoma. she survived for 10 days in an acute coma and for 257 days in partial recovery from uws. she had no further direct traumatic brain lesions. in addition to spastic hemiparesis, she developed symmetric rigidity, akinesia and amimia, but reacted to external stimuli and, finally spoke a few hardly understandable words. she died from pneumonia. neuropathology revealed residuals of the epidural hematoma but no primary traumatic lesions, subarachnoid hemorrhage or cortical contusions. yet, there were cystic necroses of the left central thalamus and contralateral medial hypothalamus next to the sn (fig. 2a), compression necrosis of the left hippocampus/parahippocampus (not shown) and symmetric cystic necroses in the caudal midbrain colliculi (fig. 2b) and extensive unilateral necrosis of the sn and pes pedunculi (fig. 2c). the location of the most frequent brainstem lesions due to transtentorial shifting and compression as well as the corresponding arterial blood supply are shown in fig. 2 d–f. figure 2. a. old cystic necroses in left medial thalamus and in contralateral medial hypothalamus next to medial sn. b. old cystic necroses in lateral and contralateral paramedian tegmentum of caudal midbrain. c. extensive cystic necroses in caudal midbrain colliculus and unilateral cystic necrosis of sn and pes pedunculi. heidenhain stain. d–f. schematics showing localization of compression lesions in the brainstem due to transtentorial herniation and their corresponding arterial blood supply (in bold). (b–f from [25] with permission from the publisher taylor & francis ltd) a 39-year-old man sustained a drunken fall on his occiput. no skull fracture, intracerebral hemorrhages or cortical contusions were found. following a deep coma lasting four days, he died after 456 days in uws with moderate clinical improvement, spasticity of his extremities, double vision (paresis of the right third brain nerve), bilateral rigidity, akinesia and hypomimia without tremors. cerebral blood flow measurement at 100 days post tbi showed considerable reduction of gray matter perfusion, which significantly correlated with the anatomical brainstem lesion. after about 6 months, he was extubated, and later began to speak single words and to react to commands. ct showed cystic lesions in bilateral basal ganglia and dorsolateral brainstem. levodopa was administered first through gastric tube and later on orally with levodopa / benserazide 100 + 25 mg three times per day. levodopa treatment led to slow reduction of rigidity and amimia but did not change spasticity. the patient was transferred to a chronic care center, where his general state did not change considerably. he died from acute cardiac infarction. neuropathology revealed no superficial traumatic residuals but diffuse wm lesions and old cystic necroses in globus pallidus and thalamus due to compression of the left anterior choroidal and right thalamo-perforant arteries. in addition to an old hemorrhagic infarction in the right occipital lobe, extensive lesions were found in the hippocampus/parahippocampus with unilateral predominance. there were small cystic necroses in the left dorsolateral midbrain tegmentum around the aqueduct, in the nucleus ruber and in the ipsilateral sn (fig. 3a). old hemorrhagic compression necroses in the pontine tegmentum surrounded the enlarged aqueduct (fig. 3b), while extensive necroses were found in the pontine tegmentum affecting the rostral reticular formation and central tegmental tract (fig. 3c). in the sn, small cystic lesions and focal loss of neurons were seen (fig. 3d). figure 3. a. multiple small necroses in caudal midbrain tegmentum around aqueduct, in unilateral red nucleus and sn. heidenhain stain. b. old hemorrhagic compression necroses in pontine tectum and tegmentum, enlarged aqueduct. c. old bilateral necroses in pontine tegmentum involving both the rostral reticular formation (nuclei pontis centralis oralis) and the central tegmental tract. luxol fast blue. d. cystic lesion and focal neuron loss in the sn. h&e stain. a 36-year-old motorcyclist, hit by a car, received a blow to the right forehead, causing no skull fracture, intracranial hematoma or cortical contusions. he was in deep coma for around 30 days and died after 301 days, transition from uws to klüver-bucy syndrome. in addition to left-sided spastic hemiparesis, he developed parkinsonian symptoms with almost bilateral akinesia, rigidity, hypomimia and slurred, hardly understandable speech. he was given levodopa via gastric tube and later on orally, which was followed by incomplete reduction of rigidity and akinesia without changes of spastic hemiparesis. he died from pneumonia. neuropathology revealed neither skull fracture nor residuals of intracranial hemorrhage or cortical contusions, but diffuse wm changes, partial rupture of corpus callosum and severe hippocampal/parahippocampal lesions with left preponderance. the basal ganglia, walls and bottom of the third ventricle as wells as the periventricular gray matter and oral midbrain were preserved. old hemorrhagic necroses involved however the dorsal and dorsolateral tegmentum (fig. 4a and c). a small necrosis was seen in the right dorsolateral pontine tegmentum (fig. 4b), while extensive compression necroses were seen in the right dorsolateral pontine tegmentum and adjacent cerebellar gyri (fig. 4d). pictures of the bilateral sn lesions have unfortunately been lost. figure 4. a. old hemorrhagic necroses in the dorsal and dorsolateral pontine tectum and tegmentum. b. small necrosis in the right dorsolateral pontine tegmentum. c. compression necroses of the right dorsolateral pontine tegmentum and the adjacent cerebellar gyri. luxol fast blue. d. extensive, mainly unilateral, old necroses of the dorsal and dorsolateral pontine tegmentum. luxol fast blue. (a–c from [25] with permission from the publisher taylor & francis ltd) five subjects survived tbi for periods ranging from 22 to 293 days in various recovery states from uws, presenting with mcs associated with spasticity and moderate to mild parkinsonian symptoms. neuropathology revealed residuals of subdural hemorrhage (n = 1) and cortical contusions (n = 2), callosal damage (n = 1) and diffuse wm lesions (n = 3), unior bilateral lesions of globus pallidus and/or thalamus with or without lesions to the sn were seen in one case each, as well as hippocampal/parahippocampal lesions (n = 2). brainstem lesions were restricted to old unior bilateral necroses in dorsolateral pontine tegmentum (fig. 5a) that were associated with diffuse pontine gliosis in one case. a 30 year-old man, a motorcyclist hit by a car, survived after evacuation of a subdural hematoma for 301 days, first 20 days in deep coma and subsequently with moderate improvement in uws. in addition to right-sided spasticity, he developed almost symmetrical akinesia, bilateral rigidity and hypomimia with mild unilateral resting tremor. he died in 1968 hence no mri was possible. he also received levodopa through gastric tube and later orally, which was followed by reduction of rigidity and hypomima without changes of spasticity. he died from pneumonia. neuropathology revealed a brain fracture and residuals of subdural hematoma and superficial cortical contusions as well as bilateral partial necrosis of striatum and globus pallidus, diffuse wm lesions and considerable hippocampal lesions with left-sided predominance. the floor of the 3rd ventricle, the periaqueductal gray matter and the midbrain tegmentum were relatively preserved, while old necroses involved the dorsal and dorsolateral pontine tegmentum and the brachium conjunctivum (fig. 5a). another vascular necrosis in the dorsolateral tegmentum of the rostral pons (fig. 5b) was associated with partial degeneration of the pyramidal tracts in the pontine basis (fig. 5c). figure 5. a. bilateral vascular compression necroses in the dorsolateral tegmentum of the upper pons and in the brachium conjunctivum. b. unilateral vascular necroses of the dorsolateral tegmentum of the upper pons and bilateral incomplete degeneration of the pyramidal tracts. c. necroses in the lateral and ventral pontine tegmentum and bilateral partial degeneration of the pyramidal tracts in the pontine basis. all heidenhain stain. discussion parkinsonian symptoms are well recognized as a component of posttraumatic encephalopathy in boxers and in other sport-related traumatic brain injuries (tbi) [42–44]. the incidence of parkinsonism after tbi (ptp) has been estimated to 3.07 % overall and to 11.59 % in tbi patients aged over 65 years [45]. almost 100 years ago, the minimal criteria for the diagnosis of parkinson's syndrome following acute head injury (ptp) were that (1) the trauma should be severe and should have led to concussion or unconsciousness, (2) there should be a close temporal relation between the acute trauma and the onset of parkinsonian features and the course of parkinsonian features should be uninterrupted [46]. the number of ptp cases after acute tbi in the earlier literature is limited [20, 47, 48]. a man aged 36 years with a skull fracture and unconsciousness for 24 hours developed over six weeks a predominantly right-sided, slowly progressive, levodopa-unresponsive parkinsonian syndrome. neuroimaging disclosed a cerebral infarction in the left caudate and lenticular nucleus, suggesting impairment of nigro-striato-frontal circuits [49]. other studies suggested that in ptp, supplementary motor area impairment seems important [50]. in a series of 54 patients who died in prolonged coma after closed tbi, a man aged 30 years died 301 days after a motorcycle accident in a partial recovery state from pvs, following a coma of 40 days. he had no skull fracture, cortical contusions or intracerebral hemorrhage but presented with parkinsonian features in addition to chronic decerebration. autopsy revealed extensive brainstem lesions due to tentorial compression of the rostral brainstem as complications of increased icp [22]. matsuda and colleagues [51] reported three patients with pvs after severe tbi who, after recovering from prolonged loss of consciousness, presented parkinsonian features with mainly rigidity and hyperkinesia, which improved after levodopa treatment. t2-weighted mri studies showed lesions in the dorsolateral midbrain and the cerebral peduncle, suggesting axonal injury involving the dopaminergic system (sn and ventral tegmental area). these findings implied that the midbrain was injured by tentorial compression induced by translatory and rotatory acceleration when the cranium was struck in its sagittal axis, or by posterolateral damage [52, 53]. this conclusion was based on neuroradiological and neuropathological studies on pvs after tbi, indicating that the most common structural lesions were diffuse axonal injury involving the corpus callosum, the thalamus and the dorsolateral aspects of the rostral brainstem [26, 54]. a study using voxel-based morphometry on structural mri involving 61 patients with doc of traumatic origin found widespread structural brain injury in the brainstem, midbrain, thalamus, hypothalamus, basal forebrain, cerebellum, and posterior corpus callosum. potential structural differences were found between uws and mcs, especially at the individual level [7]. primary traumatic brainstem lesions after severe tbi inducing parkinsonian symptoms are either caused by hyperextension of the head, tearing forces or rotation mechanisms leading to contusions, fatal tears and hemorrhages of the rostral brainstem [55] or by shearing motion due to a rotatory mechanism and cavitation forces inducing hemorrhages and strains in the upper brainstem [23]. this is documented by two personal observations: first, a 19-year-old motorcyclist who died after 18 hours in deep coma after a rear-end collision crashing into a tree showed rupture of the diencephalon and hemorrhages in the sn; second, a 37-year-old man who survived 10 days in deep coma after a drunken fall with multiple fractures of the calvaria showed multiple cortical lacerations, deep diencephalic ruptures and hemorrhages in midbrain sn and rostral pons [56]. rare cases of patients with traumatic sn lesions who survived for a longer time with or without developing a parkinsonian syndrome have been reported, such as a subject who survived a severe tbi with symmetric sn necroses for 32 years without parkinsonism [57]. the findings in the present study of patients who developed parkinsonian symptoms in the course of long-term uws after blunt tbi and in an earlier study of a larger group of patients with prolonged uws after severe tbi [25] are in favor of a secondary traumatic cause of morphological lesions related to prolonged posttraumatic uws, recovery from uws and defective states including posttraumatic parkinsonian symptoms. this conclusion is based on the following facts: (1) translatory and rotational acceleration following tbi is usually followed by extensive cortical contusions/lacerations at the contralateral side of the focal impact, often associated with lesions or rupture of the corpus callosum, which was seen in only 13.3 % of cases in the present series. (2) direct or "primary traumatic" lesions to the rostral or caudal brainstem due to rotational shearing injury or rotatory acceleration forces are usually associated with acute death [56, 58, 59]. however, in rare patients with long survival following acute tbi, symmetrical necrosis of the sn without clinical parkinsonian symptoms has been reported [57]. in the majority of cases with prolonged survival in uws and recovery states, the morphological lesions are restricted to the dorsal and dorsolateral pontine tegmentum, with preservation of the upper brainstem. (3) when comparing the frequency of brainstem lesions and morphological signs of increased icp, there is strong association between both parameters. this is particularly true for the uws group, when considering the presence of intracranial expanding lesions, the history of elevated icp and the morphological sequelae of elevated icp in various cerebral regions, e.g., compression necrosis in the hippocampus, which is seen in up to 80 % of fatal tbis [25, 60–62]. patients surviving tbi in deep coma, in addition to frequently showing both intracranial hemorrhages, cortical contusions (about 80 %) or hippocampal lesions (only 50 %), showed multiple brainstem lesions mostly involving the floor of the 3rd ventricle, the periaqueductal gray matter and the brainstem tegmentum, involving the ascending arousal system (aas) or the ascending arousal network (aan) [29, 37, 58, 63]. these lesions are frequently associated with diffuse axonal injury [28, 64, 65], disrupting the connection between brainstem, thalamus, forebrain, and substantially impairing the activation of the default mode network [66], the resting state and other functional networks [67]. while some patients will irreversibly remain in uws, particularly due to severe damage to central areas of the upper brainstem, usually involving sequelae of increased icp and transtentorial compression of the upper brainstem, other patients evolve to a minimally responsive condition or mcs [68, 69]. in these cases, brainstem lesions are usually restricted to dorsolateral/peripheral parts of the pontine tegmentum with preservation of the aas [17]. as illustrated in a former and in the present case series, these patients, irrespective of occasionally frequent supratentorial lesions, may show involvement of the dorsolateral pontine tegmentum, the cerebral peduncles and the sn, with preservation of the central parts of the rostral brainstem. there is evidence that during recovery from traumatic coma, there is increase in brainstem-thalamic or aan connectivity [70]. the distribution pattern of the brainstem lesions in the present series of posttraumatic uws and residual parkinsonian symptoms, i.e., superficial softening in the dorsolateral tegmentum of the rostral pons and the velum medullare anterior, corresponds to the supply area of branches of the long lateral midbrain and pontine vessels, the superior cerebellar and posterior perforating arteries or of the basal and internal cerebral veins. focal lesions in the dorsolateral pontine tegmentum affecting the superior cerebellar peduncle and the lateral lemniscus correspond to the supply areas of branches of long circumferential and superior cerebellar arteries or the drainage territory of precentral cerebellar veins as well as of the lateral sulcus veins. occasional small scattered necroses and hemorrhagic scars in the pontine tegmentum result from obstruction of branches of circumferential vessels or the superior cerebellar artery (fig. 2 d–f). these circulatory disorders result from compression of the brainstem tegmentum, leading from increased icp to free tentorial edge / transtentorial shifting. the lesion pattern of posttraumatic parkinsonian symptoms and the therapeutic efficacy of long-term levodopa treatment suggest a dysfunction of the nigro-striato-cortical catecholaminergic system [14, 71], which may, however, progressively decompensate in some patients with long-lasting uws after severe tbi. the limitations of the present study based on archival material are threefold: the absence of mri data and / or spect and pet data on striatonigral dysfunction [16], the lack of immunohistochemical data on alzheimer-related lesions, in particular of analyses of p-tau and β-amyloid to exclude changes akin to chronic traumatic encephalopathy [72, 73] and the lack of data on amyloid precursor protein to detect axonal injury [74], which was partly detected in the present study by bodian silver impregnation. another limitation is the fact that disorders previously diagnosed as "apallic syndrome" or pvs had to be reclassified using current criteria (uws). the essential point of the present study is to demonstrate the morphological basis of rare parkinsonian symptoms in the course of uws following fatal tbs. this endeavour appears important for further clinical and prognostic studies aiming to improve assessment of brain (dys)functions in prolonged doc / uws. here, modern neuroimaging and other markers may provide valuable signs, not only documenting changing nosology but also offering further insights into the pathophysiology of posttraumatic parkinsonism. acknowledgements the author thanks mr. e. mitter-ferstl for secretarial and editorial work. funding statement the study was funded by the society for the promotion of research in experimental neurology, vienna, austria. conflict of interest statement the author declares that he has no conflict of interest. ethics approval this is a retrospective analysis of archival 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(2023) identifying the phenotypes of diffuse axonal injury following traumatic brain injury. brain sci 13:1607. https://doi.org/10.3390/brainsci13111607 copyright: © 2025 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. neurodegeneration: 2023 update feel free to add comments by clicking these icons on the sidebar free neuropathology 4:13 (2023) review neurodegeneration: 2023 update john f. crary department of pathology, nash family department of neuroscience, department of artificial intelligence & human health, neuropathology brain bank & research core, ronald m. loeb center for alzheimer's disease, friedman brain institute, icahn school of medicine at mount sinai, new york, ny, usa corresponding author: john f. crary · icahn school of medicine at mount sinai · 1 gustave l. levy place box 1194 · new york, ny 10029 · usa john.crary@mountsinai.org submitted: 06 june 2023 accepted: 10 august 2023 copyedited by: aivi nguyen published: 04 september 2023 https://doi.org/10.17879/freeneuropathology-2023-4899 keywords: neurodegeneration, neuropathology, aging, alzheimer disease, tauopathy, α-synucleinopathy, tdp-43 proteinopathy, traumatic brain injury abstract this paper reviews ten highly impactful studies published in the previous year selected by the author from the neurodegenerative neuropathology literature. as in previous years, the focus is to highlight human tissue-based experimentation most relevant to neuropathologists. a concerted effort was made to balance the selected studies across disease categories, approaches, and methodologies to capture the breadth of the research landscape. studies include an integrated proteomic and transcriptomic study of alzheimer disease (ad) and new consensus diagnostic neuropathological criteria for progressive supranuclear palsy. a number of studies looking at tar dna-binding protein 43 (tdp-43) are highlighted. one examined interaction between ad and limbic age-related tdp-43 encephalopathy (late) and yet another demonstrated how tdp-43 represses cryptic exon inclusion in unc13a, suggesting a novel pathogenic mechanism. most surprisingly, three cryogenic electron microscopy (cryo-em) studies showed that tmem106b filaments form the core of tdp-43-positive inclusions. cryo-em revealed a prion protein amyloid structure from aggregates in gerstmann-sträussler-scheinker disease. there was an elegant functional genomic study cataloging microglial gene expression in the human brain. a study shed light on how apoe influences chronic traumatic encephalopathy. a pathoanatomical study tested the dual hit hypothesis of lewy body progression throughout the nervous system. and finally, deep learning continues to show its promise with application of a weakly supervised multiple instance learning paradigm to assess aging post-mortem brains. 1. an integrated proteomic and transcriptomic analysis of alzheimer disease reveals unique pathways beyond mrna powerful transcriptomic approaches are increasingly being applied to post-mortem alzheimer disease (ad) tissues and other diseases. historically, methods included hybridization-based microarrays and bulk rna-sequencing, but increasingly single cell rna profiling and spatial transcriptomics are becoming routine. proteins, owing to their increased complexity, are more challenging to assess, but are ostensibly more proximate to the relevant alterations that drive neurodegeneration. a study by johnson et al. entitled “large-scale deep multi-layer analysis of alzheimer's disease brain reveals strong proteomic disease-related changes not observed at the rna level” analyzed proteomes of more than 1,000 brain tissues with a goal of trying to better understand the biological processes disrupted in ad (johnson et al., 2022). the investigators deployed tandem mass tag mass spectrometry (tmt-ms), an approach used to quantitatively analyze and compare protein expression levels that uses isobaric tagging to enable multiplexed quantification of peptides and proteins. tmt-ms offers several advantages, including high multiplexing capacity, accurate and reproducible quantification, and the ability to analyze complex proteomes. using this methodology, the research team identified new ad-related protein co-expression modules that were highly preserved across cohorts and brain regions. remarkably, nearly half of the protein co-expression modules were not observed in mrna networks. two notable ad-associated modules that were unique to the proteomic network were related to mitogen-activated protein kinase (mapk) signaling and the matrisome (figure 1). the matrisome refers to the collection of extracellular matrix (ecm) proteins and associated factors and is a complex network of proteins and other molecules that provide structural support and regulate various cellular processes. the mapk/metabolism module was associated with the rate of cognitive decline whereas the matrisome module was influenced by the apoe ε4 allele. these results indicate that pathways that are unique to the proteome could lead to therapeutic targets and biomarkers. figure 1. tandem mass tag mass spectrometry (tmt-ms) derived alzheimer disease protein network contains modules that are not present in transcriptomic analysis. a. experimental overview. b. modules that had a zsummary score greater than or equal to 1.96 (or q = 0.05, blue dotted line) were considered to be preserved. unique modules included mapk signaling and the matrisome (reproduced from johnson et al., 2022 under the creative commons license). 2. revision of the neuropathological criteria for progressive supranuclear palsy the national institute of neurological disorders and stroke previously advanced neuropathologic criteria for progressive supranuclear palsy (psp), which have been the standard for nearly 30 years. however, the complex evaluation process and dependence on non-uniform staining methods limited the utility of the criteria. since then, advancements in immunohistochemistry and the availability of specific antibodies targeting pathological tau proteins, especially phosphorylation-dependent epitopes, have improved the recognition of key psp neuropathological features, especially tufted astrocytes. further, new diagnostic entities have emerged, including chronic traumatic encephalopathy (cte), aging-related tau astrogliopathy (artag) and globular glial tauopathy ggt, which required better delineation from psp. to update the neuropathologic criteria for psp, an international team of neuropathologists, supported by the rainwater charitable foundation / tau consortium proposed criteria that incorporate recent advancements in tauopathies and are easier to apply in non-specialized clinical settings (roemer et al., 2022). the goal was to develop criteria with high sensitivity, specificity, and inter-rater agreement, even for atypical variants of psp, while being independent of clinical information and suitable for non-specialized neuropathology services. the investigators evaluated digital slides from 10 brain regions stained with hematoxylin and eosin and phosphorylated tau (at8) immunohistochemistry for 15 typical and atypical psp cases and 10 other tauopathies. blinded to clinical and neuropathological information, 14 expert neuropathologists provided a categorical diagnosis (psp or not-psp) based on provisional criteria requiring neurofibrillary tangles or pretangles in two of three regions and tufted astrocytes in one of two regions. the provisional criteria had high sensitivity (0.97) and specificity (0.91), as well as almost perfect inter-rater reliability for diagnosing psp and differentiating it from other tauopathies. the rainwater charitable foundation criteria for the neuropathologic diagnosis of psp feature a simplified diagnostic algorithm based on phosphorylated tau immunohistochemistry and incorporate tufted astrocytes as an essential diagnostic feature (figure 2). figure 2. rainwater neuropathological criteria for progressive supranuclear palsy. a. the minimum requirements are demonstration of neurofibrillary tangles in two any of three classically involved regions (e.g., the subthalamic nucleus, globus pallidus, and substantia nigra) alongside tufted astrocytes in the putamen or peri-rolandic cortex. b. examples of neurofibrillary tangles and tufted astrocytes on phospho-tau (at8 antisera) immunohistochemical staining (reproduced with permission from roemer et al., 2022 under the creative commons license). 3. limbic-predominant age-related tdp-43 encephalopathy and alzheimer disease: the impact of coexistence on cognition limbic-predominant age-related tdp-43 encephalopathy neuropathologic change (late-nc) and alzheimer disease neuropathologic change (adnc) are both implicated in cognitive impairment among older individuals. however, the frequency of late-nc across the entire range of adnc remains uncertain. to address this gap in knowledge, nelson et al. performed a comprehensive analysis from 13 high-quality longitudinal studies, encompassing a total of 6,196 participants from diverse countries (nelson et al., 2022). among individuals with documented cognitive status prior to death, 43.0% were found to be cognitively normal, 14.9% had mild cognitive impairment, and 42.4% had dementia, consistent with epidemiological data for this age range. in cases with available cerad neuritic amyloid plaque score data, 39.4% were confirmed to have late-nc at any stage based on autopsy findings. notably, among brains with "frequent" neuritic amyloid plaques, 54.9% exhibited concurrent late-nc, while 27.0% of brains lacking detectable neuritic amyloid plaques displayed late-nc. remarkably, late-nc was present in nearly 40% of participants and often coexisted with adnc. detailed neurocognitive assessments conducted proximate to death across 10 cohorts demonstrated a tendency for worse cognition in individuals with late-nc across the entire range of adnc severity. none of the participants received a clinical diagnosis of definite frontotemporal dementia or a pathological diagnosis of frontotemporal lobar degeneration with tdp-43 inclusions. these findings provide valuable insights into the prevalence and interplay between late-nc and adnc, highlighting their impact on cognitive function in the aging population. figure 3. frequency of limbic-predominant age-related tdp-43 encephalopathy neuropathologic change (late-nc) across the entire range of alzheimer disease neuropathologic change (adnc). in participants with minimal adnc, ~25% have late-nc, suggesting adnc-independent mechanisms. late-nc is associated with more severe primary age-related tauopathy pathology, indicating potential synergy. in subjects with severe adnc, approximately 50% of participants had late-nc that may be driven by genetic factors (e.g., apoe genotype) or other factors. 4. tdp-43 represses cryptic exon inclusion in unc13a most amyotrophic lateral sclerosis (als) and a subset of frontotemporal lobar degeneration cases display tar dna-binding protein 43 (tdp-43) pathology. this is neuropathologically reflected by translocation of phosphorylated forms of the protein from the nucleus to the cytoplasm, a phenomenon that is readily visible on immunohistochemical stains. the significance and functional implications of this translocation are not clear, but may result in loss of tdp-43 function in the nucleus. tdp-43 is an rna-binding protein with numerous roles, including repression of the inclusion of cryptic exons during splicing of heterogeneous rna while in the nucleus. previous studies have highlighted several genes, including poldip3 and stmn2 that exhibit aberrant splicing following tdp-43 depletion. in a paper by ma et al., investigators sought to identify additional genes that exhibit cryptic splicing changes that are regulated by tdp-43 (ma et al., 2022). using post-mortem brain tissues from patients with frontotemporal lobar degeneration (ftld) and als, the authors used fluorescence-activated cell sorting to isolate nuclei with nuclear tdp-43 protein expression from nuclei lacking tdp-43 and performed rna-seq. they found 66 genes with altered splicing using this analysis, including unc13a, which had previously been identified in genome wide association studies with unclear mechanisms of increased risk. specifically, they found a cryptic exon that is retained in cells with tdp-43 pathology that could be detected with exon splice site specific probes (figure 4). further cellular and histological validation supported these findings. it is known from studies in c. elegans and other systems that unc13a encodes for a synaptic multidomain protein that participates in vesicle priming prior to fusion. these findings provide a direct mechanistic link between loss of tdp-43 function and a strong genetic risk factor for als-ftd. figure 4. unc13a cryptic splicing is associated with loss of nuclear tdp-43 in patients with ftd and motor neuron disease. a. basescope probe design b. in situ hybridization using the unc13a e20/ce probe, combined with immunofluorescence for tdp-43 and neun illustrating the presence of unc13a ce (arrowheads) in neurons showing depletion of nuclear tdp-43. scale bar, 10 μm. (reproduced from ma et al., 2022 under the creative commons license). 5. a surprising turn in frontotemporal dementia ftld is the third most common neurodegenerative disorder after alzheimer’s and parkinson’s disease, with one major ftld subtype characterized by pathological neuronal inclusions with tdp-43 immunoreactivity. determining the atomic structure of these inclusions is a high research priority given the expected utility for facilitating biomarker and therapeutic discovery. using cryogenic electron microscopy (cryo-em), three independent groups addressed this gap in knowledge with all making an astonishing discovery: the extracted amyloid fibrils were composed of fragments of transmembrane protein 106b (tmem106b), a lysosomal membrane protein previously implicated as a genetic risk factor for ftld-tdp (chang et al., 2022; jiang et al., 2022; schweighauser et al., 2022). while details among the studies differ, in essence the inclusions consist of a normally intraluminal 135-residue fragment of tmem106b that is likely shed before assuming this abnormal configuration. the aggregates, which are derived from the c-terminus, were likely missed in previous studies because most antisera target the n-terminus of the molecule. these findings raise a flood of new questions about tmem106b and abnormal lysosomal activity in neurodegeneration. figure 5. tmem106b cryo-em doublet fibril. (reproduced with permission from chang et al., 2022 under the creative commons license). 6. the cryo-em structure of prion protein amyloid there are a number of dominantly inherited prion protein (prp) amyloidoses, including gerstmann-sträussler-scheinker disease (gss), prp cerebral amyloid angiopathy, and prion amyloidosis with variable phenotypes. all are caused by mutations in the prion protein gene (prnp). gss is notable in that typical extracellular deposits made of prp amyloid (aprp) are associated with abnormal intraneuronal tau inclusions that are identical to those observed in classical ad, which raises fundamental questions as to how amyloid-beta causes tau pathology and whether there are shared pathogenic mechanisms. understanding the atomic structure of the aprp has the potential to shed light on how extracellular amyloid deposition in general might trigger tau pathology independently and facilitate the development of positron emission tomography (pet) tracers as well as therapeutic interventions for prion diseases. this is possible with cryo-em. in a study entitled “cryo-em structures of prion protein filaments from gerstmann-sträussler-scheinker disease”, investigators studied brain fibrils isolated from two patients with the prnp f198s mutation (hallinan et al., 2022). one patient was homozygous (vv) and the other homozygous (vm) at codon 129, which is known to influence disease onset and progression, allowed them to compare this important clinically relevant difference. looking at the first vv homozygous patient, they found that the aprp was polymorphic, with mainly two, three or four intertwined protofilaments (figure 6). the structures better delineated the location of the v residue at codon 129, which is buried deep in the amyloid core, protected from solvent exchange and tightly packed, suggesting that substitution with a methionine would affect fibril formation. these new structural data human brain aprp filaments have the potential to open new avenues for diagnosis and treatment of prion disease. figure 6. analysis of the m/v 129 residue region of prion protein amyloid (aprp). a,b. atomic models from patient 1 and 2. the 129 v residue (green) is tightly packed. c, d. this leaves little space for the larger methionine residue (reproduced with permission under the creative commons license from hallinan et al., 2022). 7. apoe's role in chronic traumatic encephalopathy chronic traumatic encephalopathy (cte) results from mild yet repetitive traumatic brain injury that is often sustained during contact sports or in other contexts. while the association between this exogenous risk factor and resultant tauopathy and neurodegeneration is well established, not all participants in contact sports develop cte. this suggests that other factors, perhaps genetic, might play a moderating role. one gene that has been implicated is apoe, which encodes the most abundant cholesterol transporter in the brain that is also the strongest genetic risk factor for sporadic ad. previous studies have suggested that the apoeε4 allele may confer risk for a poor outcome following tbi, but the risk for cte remains unclear. in a study by atherton et al., investigators performed the largest study to date investigating the potential association between apoe and cte (atherton et al., 2022). the study was a cross sectional association analysis of 364 brain donors with repetitive head impacts exposure (i.e., contact sports or military service), with 294 positive and 70 negative for cte. in addition to disease status, the investigators were able to explore a series of diseases relevant endophenotypes, including quantitative and semiquantitative tau pathology burden assessments. they found an association between tau pathology burden across multiple cortical regions and the amygdala as well as cte stage among brain donors over 65 years of age at death. the presence of a apoeε4 was equivalent to 7 years of playing football. this association remained even after accounting for amyloid plaque status. these findings provide the strongest evidence to date that apoeε4 is a risk factor for cte. 8. unraveling genetic influences on microglia in neurodegeneration understanding the contribution of microglia to the pathogenesis of degenerative brain disorders continues to increase in relevance. microglia mediate numerous essential functions such as inflammation, injury repair, maintenance of brain networks, and neurodevelopment. they exhibit dynamic characteristics and can have distinct phenotypes and functions depending on environmental signals, brain regions, age, and disease pathologies. previous studies have documented changes in microglial density, morphology, and gene expression in postmortem brain tissue from individuals with neurological and psychiatric disorders, suggesting their involvement in numerous disease processes. recent genetic evidence has also strongly implicated myeloid cells, including microglia. to better understand the potential causal role of microglia and perhaps identify therapeutic targets, it is crucial to determine the genes influenced by disease-associated genetic variants. however, this is challenging due to the complex regulatory mechanisms involved, especially when considering variants located outside protein-coding regions. integrating genetic and transcriptomic analyses can help identify quantitative trait loci (qtls) and elucidate gene-gene variant relationships. although recent efforts have detected expression qtls (eqtls) in microglia and their overlap with ad loci, larger sample sizes are needed. in a study conducted by lopes et al., a team of researchers performed transcriptome analysis on 255 primary human microglial isolates obtained from multiple brain regions of 100 individuals obtained fresh at autopsy (lopes et al., 2022). they investigated factors contributing to microglial heterogeneity, such as brain region and aging. through mapping of expression and splicing quantitative trait loci, the study revealed that numerous susceptibility loci for neurological diseases are mediated by gene expression or splicing in microglia. notably, the researchers identified candidate causal variants within microglia-specific enhancers, including associations between microglial expression of usp6nl and ad, as well as p2ry12 and parkinson’s disease. overall, this study provides a comprehensive catalog of genetic effects on the microglial transcriptome and presents potential functional variants relevant to neurological and psychiatric disorders. 9. a postmortem study examines the dual-hit hypothesis of parkinson’s disease pathoanatomically mapping the initiation and progression of lewy body pathology through the brain is a critical aspect of our understanding of neurodegeneration. one hypothesis, first suggested by braak and colleagues, is that there is a dual-hit, with simultaneous involvement of the olfactory bulb and dorsal motor nucleus of the vagus that underlies hyposmia and autonomic symptoms, respectively. early studies had mandatory inclusion of lewy pathology in the dorsal motor nucleus as an inclusion criterion, potentially biasing against cases with lewy bodies in the olfactory bulb alone. nevertheless, this hypothesis is debated and yet has spawned many clinical studies aimed at understanding potential initiating factors. little work has been done to test the hypothesis in the neuropathological setting. in a study by borghammer et al., investigators re-analyzed two large human post-mortem autopsy datasets that included mild lewy body disease cases (borghammer et al., 2022). they found that cases with alpha-synuclein immunopositivity in the peripheral autonomic nervous system and lower brainstem infrequently displayed pathology in the olfactory bulb (figure 7). conversely, cases with mild amygdala-predominant lewy body pathology essentially always showed olfactory bulb pathology. together, these findings suggest that the initiation of lewy body pathology starts either in the olfactory bulb or enteric nervous system but rarely simultaneously. there are a number of important caveats: these cases with mild lewy pathology were in a prodromal phase but might alternatively have not progressed. nevertheless, these findings are an important contribution to our understanding of prion-like spreading of lewy neuropathology and may lead to better diagnostics and advance our mechanistic understanding. figure 7. single-hit hypothesis of “prion-like” propagation in lewy body disease. a. in the “brain-first/olfactory bulb-first” subset, pathology arises in the olfactory bulb or amygdala and rapidly spreads to forebrain structures ipsilaterally and later bilaterally. b. in the “body-first” subset, pathology spreads to the medulla bilaterally and progresses superiorly. (reproduced with permission under the creative common license from broghammer et al., 2022). 10. deep learning continues to reshape histological assessments numerous studies continue to apply deep learning to digital whole slide images of histological sections of human brain tissue. this approach has been fueled by the more widespread availability of whole slide image scanners. combined with both next generation deep learning image analysis models and powerful graphical processing units, there is fertile ground for innovation. a convolutional neural network (cnn) is a variety of model that is fine tuned for processing image data. cnns have been applied to the detection of amyloid plaques, neurofibrillary tangles, and other features for example. these models work in a supervised fashion, requiring large numbers of annotated images for training. this is highly burdensome and acquiring this ground truth for model training represents a major barrier. in a study by mckenzie et al. (led by the author of this review), investigators applied a multiple instance learning model to a collection of whole slide images from aged individuals in a manuscript entitled “interpretable deep learning of myelin histopathology in age-related cognitive impairment” (mckenzie et al., 2022). this model uses slide level labels, rather than annotations on patches or pixel level segmentation of individual cells (figure 8). the investigators found that the ai was capable of predicting cognitive impairment based on a luxol-fast blue counterstained hematoxylin & eosin-stained section alone with an accuracy of 0.65. it should be noted that this is better than chance and this is a task not currently possible by a human observer. further analysis of the ai model found highly informative attention tiles, image patches of the whole slide image that contain the most salient aspects of the slide, were concentrated within white matter regions, not gray matter that contains well recognized structures including amyloid plaques and tangles that contribute to age-related impairment. this study paves the way towards more advanced models for analyzing relevant histological features of neurodegeneration in human autopsy brain tissues. figure 8. multiple instance learning pipeline for whole slide image analysis. a. the whole slide image dataset was derived from aged human brain donors. b. whole slide images are segmented into tiles and processed through a multiple instance learning pipeline. c. attention values are used to map salient features. (reproduced with permission under the creative commons license from mckenzie et al., 2022). acknowledgements this work was supported by funding from the nih (r01ag054008, r01ns095252, r01ag062348, r01ns086736, u54ns115266, u54ns115322) as well as the rainwater charitable trust/tau consortium, a alexander saint amand scholar award, and a karen strauss cook research scholar award. images have been reproduced under the creative commons license: visit http://creativecommons.org/licenses/by/4.0/. references 1. atherton, k., han, x., chung, j., cherry, j. d., baucom, z., saltiel, n., nair, e., abdolmohammadi, b., uretsky, m., khan, m. m., shea, c., durape, s., martin, b. m., palmisano, j. n., farrell, k., nowinski, c. j., alvarez, v. e., dwyer, b., daneshvar, d. h., … mez, j. 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(2022). age-dependent formation of tmem106b amyloid filaments in human brains. nature, 605(7909), 310–314. https://doi.org/10.1038/s41586-022-04650-z copyright: © 2023 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. multiple sclerosis: 2023 update feel free to add comments by clicking these icons on the sidebar free neuropathology 4:3 (2023) review multiple sclerosis: 2023 update tanja kuhlmann1,2, jack antel2 1 institute of neuropathology, university hospital münster, 48149 münster, germany 2 neuroimmunology unit, montreal neurological institute, mcgill university, 3801 québec, canada corresponding author: tanja kuhlmann · institute of neuropathology · university hospital münster · pottkamp 2 · 48149 münster · germany tanja.kuhlmann@ukmuenster.de submitted: 02 february 2023 accepted: 01 march 2023 copyedited by: monica miranda published: 09 march 2023 https://doi.org/10.17879/freeneuropathology-2023-4675 keywords: multiple sclerosis, ebv, remyelination, slowly expanding lesions, spatial transcriptomics abstract multiple sclerosis (ms) is the most frequent inflammatory and demyelinating disease of the central nervous system (cns). significant progress has been made during recent years in preventing relapses by using systemic immunomodulatory or immunosuppressive therapies. however, the limited effectiveness of such therapies for controlling the progressive disease course indicates there is a continuous disease progression independent of relapse activity which may start very early during the disease course. dissecting the underlying mechanisms and developing therapies for preventing or stopping this disease progression represent, currently, the biggest challenges in the field of ms. here, we summarize publications of 2022 which provide insight into susceptibility to ms, the basis of disease progression and features of relatively recently recognized distinct forms of inflammatory/demyelinating disorders of the cns, such as myelin oligodendrocyte glycoprotein antibody-associated disease (mogad). introduction multiple sclerosis (ms) is the most frequent inflammatory demyelinating disease of the central nervous system (cns) and a leading cause for permanent neurological disability in young adults. the variability in disease course amongst cases is well known. significant progress has been made during recent years in using systemic immune-system directed therapies to prevent relapses that characterize the initial disease course. however, the limited effectiveness of such therapies for the later-recognized progressive disease course indicates there is a continuous disease progression independent of relapse activity which may start very early during this condition 1,2. we select articles published in 2022 that provide insight into susceptibility to ms, the basis of disease progression, and features of relatively recently recognized distinct forms of inflammatory/demyelinating disorders of the cns (figure 1). ms has a strong hereditary component, but also environmental factors play an important role, as shown in studies of monozygotic twins discordant for the disease. several articles published in 2022 support that epstein-barr virus (ebv), which has long been implicated in ms, plays an important role in the development of ms. this raises the tantalizing possibility that vaccination against ebv may eliminate ms. another topic, which continues to be heavily discussed in the ms field, is the impact of the microbiome, not only in the gut but also in the lung, on ms disease course. potential mechanisms driving disease progression include persistent focal inflammation resulting in slowly expanding lesions, meningeal inflammation causing cortical demyelination and neuronal injury, and inflammation induced diffuse changes in white matter (wm) as well as loss of compensatory mechanism, such as remyelination and brain plasticity 3. we summarize several pathological and imaging studies, which aim to dissect the underlying mechanisms driving disease progression. however, these mechanisms most likely vary over time, adding another layer of complexity for the successful development of new treatment strategies to prevent or stop disease progression. furthermore, individual patient specific factors may modulate ms pathology significantly. this is supported by results from a single nuclei rna sequencing (snrnaseq) study of human ms tissue samples which reveals that the variability in gene expression between patients is higher than between lesions 4. based on findings from animal studies, it is assumed that promotion of remyelination can prevent disease progression. however, the evidence that remyelination in ms is neuroprotective is relatively sparse. in 2022, an imaging study provides first evidence that lack of remyelination in ms is associated with increased brain atrophy, a maker of neurodegeneration 5. figure 1: insights into disease course of ms. we selected articles published in 2022 providing insights into the susceptibility to ms, potential modulators of the disease course and new technologies to monitor the disease or to dissect underlying pathways. finally, we discuss the most recent findings in myelin oligodendrocyte glycoprotein antibody-associated disease (mogad), a relatively recently newly defined inflammatory and demyelinating disease recognized by the presence of anti-myelin oligodendrocyte glycoprotein (mog) antibodies in the serum. (i) ms susceptibility and disease course – population based studies role of ebv in susceptibility to ms epidemiologic based studies in 2022 bolstered the evidence for ebv infection being a necessary albeit, not sufficient trigger, for development of ms. bjornevik et al. examined the records of a cohort of more than 10 million active us military personnel in which 801 of 955 cases that developed ms could be evaluated with regard to ebv serology 6. all but one were serologically positive at the time of diagnosis. although 35 had initially been serologically negative, 34 converted prior to diagnosis. seroconversion rate was 57% in the non-ms cohort. no enhanced conversion rate was found for other viruses; cytomegalovirus (cmv) conversion may have been protective. in a retrospective cohort study of 2,576,011 individuals born in denmark between 1971 and 2018, rostgaard et al. found that having a sibling 0–2 years younger reduces the probability of ms by 25% compared to having no siblings. furthermore, having a second sibling 6–8 years younger reduces this probability by 30% 7. they concluded that having younger siblings reduces the risk of infectious mononucleosis and the risk of ms in a mirror-like manner, suggesting that the reduction in ms risk results from earlier primary ebv infection. the basis whereby ebv infection links to disease susceptibility and/or disease course remains under investigation. ebv is known to infect b cells and these cells have been detected in ms lesions in some studies, but still require unequivocal confirmation. b cells are implicated in producing factors that contribute to underlying tissue injury. support for the postulate that ebv specific humoral and/or cellular immune mediators contribute to the disease pathology is supported by the findings by lanz et al., regarding cross-reactivity between ebv and neural tissue components 8. they demonstrated high affinity molecular mimicry between the ebv transcription factor epstein–barr nuclear antigen 1 (ebna1) and the cns protein glialcam, as well as presence of cross reactive antibodies in the cerebrospinal fluid (csf) of ms patients. interestingly, these cross-reactive antibodies bound to rodent oligodendrocytes and astrocytes, glia limitans and, likely, perivascular glial cells in the hippocampus and brainstem, a pattern reminiscent of the expression pattern of aquaporin-4 (aqp4). anti-aqp4 antibodies are the hallmark of neuromyelitis optica (nmo), another inflammatory demyelinating disease with pathological features distinct from ms 9,10. further support for the potential relevance of ebv specific humoral and/or cellular immune mediators in ms pathogenesis is the study by schneider-hohendorf et al. conducted on ms disease discordant monozygotic twins, showing a broader ebv-specific t cell receptor (tcr) repertoire in the affected one of the pair 11. these combined population and laboratory based studies raise the issue of potential benefit of ebv directed vaccines in the very young population. however, one need consider the potential risk of such vaccination, including inducing a neural-target directed cross reactive immune response and persistent modulation of overall immune-regulatory mechanisms which may have protective purposes. microbiome impacts on disease susceptibility and course in ms having identified that an array of environmental factors contribute to the susceptibility and course of ms, the challenge remains to understand the underlying mechanisms for such effects. further research is needed to find out how these factors can act via modulating the systemic immune system to acquire pro-inflammatory properties. to dissect the influence of genetic predisposition versus environmental factors on shaping the immune system, ingelfinger et al. applied a combination of antibody-based and single-cell technologies to define the peripheral immune signatures of 61 monozygotic (mz) twin pairs discordant for ms (+ healthy mz twin and dizygotic twin pairs) 12. they could discern both genetically (cd25 expression by naïve helper t cells) and environmentally (helper t cells with a dysregulated cd25-il-2 axis) determined features of an ms-associated immune signature. the microbiome serves as a major interaction of a host with the environment. the gut is long recognized as a major resident niche for the immune system, being a major site of host immune interaction with the enormous population of resident microbes. the results of the international multiple sclerosis microbiome study (imsms), derived from an analysis of the gut microbiome of 576 ms patients (36% untreated) and genetically unrelated household healthy controls (1,152 total subjects), indicated that the phytate degradation pathway was over-represented in untreated ms, while pyruvate-producing carbohydrate metabolism pathways were significantly reduced 13. microbiome composition, function, and derived metabolites were all modified in response to disease-modifying treatments. as pointed out by hosang et al., the lung (airway) also serves as a niche for immune cells supporting their long-term survival and maturation into migration-competent effector t cells 14. smoking is a recognized risk factor for the development of ms. hosang et al. demonstrated the potential for the interaction of this niche with the environment to influence the susceptibility to autoimmune neurologic disease, using the experimental autoimmune encephalomyelitis (eae) model. treating animals with antibiotics (neomycin) shifted the microbiota towards lipopolysaccharide (lps)-enriched phyla without an effect on the gut microbiome. the underlying mechanism of effect implicated was that lps would usually induce a type 1 interferon response in microglia and reduce immune activation within the cns. however, lps is also used to induce a pro-inflammatory response in human microglia 15. the above studies indicate the potential for the microbiome in multiple niches to impact on ms throughout its disease course. microbiome influences on adaptive immune cells would impact on their initial activation and migration into the cns as part of new lesion formation. modulation of the endogenous glia cells populations (astrocytes, microglia) by products derived from the microbiome would impact on the extent to which these glial cells participate in tissue injury and repair processes throughout the ms disease course. progression independent of relapse activity the natural history of multiple sclerosis has been considered in terms of relapsing and progressive categories; the latter could occur in those with previous clinical relapses (secondary progressive (sp)) or not (primary progressive (pp)). specific criteria have been defined for diagnosis of either relapsing or pp forms of the disease that now include paraclinical measures (csf, imaging) as well as clinical features 16. progression needs not be sustained in either sp or pp forms resulting in use of the term “confirmed progression” (over 3-6 months) in clinical trials. more recent focus is whether progressive forms are associated with ongoing disease activity, as defined by imaging or clinical activity, introducing the term progression independent of relapse activity (pira) 1,2,17. this designation has acquired increased significance in that regulatory approval for agents in sp ms has been denied for cases lacking of documented activity. lublin et al. used the novartis-oxford multiple sclerosis (no.ms) data pool, spanning all multiple sclerosis phenotypes and paediatric multiple sclerosis, to evaluate ∼200,000 expanded disability status scale (edss) transitions from >27,000 patients with ≤15 years follow-up 18. their conclusions were that although relapses contribute to the accumulation of disability, primarily early in multiple sclerosis, pira started early in the disease process, occurred in all phenotypes and became the principal driver of disability accumulation in the progressive phase of the disease. tur et al. could show that pira, after a first demyelinating event, was not uncommon and suggested an unfavorable long-term prognosis, especially if it occurs early in the disease course 19. focal inflammation as driver of disease progression the observation that disease progression is driven by mechanisms other than peripherally-driven acute focal inflammatory lesions, raise the issue of the pathogenic mechanisms responsible for progression. this has become a hot topic for both histopathologic and serial neuroimaging studies, with a challenge of how to reconcile these two approaches. both have implicated pathogenic processes in the white matter, cortical grey, periventricular, and spinal cord regions of the cns. as regards focal white matter lesions, beynon et al. examined chronic lesion activity (cla) ( assessed by t1-hypointense lesion volume increase within baseline t2-non-enhancing lesions identified as either slowly expanding (sels) or not slowly expanding (non-sels)) in a 108 week phase 3 clinical trial of natalizumab (ascend) in sp ms patients 20. they found that cla in both sels and non-sels was greater in patients with spms and confirmed disability progression than in those with no progression. sel prevalence was lower in the absence of acute lesion activity (ala) (5% vs. 19% in subjects with ala), while cla remained associated with disability progression. in view of the association of ala with sel prevalence, the authors suggest that the effect of natalizumab on cla may be related to its effect on ala and the suppression of ala in mixed active/inactive lesions. further studies are required to understand whether and to which extent the established immunosuppressive and immunomodulatory ms treatments target sel (with or without paramagnetic rims). (ii) ms susceptibility and disease course – tissue based studies meningeal inflammation correlates with lesion activity the meninges consists of the dura mater and the leptomeninges comprising pia mater and arachnoid. during recent years, a number of papers have elucidated the relationship between leptomeningeal inflammation and subpial cortical lesions 21-23. subpial lesions are more frequently found in close proximity to meningeal infiltrates and, subpial demyelination is associated with neuronal, oligodendroglial and astrocytic injury and loss; cortical pathology is now considered as a driver of disease progression 22. subpial lesions are characterized by reduced extent of grey matter (gm) inflammation compared to white matter lesions; therefore, the question occurs whether additional mechanisms may contribute to the development of these lesions. an obvious possibility would be that soluble factors secreted by the inflammatory cells contribute to subpial demyelination and cellular injury. earlier pathological studies could not identify a correlation between meningeal inflammation and number of wm lesions 23,24. ahmed et al. addressed the question whether meningeal infiltrates correlate with wm lesion activity 25. they analyzed the density of meningeal lymphocytes and their topographical association with grey (cortical) and white matter lesions in 27 patients with progressive ms and nine controls. numbers of t and b cells per unit length of meninges were significantly increased in ms compared to controls and number of meningeal lymphocytes correlated with extent of subpial demyelination, confirming earlier studies 22,23,24,26. patients with high meningeal lymphocytic counts had more cortical lesions compared to patients with low lymphocytic counts, but no difference in the percentage of subpial, leukocortical or intracortical lesions. importantly, patients with a high density of meningeal lymphocytic infiltrates had a higher proportion of active and mixed lesions and a lower percentage of inactive or remyelinated lesions than patients with a low density of meningeal infiltrates. interestingly, no correlation between meningeal myeloid infiltrates and subpial demyelination or wm lesion activity was detected. the authors speculated that the correlation of the extent of meningeal inflammation and the presence of higher proportions of active and mixed lesions may represent two independent pathogenic mechanisms which reflect an overall higher disease activity. in line with this observation are studies which demonstrated a correlation between numbers of leptomeningeal b cells and perivascular t and b cells in wm or brain stem lesions 27,28. cellular trafficking to and from the meninges the bi-directional communication between the systemic and cns compartments is a central process contributing to the disease course of multiple sclerosis. to be considered are the connections whereby systemic immune constituents can access the cns and how materials released within the cns are cleared from this compartment and transferred back to the systemic compartment. in context of immune-brain interactions, entry via the blood-brain barrier (bbb) has been the classic pathway. blocking immune cell passage with adhesion molecule directed inhibitors or antibodies is an effective therapy in the eae model and translates into clinical therapy (natalizumab is an approved therapy for relapsing ms). a further pathway involves blood-csf trafficking that underlies immune seeding within the meninges. the observed pathologic changes in ms in the subpial grey matter and periventricular regions support the impact of immune constituents from the csf-meninges acting on the brain parenchyma. drieu et al., taking advantage of the capacity to pharmacologically or genetically delete selective cell populations in mice, showed that populations of perivascular and leptomeningeal macrophages, referred to as parenchymal border macrophages (pbms), regulate arterial motion that drives csf flow 29. the cns 'waste clearance' system is defined as the glymphatic system. carotenuto et al. used an array of different magnetic resonance imaging (mri) techniques to identify dysfunction of the glymphatic system function in multiple sclerosis and found an association with clinical disability, disease course, demyelination and neurodegeneration 30. transport of neural tissue breakdown products, specifically myelin constituents, back to regional lymph nodes has previously been documented by showing their presence in cervical lymph nodes of ms cases 31.the detection of lymphatic dural vessels, which connect csf and draining cervical lymph nodes, raises the question of the relevance of these vessels and dural inflammation for autoimmune disease such as multiple sclerosis 32. merlini et al. have studied the functional role of dural and leptomeningeal inflammation in eae using intravital microscopy to address this question 32. they could show that the leptomeninges were highly inflamed in acute and chronic eae, whereas the dura was only minimal affected. t cells adhered more weakly, antigen presentation was less efficient and activation of autoreactive t cells was lower in the dura compared to the leptomeninges suggesting that the dura plays only a minor role in autoimmune inflammation. these observations were matched by findings in ms brains. histological studies analyzing the extent of inflammation in leptomeninges compared to the dura observed significant t cell infiltrates only in the leptomeninges, but not in the dura. diffuse white mater inflammation and it consequences for the myelin-axon unit the pathogenic mechanisms driving disease progression independent of new lesions are, so far, only incompletely understood. potential mechanisms include the expansion of focal lesions, as described above, but also diffuse changes in normal appearing white matter in ms. in their study, van den bosch et al. elucidate the consequences of diffuse microglia activation and low-level t cell infiltration for axon-myelin units in the normal appearing white matter (nawm), using high-resolution immunohistochemistry (ihc) and transmission electron microscopy (tem) 33. they compared the ultrastructural characteristics of the axon-myelin unit in non-lesional optic nerves from 8 people with ms and 8 controls. in the optic nerves of ms patients, they observed, as expected, more activated and phagocytic microglia and more t cells than in controls. they analyzed the structure of the nodes of ranvier and found an elongation of paranodes and juxtaparanodes, as well as an increased overlap between paranodal and juxtaparanodal regions, confirming the results of earlier studies 34,35. when analyzing the structure of the myelin sheath, they found a decrease in the g-ratio compared to controls, suggesting a thicker myelin sheath in ms optic nerves. however, more detailed analyses demonstrated a loss of myelin compaction as the underlying cause for the decreased g-ratio. tem analyses revealed a higher percentage of axons containing mitochondria in patients with ms, which was confirmed by high-resolution ihc. the overlap of paranodes and juxtaparanodes, less compacted myelin and mitochondrial frequency in axons correlated with the number of activated microglia. these findings suggest that activated microglia contribute to nodal and myelin disorganization in nawm in ms patients, which may result in a higher axonal energy demand and increased mitochondrial numbers to compensate it. further studies are required to determine whether the same changes occur throughout the cns in patients with ms, when these changes start and whether these pathological changes correlate with pira. (iii) ms susceptibility and disease course new technologies in vivo imaging to assess consequences of demyelination and presence of re-myelination in ms emerging concepts suggest that a combination of persisting focal and diffuse inflammation within the cns and a gradual failure of compensatory mechanisms, including remyelination and brain plasticity, result in disease progression. in demyelinating animal models, remyelination reduces axonal as well as neuronal damage and is associated with clinical recovery 36-38. therefore, promotion of remyelination appears to be a promising new treatment approach in ms to prevent disease progression. however, despite increasing knowledge regarding the molecular mechanisms regulating deand remyelination in animal models, the development of new remyelination promoting drugs is still a challenge. to date, clinical trials testing experimentally identified drugs have had limited success. the failure of these clinical trials might be at least partly explained by our still limited knowledge about the natural course and clinical effects of remyelination in ms patients. tonietto et al. combined voxel based positron emission tomography (pet) and magnetization transfer mri (mtr) approaches to study the influence of lesion location on myelin repair and the correlation between remyelination and grey matter atrophy 5. at baseline, the probability of demyelinated voxels was highest close to the ventricle and, increasing distance from the ventricle was associated with an increased myelin content in lesions. they also reported that the probability of a demyelinated voxel to remyelinate in the follow-up period correlated with the distance from the ventricle. these findings are in line with histopathological studies, observing a lower extent of remyelination in periventricular compared to subcortical lesions 39,40. interestingly, tonietto et al. also observed that failure of remyelination was associated with lower thalamic volume, an imaging marker of neurodegeneration 5. additionally, they found a weak association between periventricular remyelination failure and regional cortical atrophy depending on the number of cortex-derived tracks passing through periventricular white matter. these finding suggest that lack of periventricular remyelination contributes to cortical damage in ms. however, the small sample size and the relatively short follow-up are limitations of these findings. further studies are required to determine the relationship between remyelination (failure) and brain atrophy. arnold et al. reported that siponimod therapy (expnd trial) improved magnetic transfer ratios (mtr), a measure of myelination within new lesions and across grey and white matter 41. this was associated with reduced loss of cortical grey matter, thalamic, and total brain volume, again suggesting a beneficial effect of remyelination for brain atrophy. as mentioned, the challenge remains to distinguish therapeutic effects acting via impact on inflammation from neuroprotective and repair promoting effects. to date, no therapy has received regulatory approval based entirely on the latter. a challenge remains how to use advances in imaging (and other para-clinical measures (electrophysiology, biomarkers)) to rapidly predict meaningful efficacy of therapies aimed at the latter, as so successfully done for immune-therapies for relapsing ms. new imaging techniques may help to differentiate repair and injury mechanisms. rahmandazeh et al. combined cross-sectional, longitudinal and post-mortem histopathology-imaging validation studies to evaluate, for the first time, the usefulness of quantitative susceptibility mapping (qsm) for the identification of different ms lesion types, including remyelinated lesions 42. qsm quantifies the distribution of magnetic susceptibility in tissues and is sensitive to myelin content and iron accumulation in the brain 43,44. due to its ability to detect iron, it has been used in the past for the detection of paramagnetic rim lesions (prls), which histopathologically, at least partly, correspond to mixed active/inactive lesions (= chronic active lesions). in this cross-sectional study, the authors analyzed 1,621 lesions and classified them in the qsm maps as either isointense (29 %), hypointense (4 %), hyperintense (52 %), lesions with a hypointense rim (1 %) and prls (13 %). the qsm histopathological correlation study revealed that iso/hypointense lesions mostly corresponded to remyelinated lesions, hyperintense lesions to inactive lesions and prls mostly to mixed active/inactive lesions. however, one caveat is that hypo-intensity in qsm indicates complete remyelination in the absence of ongoing inflammation; therefore, it appears less suitable for the detection of ongoing remyelination during the first five to six months after lesion formation 45. further studies comparing different imaging technologies with histopathological findings are required to evaluate the advantages and limitations of these approaches in the follow-up of remyelination in people with ms. novel molecular technologies to dissect ms pathogenesis in human tissue sections a number of studies so far have analyzed human ms tissue samples by snrnaseq 46-48. however, these studies included only a relatively small number of tissue samples from few patients. macnair et al. analyzed in their not yet peer-reviewed study the largest cohort of ms and control tissue samples by snrnaseq 4. they analysed 740,000 snrnaseq profiles from 165 samples including wm lesions (n = 62), normal appearing wm (n = 17), gm lesions (n = 39) and normal appearing gm (n = 16) from 55 ms patients, as well as, 15 wm and 16 gm tissue samples from 28 controls. they reached a median sequencing depth of 4,194 nuclei/sample, 3,154 reads/nucleus and 1,702 genes per nucleus. unsurprisingly, the authors observed highly cell-type specific changes in gene expression in wm and gm lesions in ms. interestingly, cell-type specific changes were largely shared across lesions, and patient identity was a much stronger driver of variability in cell type-specific expression for many genes than lesion type. nevertheless, since the composition of ms lesions is heterogeneous and the authors did not dissect specific lesion areas, for example the rim of mixed lesions, more specific signatures may have been overlooked. the authors observed an enrichment of ms risk genes differentially expressed in immune cells, but also in neurons, astrocytes and oligodendrocytes, supporting the notion that not only immune cells but also neural cells play a role for ms disease risk and progression. they also found an enrichment of different oligodendroglial subpopulations in lesions and nawm in ms compared to control wm; however, again donor id was a stronger driver of variability than lesion type. they identified three different oligodendroglial patterns. the first pattern was similar to controls, the second was characterized by a high proportion of an oligodendroglial subtype upregulating cellular stress genes and, the third pattern showed high proportions of early oligodendrocytes and reduced levels of more mature oligodendrocytes, suggestive of an arrest of oligodendroglial maturation or differentiation. these data suggest that patients may respond differently to potential remyelination promoting drugs. the challenge for the future will be to develop measure to identify patients who will especially benefit from remyelination promoting drugs. one limitation of snrnaseq is the lack of spatial resolution. this is of special interest for ms lesions, which may be spatially heterogeneous with respect to extent and composition of inflammation (such as mixed lesions) or remyelination (for example, partially remyelinated lesions). spatial transcriptomics allows transcriptomic analyses in a spatially highly defined manner. kaufmann et al. combined this technology with high sensitivity proteomics, published single cell-rna sequencing data and in vivo perturbation model data to dissect underlying disease mechanisms of progressive ms and to identify new drug targets 49. they focussed their analysis on cortical grey matter from 13 progressive ms patients. there was a heterogenous distribution of neurodegeneration within the tissue and across patients, and pseudotemporal analyses allowed the identification of early neurodegenerative pathways in cortical ms tissue. interestingly, these pathways were cell-type specific; however their regulation was highly connected between cell types suggesting multicellular pathological networks driving neurodegeneration. kaufmann et al. confirmed earlier studies describing significant synaptic loss in cortical ms lesions and normal appearing grey matter (nagm) 50. they also found an inverse correlation between genes regulating synaptic plasticity and maintenance and genes regulating inflammation and tissues remodelling expressed by astrocytes and innate immune cells. interestingly, their data suggested that chronic inflammation only in combination with local failure of neurotrophic support result in full extent of multicomponent neurodegeneration. one caveat might be that the authors used pseudotemporal biostatistical approaches to identify early neurodegenerative events. it would be desirable to validate these results in tissue samples from ms patients with ongoing cortical demyelination; but these lesions are rarely available in post-mortem tissue collections. using complex and elegant biostatistical analyses including in vivo cns disease model data and their own spatial transcriptomic and proteomic results they identified and prioritized cns enriched receptors as well as new pharmacological drug targets. (iv) other demyelinating diseases multiple sclerosis remains a diagnosis based on carefully considered clinical and mri based criteria plus csf findings of immunoglobulin (ig) synthesis and oligoclonal bands. although considered as an autoimmune disorder, no specific antigen-directed immune response is yet incorporated into the diagnostic criteria. however, over the last years, antigen specific immune responses have been identified in patients who would previously have been included within the ms umbrella. the first of these was nmo featuring aqp4directed antibodies. consensus clinical syndromes and mri findings related to optic nerve, spinal cord, area postrema, other brainstem, diencephalic, or cerebral presentations are now recognized 51. more recent has been recognition of monophasic or recurrent syndromes linked to presence of anti-mog antibody. these expanding syndromes now include optic neuritis (on), transverse myelitis (tm), encephalitis, and seizures, as well as aseptic meningitis and peripheral nervous system demyelination (reviewed in 52). mogad can be presented across the age spectrum. a specific question of interest addressed by gaudioso et al. is the frequency of mog-igg and aqp4-igg among patients with pediatric-onset multiple sclerosis (poms) 53. in their review of 493 cases, mog-igg was positive in 30 cases (6%) and zero controls; none were nmo-antibody positive. twenty-five of the 30 positive patients were judged to have mogad clinical criteria, 5 maintained a diagnosis of multiple sclerosis (ms). only 44% had serum epstein-barr virus (ebv) positivity and only 20% had cerebrospinal fluid oligoclonal bands. these distinct “ms-related” disorders are receiving increasing attention with regard to optimal treatment paradigms, both for acute episodes and for prevention of recurrences. cerebral cortical encephalitis (cce) is a recently recognized phenotype of mogad. it is characterized by cortical t2-fluid-attenuated inversion recovery hyperintensity on mri 54. valencia-sanchez et al. presented the clinical, imaging and pathological features of 19 cce patients, identified by screening their cohort of 285 mogad patients 55. the majority of cce occurred in patients with childhood onset of mogad and many patients presented with epilepsy. pathology details were available in two patients. in one patient inactive perivascular demyelinated lesions in cortex and subcortical white matter were observed. the second biopsy was characterized by marked meningeal inflammation, extensive macrophage and microglia activation in the meninges as well as cortex and extensive subpial demyelination. the cd4/cd8 ratio was variable in these two cases and varied between 2.4 and 0.7. few b cells aggregates were found in the meninges of one patient; terminal complement activation products were absent. extensive subpial demyelination has been described to be specific for multiple sclerosis 56. this recent report, together with earlier mogad case reports demonstrate that subpial demyelination can also be found in other inflammatory demyelinating diseases and raises speculation regarding the role of humoral immune constituents and soluble inflammatory mediators as contributors 57,58. a further recently addressed issue is whether sars-cov-2 vaccination programs could result in increased rates of mogad. francis et al reported a total of 25 cases referred to specialized mogad centers in the uk after the introduction of the covid vaccination program; their conclusion was “these observations might support a causative role of the chadox1s vaccine in inflammatory cns disease and particularly mogad” 59. summary the development of successful systemic immunomodulatory or anti-inflammatory treatment options for ms within the last two decades resulted in the discovery of progression independent of relapse activity. to dissect the underlying mechanisms driving this progression represents currently the biggest challenge in the field of ms. no 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these icons on the sidebar free neuropathology 5:20 (2024) review neurodevelopmental disorders: 2024 update maría martínez de lagrán1, karen bascón-cardozo1, mara dierssen1,2,3,4 center for genomic regulation (crg), the barcelona institute of science and technology, barcelona 08003, spain universitat pompeu fabra (upf), barcelona 08002, spain biomedical research networking center for rare diseases (ciberer), barcelona 08003, spain hospital del mar research institute, barcelona 08003, spain corresponding author: mara dierssen · centre for genomic regulation (crg) · the barcelona institute of science and technology · dr. aiguader 88 · barcelona 08003, spain mara.dierssen@crg.eu submitted: 22 july 2024 accepted: 12 august 2024 copyedited by: félicia jeannelle published: 05 september 2024 https://doi.org/10.17879/freeneuropathology-2024-5734 keywords: neuronal differentiation, epigenetics, retrotransposons, neurogenesis, gene regulation, down syndrome abstract neurodevelopmental disorders encompass a range of conditions such as intellectual disability, autism spectrum disorder, rare genetic disorders and developmental and epileptic encephalopathies, all manifesting during childhood. over 1,500 genes involved in various signaling pathways, including numerous transcriptional regulators, spliceosome elements, chromatin-modifying complexes and de novo variants have been recognized for their substantial role in these disorders. along with new machine learning tools applied to neuroimaging, these discoveries facilitate genetic diagnoses, providing critical insights into neuropathological mechanisms and aiding in prognosis, and precision medicine. also, new findings underscore the importance of understanding genetic contributions beyond protein-coding genes and emphasize the role of rna and non-coding dna molecules but also new players, such as transposable elements, whose dysregulation generates gene function disruption, epigenetic alteration, and genomic instability. finally, recent developments in analyzing neuroimaging now offer the possibility of characterizing neuronal cytoarchitecture in vivo, presenting a viable alternative to traditional post-mortem studies. with a recently launched digital atlas of human fetal brain development, these new approaches will allow answering complex biological questions about fetal origins of cognitive function in childhood. in this review, we present ten fascinating topics where major progress has been made in the last year. abbreviations abcd® adolescent brain cognitive development℠ study; abide autism brain imaging data exchange; ados autism diagnostic observation schedule; asd autism spectrum disorder; bbbblood-brain barrier; bold blood-oxygen-level-dependent; cbp creb-binding protein; cp cortical plate; crebbp creb-binding protein; crispr-cas9 clustered regularly interspaced short palindromic repeats and crispr-associated protein 9; daxx death-associated protein; ds down syndrome; dti diffusion tensor imaging; dwi diffusion-weighted images; enet elastic net; ep300 adenovirus e1a-associated cellular p300; er endoplasmic reticulum; esam endothelial cell adhesion molecule; fmri functional-mri; gbss gray matter-based spatial statistics; h3k4me histone h3 lysine 4; hcs human cortical spheroids; hnrnps heterogeneous nuclear ribonucleoproteins; hpscs human pluripotent stem cells; hsa21 homo sapiens chromosome 21; id intellectual disability; ipscs induced pluripotent stem cells; isovf isotropic volume fraction; jams junctional adhesion molecules; kdm5c lysine (k)-specific demethylase 5c; kdm6a lysine (k)-specific demethylase 6a; kdm7b lysine (k)-specific demethylase 7b; kmt2b lysine methyltransferase 2b; lines‑1 long-interspersed elements class‑1; lncrna long non-coding rna; lnpk lunapark; ltr long-terminal repeat; mecp2 methyl-cpg binding protein 2; ml machine learning; mri magnetic resonance imaging; ndd neurodevelopmental disorder; ndi neurite density index; noddi neurite orientation dispersion and density imaging; nvu neurovascular unit; ocln occludin; odi orientation dispersion index; orgs outer radial glia; osvz outer subventricular zone; p300 e1a-associated protein p300; pbmc peripheral blood mononuclear cell; pcp preprocessed connectomes project; rbps rna-binding proteins; rbfox1 rna-binding fox protein 1; roi regions of interest; rsi restriction spectrum imaging; sfari simons foundation autism research initiative; shap shapley additive explanations; shh sonic hedgehog; snatacseq single nucleus assay for transposase-accessible chromatin using sequencing; snhg11 small nucleolar rna host gene 11; snrna-seq single nucleus rna-sequencing; snrnps small nuclear rnps; svm support vector machines; svr support vector regression; swi/snf switch/sucrose non-fermentable; td typically developing; tes transposable elements; tnd total neurite density; vz ventricular zone; xlmr human x-linked mental retardation. introduction in this new collection featuring the most relevant findings in neurodevelopmental disorders (ndds) from 2023 and early 2024, we aimed to encompass a wide range of aspects that we believe will be of interest to neuropathologists. the selected topics range from the importance of understanding genetic and epigenetic contributions to the establishment of ndds to the presentation of new technologies and tools for studying brain development, with a high impact on disentangling the origin of ndds. first, we highlight genetic elements that have been historically understudied but have become in recent years more relevant in the pathogenesis of neurodegenerative disorders, new evidence suggesting their involvement in neurodevelopment. new studies suggest that de novo retrotransposition events occur during early embryogenesis and transposable elements are involved in several ndds, such as down syndrome (ds) (1). although still poorly studied, we expect that their understanding of their role in the onset and evolution of ndds will increase in the next few years. another intriguing genetic element with a possible impact on ndds that we spotlight in this review is the non-coding rna, particularly the long non-coding rna (lncrna). lncrnas are epigenetic regulators and thus can be determinants of the identity and functionality of postmitotic neuronal cells and the organization of the nervous system development. around 40 % of the lncrnas are specifically expressed in the brain, suggesting brain-specific roles and a link to neurobiological processes and ndds. for example, the lncrna snhg11 is involved in ds patients with neurogenesis deficits (2,3). these findings underscore the importance of understanding genetic contributions beyond protein-coding genes. going beyond the elements of the genome, we emphasize key regulatory mechanisms such as rna transcription. during the pre-mrna transcription, alternative splicing takes place to remove introns and there is increasing evidence that a spliceosome malfunction drives altered neuronal differentiation and function. in fact, a 2023 discovery shows that de novo mutations in u2af2 and prpf19 which encode spliceosomal subunits, are associated with intellectual disability (id) and autism spectrum disorder (asd) (4). a revolution in the management of big data has occurred in the last decade providing new analytical tools for disentangling complex imaging data. this is the case of the first digital atlas of human fetal brain development in which 1,059 optimal quality, three-dimensional (3d) ultrasound brain volumes from 899 fetuses have been analyzed using an automated pipeline (5). the outcome is a comprehensible tool to explore brain maturation and the origin of cognitive function in childhood. in other recent studies, new strategies to combine imaging data coming from different resources such as magnetic resonance imaging (mri) or diffusion tensor imaging (dti) have shed new light on the existing discrepancy in the cortical thickness across various brain regions in asd (6,7). finally, we discuss the use of advanced machine learning and image processing tools with different purposes such as the construction of a map showing how the fetal brain matures as pregnancy advances or the diagnosis of asd taking into account the brain network organization (8). another 2023 highlight is the new concept of “tightjunctionopathies”. this refers to the importance of the integrity of tight junctions in the neurovascular unit for maintaining blood-brain barrier (bbb) function and protecting the brain during development. disruption or dysfunction of the bbb has been implicated in various ndds, in some cases due to variants in cell adhesion proteins such as esam (endothelial cell-selective adhesion molecule) (9). another relatively poorly studied aspect of ndds is the region-specific contribution to those pathologies. the cerebellar development requires a long maturation during early childhood, becoming especially susceptible to early perturbations increasing the risk of ndds. new findings from a single-cell genomics study at early postnatal ages indicate that early childhood inflammation prevents specific cerebellum neurons from reaching complete maturation. the authors suggest that inflammation might lead to the premature down-regulation of developmental gene expression programs with putative consequences in the onset of ndds (10). finally, we address the relevance of the new genes discovered through whole-exome sequencing and genome-wide association studies on brain development and ndds, and how new in vitro experimental approaches are gaining relevance. recent advances have combined human induced pluripotent stem cells (ipsc)-derived cerebral organoids and crispr-cas9-mediated genome editing, achieving a powerful tool for investigating in vitro the role of candidate ndd genes in brain development. in this review, we have underlined two innovative studies that allow respectively to determine cell-type-specific contributions in cortical development to genetic disorders (4) and the impact of some ndd genes in the cellular migration and maturation during brain development (11). finally, we have provided several studies using organoids and ipsc to disentangle the origin of schizophrenia by injuries during brain development (12,13). this set of studies emphasizes the importance of examining beyond neurons to understand risk genes. 1. contribution of retrotransposition to developmental disorders millions of transposable elements (tes) have been accumulated throughout evolution, comprising up to 70 % of the human genome (14). although mostly silenced via different epigenetic mechanisms, such as histone modifications and dna methylation, the tes activity impacts genome structure, function, and chromatin dynamics (15). tes are classified by their mechanism of transposition into retrotransposons and dna-transposons (16). retrotransposons, so-called due to their "copy-and-paste" mechanism involving an rna intermediate, are subdivided into long-terminal repeat (ltr) and non-ltr elements (17,18). one ltr subtype in particular, the long-interspersed elements class‑1 (lines‑1), constitutes nearly 20 % of the human genome (17,18) and is the only autonomous retrotransposon encoding essential proteins for its mobility (19). lines‑1 are involved in diverse biological processes. during early neurodevelopment, studies have shown that active lines‑1 influence neural progenitor cell differentiation, contributing to neuronal diversity and brain somatic mosaicism formation (20) (figure 1). lines‑1 enrichment was evidenced around genes involved in neuronal lineage commitment and function, altering gene expression levels and chromatin compaction (21). although studies in cell cultures and animal models suggested that most de novo lines‑1 retrotransposition events accumulate during early embryogenesis (20), new lines‑1 insertions can also accumulate in adult brain tissues (22). moreover, it was evidenced that experience-dependent lines‑1 retrotransposition may contribute to neuronal plasticity (23). even though most lines‑1 are inactive copies unable to retrotranspose (17), they can become active. studies have shown that during senescence, line‑1 elements become de-repressed and actively transpose (24), showing misregulated retrotransposition that can lead to pathological outcomes. lines‑1 are also dysregulated in ndds (25,26) such as rett’s syndrome (22), asd, or fragile x syndrome (27)..in recent studies, altered lines‑1 were detected in a ds mouse model. their altered activity was blocked by a reverse transcriptase inhibitor thus rescuing hippocampal‐dependent recognition memory (1,28). this may be related to the role of lines‑1 in neurogenesis which is altered in ds mouse models (3,29). in summary, despite tes comprising more than half of the human genome, their role in brain disorders is understudied. their pervasive presence and activity, especially lines‑1, seem to be essential for proper brain development and function. tes dysregulation generates gene function disruption, epigenetic alteration, and genomic instability which may lead to ndds. with advances in sequencing technologies and gene editing approaches, we will certainly be able to understand better the role of tes in the future. figure 1. transposable elements (tes) involved in neurogenesis and cell differentiation. scheme showing insertion and activation of lines‑1 at early stages in neurodevelopment. in differentiated neurons at mature stages, lines‑1 tend to be less active or repressed (created with biorender.com). 2. new epigenetic players in neurodevelopmental disorders epigenetic mechanisms are key determinants of the identity and functionality of postmitotic neuronal cells and the organization of the nervous system development. it is thus not surprising that mutations of genes involved in chromatin regulation and genome organization lead to ndds. this is illustrated by monogenic syndromes like the α-thalassemia mental retardation syndrome, caused by the mutation of the atrx, a chromatin-remodeling enzyme of the swi/snf family that cooperates with the histone chaperone daxx to promote nucleosome exchange (30). histone demethylases are involved in neural development, hence mutations in kdm5c and kdm7b are found in human x-linked mental retardation (xlmr), such as the claes-jensen disorder (31,32). kabuki syndrome is a mendelian disorder characterized by id and postnatal growth deficiency that has been related to variants in genes encoding for histone demethylase kdm6a (33) and histone methyltransferase kmt2d (34). similarly, the mutation of the x-linked gene encoding methyl-cpg binding protein 2 encoded by mecp2 results in rett syndrome, one of the most common causes of id in young girls (35). at the histone acetylation level, the mutation of crebbp or ep300 transcriptional co-activator genes leads to rubinstein-taybi syndrome, characterized by moderate to severe id (36). the dysfunctions manifested in these syndromes likely arise from the alteration of the epigenetic patterns required for cell fate commitment during development and neuronal maturation. in recent years, the advent of next-generation sequencing has profoundly transformed epigenetics and rna biology with the discovery of a multitude of non-coding rnas. among these intriguing molecules, lncrna are epigenetic regulators (37), acting as biological decoys, chromatin modifiers and scaffolds. albeit lncrnas can be found throughout the body, around 40 % are specifically expressed in the brain, suggesting brain-specific roles (38). although these roles have only recently been investigated, multiple lncrnas have been functionally and mechanistically linked with neurobiological processes and ndds (39). sierra et al. (3) showed the involvement of the lncrna snhg11 in ds, the most common genomic cause of id caused by trisomy of homo sapiens chromosome 21 (hsa21) (figure 2). hsa21 is among the poorest chromosomes for non-coding rnas, including microrna-encoding genes, with the relevant exception of lncrna genes, which are very enriched in hsa21 and are abnormally expressed in ds ipscs (40). lncrnas have been associated with neuronal proliferation or migration (2), but little is known about their direct mechanisms. a study proposes that lncrnas modulate their ability to differentiate, or the migration of newly born cells to their proper location within the brain (2). interestingly, lncrnas might also be critical for the adult hippocampal gabaergic circuit (liu et al., 2022), thus covering several of the ds pathogenicity mechanisms. single-cell transcriptomics revealed that lncrnas are abundantly expressed in individual cells and are highly cell type-specific (3,41). using single nuclei rna-sequencing (snrna-seq), the authors identified snhg11 to be specifically downregulated in the dentate gyrus of a ds mouse model and post-mortem brains of ds individuals (3). moreover, through in vivo knockdown strategies they showed snhg11 involvement in hippocampal-dependent cognitive phenotypes, possibly contributed by impaired adult neurogenesis. this recapitulates findings in human embryonic stem cells, in which knocking down lncrnas blocked neurogenesis and differentiation (42). these findings underscore the importance of understanding genetic contributions beyond protein-coding genes and emphasize the role of rna molecules and non-coding dna in ndds. figure 2. snhg11, a long non-coding gene involved in down syndrome. the left panel shows ds brain neuropathology due to the excess dosage of hsa21 products. the right panel shows the impact of a long non-coding gene (shng11) on neurogenesis at the early stages of neurodevelopment. (created with biorender.com, modified from cesar sierra doctoral thesis presentation) 3. neurodevelopmental disorders caused by spliceosome malfunction most human protein-coding genes are transcribed as pre-mrnas containing exons and introns, with introns removed through pre-mrna splicing before translation. this precise process involves numerous elements and protein factors, including heterogeneous nuclear ribonucleoproteins (hnrnps), uridine-rich small nuclear rnps (snrnps), the prp19 complex, and various rna-binding proteins (rbps)(43). dysregulation of these elements can significantly impact neuronal differentiation, patterning, and synaptic function, although germline variants in core spliceosome components are rarely implicated in ndds (44). during the last year, de novo variants in spliceosome machinery have been incorporated into the list of traits related to ndds (45–47). li et al. (48) integrated clinical phenotyping, exome/genome sequencing, protein structure analysis, and modeling in flies and human pluripotent stem cells (hpscs) to explore the roles of three ndd-associated genes. the authors identified 46 and 6 individuals with de novo variants in u2af2 and prpf19, respectively, which encode spliceosomal subunits. these variants led to neurodevelopmental phenotypes like developmental delay, id, and asd. neuritogenesis was reduced in human neurons differentiated from hpscs carrying two u2af2 hyper-recurrent variants. neural loss of function of the drosophila orthologs u2af50 and prp19 led to lethality, abnormal mushroom body patterning, and social deficits. transcriptome profiling of deficient fly brains with crispr/cas9 knock-in, and patient-derived ipscs revealed potential splicing substrates and downstream effectors, including rna-binding fox protein 1 (rbfox1). overexpression of these effectors partially rescued neural defects in deficient flies, establishing a hierarchical genetic network of ndd-associated susceptibilities. additionally, 6 individuals with de novo rbfox1 missense variants exhibited similar neurodevelopmental features, highlighting a conserved genetic hotspot crucial for neurodevelopment and function. 4. first digital atlas of human fetal brain development the first digital atlas, launched in 2023 (5), illustrates the dynamics of normative maturation of each hemisphere of the fetal brain during a critical period of human development, improving the understanding of fetal brain maturation. previous studies were limited as those were mainly carried out by a single laboratory, with usually small sample sizes, methodological heterogeneity and lack of postnatal follow-up. the normative atlas now launched is based on a prospective international cohort of healthy pregnant women, whose fetuses were accurately dated in the first trimester, with normal growth and neurodevelopment from early pregnancy to 2 years of age (49). the atlas was produced using 1,059 optimal quality, 3d ultrasound brain volumes from 899 fetuses and an automated analysis pipeline (50,51). importantly, the study shows remarkably similar patterns of fetal brain growth and development across diverse populations, an important scientific advance in the neuroscience field. even being much larger than previous efforts, and pooling data from the 8 geographically diverse study sites, the between-study site variability represented less than 8.0 % of the total variance of all brain measures. this result is consistent with the previously reported findings of the same intergrowth-21st cohorts, for fetal skeletal growth, newborn size and infant neurocognitive development. moreover, the atlas reproduces the findings of published mri data (52), but with finer anatomical details in deep grey matter. the cohort used by namburete et al. (50) included also a postnatal follow-up study to 2 years of age, so this atlas provides a unique spatiotemporal benchmark of fetal brain maturation from a large cohort with normative preand postnatal growth and neurodevelopment. the work revealed significant asymmetries in brain maturation since 14 gestational weeks. the most distinctive region to show asymmetry in this study was the superior frontal, which is formed by the relative overgrowth of the frontal and temporal lobes. in addition, the cerebral hemispheres follow separate maturational programs, with greater rightwards dominance in regions associated with language development without any differences between the sexes, suggestive of prioritization of language readiness. these patterns were validated in 1,487 3d brain volumes from 1,295 different fetuses. this atlas will allow answering complex biological questions about the fetal origins of cognitive function in childhood, such as how language is acquired. using the atlas in combination with the soon-to-be-published international standards describing the complementary growth of the fetal brain will be a valuable clinical tool in specialized, referral centers when brain development appears abnormal on ultrasound. 5. cortical microstructural imaging in neurodevelopmental disorders one of the most consistent morphological parameters for assessing abnormal brain structures in patients with asd is changes in the cortical thickness within the frontal, temporal, parietal, and occipital lobes. these changes often correspond to specific clinical features observed in individuals with asd (53), with core symptoms of asd being associated with prefrontal cortex abnormalities. in fact, using cortical thickness features extracted from a small subset of brain regions as input for a classifier to identify asd subjects, yielded accuracies of over 84.2 % when concatenating 8 regions (54). however, research in this area has yielded contradictory findings regarding the cortical thickness across various brain regions in asd, with some reporting increases and others decreases. to complicate things even more, the spatial distribution of abnormal brain development has a high heterogeneity, even with similar pathophysiology (55). cortical development shows an aberrant trajectory in asd, being associated with cortical hyperplasia in early childhood, followed by a cortical plateau and subsequent decline in later stages of development (56,57). the atypical cortical developmental trajectory likely underlies structural and functional anomalies in the brain’s connectivity network (58), with cortico-cortical connectivity abnormalities not restricted to the white-matter connections but also affecting the intrinsic gray-matter architecture and connectivity within the cerebral cortex (59). arai et al. (6) in a recent investigation focused on examining microstructural changes in gray matter among adults with asd compared to typically developing (td) individuals. the work employed neurite orientation dispersion and density imaging (noddi) to measure neurite density index (ndi), orientation dispersion index (odi), and isotropic volume fraction (isovf). these measures, along with dti metrics and surface-based cortical thickness, were compared between the asd and td groups using voxel-wise gray matter-based spatial statistics (gbss). additionally, the study explored correlations between mri-based metrics and asd-related scores, such as the asd-spectrum quotient, empathizing quotient, and systemizing quotient, within specific regions of interest (roi). the analysis revealed a notable decrease in neurite density in individuals with asd compared to the td group, predominantly in the left prefrontal cortex regions, including the caudal middle frontal, lateral orbitofrontal, pars orbitalis, pars triangularis, rostral middle frontal, and superior frontal areas. furthermore, there was a significant correlation between reduced empathy and lower neurite density in the left rostral middle frontal cortex, superior frontal and frontal pole areas within the asd group. interestingly, in another related study (7), researchers identified variable neuronal density in the cerebral cortex of individuals with asd. this study involves a comprehensive analysis of magnetic resonance diffusion-weighted images (dwi) from the adolescent brain cognitive development℠ (abcd®) study. they employed a restriction spectrum imaging (rsi) framework to assess total neurite density (tnd). the analysis revealed a significant reduction in neurite density within the right cerebellar cortex, but an increase throughout anterior fronto-temporal and basal ganglia regions. these findings reinforce the notion that asd patients show atypical brain growth and connectivity patterns from an early age. these recent developments in analyzing neuroimaging now offer the possibility of characterizing neuronal cytoarchitecture in vivo, presenting a viable alternative to traditional post-mortem studies. 6. neuroimaging in the era of “big data” over the past twenty years, there has been a notable increase in the generation, acquisition, and storage of data, characterizing the era known as "big data". in neuroscience, one example is the human brain project, which provides different datasets, including a comprehensive, multilevel atlas of the human brain. this atlas combines data on cytoarchitecture, connectivity, chemoarchitecture, genetics, and brain function. it integrates information from both macroscopic and cellular perspectives into a unified framework and a highly detailed 3d model with microscopic resolution, distinguishing individual cortical layers and cells of the healthy brain. this significant growth in data volume, velocity, and variety along with the proliferation of machine learning (ml) toolkits has created unique opportunities for researchers. numerous studies have introduced ml methods applied to neuroimaging data to explore various aspects of asd. most have focused on supervised ml algorithms, mainly based on support vector machines (svm) to distinguish between asd and td. for example, using datasets sourced from the autism brain imaging data exchange (abide, analyzed with support vector regression (svr) and elastic net (enet)) (60,61) penalized linear regression scores on cortical thickness measurements for predicting the severity of asd symptoms assessed with the autism diagnostic observation schedule (ados) (62). an international dataset of 3d ultrasound scans, previously collected using standardized methods and equipment (63), has been analyzed with advanced ml and image processing tools to construct a map showing how the fetal brain matures as pregnancy advances. the use of newly developed ml methods allowed improved visibility of brain structures in 3d ultrasound images. this results in the generation of an average representation of each cerebral hemisphere with quantification of intracranial volume variability and growth patterns between 14 and 31 gestation weeks, a critical time window of active cell proliferation, migration and synaptogenesis, as well as macroscopic changes. despite significant advances in ml methods, several challenges remain. many ml classification methods lack interpretability, which is particularly problematic for medical data analysis. additionally, small datasets are prevalent, which can lead to unreliable results. recent techniques like shapley additive explanations (shap) values have been developed to enhance the interpretability of ml results by identifying key features in a model. to address the issue of small datasets, data augmentation methods such as sliding windows, which segment data into smaller overlapping sections, can be employed. however, this can sometimes result in information loss that overlapping windows techniques can help mitigate. in this context, alves et al. (8) propose a new method for diagnosing asd that is both interpretable and suitable for small datasets. they used the preprocessed version of abide, which consists of 1,112 datasets comprising 539 asd and 573 td with 300s bold (blood oxygen dependent) time series and provided by the preprocessed connectomes project (pcp) dataset. much of the recent research proposes methods based on ml but uses a single statistical parameter, ignoring brain network organization. their approach involves classifying functional-mri (fmri) time series using a connectivity matrix input, which produces more accurate results compared to previous studies and is the main innovation featured by this study. they also used complex network measures to characterize brain organization differences between asd and td individuals and employed shap values for the biological interpretation of brain region connections. additionally, they utilized a sliding window data augmentation strategy to expand the sample size, splitting time series into overlapping sections to maintain data integrity and improve model robustness. the analysis of fmri data highlighted changes in certain brain regions associated with cognitive, emotional, learning and memory processes. the cortical networks of asd patients displayed more segregation, less distribution of information and less connectivity compared to td individuals. this methodology will contribute to the understanding of cerebral differences in asd and will be useful in future to assist specialists, especially in cases involving diagnostic uncertainty. 7. tightjunctionopathies: a new class of diseases affecting the blood-brain barrier the bbb regulates the passage of substances from the bloodstream into the brain, thereby maintaining the brain's microenvironment. it is composed of endothelial cells, astrocyte end-feet, pericytes, and a basement membrane. the bbb's integrity and functionality are crucial for normal brain development and function. disruption or dysfunction of the bbb has been implicated in various ndds. the bbb is a component of the neurovascular unit (nvu), a complex and dynamic interface between the brain and the blood vessels, comprising endothelial cells, pericytes, astrocytes, neurons, and the extracellular matrix. tight junctions within the endothelial cells of the nvu are critical for maintaining the integrity of the bbb, regulating the passage of molecules and ions, and protecting the brain from potentially harmful substances. disruption of these tight junctions is increasingly recognized as a contributing factor to ndds (64). tight junctions are composed of various proteins, including occludin, claudins, junctional adhesion molecules (jams), and cytoplasmic scaffolding proteins. these proteins interact to form a barrier that regulates paracellular permeability and maintains the homeostasis of the brain's microenvironment (65). studies have shown changes in the expression of tight junction proteins in individuals with asd. for instance, reduced levels of occludin (encoded by the ocln gene, also involved in polymicrogyria), have been observed in asd, suggesting a compromised bbb (66). the esam gene encodes a cell-cell attachment protein belonging to the immunoglobulin receptor family. this cell-to-cell junction is of vital importance in cells that form part of the bbb and thus, esam protein is essential for its integrity. lecca et al. (9) have now identified a new rare disease in humans associated with homozygous loss-of-function variant alleles of esam. the study was carried out in 13 individuals including 4 fetuses, from 8 unrelated families from all over the world. the c.115del (p.arg39glyfs∗33) variant severely impaired the in vitro tubulogenic process of endothelial colony-forming cells, and caused a lack of esam expression in the capillary endothelial cells of the damaged brain. affected individuals with bi-allelic esam variants presented disruption of the bbb integrity and suffered profound global developmental delay/unspecified id, epilepsy, absent or severely delayed speech, varying degrees of spasticity, ventriculomegaly and intracranial hemorrhage/brain calcifications. dilation of lateral ventricles, thinning of the corpus callosum, hydrocephalus, and focal white matter lesions were the most frequently observed neuroimaging abnormalities. also, hydrocephalus was detected in all fetuses as previously reported in a patient with an esam homozygous nonsense variant (67). intracranial hemorrhages, frequently associated with cerebral calcifications, were observed in all patients. the features of individuals with bi-allelic esam variants resemble those of other known conditions characterized by endothelial dysfunction due to mutation of genes encoding tight junction molecules, namely the jam2 (68), jam3 (69), and ocln (66) genes. in summary, the integrity of tight junctions in the neurovascular unit is crucial for maintaining bbb function and protecting the brain during development. disruption of these tight junctions can lead to increased permeability, neuroinflammation, and exposure to harmful substances, all of which contribute to the pathophysiology of various ndds. because of the importance of cell adhesion in the neurovascular unit, the authors propose to rename this group of diseases as “tightjunctionopathies“. 8. zooming-in into cerebellar involvement in neurodevelopmental disorders through single-cell analysis in recent years, animal models have revealed new functions and increasing complexity of the cerebellum (70,71). however, the roles of the cerebellum in human pathology, particularly during preand postnatal brain development remain understudied compared to other brain regions. the human cerebellum undergoes a long maturation during early childhood being one of the first brain regions to begin developing and one of the last to reach maturity. this makes it especially susceptible to perturbations contributing to the risk of developing ndds (72). early-life inflammation is a clinically established risk factor for asd and schizophrenia, but its impact on human cerebellar development is poorly understood (73). using single-cell genomics, a study published last year found that early childhood inflammation prevents specific cerebellum neurons from reaching complete maturation (10). ament et al. characterized the cell type-specific effects of early childhood inflammation in post-mortem cerebellar brain samples from 17 children aged one to five years, who died from inflammatory conditions, such as bacterial or viral infections or asthma. they compared them with those who died from sudden accidents but with no neurological disorder. the two groups were similar in age, gender, race/ethnicity, and time since death. the first interesting finding was that despite considerable variation among samples in the sources of inflammation, ranging from meningitis to asthma, there was a high degree of congruence in transcriptional effects across children experiencing inflammation at the time of death. the characterization of postnatal cerebellar neuronal and glial development revealed that inflammation is associated with cerebellar maturation during the postnatal period through significant, overlapping transcriptional changes in 2 types of inhibitory neurons: purkinje and golgi neurons. alterations in purkinje neuron morphology have been documented in ndds, including asd and schizophrenia, with the most consistent finding being a reduction in soma size (74) and a decrease in the number and density of purkinje neurons (75). throughout development, purkinje neurons establish long-range synapses that connect the cerebellum to brain regions involved in cognition and emotional regulation, whereas golgi neurons coordinate neural communication within the cerebellum. ament et al. performed an immunohistochemical examination of post-mortem cerebellar samples that did not reveal changes in purkinje neuron soma size but showed increased microglial activation in children who had experienced inflammation. the authors then constructed a gene regulatory network model for purkinje neuron maturation by integrating their cell type-specific gene expression (snrna-seq) and chromatin accessibility (single nucleus assay for transposase-accessible chromatin (snatacseq)) datasets and identified seven temporally specific gene networks in purkinje neurons. they suggested that inflammation might lead to the premature down-regulation of developmental gene expression programs. genes involved in ndds such as id, asd, and schizophrenia (76,77) were part of the maturationand inflammation-associated gene expression changes. preterm birth can also lead to cerebellar underdevelopment and is often associated with chorioamnionitis with an increased risk of ndds, such as autism. using a preclinical experimental approach involving snrna-seq (78) has established the role of chorioamnionitis by decreased sonic hedgehog (shh) signaling on disrupted cerebellar maturation associated with preterm birth. those works highlight the importance of using single-cell sequencing to zoom into the neuropathology of ndds. 9. new cellular models are uncovering the function of neurodevelopmental disorder genes over the past decade, whole-exome sequencing and genome-wide association studies have uncovered hundreds of asd and other ndd genes. as promising as these discoveries are, how defects of these genes impair brain function remains largely unclear for most of those genes. this is due to the difficulties in accessing human brain tissue from ndd patients. one approach to address this question would be to investigate key features of neocortex development, using appropriate model systems and focusing on the actions of key genes that lead to ndds. recent advances in human ipsc-derived cerebral organoids and crispr-cas9-mediated genome editing can be combined into a powerful tool for investigating molecular mechanisms involved in ndds in in vitro systems (79). human brain organoids recapitulate the architectural and functional features of developmental brain stages, containing apical radial glia in the ventricular zone (vz), human-specific outer radial glia (orgs), outer subventricular zone (osvz), and deep-layer and upper-layer neurons in the cortical plate (cp) (80). several research groups have established cerebral organoids to clarify the pathogenesis of autism, fragile x syndrome, rett syndrome, ds, and schizophrenia (81). last year several works offered new advanced cell models that may be instrumental in identifying the mechanisms by which candidate genes produce brain alterations. li and collaborators (82) have developed an organoid system that couples cutting-edge technologies such as crispr perturbations in organoids with single-cell transcriptomic readout (choose system). through loss-of-function assays of 19 high-risk asd genes known to be involved in epigenetic regulation, the authors have established a developmental and cell type-specific phenotypic database. asd genetic perturbations particularly have a strong impact on progenitors that differentiate into interneurons and oligodendrocytes and l2/3 excitatory neurons, suggesting that asd genes are enriched in upper-layer neurons during development. the choose system opens a new frame to determine cell-type-specific contributions to genetic disorders, including ndds (figure 3). going further, pasca and colleagues (11) combined assembloids and crispr to identify a set of 46 genes, out from 425 associated with asd and other ndds, based on genetic studies of patients and the simons foundation autism research initiative (sfari) database. the authors had previously developed a platform to study interneuron development and migration in subpallial and forebrain assembloids (83). this 3d subdomain-specific forebrain spheroids can be assembled in vitro from human hpscs and resemble either the dorsal or ventral forebrain and contain cortical glutamatergic or gabaergic neurons, thus being able to recapitulate the saltatory migration of gabaergic neurons from ventral to dorsal forebrain and their integration into cortical circuits observed in the fetal forebrain. now, the authors have integrated those assembloids with crispr screening to investigate the involvement of ndd genes in specific aspects of interneuron development. using this method, they discovered a group of genes that impair the generation or migration of interneurons to the cp. notably, the loss of an endoplasmic reticulum (er)-shaping protein, lnpk disrupts interneuron migration, indicating a previously unappreciated role of er dynamics. the platform developed by pasca and collaborators allows systematically map loss-of-function phenotypes for ndd genes based on stages of human interneuron development. this approach can accelerate the search for common functional deficits caused by disparate genes, enabling the grouping of neurodevelopmentally impaired patients for faster, more meaningful clinical trials and speeding treatments. however, to prevent unintended genomic alterations produced by gene editing strategies and long-term culture, the integration of advanced methods like optical genome mapping to existing protocols can provide a more thorough understanding of the genomic integrity of the in vitro cellular systems with consequences in the reliability of the research (84). figure 3. new cellular models are uncovering the function of neurodevelopmental disorder genes. a new organoid system that couples cutting-edge technologies such as crispr perturbations in organoids with single-cell transcriptomic readout (choose system) allows systematic loss-of-function assays of high-risk ndd genes (created with biorender.com). 10. the developmental origin of schizophrenia schizophrenia's complex biology highlights two crucial themes: the excessive elimination of glutamatergic synapses during development and disruptions in their signaling properties. these issues may impair circuit function, contributing to the symptoms and cognitive deficits seen in schizophrenia. since some years ago, schizophrenia has been thought to involve adverse neuropathological events beginning in early brain development (85), when disturbances in neural stem cell proliferation, neural differentiation, and synapse formation may lead to ndds. this is the reason for using brain organoids as an interesting approach. previous studies using ipscs to explore neurodevelopmental processes in schizophrenia have either focused on a single stage of organoid maturation (86), or generated brain organoids from only healthy individuals. additionally, donor variability in ipsc models can obscure case-control differences, a critical issue often overlooked (87). moreover, schizophrenia is highly polygenic making it challenging to understand basic disease mechanisms. to preserve the disease-specific genetic context (12) decided to generate ipsc-derived organoids, called human cortical spheroids (hcs), from a genetically stratified sample of schizophrenia cases and ageand sex-matched controls. in this study, the authors conducted an extensive transcriptional profiling in human cortical spheroids derived from ipscs by at 4 stages of hcs maturation, which enabled them to establish the persistent nature of the disturbances throughout development. the organoids grown from patients and controls differed in cell type composition but more importantly, in the expression of thousands of genes, in line with the finding that the genetic influences on schizophrenia are many and very small. however, axonal genes showed persistent dysregulation shedding light on underlying disease mechanisms. in a second study, another research group focused on the schizophrenia risk locus 15q11.2, containing 4 genes, that has over 10 % penetrance and doubles the risk for schizophrenia in individuals with unusual copy numbers in this region (13). one gene in this locus, cyfip1, is linked to synaptic function and increases the risk for ndds like schizophrenia and autism. although cyfip1 is highly expressed in microglia, the brain's immune cells, its role there remains unclear. sheridan et al. (13) collected blood cells from healthy volunteers, isolated ipscs, and differentiated them into microglia-like cells. microglia are known for synaptic pruning, essential for brain development. using crispr, they removed functional cyfip1 from these cells and detected changes in microglial behavior and function that could impact microglial processes like synaptic pruning and neuronal maintenance, crucial for brain development and function. this dysfunction could contribute to cyfip1-related neurodevelopmental and psychiatric disorders, including autism and schizophrenia. this study emphasizes the importance of examining beyond neurons to understand risk genes. identifying risk loci is just the beginning; determining the relevant cell types and gene functions is essential for progressing from genetic associations to potential treatments. acknowledgements the lab of md is recognized by the secretaria d’universitats i recerca del departament d’economia i coneixement de la generalitat de catalunya (grups consolidats 2023). we acknowledge the support of the agencia estatal de investigación (pid2022-141900ob-i00). we acknowledge the support of the spanish ministry of science and innovation through the centro de excelencia severo ochoa (cex2020-001049-s, mcin/aei13 /10.13039/501100011033), the generalitat de catalunya through the cerca programme and to the embl partnership. funding statement the lab of m.d. acknowledges financial support from the spanish ministry of science and innovation (projects cpp2022-009659 and rtc2019-007329-1), the spanish state research agency (project into-ds, reference pid2022-141900ob-i00), the marato-tv3 foundation (202212-30-31-32), and the european comission horizon 2020 programme (projects h2020-899986 and go-ds21-848077). the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. the crg acknowledges the support of the spanish ministry of science and innovation to the embl partnership, the centro de excelencia severo ochoa, and the cerca programme/generalitat de catalunya. the ciber of rare diseases (ciberer) is an initiative of isciii. conflicts of interest statement the authors declare no conflicts of interest. all co-authors have reviewed and approved the contents of the manuscript as well as the requirements for authorship have been met. references 1. borgognone a, casadellà m, martínez de lagrán m, paredes r, clotet b, dierssen m, et al. lamivudine modulates the expression of neurological impairment-related genes and line-1 retrotransposons in brain tissues of a down syndrome mouse model. front aging neurosci 2024 jul 19;16:1386944. https://www.doi.org/10.3389/fnagi.2024.1386944 2. alammari f, al-hujaily em, alshareeda a, albarakati n, al-sowayan bs. hidden regulators: the emerging roles of lncrnas in brain development and disease. front neurosci. 2024 may 22;18:1392688. https://www.doi.org/10.3389/fnins.2024.1392688 3. sierra c, sabariego-navarro m, fernández-blanco á, cruciani s, zamora-moratalla a, novoa em, et al. the lncrna snhg11, a new candidate contributing to neurogenesis, plasticity and memory deficits in down syndrome. res sq. 2023 sep 25;rs.3.rs-3184329 4. li d, wang q, bayat a, battig mr, zhou y, bosch dgm, et al. spliceosome malfunction causes neurodevelopmental disorders with overlapping features. j clin invest. 2024 an 2;134(1):e171235. https://www.doi.org/10.1172/jci171235 5. namburete ail, papież bw, fernandes m, wyburd mk, hesse ls, moser fa, et al. normative spatiotemporal fetal brain maturation with satisfactory development at 2 years. nature. 2023 nov;623(7985):106–14. https://www.doi.org/10.1038/s41586-023-06568-6 6. arai t, kamagata k, uchida w, andica c, takabayashi k, saito y, et al. reduced neurite density index in the prefrontal cortex of adults with autism assessed using neurite orientation dispersion and density imaging. front neurol. 2023;14:1110883. https://www.doi.org/10.3389/fneur.2023.1110883 7. christensen zp, freedman eg, foxe jj. autism is associated with in vivo changes in gray matter neurite architecture. biorxiv; 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changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. a novel fus::bend2 fusion expanding the molecular spectrum of astroblastomas feel free to add comments by clicking these icons on the sidebar free neuropathology 5:34 (2024) letter a novel fus::bend2 fusion expanding the molecular spectrum of astroblastomas arnault tauziède-espariat1,2, benjamin bonhomme3, nathalène truffaux3, volodia dangouloff-ros4, nathalie boddaert4, kévin beccaria5, lauren hasty1, alice métais1,2, pascale varlet1,2 department of neuropathology, ghu paris psychiatry and neuroscience, sainte-anne hospital, paris, france institut de psychiatrie et neurosciences de paris (ipnp), umr s1266, inserm, ima-brain, paris, france department of biopathology, institut bergonié, bordeaux, france pediatric radiology department, hôpital necker enfants malades, ap-hp, university de paris, paris, france department of pediatric neurosurgery, necker hospital, aphp, université paris descartes, sorbonne paris cité, paris, france corresponding author: arnault tauziède-espariat · department of neuropathology · sainte-anne hospital · 1, rue cabanis, 75014 · paris · france a.tauziede-espariat@ghu-paris.fr additional resources and electronic supplementary material: supplementary material submitted: 23 october 2024 accepted: 21 november 2024 copyedited by: shino magaki published: 10 december 2024 https://doi.org/10.17879/freeneuropathology-2024-5983 keywords: fus, bend2, astroblastoma introduction the name astroblastoma was modified to include “mn1-altered” in the latest world health organization (who) classification of tumors of the central nervous system (2021) [1], after the discovery of mn1 fusions (with bend2 as the main partner gene) [2]. the presence of this alteration constitutes an essential diagnostic criterion for this tumor type, and for unresolved lesions, a dna-methylation profile is necessary to confirm this diagnosis. the recent literature has concluded that bend2 fusions (without the mn1 gene) may occur in this tumor type. herein, we report one case of astroblastoma with a novel fus::bend2 fusion. we compare its clinical, histopathological, immunophenotypic, genetic, and epigenetic features with those previously described in astroblastomas, mn1 and/or bend2 fusion-positive. figure 1: radiological and histopathological features of the tumor (a–b) axial unenhanced computerized tomodensitometry images of the patient, showing a hyperdense and calcified mass extending from the third to the fourth ventricle with hydrocephalus and intraventricular hemorrhage. (c) an ependymoma-like tumor with astroblastic pseudorosettes (hps, magnification x100 and x400 for insert). (d) expression of gfap by tumor cells (magnification x400). (e) ema immunoexpression by tumor cells (magnification x400). scale bars represent 60 μm for figures c–e. hps: hematoxylin phloxin saffron. case presentation in 2007, a 4-year-old girl presented with a hyperdense and calcified mass extending from the third to the fourth ventricle with hydrocephalus and intraventricular hemorrhage (figure 1 a–b). the tumor was partially resected, and a diagnosis of ependymoma was made according to the 2007 who classification. the tumor recurred 14 months later, and the patient died 69 months after the initial diagnosis. in 2024, a review of the diagnosis was performed. microscopically, the re-examination showed a tumor having an ependymoma-like pattern with astroblastic pseudorosettes (figure 1c). there was no clear cell component, no hyalinized fibrous stroma nor pas-positive eosinophilic granular bodies. there was no microvascular proliferation or necrosis. mitotic figures were scarce, and the mib1-labeling index was low (5 %). the expression of brg1, ini1 and h3k27me3 was retained. the tumor cells expressed gfap and ema with a dot-like and a microlumen pattern (figure 1 d–e). there was no immunoreactivity for olig2, ck18, lin28a, nfκb, l1cam or neurofilaments. fish analysis of the mn1 gene failed to reveal a rearrangement, and rna sequencing evidenced a fus::bend2 gene fusion (figure 2a–b). additionally, dna-methylation analysis was conducted. the tumor was not classifiable using the heidelberg brain tumor classifier (v12.8) and the bethesda brain tumor classifier (v2), but clustered within astroblastoma, ewsr1::bend2 methylation class using the uniform manifold approximation and projection (umap) method (supplementary figure 1). because of the histopathological features and the presence of a bend2 fusion, the integrated diagnosis was an astroblastoma, bend2 fusion-positive. figure 2: genetic features (a) rnaseq analysis highlights a fusion between fus (pink) and bend2 (blue) genes, respectively located on chr16p11.2 and chrxp22.13. (b) chimeric protein between fus and bend2 with two retained ben protein domains of bend2. discussion and conclusions the current literature review includes 92 cases of astroblastomas that either carried a mn1 fusion or had a methylation profile compatible with astroblastoma, mn1-altered [2–28] (see supplementary table 1 for literature review). the majority of cases are supratentorial (84 %, 72/86 tumors with available data) [2–28]. most patients are pediatric (78 %, 67/86 cases with available data) [2–28]. however, this tumor occurs in a wide age range (0–60 years) [11,23]. typical histopathological features include an ependymoma-like pattern, astroblastic pseudorosettes and stromal sclerosis. as ependymomas, tumor cells frequently express gfap [3,4,6–11,14–19,22–25,27] and ema [3,4,6,8–11,14,16–19,21,22,25,28] with a variable olig2 immunopositivity [3,4,7,9–11,11,13,15–17,19,21–25,28]. a mn1 gene fusion is found in 84 % of astroblastomas (54/64 reported cases with genetic analyses, the 28 remaining cases being only diagnosed using dna-methylation profiling analysis), mainly in association with the bend2 gene (94 %, 17/18 cases with rna-sequencing data, the 36 remaining cases being only diagnosed using fish mn1) [2–4,6–16,18–28]. one tumor harbored a mn1::cxxc5 fusion [2]. alternative fusions including the bend2 gene without mn1 have been reported in ten tumors (7 with ewsr1, 2 with mamld1, and one with yap1) [3,4,8,19,22,23,27]. moreover, an ewsr1::ezhip fusion was identified in an astroblastoma; however, no methylation-based classification was available for this tumor [29]. in this study, we report for the first time a fus::bend2 fusion in astroblastoma. this novel gene rearrangement results in an in-frame fusion of exons 1 to 5 of the fus gene and exons 2 to 14 of the bend2 gene, the same breakpoint as previously reported [3,4]. in the literature, we identified two reports of a nasopharyngeal and soft tissue sarcoma harboring a fus::bend8 (another gene encoding a ben domain containing protein) fusion [30,31]. the fus gene belongs to the fet family of genes which encode for rna-binding proteins, like ewsr1 [32]. the predicted fus-bend2 chimeric protein maintained two ben domains in the c-terminus encoded by bend2, which are involved in transcription and chromatin regulation [33]. as was found for our case, clinical findings seem to be different for patients who have an astroblastoma, bend2 fusion-positive. they tend to present in a younger population (median age of 6 years-old at diagnosis), affect females less frequently (with a female-to-male ratio of 1.75), and be mainly infratentorial (73 % of cases) compared to the astroblastomas, mn1-altered (presenting at a median age of 10 years, a female-to-male ratio of 7.5 and only making up 8 % of infratentorial tumors) [2–4,6–16,18–28]. most likely due to the tumor location, the prognosis of patients with astroblastomas, bend2 fusion-positive is worse (33 %, 3/9, patients died with a median overall survival of 25 months [1;276]) than those with astroblastomas, mn1-altered (14 % of patients, 9/62, died with a median overall survival of 70 months [3;324]). the analysis of the recent literature seems to suggest that it may be bend2, rather than mn1, which has a more critical functional role in the oncogenesis of astroblastomas [34]. in conclusion, we expanded the astroblastoma genetic spectrum with one novel fusion fus::bend2. reminiscent of the terminological history of supratentorial ependymomas, originally referred to as “rela-fusion positive” and afterward “zfta-fused”, our case supports the consideration of a bend2 fusion as the defining alteration for astroblastoma, rather than the mn1 rearrangement. ethics approval this study was approved by the ghu paris psychiatrie neurosciences, sainte-anne hospital’s local ethic committee. consent for publication the patient signed informed consent forms before treatment was started. competing interests the authors declare that they have no conflicts of interest directly related to the topic of this article. funding statement the authors declare that they have not received any funding. authors’ contributions ate, vdr, kb and nb compiled the mri and clinical records; ate, am and pv conducted the neuropathological examinations; bb conducted the molecular studies; ate, lh, and pv drafted the manuscript. all authors reviewed the manuscript. acknowledgements we would like to thank the laboratory technicians at the ghu paris neuro sainte-anne for their assistance.   supplementary material supplementary table 01 (download) literature review (pdf) supplementary figure 01 (download) uniform manifold approximation and projection (umap) analysis (pdf) references 1. brat dj, solomon da, rosenblum mk, aldape kd, idbaih a, kool m, et al. astroblastoma, mn1-altered. in: who classification tumours editorial board. central nervous system tumours. lyon (france): international agency for research on cancer; 2021. 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neural transcriptional repressors. genes dev. 2013;27:602–14. https://doi.org/10.1101/gad.213314.113 34. lehman nl. early ependymal tumor with mn1-bend2 fusion: a mostly cerebral tumor of female children with a good prognosis that is distinct from classical astroblastoma. j neurooncol. 2023;161:425–39. https://doi.org/10.1007/s11060-022-04222-1 copyright: © 2024 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. histopathologic evidence of intimal hyperplasia in carotid artery webs associated with stroke feel free to add comments by clicking these icons on the sidebar free neuropathology 6:16 (2025) case report histopathologic evidence of intimal hyperplasia in carotid artery webs associated with stroke farhan khan1, alisa nobee2, skylar lewis1, john e. donahue2, shadi yaghi1 department of neurology, brown university, providence, ri 02903, usa department of pathology, brown university providence, ri 02903, usa corresponding author: farhan khan · brown university · department of neurology · 593 eddy street · providence, ri 02903 · usa farhankhan.md@outlook.com submitted: 06 june 2025 accepted: 05 august 2025 copyedited by: jerry lou published: 26 august 2025 https://doi.org/10.17879/freeneuropathology-2025-7139 keywords: carotid artery web, stroke, intimal hyperplasia abstract background: carotid artery webs (cws) are an underrecognized cause of ischemic stroke, particularly in younger patients who lack conventional vascular risk factors. cws are thought to represent an intimal variant of fibromuscular dysplasia (fmd); however, histopathologic data supporting this hypothesis remain limited. we report a case series of three patients with cw-related ischemic stroke who underwent carotid endarterectomy (cea), allowing for histological analysis of the resected specimens. methods: we retrospectively reviewed patients admitted to a comprehensive stroke center between january 2015 and april 2025 with ischemic stroke or transient ischemic attack attributed to an ipsilateral carotid web who subsequently underwent carotid endarterectomy. clinical data, imaging findings, and histopathologic features were analyzed. all cases met criteria for embolic stroke of undetermined source (esus) prior to surgery. results: three patients with cw-related stroke underwent carotid endarterectomy following recurrent events or high embolic risk. in two cases, superimposed thrombi led to initial misdiagnoses such as soft plaque or dissection. histopathologic analysis consistently demonstrated fibrovascular tissue with intimal fibroid hyperplasia and myxoid degeneration, without lipid-rich plaques or inflammatory infiltrates. no patients experienced recurrent stroke or tia by the time of their last documented follow-up. conclusions: cws represent a distinct non-atherosclerotic pathology characterized by intimal hyperplasia and myxoid degeneration. superimposed thrombus may complicate diagnosis, often mimicking plaque or dissection. advanced imaging, including mr vessel wall imaging and intravascular optical coherence tomography (oct), can aid in accurate identification. carotid revascularization may be effective in selected patients, particularly those with recurrence or esus. prospective studies are needed to inform standardized diagnostic and therapeutic strategies. introduction carotid artery web (cw) is an underrecognized cause of ischemic stroke1. cws appear as shelf-like projections arising from the posterior wall of the proximal internal carotid artery3. cws have a reported prevalence of 1–3 % among patients with large vessel occlusion and is more commonly observed in black women2. hemodynamic studies suggest that this morphology leads to increased recirculation time and high wall shear stress, which promotes blood stasis and endothelial injury, thereby facilitating thrombogenesis4. because of their subtle appearance on imaging, cws are frequently overlooked, resulting in delayed or missed diagnoses5. despite medical treatment, cws are associated with a high rate of recurrent stroke2,5,6. these recurrence rates are higher than those reported in large randomized controlled trials (rcts) studying embolic stroke of undetermined source (esus), supporting the consideration of more aggressive interventions, such as carotid revascularization, for secondary prevention7,8. a recent meta-analysis reported recurrence rates as high as 32 % in patients with ipsilateral cws and found no significant difference in efficacy between carotid stenting and endarterectomy9. prior literature suggests that cws may represent a variant of fibromuscular dysplasia (fmd), involving intimal hyperplasia, in contrast to the more common medial hyperplasia seen in classical fmd. due to limited data on the histopathology of carotid artery webs, we present a case series of patients who underwent carotid endarterectomy at our institution. methods clinical data the data supporting the findings can be obtained from the corresponding author upon reasonable request. the study was approved by the local institutional review board, and informed consent was waived due to the nature of the study. this retrospective observational case series includes patients who developed clinical and neuroimaging evidence of ischemic stroke and underwent carotid endarterectomy. we only included patients admitted to the comprehensive stroke center with strokes occurring between january 2015 and april 2025, where the ischemic stroke was attributed to an ipsilateral carotid web. carotid webs were identified by reviewing ct angiograms of the neck in multiple planes (axial, sagittal, and coronal) by treating vascular neurologists, neuroradiologists, and neurosurgeons. carotid web revascularization was performed only when no alternative stroke etiology was identified, as these strokes were classified as esus (embolic stroke of undetermined etiology) as per toast criteria by board-certified vascular neurologists. carotid revascularization was performed according to institutional protocols. demographic data, clinical presentation, laboratory and radiologic results were recorded by approved personnel through chart review. pathology all surgical specimens obtained during carotid endarterectomy (cea) were submitted for gross and histopathological examination by board-certified neuropathologists. specimens were fixed in 10 % neutral-buffered formalin, processed, and paraffin-embedded using standard protocols. tissue blocks were sectioned at 4–5 microns and stained with hematoxylin and eosin (h&e). gross examination included an evaluation of specimen size, color, texture, and the presence of thrombus, calcification, or fibrous thickening. all slides were reviewed by at least one board-certified neuropathologist blinded to clinical imaging findings. representative photomicrographs were captured at low (2×) and high (20×) magnification using a digital slide scanner. results case 1 a woman in her 20s without known vascular risk factors presented with dysarthria and numbness in her left hand. a non-contrast head ct scan was normal. at an outside hospital, an initial ct angiogram of the head and neck raised concern for a dissection of the right proximal internal carotid artery, and she was transferred to our institution for further evaluation. her initial nihss was 1. brain mri showed an embolic-appearing ischemic stroke in the right frontal lobe. tte, outpatient cardiac monitoring, and hypercoagulable workup including antiphospholipid antibody syndrome (aps) were negative. upon reviewing the neck imaging with a neuroradiologist, the lesion was interpreted as a focal artery dissection versus a possible carotid artery web with superimposed thrombus. she was started on oral anticoagulation. six months later, she underwent a diagnostic cerebral angiogram, which confirmed the presence of a carotid web in the proximal right internal carotid artery (figure 1) and later right carotid endarterectomy for secondary stroke prevention. gross examination revealed a single soft tissue fragment 1.3 x 0.5 x 0.3 cm. examined histologic sections demonstrated fragments of fibrovascular tissue consisting of vascular smooth muscle cells in a background of myxoid degeneration, no lipid or inflammatory component was identified (figure 2). at her most recent follow-up, 10 months after the initial event, she had not experienced a recurrent ischemic stroke. figure 1. imaging findings of a right carotid artery web with superimposed thrombus and interval resolution. (a) axial cta demonstrating a shelf-like projection along the posterior wall of the right internal carotid artery (ica) with superimposed thrombus (green arrow). (b) corresponding sagittal cta confirming the presence of thrombus overlying a carotid web in the right ica (orange arrow). (c) follow-up sagittal cta performed after anticoagulation shows resolution of thrombus with a small residual carotid web in the proximal right internal carotid artery (red arrow). (d) digital subtraction angiography (dsa) confirms the presence of a subtle right carotid artery web without flow-limiting stenosis (blue arrow). figure 2. histological findings of carotid artery web resected via endarterectomy (hematoxylin and eosin stain, elastic stain). (a) low-power (10×) microscopic view showing a single fragment of fibrovascular tissue, without evidence of thrombus formation, lipid deposition or inflammatory infiltrate. (b) elastic stain (10×) demonstrating the absence of an internal elastic lamina (red arrow). (c) trichrome stain (10×) showing increased fibrosis (black arrow). (d) high power (20×) view highlighting areas of myxoid degeneration (blue arrow). case 2 a man in his 50s with no significant past medical history presented with signs of a right middle cerebral artery (mca) syndrome, three weeks after a transient ischemic attack (tia) affecting the same hemisphere. his admission nihss was 6. a non-contrast head ct was negative for hemorrhage, and ct angiography of the head and neck revealed an occlusion of the right m1 segment. he underwent mechanical thrombectomy with successful tici 2c recanalization. cardiac workup, including transthoracic echocardiography (tte), telemetry, and hypercoagulable testing, was unremarkable. vascular imaging revealed a small right internal carotid artery (ica) web with superimposed thrombus (figure 2a and b), which had not been present on prior imaging performed during his initial tia evaluation (figure 3c). due to recurrent vascular events ipsilateral to cw, he underwent a right carotid endarterectomy (cea) during the same hospitalization. gross examination of the specimen revealed multiple fragments of tan soft tissue 1.1 x 1.1 x 0.2 cm in aggregate. examined histologic sections demonstrated fragments of fibrovascular tissue consisting of vascular smooth muscle cells in a background of myxoid degeneration and increased fibrosis. in this case, no lipid or inflammatory component was identified either (figure 4). at his most recent follow-up, 11 months after the initial event, he had not experienced a recurrent ischemic stroke. figure 3. interval development of thrombus over a carotid artery web in a patient with recurrent ischemic events. (a) axial cta at the time of large vessel occlusion demonstrates a carotid web in the right internal carotid artery with superimposed thrombus (orange arrow). (b) corresponding sagittal view confirms the presence of thrombus overlying the web in the proximal right internal carotid artery (green arrow). (c) imaging performed three weeks earlier during workup for tia shows the same carotid web without thrombus (red arrow). figure 4. histological findings of carotid artery web were collected via endarterectomy (hematoxylin and eosin stain, elastic stain). (a) low-power (4×) microscopic view showing a single fragment of fibrovascular tissue, without evidence of thrombus formation, lipid deposition or inflammatory infiltrate. (b) elastic stain (4×) demonstrates the absence of staining. (c) trichrome stain (4×) showing increased fibrosis (black arrow). (d) high power (20×) view highlighting areas of myxoid degeneration (blue arrow). case 3 a man in his 60s with a history of dyslipidemia presented with a left middle cerebral artery (mca) syndrome. his nihss score was 14. a non-contrast head ct was negative for hemorrhage or a large ischemic core. given that his last known well was within 4.5 hours of symptoms onset, he received intravenous thrombolytic (tenecteplase). ct angiography of the head and neck revealed an occlusion of the m1 segment of the left mca with proximal carotid artery web (figure 5), and he underwent mechanical thrombectomy with successful tici 3 recanalization. on hospital day 2, he developed worsening right arm weakness and aphasia. imaging revealed a new proximal left m2 occlusion, and he underwent a repeat mechanical thrombectomy with tici 2c recanalization. further stroke workup, including tte and age-appropriate cancer screening, was negative. during the same hospitalization, he underwent resection of a left internal carotid artery (ica) web without stroke recurrence of tia or ischemic stroke until his last follow up (4 months). gross examination of the specimen revealed a 2.3 x 1.0 x 0.9 cm portion of tan tissue. examined histologic sections demonstrated fibrovascular tissue with intimal fibroid hyperplasia, focal calcifications, and myxoid degeneration. like the other cases, no lipid or inflammatory component was identified (figure 6). figure 5. radiographic imaging of a carotid artery web (cw) without evidence of superimposed thrombus in a patient with recurrent large-vessel occlusions (case 3). (a) axial cta demonstrates a shelf-like projection along the posterior wall of the left internal carotid artery, consistent with a carotid web in the proximal left ica (yellow arrow). (b) corresponding sagittal cta confirms the non-stenotic web morphology in the absence of atherosclerosis (green arrow). (c) digital subtraction angiography (left common carotid contrast injection) reveals a subtle web-like filling defect in the proximal consistent with a carotid artery web (black arrow). figure 6. histological findings of carotid artery web resected via endarterectomy (hematoxylin and eosin stain, elastic stain, trichrome stain). (a) low-power (4×) microscopic view showing a single fragment of fibrovascular tissue, with shelf-like projections into the luminal aspect of the specimen (green arrows). there are focal calcifications (orange arrow), however there is no evidence of thrombus formation, lipid deposition or inflammatory infiltrate. (b) elastic stain (4×) demonstrating the absence of an internal elastic lamina (red arrow). (c) trichrome stain (4×) showing increased fibrosis in these shelf-like areas (black arrow). (d) high power (20×) view highlighting areas of myxoid degeneration (blue arrow). discussion histopathologic examination of the carotid endarterectomy specimens in this series consistently revealed fibrovascular tissue composed of vascular smooth muscle cells, intimal fibroid hyperplasia, and areas of myxoid degeneration10. these findings support the hypothesis that carotid webs represent an intimal variant of fibromuscular dysplasia rather than an atherosclerotic process. notably, none of the cases demonstrated a lipid core or significant inflammatory infiltrate, distinguishing these lesions from classic atheromatous plaques11. the presence of myxoid degeneration and intimal hyperplasia may contribute to a prothrombotic milieu by disrupting local flow dynamics, consistent with prior imaging studies showing flow stasis and turbulence in the region of the web12. the presence of superimposed thrombus was observed in multiple cases and played a critical role in diagnostic uncertainty. in acute clinical settings, thrombus layering on a carotid web may mimic the imaging appearance of soft plaque or a focal dissection, particularly when evaluated without high-resolution imaging or expert review. this can lead to initial misclassification, as illustrated by the first case in which the lesion was initially suspected to be a dissection. advanced imaging modalities can aid in more accurate differentiation. high-resolution magnetic resonance vessel wall imaging (mr vwi) may provide indirect evidence of myxoid degeneration and help distinguish non-atherosclerotic lesions from lipid-rich plaques13. additionally, intravascular optical coherence tomography (oct) offers high-resolution cross-sectional visualization of the intimal surface and has been shown to reliably differentiate carotid webs from both soft atheromatous plaque and dissection flaps14. incorporating these advanced imaging techniques can enhance diagnostic accuracy and guide appropriate management decisions in ambiguous cases. revascularization of the affected carotid artery through endarterectomy appears to be an effective strategy in selected patients, particularly those with recurrent ischemic events and no identifiable alternative stroke mechanism. in all three cases presented, surgical intervention was pursued after recurrence or persistent thromboembolic risk was established, with no recurrent stroke reported during follow-up. these findings are consistent with a recent meta-analysis, which demonstrated significantly reduced rates of recurrent ischemic stroke in patients with carotid artery webs who underwent carotid revascularization compared to those treated with medical management alone9. despite this emerging evidence, clinical practice remains highly variable. a nationwide survey comparing vascular neurologists and neurointerventionalists found that neurointerventionalists were substantially more likely to favor revascularization, even after a first-ever ischemic stroke, whereas vascular neurologists tended to recommend continued medical therapy15. this variability highlights the absence of randomized controlled trials and consensus guidelines, reinforcing the need for prospective studies to establish standardized treatment pathways and better define which patients are most likely to benefit from intervention. conclusion carotid artery webs represent a distinct, non-atherosclerotic cause of ischemic stroke, characterized histologically by intimal fibroid hyperplasia and myxoid degeneration without lipid or inflammatory components. the presence of superimposed thrombus can complicate imaging interpretation, often mimicking soft plaque or dissection. in selected patients, particularly those with recurrent ischemic events and no other identifiable etiology, carotid revascularization appears to be an effective preventive strategy. while emerging evidence, including histopathological findings and meta-analytic data, supports intervention, significant variability in clinical practice persists. prospective, multicenter studies are urgently needed to clarify patient selection criteria and establish standardized guidelines for the management of carotid artery webs. limitations this study has several limitations due to its retrospective observational design. first, the small sample size and single-center setting may limit the generalizability of our findings. second, carotid web diagnosis was based on imaging review by subspecialists, which, while thorough, may still be subject to interobserver variability, especially in cases with superimposed thrombus. although we included histopathological confirmation, not all patients with suspected carotid webs undergo surgical resection, potentially introducing selection bias toward more severe or recurrent cases. additionally, the absence of a control group receiving medical management alone precludes direct comparison of outcomes between treatment strategies. finally, long-term follow-up was limited to less than one year. conflict of interest statement the authors declare no competing interests. funding statement the authors declare that they have not received any funding. author contributions f.k. drafted the manuscript. a.n. co-drafted, revised the manuscript, and prepared the histological slides. s.l., j.d., and s.y. critically revised the manuscript. references 1. brinster cj, vidal g, bazan h, et al. carotid web is an under-recognized and devastating cause of ischemic stroke. journal of vascular surgery 2021;73(3):43. https://doi.org/10.1016/j.jvs.2020.12.032 2. guglielmi v, compagne kcj, sarrami ah, et al. assessment of recurrent stroke risk in patients with a carotid web. jama neurol 2021;78(7):826–833. https://doi.org/10.1001/jamaneurol.2021.1101 3. choi pmc, singh d, trivedi a, et al. carotid webs and recurrent ischemic strokes in the era of ct angiography. ajnr am j neuroradiol 2015;36(11):2134–2139. https://doi.org/10.3174/ajnr.a4431 4. el sayed r, lucas cj, cebull hl, et al. subjects with carotid webs demonstrate pro-thrombotic hemodynamics compared to subjects with carotid atherosclerosis. sci rep 2024;14:10092. https://doi.org/10.1038/s41598-024-60666-7 5. khan f, kala n, chang k, et al. in-hospital recurrent stroke in ipsilateral carotid web patients undergoing thrombectomy. ann clin transl neurol 2024;11:2450–6. https://doi.org/10.1002/acn3.52161 6. choi pm, singh d, trivedi a, et al. carotid webs and recurrent ischemic strokes in the era of ct angiography. ajnr am j neuroradiol 2015;36(11):2134–9. https://doi.org/10.3174/ajnr.a4431 7. hart rg, sharma m, mundl h, et al. rivaroxaban for stroke prevention after embolic stroke of undetermined source. new england journal of medicine 2018;378(23):2191–2201. https://doi.org/10.1056/nejmoa1802686 8. dabigatran for prevention of stroke after embolic stroke of undetermined source. n engl j med 2019;380:1906-1917. https://doi.org/10.1056/nejmoa1813959 9. khan f, gujjar ar, fletcher l, et al. carotid revascularization vs. medical management for ischemic stroke with ipsilateral carotid web: a systematic review and meta-analysis (s25.005). neurology 2025;104(7_supplement_1):2690. https://doi.org/10.1212/wnl.0000000000210516 10. wang lz, calvet d, julia p, et al. is carotid web an arterial wall dysplasia? a histological series. cardiovasc pathol 2023;66:107544. https://doi.org/10.1016/j.carpath.2023.107544 11. stary hc, chandler ab, dinsmore re, et al. a definition of advanced types of atherosclerotic lesions and a histological classification of atherosclerosis. a report from the committee on vascular lesions of the council on arteriosclerosis, american heart association. circulation 1995;92(5):1355–1374. https://doi.org/10.1161/01.cir.92.5.1355 12. compagne kcj, dilba k, postema ej, et al. flow patterns in carotid webs: a patient-based computational fluid dynamics study. ajnr am j neuroradiol 2019;40(4):703–708. https://doi.org/10.3174/ajnr.a6012 13. su y, stein la, cucchiara bl, song jw. vessel wall imaging of a carotid web. annals of neurology 2022;92(2):335–336. https://doi.org/10.1002/ana.26424 14. al-bayati ar, nogueira rg, sachdeva r, et al. optical coherence tomography in the evaluation of suspected carotid webs. j neurointervent surg 2023;jnis-2023-020813. https://doi.org/10.1136/jnis-2023-020813 15. khan f, mallick d, wolman d, et al. management of carotid artery web: a nationwide survey of vascular neurologists versus neurointerventionalists [internet]. journal of neurointerventional surgery 2025;[cited 2025 may 21 ] available from: https://jnis.bmj.com/content/early/2025/05/16/jnis-2025-023232. https://doi.org/10.1136/jnis-2025-023232 copyright: © 2025 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. 64th annual meeting of the canadian association of neuropathologists association canadienne des neuropathologistes (canp-acnp) feel free to add comments by clicking these icons on the sidebar free neuropathology 6:1(2025) meeting abstracts 64th annual meeting of the canadian association of neuropathologists association canadienne des neuropathologistes (canp-acnp) meeting abstracts october 3rd–5th, 2024 toronto, on submitted: 04 december 2024 accepted: 05 december 2024 published: 29 january 2025   the canadian association of neuropathologist – association canadienne des neuropathologistes (canp-acnp) held their 64th annual meeting at the sickkids – peter gilgan centre for research and learning, in toronto, ontario, from october 3rd-5th, 2024, under the leadership of dr. robert hammond, president of the canp-acnp, dr. peter schutz, secretary treasurer of the canp-acnp, and with technical support from canp administrator colleen fifield. the academic program comprised 13 scientific abstracts, 9 unknown cases, the neuropathology in practice forum on molecular testing of brain tumours in canada, and the presidential symposium on looking to the future – digital pathology and spatial transcriptomics. digital pathology images from the 9 unknown cases were available for viewing online (www.canp.ca). the unknown case session was moderated by dr. andrew gao. the presidential symposium 2024 on looking to the future – digital pathology and spatial transcriptomics featured the david robertson lecture given by dr. a. kriegstein on transcriptomic cytoarchitecture of the brain, and the jerzy olszewski guest lecture given by dr. m. faiz entitled an integrated single-cell and spatial transcriptomics approach reveals the heterogeneity of the astrocyte response to stroke. the program was completed by three invited presentations with dr. brittany dugger presenting on digital pathology applications in neurodegeneration, dr. s. camelo-piragua presenting on digital pathology applications in neuro-oncology, and dr. matthew cecchini presenting on optimizing digital pathology with ai: strategic approaches for modern clinical practice. the mary tom award for best clinical science presentation by a trainee went to dr. christopher newell (supervisor dr. christopher dunham), and the morrison h. finlayson award for best basic science presentation by a trainee was won by dr. erin stephenson (supervisor dr. voon wee yong). the following abstracts were presented at the 64th annual meeting of the canadian association of neuropathologists – association candienne des neuropathologistes (canp-acnp) in october 2024.   https://doi.org/10.17879/freeneuropathology-2025-6284 keywords: canadian association of neuropathologists, canp, meeting abstracts, 64th meeting oct. 2024 contents abstract 1: distinctive pattern of cerebral amyloid angiopathy in the cerebellum abstract 2: nigral astrocytic tau pathology in psp abstract 3: keratan sulfate is a chemical template for neuroblast migratory pathways and axonal fascicles in human fetal forebrain abstract 4: unique neuropathological mechanisms underlying cognitive impairment in schizophrenia abstract 5: recent advances in spatial transcriptomics of the extracellular matrix abstract 6: an anatomic transcriptional atlas of focal cortical dysplasia abstract 7: transcriptomic profiling reveals hippocampal white matter inflammation as potential epileptogenic focus in temporal lobe epilepsy abstract 8: global proteomics reveals bidirectional integrin signaling as a driver of glioblastoma invasion abstract 9: integration of molecular and morphometric features improves prognostication of diffuse gliomas abstract 10: a 13 year review of non-diffuse large b cell lymphomas of the central nervous system at a tertiary hospital: a case series study abstract 11: cost-benefit analysis of fluorescence in situ hybridization testing in the diagnosis of idh-mutant 1p/19q-codeleted oligodendrogliomas abstract 12: infant-type hemispheric glioma is mostly relatively well-demarcated tumor and not diffusely infiltrative abstract 13: features of central nervous system metastases by neoplasms of gynecological origin     abstract 1 free neuropathol 6:1:4 distinctive pattern of cerebral amyloid angiopathy in the cerebellum david g. munoz1,3, julia keith2,3, sara mitchell4, mario masellis4, alyona ivanova5, sandra black4 department of laboratory medicine, st. michael’s hospital, unity health toronto, toronto, on, canada department of anatomic pathology, sunnybrook health sciences center, toronto, on, canada department of laboratory medicine & pathobiology, university of toronto, toronto, on, canada division of neurology, sunnybrook health sciences center, toronto, on, canada institute of medical science, university of toronto, toronto, on, canada we aimed to compare the patterns of cerebral amyloid angiopathy (caa) in the cerebral and cerebellar cortices. we studied 24 brains from the pathology departmental archives at sunnybrook, ages 56–93. diagnoses included alzheimer’s disease and vascular dementia. meningeal, parenchymal, and capillary caa were evaluated in frontal, occipital, and basal temporal cortices, and cerebellum. caa and plaques were scored 0–3 as per love 2014. caa was identified in 21/24 cases. cerebellum caa was present in 17/24 cases; it was never present without some caa in one of the neocortical regions. however, the cerebellar caa score exceeded the score in any neocortical region in 4 cases, was equal in 9, and less than at least one neocortical region in 7. in the cerebellum meningeal exceeded parenchymal scores in 12 cases, and parenchymal exceeded meningeal in 5 cases. cerebellar caa with scores of up to 3 was present in cases without cerebellar plaques; likewise cerebellar plaque scores of 2 were present in a case without cerebellar caa. there never was capillary caa in the absence of parenchymal caa in any region. there was no correlation between meningeal and parenchymal caa scores in the cerebellum, whereas they were highly correlated in neocortical areas. we conclude that cerebellar caa is common, often equal or more severe than neocortical caa. in the cerebellum, unlike in the neocortex, meningeal and parenchymal caa are not correlated, suggesting that they are responses to largely independent pathophysiological mechanisms.   abstract 2 free neuropathol 6:1:5 nigral astrocytic tau pathology in psp hidetomo tanaka1, seojin lee1, shelley l. forrest1, gabor g. kovacs1 department of laboratory medicine and pathobiology and tanz centre for research in neurodegenerative disease, university of toronto, toronto, on, canada background: astrocytic tau pathology is a major feature of primary tauopathies and aging-related tau astrogliopathy (artag). although tau pathology in the substantia nigra (sn) is usually neuronal, we recently reported cases with prominent nigral tau astrogliopathy (nitag). interestingly, despite diverse primary diseases, nitag exhibited shared distinctive features: abundant tau-positive astrocytes with fewer tau-positive neuronal cytoplasmic inclusions (ncis) and mild neuronal loss in the sn, and some astrocytic lesions in the other brainstem regions. method: we investigated the nigral astrocytic tau pathology in 39 progressive supranuclear palsy (psp) cases. results: compared to the cases with prominent nitag, the severity of nigral astroglial tau pathology was not as high and was present in 36 of the 39 psp cases, with varying amounts. we classified psp cases into high and low groups based on the number of tau-positive astrocytes in the sn and compared clinicopathological features. morphologically, nigral tau-positive astrocytes in psp were distinct from typical tufted astrocytes and resembled those seen in nitag. unlike nitag, both groups exhibited more than moderate neuronal loss and ncis in the sn, with no significant difference. however, the extent of tau-positive astrocytes in midbrain regions outside the sn was significantly higher in the high-group. conclusion: we stratified psp cases based on the presence and severity of nitag-type pathology and identified distinct features supporting the notion that psp might have different pathological subtypes eventually reflect distinct pathogenesis.   abstract 3 free neuropathol 6:1:6 keratan sulfate is a chemical template for neuroblast migratory pathways and axonal fascicles in human fetal forebrain harvey b. sarnat1,2, weiming yu3 departments of paediatrics, pathology (neuropathology) and clinical neurosciences, university of calgary cumming school of medicine, calgary, alberta, canada alberta children’s hospital research institute (owerko centre), calgary, alberta, canada department of pathology and laboratory medicine, alberta children’s hospital, calgary, alberta, canada objectives. timing of keratan sulfate (ks) during morphogenesis in normally developing human fetal forebrain structures was studied. ks is a proteoglycan secreted in fetal brain by astrocytes and radial glia into extracellular parenchyma as granulofilamentous deposits. it envelops neurons except dendritic spines, repels glutamatergic and facilitates gabaergic axons. the same genes are expressed in both neuroblast migration and axonal growth. methods. twenty normal human fetal brains from 9–41 weeks gestational age were studied at autopsy. ks was examined by immunoreactivity in formalin-fixed paraffin sections, plus other markers including synaptophysin, s-100β protein, vimentin and nestin. results. radial and tangential neuroblast migratory pathways from subventricular zone to cortical plate were marked by ks deposits as early as 9wk ga, shortly after neuroblast migration initiated. during later gestation this reactivity gradually diminished and disappeared by term. long axonal fascicles of the internal capsule and short fascicles of intrinsic bundles of globus pallidus and corpus striatum also appeared as early as 9–12wk, as fascicular sleeves before axons even entered. intense ks occurs in astrocytic cytoplasm and extracellular parenchyma at 9wk in globus pallidus, 15wk thalamus, 18wk corpus striatum, 22wk cortical plate, and hippocampus postnatally. corpus callosum and anterior commissure do not exhibit ks at any age. optic chiasm shows peripheral reactivity but not around intrinsic subfasciculi. cross-immunoreactivity with aggrecan may confuse molecular distinctions. conclusion. ks forms a putative chemical template (but not a structural scaffold) for many long and short axonal fascicles before axons enter and for neuroblast migratory pathways at initiation of migration.   abstract 4 free neuropathol 6:1:7 unique neuropathological mechanisms underlying cognitive impairment in schizophrenia naomi c. futhey1, elizabeth gregory1,3, belen arranz4, josep maria haro4, fidel vila-rodriguez3,5, mark s. cembrowski2,5–7, veronica hirsch-reinshagen8 md/phd program, university of british columbia, vancouver, canada department of cellular and physiological sciences, life sciences institute, university of british columbia, vancouver, canada non-invasive neurostimulation therapies laboratory, department of psychiatry, university of british columbia, vancouver, canada sant joan de déu, serveis de salut mental, barcelona, spain school of biomedical engineering, university of british columbia, vancouver, canada djavad mowafaghian centre for brain health, university of british columbia, vancouver, canada department of mathematics, university of british columbia, vancouver, canada department of pathology and laboratory medicine, university of british columbia, vancouver, canada cognitive impairment is a core tenet of chronic schizophrenia, impacting as many as 98 % of patients. it is not targeted by any currently available therapies, and the underlying neuropathology only occasionally includes neurodegenerative processes such as alzheimer’s disease. this study seeks to understand the cellular mechanisms for cognitive impairment which may be unique to schizophrenia. demographic, clinical, and autopsy data were obtained for 55 elderly patients with chronic schizophrenia and post-mortem neuropathological evaluation, with additional neuropsychological testing conducted during life in a subset. standard evaluation of neurodegenerative conditions and exploratory data analyses including clinical-pathological correlations were performed. further dimensionality reduction and clustering analysis were conducted using uniform manifold approximation and projection (umap) and k-means clustering to illustrate characteristics of patient subpopulations. on average, patients experienced multiple decades of symptoms, with marked disruption of reality by hallucinations and delusions. scoring of negative symptoms at this advanced disease stage outweighed that of positive symptoms, although both were present. significant cognitive impairment was noted across different neuropsychological testing modalities. only 56 % of patients had sufficiently severe neuropathological changes to explain their cognitive impairment, and distinct subpopulations were highlighted in clustering analysis. together, this suggests the presence of additional mechanisms underlying cognitive impairment in chronic schizophrenia. better understanding of the pathophysiology of this impairment may inform our knowledge of schizophrenia as a whole and provide the basis for novel precision medicine applications.   abstract 5 free neuropathol 6:1:8 recent advances in spatial transcriptomics of the extracellular matrix erin l. stephenson1, rajiv w. jain2, samira ghorbani2, rianne p. gorter2, charlotte d’mello2, voon wee yong2 department of pathology and laboratory medicine, university of calgary, calgary, ab, canada hotchkiss brain institute and department of clinical neurosciences, university of calgary, ab, canada the extracellular matrix (ecm) of the central nervous system (cns) is an interconnected network of proteins and polysaccharides with critical roles in maintaining brain homeostasis and modulating responses to injury. in neurological diseases, alterations of the ecm can be beneficial or detrimental depending on the constellation of changes. however, there is a lack of data investigating how ecm members change in relation to each other or the relevance of location following injury. the purpose of this research was to investigate how the ecm is altered in multiple sclerosis and glioblastoma, and the spatial heterogeneity of ecm changes. active and inactive demyelinated lesions from multiple sclerosis were investigated with a combination of spatial mrna-sequencing, single-nucleus rna sequencing, and immunohistochemistry. there were widespread changes in the ecm and distinct ecm profiles within inactive cores, lesion rims, and surrounding white matter (int j mol sci 2024; 25(2):1240). results uncovered multiple novel ecm targets, including the sparc family, which have the capacity to affect immune cell activation. brain parenchyma of genetically engineered mice invaded by glioblastoma were separated into tumour core, tumour edge, and infiltrated cortex and analysed using spatial transcriptomics. there were differentially expressed ecm genes that defined tumour regions and unique expression was associated with different tumour molecular drivers. the profound spatial changes of the ecm deserve more scrutiny to appreciate the impact on neuroinflammation, injury, and repair.   abstract 6 free neuropathol 6:1:9 an anatomic transcriptional atlas of focal cortical dysplasia ameesha paliwal1,2, kevin faust2, okty a. borhani2, parsa b. zadeh2, phedias diamandis1–4 department of laboratory medicine and pathobiology, university of toronto, toronto, on m5s 1a8, canada princess margaret cancer centre, 101 college street, toronto, on m5g 1l7, canada department of medical biophysics, university of toronto, 101 college st, toronto, on m5g 1l7, canada laboratory medicine program, department of pathology, university health network, 200 elizabeth street, toronto, on m5g 2c4, canada focal cortical dysplasia (fcd) is a severe neurodevelopmental malformation that manifests in medically refractory epilepsy. fcd presents with classic histological hallmarks, such as cytomegalic dysmorphic neurons, that are presumed to be key anatomical drivers of disease. the relatively low frequency and interspersed patterns of dysmorphic neurons within resected lesions has challenged traditional molecular characterization approaches and consequently, therapeutic options. therefore, the objective of this study is to systematically identify the key anatomical components of fcd in multiple tissue sections in order to perform molecular sequencing on dysmorphic neuron-enriched samples. to accomplish this, we leveraged unsupervised deep learning approaches to map the conventional pathological features of this disease for spatially-conserved rna-sequencing. using this approach, we present the highest resolution molecular catalog of fcd to date. our analysis has uncovered non-canonical signaling and neurotransmitter pathways in dysmorphic neurons that could serve as new targets for this debilitating disorder. specifically, we have found aberrant enrichment of an inhibitory glycinergic neurotransmission regulator, glycine transporter 2 (glyt2). immunohistochemical staining in eight additional cases of fcd revealed precise enrichment for glyt2 in dysmorphic neurons. glyt2 inhibitors have demonstrated efficacy in treating central nervous system disorders by reducing excessive excitatory signaling, making this transporter a compelling therapeutic approach for further research in fcd. importantly, we have generated a comprehensive resource for discovery in fcd, which reflects the key pathologic features of this disease including cytomegalic dysmorphic neurons, balloon cells, and rarefacted white matter. we anticipate that the research and resource produced here will improve outcomes in fcd.   abstract 7 free neuropathol 6:1:10 transcriptomic profiling reveals hippocampal white matter inflammation as potential epileptogenic focus in temporal lobe epilepsy xuan wang1, rober abdo1, carolyn twible1, chelsey zhao1, qi zhang1,2 department of pathology and lab medicine, western university, london, ontario, canada department of pathology and lab medicine, london health sciences center, london, ontario, canada rationale: hippocampal sclerosis (hs) is the most common pathological finding in surgically resected brain tissue in mesial temporal lobe epilepsy (mtle). the histopathologic hallmark of hs is pyramidal neuronal loss within the cornu ammonis (ca) sectors. twenty percent of tle cases have no significant neuronal loss (no-hs, ilae type 4). about 40 % of no-hs patients achieve seizure freedom post-operatively. objectives: we performed spatial transcriptomic profiling of no-hs hippocampi to identify molecular changes associated with post-operative outcomes. method: eight no-hs hippocampi were obtained from the department of pathology at lhsc. five patients had engel 1a outcome (seizure free, sf); three patients with engel outcome 2–4 (non-seizure free, nsf). six samples (3 sf and 3 nsf) were selected based on anatomical integrity and rna quality. spatial transcriptomic profiling was performed using 10x genomics visium technology. data analysis was conducted using the loupe browser and r/bioconductor packages to compare gene expression across various hippocampal regions. results: unsupervised data-driven clustering (bayesspace) correlates well with histology based manual annotation. two distinct spatial domains (cluster 7 and 8) were identified in hippocampal ca1 region. differential expression analysis identified upregulated neuroinflammation genes in the seizure free group, predominantly in the hippocampal white matter (e.g. stratum radiatum). trajectory analysis indicated disease spatial progression starting from hippocampal white matter, progressing to ca sectors, and reaching the dentate gyrus at the end. conclusions: our findings suggest molecular heterogeneity of the human ca1 sector. patient who had better post-surgical outcomes possess elevated neuroinflammatory signature in the hippocampal white matter.   abstract 8 free neuropathol 6:1:11 global proteomics reveals bidirectional integrin signaling as a driver of glioblastoma invasion rifat s. sajid1, lennart j. van winden2, nakita gopal1, evelyn r. kamski-hennekam2, okty abbasi borhani2, ameesha paliwal1, phedias diamandis1–4 department of laboratory medicine and pathobiology, university of toronto, toronto, canada princess margaret cancer centre, toronto, canada laboratory medicine program, university health network, toronto, canada department of medical biophysics, university of toronto, toronto, canada glioblastoma (gbm) is the most aggressive form of adult brain cancer with a median survival of 15 months despite multimodal treatment. a significant treatment challenge is the ability of tumour cells to infiltrate into surrounding brain tissue. recent studies have revealed an enrichment of hypoxia-associated signatures in invasive gbm cells and that tumour cells actively migrate away from hypoxic areas to distant brain regions. while these studies have advanced our understanding of gbm invasion, the significant heterogeneity of clinical tissue samples makes it difficult to prioritize migratory signaling pathways for therapeutic targeting. therefore, we utilized a diverse cohort of patient derived gbm stem cell (gsc) lines and experimental systems to stimulate more controlled invasion models and identify targets to impede tumour infiltration. to investigate these signaling pathways, we leveraged mass spectrometry-based proteomics to profile gscs cultured under varying oxygen concentrations. this analysis revealed the enrichment of integrin-related signaling pathways in hypoxia. next, we utilized the gsc and cerebral organoid (glico) co-culture model of gbm brain infiltration to investigate hypoxia-independent gbm invasion and discovered the upregulation of proteins involved in both outside-in and inside-out integrin signaling pathways. we then spatially correlated this increased expression in gscs through spatial transcriptomics. finally, we functionally evaluated the role of integrin signaling in gbm invasion through pharmacological inhibition in glicos, which resulted in significantly reduced gsc infiltration. therefore, this study highlights the pivotal role of bidirectional integrin signaling as a driver of gbm invasion, and further exploration of key regulators presents a promising therapeutic avenue.   abstract 9 free neuropathol 6:1:12 integration of molecular and morphometric features improves prognostication of diffuse gliomas dimitrios g. oreopoulos1,2, ameesha paliwal2, parsa babaei-zadeh2, alberto leon2, kevin faust2, phedias diamandis2–4 temerty centre for artificial intelligence research and education in medicine, university of toronto, toronto, on, canada department of pathology, princess margaret cancer centre, 101 college street, toronto, on, m5g 1l7, canada department of laboratory medicine and pathobiology, university of toronto, toronto, on, canada department of medical biophysics, university of toronto, toronto, on, canada background: diffuse gliomas are the most common forms of adult brain cancer, but exhibit substantial heterogeneity in outcomes. while much of the clinical variability has recently been objectively resolved by molecular profiling and subclassification of gliomas into isocitrate dehydrogenase (idh)-wildtype (glioblastoma) and idh-mutant tumors, the later with and without 1p19q chromosomal codeletions (oligodendroglioma, astrocytoma respectively), these subgroups still exhibit patient-level differences in progression that challenge precision management and care. hypothesis: we hypothesized that integrating artificial intelligence-derived tissue features with modern molecular subgrouping may resolve further variation of outcome within established glioma subtypes. scientific approach: to explore this hypothesis, we retrieved the clinical cohort of gliomas from the cancer genome atlas and used a publicly available tool we previously developed, pharaoh, (biorxiv 2024), to automatically extract and analyze morphometric features of all available cases (tcga-lgg, n = 722; tcga-gbm, n= 670). we systematically analyzed 160 different nuclear features and correlated the results with accompanying clinical pathological survival data in 3–5 relevant glioma subcohorts. results: this systematic analysis revealed morphometric features that track variations in nuclear staining and cellularity as strong predictors of aggressive disease and poorer outcomes in patients with grade 3 oligodendrogliomas; a notoriously clinical heterogenous glioma subgroup. importantly, this feature extraction resource is publicly available, allowing these results to be generalized to other cohorts. conclusions: systematic characterization and validation of morphometric features in additional cohorts and clinical practice may support the development of more advanced multimodal biomarkers that can better predict outcome in this aggressive and heterogeneous disease.   abstract 10 free neuropathol 6:1:13 a 13 year review of non-diffuse large b cell lymphomas of the central nervous system at a tertiary hospital: a case series study niloofar sina1, chris heyn2, zeina ghorab1, julia keith1 laboratory medicine and molecular diagnostics, sunnybrook health sciences centre, laboratory medicine and pathobiology, university of toronto, toronto, on, canada diagnostic imaging, sunnybrook health sciences centre, university of toronto, toronto, on, canada background: non-diffuse large b cell lymphomas (non-dlbcl) of the central nervous system (cns) are rare. the rarity of such involvement poses a diagnostic challenge for neuropathologists and increases the risk of misclassification, particularly in the absence of a preceding lymphoma diagnosis. patients and methods: in this retrospective study we reviewed the clinicopathologic and imaging characteristics of seventeen cases of non-dlbcl of the cns diagnosed from 2010 to 2022 by neuropathology at our tertiary hospital. results: the seventeen cases included eight primary lymphomas and nine secondary lymphomas of the cns, fifteen neurosurgical cases and two autopsies. the tumours were extra-axial in nine and intra-parenchymal in eight cases, thirteen cases were b cell origin (including nine small mature b cell lymphomas, three large b cell lymphomas and one pleomorphic posttransplant lymphoproliferative disorder) and four cases were t cell origin (including two peripheral t cell lymphoma not otherwise specified, one anaplastic t cell lymphoma alk negative and one case of disseminated mycosis fungoides). four distinct radiographic patterns were appreciated on central imaging review. conclusion: our study highlights the heterogeneity of non-dlbcl lymphomas of cns. this associated with their rarity and variable atypical features increases the risk of misdiagnosis. reports such as this foster consensus of the diagnostic and management strategies of these rare entities.   abstract 11 free neuropathol 6:1:14 cost-benefit analysis of fluorescence in situ hybridization testing in the diagnosis of idh-mutant 1p/19q-codeleted oligodendrogliomas jacob a. houpt1, robert r. hammond1 department of pathology and laboratory medicine, london health sciences centre, london, on, canada fluorescence in situ hybridization (fish) testing has long served as a diagnostic cornerstone in confirming codeletion of the 1p and 19q chromosome arms in idh-mutant 1p/19q-codeleted oligodendrogliomas; a test necessary to distinguish the entity from other diffuse adult-type gliomas. however, fish is both costly and time-consuming relative to other ancillary tests such as next generation sequencing (ngs), requiring expensive fluorescent probes, specialized microscopy suites, and manual evaluation/counting by dedicated medical lab technologists (mlts). as institutions such as london health sciences centre (lhsc) proceed to validate ngs copy number variation assessment in place of direct fish testing for the evaluation of 1p/19q-codeletion in gliomas, we reviewed all 1p/19q cytogenetic test results at our institution to better contextualize fish testing in terms of cost-benefit ratio (cbr). from 2010 to 2024, 700 cytogenetics cases reporting 1p/19q-codeletion were retrieved. of these, 171 were positive for codeletion of 1p/19q (24.4 %). repeat testing was requested in 25 cases (3.6 %) due to borderline results and/or increased clinical suspicion, yielding a different diagnosis in at least 3 cases. conservative estimates of the per case cost of fish testing (including facility/equipment, reagents, and mlt labour/time) total at least $1400 cad. while its cbr is comparable to other oncological investigations and can be less expensive than ngs on a per test basis, ngs is often ordered in conjunction to facilitate confirmation of idh mutations and therefore is poised to save considerable time and cost if all but borderline cases of 1p/19q-codeletion can be elucidated without undertaking cytogenetic evaluation via fish.   abstract 12 free neuropathol 6:1:15 infant-type hemispheric glioma is mostly relatively well-demarcated tumor and not diffusely infiltrative murad alturkustani1 department of pathology, king abdulaziz university, jeddah, saudi arabia background: ihg has been identified as a distinct entity with unique methylation patterns and gene fusions, presenting a better prognosis than other pediatric high-grade gliomas. the current who classification presents discrepancies regarding the tumor's circumscription, necessitating further investigation. methods: the children's brain tumor network dataset of 1029 samples was accessed, selecting tumors from patients aged 0–4 years, resulting in 191 gliomas, glioneuronal tumors, and ependymomas. methylation profiles were analyzed using the heidelberg v12.5 and v12.8 classifiers. cases were selected based on diagnoses of ihg, desmoplastic infantile astrocytoma / ganglioglioma (dia/g), or high-grade gliomas with available methylation class. morphological assessments focused on tumor circumscription, low-grade areas, glial differentiation, and high-grade features. results: the final cohort included 12 cases: 4 with an initial diagnosis of dia/g, and 8 with an initial diagnosis of high-grade astrocytoma (6 diagnosed as ihg). all ihg cases were infantile tumors, while dia/g cases were in older children. both ihg and dia/g were well-circumscribed, whereas the case with diffuse infiltration was classified as diffuse pediatric high-grade glioma. the 4 dia/g cases were heterogeneous, with only one showing the methylation profile of dig. one dia/g case was reclassified as ihg. the remaining six ihg cases were initially diagnosed as high-grade astrocytoma. conclusion: ihg is predominantly a well-circumscribed tumor, contrasting with the diffuse infiltration characteristic of other pediatric high-grade gliomas. recognizing these histological features, along with molecular profiling, is crucial for accurate diagnosis and management.   abstract 13 free neuropathol 6:1:16 features of central nervous system metastases by neoplasms of gynecological origin mimi zhang1, jacob a. houpt1, lee-cyn ang1 department of pathology and laboratory medicine, london health sciences centre, london, on, canada central nervous system involvement by metastasis from extra-axial malignancies represent the most common cns neoplasms in adults, with primary lung, breast, renal, and colorectal carcinomas and cutaneous melanomas representing the majority of such. cns metastases of gynecological origin are relatively rare – making up no more than 3 % of cases of cns metastases and occurring in as few as 0.6 % of gynecological cancer patients. all previous cases of cns metastases at london health sciences centre (lhsc) were screened for keywords related to ovarian, endometrial, and cervical origin. cases where another possible primary was speculated to be more likely or cases lacking a known or suspected primary gynecological malignancy (based on previous pathology or imaging investigations) were excluded. this yielded 6 and 15 cases of metastases compatible with ovarian and endometrial primaries, respectively. of the 6 ovarian cases, 3 (50 %) were associated with poorly-differentiated ovarian adenocarcinoma primaries, 2 with high-grade serous ovarian adenocarcinomas, and a single case of mixed clear-cell and serous ovarian adenocarcinoma in which the serous component predominated in the metastasis. within the 15 cases of uterine primaries, 4 were associated with endometrioid, 1 with serous, 1 with mixed, and 4 with poorly-differentiated adenocarcinomas. the remaining cases included a leiomyosarcoma and 4 carcinosarcomas, the latter of which featured a predominating epithelial component in 2 cases, dual components in a single case, and a rhabdomyosarcoma component in the final case. relative to other primary malignancies, poorly-differentiated primary carcinomas are over-represented in metastases from gynecological origin (both in ovarian and non-ovarian cases). copyright: © 2025 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. depletion of nuclear cytoophidia in alzheimer’s disease feel free to add comments by clicking these icons on the sidebar free neuropathology 6:8 (2025) original paper depletion of nuclear cytoophidia in alzheimer’s disease alyona ivanova 1 , david g. munoz 2 , john woulfe 3,4 university of toronto, toronto, ontario, canada department of laboratory medicine, st. michael’s hospital, unity health & department of laboratory medicine and pathobiology, university of toronto, toronto, ontario, canada department of pathology and laboratory medicine, university of ottawa, ottawa, ontario, canada ottawa hospital research institute, ottawa, ontario, canada corresponding author: john woulfe · department of pathology and laboratory medicine · university of ottawa · 501 smyth road · ottawa · ontario · canada jwoulfe@toh.ca additional resources and electronic supplementary material: supplementary table 1 submitted: 28 january 2025 accepted: 26 february 2025 copyedited by: vanessa s. goodwill published: 07 march 2025 https://doi.org/10.17879/freeneuropathology-2025-6282 keywords: alzheimer’s disease, cytoophidia, phosphoribosyl pyrophosphate synthetase, intranuclear rodlets, purines abstract there is considerable evidence for a role for metabolic dysregulation, including disordered purine nucleotide metabolism, in the pathogenesis of alzheimer’s disease (ad). purine nucleotide synthesis in the brain is regulated with high fidelity to co-ordinate supply with demand. the assembly of some purine biosynthetic enzymes into linear filamentous aggregates called “cytoophidia” (gk. cellular “snakes”) represents one post-translational mechanism to regulate enzyme activity. cytoophidia comprised of the nucleotide biosynthetic enzymes inosine monophosphate dehydrogenase (impdh) and phosphoribosyl pyrophosphate synthetase (prps) have been described in neuronal nuclei (nuclear cytoophidia; ncs). in light of the involvement of purine nucleotide dysmetabolism in ad, the rationale for this study was to determine whether there are disease-specific qualitative or quantitative alterations in prps cytoophidia in the ad brain. double fluorescence immunostaining for prps and the neuronal marker map2 was performed on tissue microarrays of cores of temporal cortex extracted from post-mortem tissue blocks from a large cohort of participants with neuropathologically confirmed ad, lewy body disease (lbd), progressive supranuclear palsy, and corticobasal degeneration, as well as age-matched cognitively unimpaired control participants. the latter group included individuals with substantial beta-amyloid deposition. ncs were significantly reduced in frequency in ad samples relative to those from controls, including those with a high beta-amyloid load, or participants with lbd or 4 repeat tauopathies. moreover, double staining for prps and hyperphosphorylated tau revealed evidence for an association between ncs and neurofibrillary tangles. the results of this study contribute to our understanding of metabolic contributions to ad pathogenesis and provide a novel avenue for future studies. moreover, because prps filamentation is responsive to a variety of drugs and metabolites, they may have implications for the development of biologically rational therapies. introduction alzheimer's disease (ad) is a devastating neurodegenerative disease characterized clinically by inexorable, progressive impairments in memory, judgement, and reasoning. the disease is characterized histopathologically by the buildup of beta-amyloid plaques and the presence of neurofibrillary tangles made up of phosphorylated tau protein. although genetic evidence supports a critical role for beta-amyloid in the pathogenesis of familial forms of ad, the pathogenetic mechanisms underlying the far more common sporadic form of ad remain enigmatic. there is growing evidence for a role for metabolic dysregulation in ad, including nucleotide dysmetabolism, at early stages of ad independent of neurofibrillary tangles and β-amyloid plaques [1, 3, 26, 29–31]. there is deregulated expression of a number of purine metabolic enzymes in ad as well as significantly altered levels of metabolites including dgmp, glycine, xanthosine, inosine diphosphate, guanine, and deoxyguanosine [3]. the brain is critically dependent on an uninterrupted supply of purine nucleotides (amp, adp, atp, gmp, gdp, gtp). they are assembled into rna and dna, provide energy for cells, are used as signalling molecules, and are incorporated into co-enzymes. accordingly, inborn errors of purine nucleotide metabolism markedly, and relatively selectively impact the nervous system, resulting in severe neurodevelopmental diseases [16, 27, 38, 42, 51, 52, 66]. the synthesis of purine nucleotides is carefully regulated to calibrate supply with cellular demand. in response to purine limitation or a variety of manipulations, enzymes involved in the de novo purine pathway are assembled by liquid-liquid phase separation into microscopically visible, supramolecular membraneless organelles comprised of nine enzymes called "purinosomes" [54]. by virtue of the physical proximity of their constituent enzymes, these "metabolon" structures more efficiently catalyze the generation of amp and gmp in response to metabolic demand [53]. in addition to purinosomes, a subset of nucleotide-synthesizing enzymes are subject to an additional level of supramolecular regulation; the formation of linear filamentous assemblies as a biological regulatory mechanism to co-ordinate enzyme activity with cellular requirements [5, 28, 41, 58]. the relationship between filament formation and enzyme activity is complex as both active and inactive multimeric conformers are present within individual filaments [28]. current consensus supports a model whereby filament formation correlates with increased enzyme activity by resisting feedback inhibition by enzyme products [28]. this adds nuance to purinosome function; permitting ongoing nucleotide synthesis under conditions of increased nucleotide demand, even when purines are plentiful. individual enzyme filaments can aggregate by side-to-side lateral alignment to form larger, mesoscale filament bundles, which have been variously referred to simply as "filaments", "rods and ring structures" or "cytoophidia" (greek for "cellular snakes"; reviewed in [12, 40]). the formation of these structures at steady state and in response to metabolic perturbations has been described in a variety of cell types both in vitro and in vivo [12]. these filament bundles have been described both in the cytoplasm and the nucleus of a variety of cell types, including mammalian neurons [50]. we recently demonstrated the presence of cytoophidia immunoreactive for inosine monophosphate dehydrogenase (impdh) within the nuclei of neurons in the human brain [63, 64]. impdh is the rate-limiting enzyme in the de novo purine biosynthesis pathway. it is encoded by two genes, impdh1 and impdh2 [48]. these nuclear cytoophidia (ncs) are also immunoreactive for the nucleotide-synthesizing enzyme phosphoribosyl pyrophosphate synthetase (prps) [50, 63]. prps is a key enzyme in the de novo purine biosynthesis pathway as it catalyses one of the first steps; the production of phosphoribosyl pyrophosphate (prpp). humans contain three isoforms of prps (prps1, 2, and 3). in light of evidence for the involvement of nucleotide metabolism in ad pathogenesis cited above and the important role of cytoophidia formation in tuning metabolic enzymatic activity, we were interested in determining whether there are disease-specific alterations in the formation of prps ncs in the ad brain. materials and methods participant selection subsequent to approval by the ottawa health science network research ethics board, tissue blocks of formalin-fixed, paraffin-embedded post-mortem human hippocampus and adjacent parahippocampal gyrus were obtained from the tissue archive in the division of anatomical pathology of the ottawa hospital. the cohort included 177 participants including 82 neurologically cognitively unimpaired controls, 30 with advanced ad, 17 with lbd (11 diffuse neocortical, 4 limbic/transitional, 2 brainstem), 14 with primary tauopathy (11 with progressive supranuclear palsy (psp) and 3 with corticobasal degeneration (cbd)), and an additional 6 cognitively intact participants with significant beta-amyloid deposition. demographic details regarding the participant cohort as well as the raw data are provided in supplementary table 1. also included in the table are data indicating the presence or absence of phosphorylated tau (ptau) neurofibrillary pathology and beta-amyloid plaques in each tissue core. subjects were included based on retrospective review of post-mortem neuropathology reports. all post-mortem neuropathological diagnoses were performed by a neuropathologist (jw) according to current diagnostic and staging criteria [4, 18, 36, 47]. specifically, ad neuropathologic change (adnc) was diagnosed based on the combined analysis of cortical beta amyloid burden according to the thal staging scheme [60], braak neurofibrillary stage [9], and cortical neuritic plaque density following consortium to establish a registry for alzheimer’s disease (cerad) criteria [45] to arrive at an "abc" score as per the national institute on aging-alzheimer's association guidelines for the neuropathologic assessment of alzheimer's disease [47] (see supplementary table 1). a proportion of cases were diagnosed prior to publication of these guidelines and only those receiving a diagnosis of "severe" ad or a braak score of v or vi were included among the ad cohort. a proportion of cognitively normal controls were assessed for adnc and only those with "mild" changes were included. tissue microarray preparation and immunostaining from each of the paraffin blocks of hippocampus and mesial temporal cortex, tissue microarrays (tmas) were prepared using 2 mm punches centered on the temporal cortex at the depth of the collateral sulcus. this area was selected because it consistently displays ncs in control brains and because it was the area we analysed in our previous study [65]. tmas were sectioned at a thickness of 5 μm and mounted onto coated slides. sections were deparaffinized and pre-treated using heat mediated antigen retrieval with edta buffer (ph 9.0). slides were then rehydrated in 1x tbst buffer and blocked for 30 min with sniper (#biocare bs966l). sections were stained using dual labelling immunofluorescence by incubating overnight at 4 degrees in a cocktail of antibodies against prps (rabbit polyclonal; 1:100, proteintech 15549-1-ap) and the neuronal marker microtubule-associated protein 2 (map2; mouse monoclonal; clone b-8; santa cruz biotechnology sc-74420) or ptau (mouse monoclonal; 1:100, thermofisher #mn1020). sections were washed with 1x tbst and then incubated with goat anti-mouse 594 (#a11005, invitrogen) and donkey anti-rabbit 488 (#a21206) secondary antibodies for 2 h in the dark at room temperature. this was followed by incubation with a quencher (vector trueview autofluorescence quenching kit #sp-8400, vector labs) to decrease autofluorescence. sections were washed, incubated with 5 ug/ml of dapi (thermoscientific #62248) and coverslipped. image and data analysis images were acquired using a zeiss axioimager m2 microscope. to quantify ncs, prps/map2 stained tmas were scanned using a zeiss m1 slide scanner. image analysis was performed on digital images using highplex fl module in halo software as well as zeiss zen blue software. the number of map2-immunoreactive cells containing ncs in each core was counted manually. cases with less than 10 neurons per core were eliminated. all statistical analyses for multiple group comparisons were conducted using one-way anova followed by bonferroni’s multiple comparisons test at α = 0.05 significance. percentage of ncs present in aggregate with nfts was determined by manual count. chi-square test at α = 0.05 significance was used to determine whether there was any association between the presence of ncs and nfts. to assess beta-amyloid pathology in each core, the tmas were stained using immunohistochemistry for the detection of beta-amyloid (rabbit polyclonal anti-beta-amyloid; 1:400, novus nbp2-13075). staining was performed using the leica bond™ system. tmas were pre-treated using heat mediated antigen retrieval with edta buffer (ph 9.0, epitope retrieval solution 2) for 20 minutes. the tmas were then incubated in primary antibody for 30 minutes at room temperature and detected using an hrp conjugated compact polymer system. slides were then stained using dab as the chromogen, counterstained with hematoxylin, mounted and coverslipped. results prps ncs are present in the brain figure 1 shows prps-immunoreactive ncs in double immunostained sections of the punches of temporal cortex in the depth of the collateral sulcus. in both control and disease cases, these were most prevalent in layers ii, v, and vi. they were present in both pyramidal neurons as well as non-pyramidal neurons of various morphologies. they showed a variety of shapes, ranging from rodor thread-like linear or curvilinear structures (fig. 1a) to dot-shaped structures or large, irregular "crystalloid" sheets (fig. 1b). some of the latter showed a rhomboid shape as reported for impdh ncs in substantia nigra neurons [64]. in a proportion of neurons, the nucleolus showed intense staining for prps (fig. 1c). this was seen both in neurons with and without ncs. in addition to ncs, most neurons showed innumerable micron-sized curvilinear cytoplasmic threads filling the perikaryal and neuritic cytoplasm and contributing to a punctate staining pattern of the background neuropil (fig. 1d). fig. 1 dual-labelling immunofluorescence for prps (green) and map2 (red) in a core of temporal cortex from a control subject. a, b) neurons containing linear or curvilinear (a) or irregular sheet-like (b) ncs. c) prps staining of the nucleoli in two neurons. in both cases, there are small ncs juxtaposed to the nucleoli (arrowheads). d) higher magnification of the proximal apical dendrite of the pyramidal neuron in b showing small curvilinear cytoplasmic prps filaments within the dendrite and dot-like structures throughout the surrounding neuropil. scale bars = 20 microns. prps ncs are reduced in the ad temporal cortex the box plot in fig. 2 shows the results of our quantitative analysis. there was significant inter-individual variability in the density of ncs. quantitative analysis confirmed a significant depletion of ncs in participants with ad relative to cognitively intact age-matched controls, including those with heavy beta-amyloid deposition, as well as those with lbd and 4r tauopathies (psp/cbd) (anova p < 0.02, see figure 2 for pairwise comparisons). patients with lbd and 4-repeat tauopathies did not differ from controls (p = 0.2749 and 0.9897, respectively). the presence of heavy beta-amyloid deposition in cognitively normal individuals did not impact nc density relative to the control population (p = 0.9322). there were no discernible qualitative differences in the appearance or morphology of ncs between ad participants, any other disease groups, and controls. fig. 2 box plot comparing frequency of ncs in cores of temporal cortex from our cohort. anova for the entire group p < 0.02. pairwise comparisons conducted using bonferroni’s multiple comparisons test. comparisons reaching the significance level (p < 0.05) are shown on the graph. all other comparisons did not reach this level. ad; alzheimer's disease, amyloid; cognitively normal subjects with high density of amyloid plaques, lbd; lewy body disease, psp/cbd; progressive supranuclear palsy/corticobasal degeneration. ****p < 0.0001, *p < 0.05., **p < 0.005. prps ncs are more common in nft-bearing neurons we explored a possible relationship between ncs and neurofibrillary tangles (nfts) using double immunofluorescence staining of the tmas for prps and ptau (fig. 3a–c). a significantly higher proportion of nft-bearing neurons contained ncs relative to nft-negative neurons, suggesting an association between these two proteinaceous bodies. specifically, of 64 nft-containing neurons, 9 (14 %) contained ncs. conversely, of 4,977 neurons lacking nfts, only 145 (2.9 %) contained ncs (fig. 3d; chi-squared p < 0.00001). fig. 3 a–c) dual labelling immunofluorescence for prps (green) and ptau (red) showing curvilinear (a), thread-like (b) and dot-like (c) ncs in tangle-bearing neurons. scale bars = 10 microns. d) bar graph showing percentage of nft-positive and nft-negative neurons with (blue) and without (white) ncs. chi-square test shows a significantly higher proportion of nft bearing neurons contain ncs (p < 0.00001). discussion here we demonstrate a significant depletion of intranuclear cytoophidia comprised of the key nucleotide-synthesizing enzyme prps from neurons in the ad cerebral cortex, implicating these enigmatic structures in the pathogenesis of ad. this study confirms and extends the results of our previous study demonstrating a significant decrement in the density of intranuclear rodlets (inrs) in the temporal cortex of individuals with ad relative to cognitively intact controls and those with dementia with lewy bodies [65]. these structures correspond to the "rodlets of roncoroni" described by the classical microscopists (see [63]). the functional significance of these structures has remained enigmatic. the demonstration that they are immunoreactive for the nucleotide synthesizing enzymes prps provides a novel functional context; linking them to the bundles of metabolic enzyme filaments known as "cytoophidia" recently described by several authors (for a review, see [11]). interestingly, we have demonstrated that the prps ncs described in this study are also immunoreactive for the rate limiting enzyme in purine nucleotide synthesis, impdh [63]. co-localization of metabolic enzymes in cytoophidia is common. for example, a similar interaction between impdh and the pyrimidine-synthesizing enzyme cytidine triphosphate synthase (ctps) in cytoophidia has been implicated as a substrate to co-ordinate the cross-regulation of purine and pyrimidine nucleotide synthesis [14]. in a screen of 440 yeast metabolic enzymes, noree et al. showed the assembly of multiple metabolic enzymes into rod-shaped structures [50]. interestingly, enzymes that co-assembled, like impdh and prps, were those that acted at branch points within the de novo purine biosynthetic pathway, further suggesting that this co-localization reflects some form of regulatory mechanism in the nucleotide biosynthetic pathway. the majority of mechanistic studies have linked cytoophidia formation to the dynamic regulation of enzyme activity and thus, in the case of prps (with impdh), to the synthesis of purine nucleotides. purine nucleotides are essential for the proper development and maintenance of the brain. guanine-based purines, and especially gtp and its breakdown product guanosine, have neuromodulatory properties (reviewed in [59]) with neuroprotective, neurotrophic, and neuritogenic capacity [23, 24, 49, 59]. these are mediated through intracellular modulation of g-proteins as well as via extracellular signaling by purines themselves [59]. gtp plays a critical role in tubulin polymerization for microtubule growth [13] and is a substrate for the multitude of small gtpases involved in neurite growth and synaptogenesis [19, 61]. our findings are consistent with evidence for the involvement of nucleotide-synthesizing enzymes, and possibly their filamentation, in neurological diseases [27]. loss-of-function mutations in prps1 (encoded on the x-chromosome), culminate in a constellation of neurodevelopmental symptoms in males referred to eponymously as arts syndrome and encompassing sensorineural hearing loss, hypotonia, ataxia, developmental delay, and intellectual disability [16, 17]. similarly, mutations in impdh2 result in variable combinations of intellectual disability, speech impairment, hand tremor, gait instability, and dystonia [51, 52, 66]. why mutations in these enzymes have a relatively selective impact on the nervous system despite their widespread systemic expression has not been adequately explained. the relatively selective localization of ncs to the brain despite the widespread expression of their constituent enzymes is provocative in this regard. intriguingly, a proportion of the disease-causing mutations in both prps1, as well as impdh alter filament assembly [25, 52], suggesting a beneficial role for filament formation. consistent with this, flores-mendez and co-workers recently demonstrated a neuroprotective role for impdh2 filament formation in neurodegeneration [21]. understanding the mechanisms through which ncs might contribute to ad pathogenesis is predicated on elucidating their physiological significance in neurons, which is currently unknown. a canonical role for prps filaments in regulating enzyme activity makes sense in the cytoplasmic compartment where nucleotides are synthesized. indeed, we postulate that the small cytoplasmic filaments we describe herein are engaged in this, perhaps in association with mitochondria (see [64]), by analogy with purinosomes [22]. on the other hand, the functional significance of prps1 filament assemblies within the nuclear compartment of neurons remains to be explored. there is evidence that prps, as well as impdh, can localize to the nucleus [32, 37]. we have described the presence of impdh ncs within the nuclei of substantia nigra neurons where they display marked age-associated morphological alterations [64]. prps1-immunoreactive rods have also been described in the nucleus of neurons in vitro [50] and in vivo [63]. this is consistent with evidence for a nuclear localization and function of a growing number of metabolic enzymes [7]. we propose two non-mutually exclusive possibilities regarding the significance of prps/impdh rods in the nuclear compartment. first, it is conceivable that ncs regulate the synthesis of purines within the nucleus, providing a proximate, "hyperlocal" source of nucleotides at loci of high nucleotide demand. indeed, previous reports have shown that impdh localization is responsive to gtp demand and that this localization enhances de novo gtp synthesis [2, 6, 15, 33, 62, 67]. this model might explain the common spatial association of ncs with the nucleolus which consumes a vast quantity of purine nucleotides for the synthesis of rrna [34, 35]. by analogy, there is evidence that acetyl-coa, essential for histone acetylation, is synthesized in the nucleus by the metabolic enzymes atp-citrate lyase (acly), acyl-coa synthetase short chain family member 2 (acss2) and the pyruvate dehydrogenase complex (pdc) [8]. it has been hypothesized that these enzymes localize to nuclear foci and generate acetyl coa at sites of histone acetyltransferase activity. a role for ncs in the generation of gtp in the nuclear compartment is predicated on the local presence of downstream enzymes in this pathway to facilitate metabolic channeling. indeed, gmps, which generates gmp from impdh-generated xmp, has been described in the nucleus [56]. alternatively, there is increasing evidence that metabolic enzymes engage in moonlighting, non-enzymatic activities in the nucleus [8]. for example, in saccharomyces cerevesiae, the nuclear localization of prps isoforms has been implicated in the maintenance of cell wall integrity [57]. in drosophila, impdh enters the nucleus in response to oxidative and/or replicative stress, binds single-stranded ct-rich regulatory dna elements and functions as a transcriptional repressor [37]. in breast cancer cells, chromatin-bound impdh interacts with parp1 and has been implicated in dna repair by regulating the availability of nad+ [20]. it is tempting to speculate that neurons exploit the capacity of prps and impdh to form filaments as a mechanism to regulate the nucleoplasmic availability of these enzymes to perform these moonlighting functions. according to the beta-amyloid hypothesis of ad pathogenesis, the natural history of ad is characterized by a protracted prodromal period, lasting at least a decade, during which beta amyloid accumulates in cortical plaques without overt physiological or cognitive sequelae. at some point, amyloid is thought to catalyze the expansion of neuronal tau pathology from the basal temporal cortex, (where nfts remain confined in the normal elderly), to the neocortex, and trigger the development of dementia. our study demonstrates no definite relationship between nc loss and beta-amyloid load. the dissociation of nc loss from beta amyloid deposition is consistent with evidence that nucleotide dysmetabolism in the ad brain occurs independently of ß-amyloid plaques [1, 3, 26, 29–31]. on the other hand, our results suggest that nc loss occurs subsequent to beta-amyloid deposition, and, based on the amyloid hypothesis, at the stage of nft expansion. this is a critical phase of the ad pathogenetic cascade as nft formation correlates best with cognitive decline. notably, despite a global loss of ncs from the ad brain, our results indicate that remaining ncs tend to associate with nft-bearing neurons. this apparent contradiction may relate to the fact that nft-bearing neurons comprise a relatively small proportion of the total neuronal population in the ad cortex (1.28 % in our cohort). thus, assuming that nc loss is independent of beta-amyloid and tau deposition, the net quantitative effect is a global loss of ncs, despite their preferential localization to nft-bearing neurons. why remaining ncs tend to form in nft-bearing neurons remains to be explored. it is tempting to speculate that nft formation imposes a metabolic burden on the neuron culminating in nc formation. this would be consistent with our previous study demonstrating increased intranuclear rods in the substantia nigra in response to the neurotoxin mptp [39]. conversely, it is possible that the metabolic status of the neuron which results in nc formation predisposes neurons to tau hyperphosphorylation and nft pathology. elucidating the mechanistic substrates underlying this association may lead to novel pathogenetic concepts and therapeutic targets. our results may also have disease implications in a broader context. for example, ncs were not significantly reduced in frequency in the cortex of lbd subjects. this supports the status of nc depletion as a disease-specific biomarker for ad as opposed to lbd. in addition, we have demonstrated the presence of inrs in insulin-secreting pancreatic beta cells [55]. like ad, we have demonstrated a significant depletion of inrs from beta cells in human type 2 diabetes [68] as well as in animal models of diabetes [44]. this is intriguing in light of evidence for: 1) a shared pathogenetic mechanism underlying ad and type 2 diabetes [46] and 2) an important role for purine nucleotide metabolism in beta cell function and survival and in diabetes [43]. moreover, we have demonstrated evidence for an inverse correlation between cellular secretory activity and inr formation [63]. notably, beta-cell and neuronal hyperactivity are hallmarks of diabetes and ad [10], providing an additional, or alternative unifying explanation for nc depletion in the two diseases. nc depletion may represent a morphological signpost indicating a common metabolic substrate contributing to the pathogenesis of these two related disorders of ageing. our study has some limitations. participant recruitment was based on retrospective review of neuropathology reports. a small proportion of the ad cases were diagnosed prior to the currently recommended diagnostic criteria [47], precluding assignment of an "abc" score. only cases with "severe" adnc or a braak score of v or vi were included in the ad cohort. the objective of this study was to confirm our previous findings in the context of new knowledge regarding the possible functional significance of, and novel marker for, these intranuclear structures. consequently, the comparison of subjects with only "high" adnc with controls is dichotomous. future studies will provide a more granular understanding of the role of ncs in the ad pathogenic cascade by analyzing their status at "low" and "intermediate" adnc stages. this is being pursued in mouse studies as well. there is increasing recognition that tdp-43 proteinopathy in the form of limbic-predominant age-related tdp-43 encephalopathy (late) is a common co-pathology in adnc. we did not examine the impact of this co-pathology on nc status in the present study. n summary, we have demonstrated: 1) that neuronal prps ncs are reduced in frequency in the ad temporal cortex and 2) that remaining ncs show an association with nft-containing neurons (fig. 4). the results of this study provide an unexplored avenue for investigation with respect to ad pathogenesis. the potential involvement of prps ncs in the cellular mechanisms underlying ad-associated neurodegeneration is consistent with evidence for metabolic dysfunction in ad. future mechanistic studies addressing the functional significance of prps ncs will elucidate their role in healthy neurons and in disease. if these confirm that intranuclear filamentation of these metabolic enzymes plays a role in neuronal health and metabolism, as has been suggested, its failure in ad may provide a novel, biologically rational therapeutic target. fig. 4 schematic summary of the results of the present study. neuronal ncs (green) are reduced in frequency in the brains of ad participants. remaining ncs are relatively more frequent in nft (red)-bearing neurons. declarations author contributions conceptualization: john woulfe, david munoz; methodology: john woulfe, alyona ivanova; formal analysis and investigation: john woulfe, alyona ivanova; writing-original draft preparation: john woulfe; writing-review and editing: david munoz, alyona ivanova; resources: john woulfe, david munoz; supervision: john woulfe, david munoz. funding statement no funding was received for conducting this study. conflict of interest statement the authors have no relevant financial or non-financial interests to disclose. ethics approval this retrospective post-mortem study was conducted in accordance with the ethical standards of the ottawa hospital. data availability the data is available from the corresponding author upon reasonable request. raw data is available in supplementary table 1. acknowledgements the authors would like 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https://doi.org/10.1016/s1474-4422(20)30312-4 67. zhao h, chiaro cr, zhang l, smith pb, chan cy, pedley am, pugh rj, french jb, patterson ad, benkovic sj (2015) quantitative analysis of purine nucleotides indicates that purinosomes increase de novo purine biosynthesis. j biol chem 290: 6705-6713. https://doi.org/10.1074/jbc.m114.628701 68. zhou yy, el hallani s, balaa f, mohammad w, gray da, woulfe j (2017) depletion of beta cell intranuclear rodlets in human type ii diabetes. endocr pathol 28: 282-286. https://doi.org/10.1007/s12022-017-9499-y copyright: © 2025 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license ( https://creativecommons.org/licenses/by/4.0/ ), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver ( https://creativecommons.org/publicdomain/zero/1.0/ ) applies to the data made available in this article, unless otherwise stated. 66th meeting of the french society of neuropathology meeting abstracts , december 6th. 2024 feel free to add comments by clicking these icons on the sidebar free neuropathology 6:2 (2025) meeting abstracts 66th meeting of the french society of neuropathology meeting abstracts december 6th, 2024 hôpital pitié salpêtrière, amphithéâtre charcot the french society of neuropathology was created in 1989, succeeding the french club of neuropathology set up in 1965.   submitted: 04 december 2024 accepted: 05 december 2024 published: 29 january 2025 https://doi.org/10.17879/freeneuropathology-2025-6285 keywords: french society of neuropathology, sfnp, meeting abstracts, 66th meeting dec. 2024   free neuropathol 6:2:2 cerebrospinal fluid processing in cytology and pathology laboratories: changes of practice plu i1,2, tran s1, boluda s1,2, privat n2, seilhean d1,2 département de neuropathologie, hôpital pitié salpêtrière, gh.ap-hp sorbonne université, paris, france sorbonne université, brain institute, inserm, umrs 1127; cnrs, umr 7225, paris, france the handling and analysis of cerebrospinal fluid (csf) in cytology and pathology laboratories require safety measures, due to the potential risk of infection. the 2004 french safety regulations recommended that the csf of patients suspected of having creutzfeldt-jakob disease was subject to maximum precautions, generally implemented in specialized laboratories. since 2020, following accidental occupational contamination in research laboratories, new procedures are being developed. in november 2021, the uk’s advisory committee for dangerous pathogens revised its guidance and considers csf to be a low-risk biofluid, allowing it to be handled with only general laboratory hygiene and safety rules. based on uk guidelines, we have updated our csf handling procedure with a single circuit applicable to all clinical situations and achievable in all laboratories, bearing in mind that any patient who develops cognitive disorders, suspected of suffering from a neurodegenerative disease, must be considered as being at risk of prion disease.   free neuropathol 6:2:3 neuropathological analysis of an als patient carrying a sod1 missense mutation and a c9orf72 repeat expansion miki tomoko1, de bertier sibylle2, deret marion2, amador maria-del-mar2–4, teyssou elisa2, muratet françois2, bohl delphine2, lobsiger christian2, boillée séverine2, salachas françois2–4, millecamps stéphanie2, seilhean danielle1,2,4 department of neuropathology, pitié-salpêtrière hospital, assistance publique hôpitaux de paris (aphp), sorbonne university, paris, france institut du cerveau ‐ paris brain institute ‐ icm, inserm, cnrs, aphp, sorbonne university, pitié salpêtrière hospital, paris, france department of neurology, centre de référence sla ile de france, pitié-salpêtrière hospital, aphp, paris, france dmu de neurosciences, paris, france mutations in the sod1 and c9orf72 genes are responsible for around 10% of apparently sporadic cases of amyotrophic lateral sclerosis (als). we report the case of a-63-year-old man, with no family history of als, carrying both a p.thr55ile mutation in the sod1 gene and a c9orf72 repeat expansion. he died 15 months after developing gait disturbances. post-mortem examination confirmed typical features of als, including severe loss of spinal and medullar motor neurons. cerebral atrophy was mild, with preservation of betz cells. in addition to the widespread tdp-43-positive neuronal cytoplasmic inclusions (nci), p62-positive tdp-43-negative numerous nci were found in the cerebellar granule cells. abundant poly-ga-positive inclusions were observed in the cerebellum, neocortex and hippocampus. this neuropathology was typical of c9orf72 repeat expansion. sod1 immunochemistry showed axonal-sprouting but no nci in motor neurons. gene therapy targeting sod1 may not be beneficial to such sod1 patients deprived of typical sod1-positive nci.   free neuropathol 6:2:4 polyradiculoneuritis with central nervous system involvement: contribution of unbiased metagenomics to the diagnosis boluda s1, plu i1, miki t1, megarbane b2, malissin i2, ferroni a3, jamet a3, fourgeaud j3, pérot p4, regnault b4, dheilly n4, seilhean d1 department of neuropathology, pitié-salpêtrière hospital, aphp-sorbonne university, paris, france intensive care unit, lariboisière hospital, aphpparis cité university, paris, france department of microbiology, necker hospital, aphp-paris cité university, paris, france pathogen discovery, pasteur institute, paris, france a 69-year-old male died of rapidly progressing polyradiculoneuritis involving the central nervous system. symptoms had begun three weeks earlier, with asthenia, diarrhea and weight loss. post mortem examination revealed cerebral edema, soft consistency of the cerebellum, necrosis of the midbrain and atrophy of the gray matter of the spinal cord. microscopic evaluation revealed necrosis in the anterior horns, the cerebellum, brainstem and temporal lobe, associated with severe neuronal loss. in the cerebellum, marked neuronal loss was observed in the purkinje cell layer and in the dentate nucleus. microglial activation associated with t-lymphocyte (cd3+) infiltration with foci of neuronophagy and microglial nodules were observed in the most affected regions. in peripheral nerves, perivascular inflammatory infiltrates were seen in the endoneurium and perineurium. no cytopathogenic effects were observed. anti-rabies antibodies were negative. unbiased metagenomic analysis revealed the presence of a virus previously unknown in human encephalitis.   free neuropathol 6:2:5 cytoskeleton perturbation in a large spastic ataxia family jean-loup méreaux1,*, mariacristina capizzi1,*, marie coutelier1, claire-sophie davoine1, susana boluda1,2, léna guillot-noël1, sabrina leclere-turbant1, franck letournel3, philippe codron3, badreddine mohand oumoussa4, hélène madry4, ludmila jornea1, giovanni stevanin5, marie-lorraine monin6, sandrine humbert1, alexis brice1, danielle seilhean1,2, alexandra durr1,4 sorbonne université, institut du cerveau paris brain instituteicm, inserm, cnrs, aphp, university hospital pitié-salpêtrière, paris, france department of neuropathology, aphp. sorbonne university, paris, france centre de référence sur la sla d'angers, centre hospitalier universitaire d'angers, angers, france; laboratoire de neurobiologie et neuropathologie, centre hospitalier universitaire d'angers, angers, france; university of angers, inserm, cnrs, mitovasc, sfr icat, angers, france sorbonne université, inserm, ums production et analyse des données en sciences de la vie et en santé, pass, plateforme post-génomique de la pitié-salpêtrière, paris, france bordeaux university (université de bordeaux), equipe « neurogénétique translationnelle nrgen », incia cnrs umr5287, ephe, 33000, bordeaux, france centre de reference maladies rares « neurogénétique », service de génétique médicale, bordeaux university hospital (chu bordeaux), 33000, bordeaux, france * these authors share senior authorship hereditary spastic ataxias are a clinically diverse group of neurodegenerative disorders, primarily characterized by spasticity in the lower limbs and generalized ataxia. here, we report an extensive neuropathological and functional study on a large family affected by autosomal dominant spastic ataxia. the disease manifests around the age of 40, beginning with intermittent dystonia in the lower limbs, followed by severe, disabling cerebellar ataxia and spasticity. the condition progresses rapidly, leading to death within 15 years of the onset age. postmortem neuropathological analysis revealed purkinje cell loss, motor tract atrophy, and abnormalities in the motile cilia at the ependymal border. despite these findings, the underlying molecular mechanisms remain unclear. through a combination of transcriptomic and cellular analyses, we identified significant involvement of cytoskeletal pathways involved in the organization of actin and microtubule networks, which must be related to the molecular cause of the disease.   free neuropathol 6:2:6 rna quality in postmortem brain tissue. the neuro-ceb experience. nasr o1, leclère s2, plu i1, maraña s3, seilhean d1, boluda s1 department of neuropathology, pitié-salpêtrière hospital, aphp-sorbonne university, paris, france neuro-ceb brain bank, aphp-sorbonne university, paris, france paris brain institute, icm, cnrs umr7225 – inserm u1127 – upmc, paris, france molecular research is often carried out on postmortem brain tissue, whose usability must be guaranteed. brain ph is a good indicator of rna quality (rin). however, the procedure is time consuming. ogata et al (1986) showed that the severity of autolysis of the cerebellar granular layer (acgl) correlates significantly with brain ph and rin1. our aim was: a) to evaluate this correlation within the neuro-ceb brain bank cohort, b) to extend the study to frequently requested brain regions other than the cerebellum. we analyzed the rin in six different brain regions (cerebellum, frontal cortex, temporal cortex, hippocampus, caudate nucleus and substantia nigra) from 20 neuro-ceb postmortem cases: acgl grade 0 (n=10) and acgl grade 4 (n=10). our results indicate: a) acgl tend to correlate with the rin in the cerebellum, but not in the other brain regions,b) there is a different susceptibility to rna degradation in distinct brain regions.   free neuropathol 6:2:7–8 ai-driven digital pathology for precision medicine in primary cns lymphoma noemie barillot1, isaias hernández verdin1, lucas rincón de la rosa1, roser velasco2, fanny drieux3, elena-liana veresezan3, bertrand mathon4, eva kirasic1, noemí vidal5, eva gonzález-barca6, fina climent esteller7, patricia lópez8, yah-se abada4, michael heming9, gerd meyer zu hörste9, olivier grauer10, magali le garff-tavernier11, frédéric davi12, albert pons-escoda13,lucia nichelli4,sylvain choquet4,fabrice jardin14,caroline houillier4,karima mokhtari15, khe hoang-xuan16, agusti alentorn1 paris brain institute, paris, france neuro-oncology unit, department of neurology, hospital universitari de bellvitge institut català d'onco-logia (ico), barcelona, spain centre henri becquerel, rouen, france groupe hospitalier pitié salpêtrière, paris, france pathology department, hospital universitari de bellvitge, barcelona, spain institut català d'oncologia (ico) institut d'investigació biomèdica de bellvitge (idibell), barcelona, spain hospital universitari de bellvitge, barcelona, spain clinical hematology department, institut català d'oncologia (ico), barcelona, spain department of neurology with institute of translational neurology, university hospital münster, germany department of neurology with institute of translational neurology, university hospital münster, germany centre de recherche des cordeliers, cell death and drug resistance in lymphoproliferative disorders team, sorbonne université, department of biological haematology, ap-hp, pitiésalp, paris, france hematology department, hôpital de la pitié-salpêtrière, ap-hp, pitiésalp, paris, france radiology department, institut de diagnòstic per laimatge (idi), hospital universitari de bellvitge, barce-lona, spain centre henri bequerel, rouen, france neuropathology department, assistance publique-hôpitaux de paris, universitary hospital la pitié salpê-trière-charles foix, and sorbonne universités, paris, france sorbonne universités, upmc univ paris 06, umr s 1127, paris, france primary central nervous system lymphoma (pcnsl) is an aggressive subtype of large b-cell lymphoma (lbcl) with poor prognosis. we developed a novel digital pathology approach to classify pcnsl, detect myd88 l265p mutations, and predict outcomes using hematoxylin-eosin (h&e) and immunohistochemistry (ihc) samples. utilizing datasets from over 400 patients, our deep learning models achieved a median auroc of 0.9 [iqr 0.85–0.93] in categorizing pcnsl molecular clusters. spatial transcriptomics and single-cell rna sequencing revealed significant intra-tumoral heterogeneity. additionally, partial least squares (pls) cox models integrating cell radiomics and clinical features achieved a c-index above 0.9 across datasets. remarkably, molecular group classifications derived from multi-omics data were accurately replicated using h&e samples alone. these findings demonstrate the potential of artificial intelligence to streamline the clinical implementation of precision medicine for lbcl and pcnsl, enabling more effective and tailored therapeutic strategies.   free neuropathol 6:2:9–10 disease-defining catalytically inactive protein kinase c alpha mutation is a driver in chordoid glioma by pathway rewiring charlotte bellamy1,$, hannah tovell2,$, selina schwaighofer3, timothy r. baffi2,4,*, janan arslan1, quentin letourneur5, florent dingli6, damarys loew6, alexandr kornev2,**, julie lerond1, tiffany kao2,4, stephane liva7, brigitte izac8, muriel andrieu8, homa adle-biassette9, jean-vianney barnier10, eduard stefan3, marc sanson1,11,&, alexandra c. newton2, franck bielle1,12 paris brain institute, paris, france cruk scotland institute, glasgow, scotland, united kingdom leopold-franzens-universität innsbruck, innsbruck, tirol, austria department of pharmacology, university of california at san diego, california, usa sorbonne université, ap-hp, siric curamus, paris, france institut curie, psl research university, centre de recherche, curiecoretech spectrométrie de masse protéomique, paris, france institut curie, service bioinformatique, paris, france institut cochin, paris, france service d’anatomie et cytologie pathologiques, faculté de médecine, université paris diderot, paris, france institut neuropsi umr9197,cnrs université paris-saclay, saclay, france sorbonne université, paris, france centre de recherche de l'institut du cerveau et de la moelle epinière, sorbonne université, paris, france chordoid glioma (chg) is a rare low-grade brain tumor, characterized by a novel recurrent point mutation, d463h, in the kinase domain of protein kinase c alpha (pkcα). the mutation is always at this position and always to his suggesting it endows a unique function beyond catalytic inactivation associated with other cancer-associated mutations in pkcα. here we use in vitro and in cellulo activity assays to show that d463h is not only catalytically inactive but is dominant negative over endogenous pkcs and has a rewired interactome. specifically, phosphoproteomic, proximity-driven biotinylation, and co-immunoprecipitation mass spectrometry data from hek293 cells overexpressing pkcαd463h identify altered phosphorylation of substrates and binding to multiple proteins involved in cell-cell junctions that wt enzyme does not interact with. lastly, single nuclei rnaseq reveals that chgs derive from specialized tanycytes. our data suggest that this fully penetrant mutation promotes aberrant interaction with unnatural partners to impair cell junction function.   free neuropathol 6:2:11–12 deciphering genetic and epigenetic of high grade gliomas with braf activating alteration is supporting the discovery of a new intracranial mpnst braf positive tumor type benoît lhermitte1,2*, thibaut wolf1,2*, marlene deschuyter2, damien reita2,4, chinar salmanli2, agathe chammas5, marie-pierre chenard1,3, guillaume gauchotte6, stephanie lacomme7, julien todeschi8, andres coca8, fabien rech9, romain appay10, roland schott11, georges noel12, sophie martin2, natacha entz-werlé2,13 department of pathology, university hospitals of strasbourg, avenue molière, 67098 strasbourg cedex, france umr cnrs 7021, laboratory bioimaging and pathologies, team onko-3t – translational, transversal and therapeutic oncology, university of strasbourg university, faculty of pharmacy, 74 route du rhin, 67405, illkirch, france centre de ressources biologiques, university hospitals of strasbourg, avenue molière, 67098 strasbourg cedex, france department of cancer molecular genetics laboratory of biochemistry and molecular biology, university hospitals of strasbourg, france department of pediatric radiology, university hospitals of strasbourg, avenue molière, 67098 strasbourg cedex, france department of biopathology, chru nancy, université de lorraine, inserm u1256, ngere, f-54500 vandoeuvre-lès-nancy, france centre de ressources biologiques, bb-0033-00035, chru nancy, f-54500 vandoeuvre-lès-nancy, france neurosurgery department, university hospital of strasbourg, 67098 strasbourg, france department of neurosurgery, chru-nancy, université de lorraine, nancy, france department of pathology and neuropathology, university hospital of timone marseille, 264 rue saint-pierre, 13005, marseille, france oncology department, icans (institut de cancérologie strasbourg europe), 17 rue albert calmette, 67200, strasbourg, france radiotherapy department, icans (institut de cancérologie strasbourg europe), 17 rue albert calmette, 67200, strasbourg, france pediatric onco-hematology unit, university hospitals of strasbourg, avenue molière, 67098 strasbourg cedex, france * these authors share senior authorship braf mutant high-grade gliomas encompasses pxa who grade 3, gbm idh wt and hgap. they share many histomolecular features making differential diagnosis difficult. dna methylation profiling is useful but some tumors fail to fall in any methylation class (mc). we describe a cohort of 16 intracranial braf-altered tumors with histological signs of malignancy. clinico-radiological, morphological, genetic, epigenetic and trancriptomic features are comprehensively described. if already known concomitant molecular alterations were frequently encountered, methylation profiles seems more diverse than expected. indeed, 2 cases were assigned to the mpnst mc. these cases clustered in a combined t-sne mixing cns and sarcoma mc in a separate group of high-grade gliomas located in the vicinity of the mpnst-like cluster. transcriptomic data of these tumors also showed differences. these data suggest the existence of an intracranial tumor type epigenetically and transcriptonomically close to mpnst whose frequency and prognosis should be clarified in larger series.   free neuropathol 6:2:13 high level of cdkn2a homozygous deletion discrepancies between fluorescent in situ hybridization and dna methylation-derived copy number in a braf-mutant glioma cohort how to solve the dilemma? thibaut wolf1, damien reita2,3, marlène deschuyter3, julie buffa2, erwan pencreach2, eric jeandidier4, marie-pierre chenard1, natacha entz-werlé3–5,benoît lhermitte1,3 pathology department, university hospital of strasbourg, 67098 strasbourg, france oncobiology platform, laboratory of biochemistry, university hospital of strasbourg, 67098 strasbourg, france umr cnrs 7021, laboratory bioimaging and pathologies, onko3t team, faculty of pharmacy, 67401 illkirch, france cytogenetic departement, mulhouse hospital, 68100 mulhouse, france pediatric oncology department, university hospital of strasbourg, 67098 strasbourg, france clarifying the diagnosis and prognosis of braf-mutant gliomas requires testing for cdkn2a homozygous deletion (hd). we herein compare the performances of 5 different techniques currently available to detect it on a retrospective cohort of 20 braf-altered gliomas, including fish and dna methylation-derived cnv. a high level of cdkn2a hd detection discrepancy was observed, especially between fish and other techniques suggesting low sensitivity of fish. to solve the dilemma, an original approach using genomic alignment of dna methylation-derived cnv raw data was performed. this revealed a high rate of isolated cdkn2a hd excluding mtap among fish false negative cases, owing to non-specific hybridization of the probe, which is a very commonly used probe targeting the entire 9p21 region including cdkn2a and mtap. we therefore report a molecular proof of a previously suspected low sensitivity of fish assay among braf-altered gliomas with an original bioinformatic pipeline based on dna methylation raw data.   free neuropathol 6:2:14 fgfr1 wild-type rosette-forming glioneuronal tumours mégane le quang1, emmanuelle uro-coste2–4 pathology department, bordeaux university hospital, bordeaux, france pathology department, toulouse university hospital, toulouse, france inserm u1037, toulouse cancer research centre (crct), toulouse, france service d'anatomie et cytologie pathologiques, institut universitaire du cancer-oncopole, toulouse cedex 9, france we report three cases of rosette-forming glioneuronal tumours (rgnt), diagnosed through dna-methylation profiling, without the classical fgfr1 alteration commonly reported in this entity. desirable criteria include the presence of a fgfr1 mutation with coexisting pik3ca and/or nf1 mutation. several teams have reported the constant presence of fgfr1 mutation in their rgnt series, suggesting that this criterion should appear as essential in the next classification. nevertheless, our three cases of rgnt show that the absence of fgfr1 alteration does not eliminate this diagnosis. our finding is also important from a therapeutic point of view since two of our cases have genetic alterations likely to be treated by targeted therapy: akt2 e17k mutation by akt inhibitors currently involved in clinical trials and kiff5::erbb4 fusion by second-generation pan-erbb tyrosine kinase inhibitors.   free neuropathol 6:2:15 cerebral inflammation in a patient with kabuki syndrome tommaso nicoletti1,2, magdeldin elgizouli3, klaus warnatz4,5, patrick roth1, regina reimann6 department of neurology, university hospital zurich and university of zurich, zurich, switzerland department of neuropathology, university hospital essen, essen, germany institute of medical genetics, university of zurich, zurich, switzerland department of clinical immunology, university hospital zurich, zurich, switzerland department of rheumatology and clinical immunology, medical center university of freiburg, freiburg, germany institute of neuropathology, university hospital zurich, zurich, switzerland here we report a 35-year-old man with common variable immunodeficiency (cvid) who presented after a seizure. he had undergone surgery for a congenital heart defect at the age of seven and had developed splenomegaly, lymphadenopathy, interstitial lung disease and autoimmune cytopenias since the age of 28. clinical examination revealed subtle facial dysmorphic features but no focal neurological deficits. brain magnetic resonance imaging showed a right frontal-insular lesion with contrast enhancement. a biopsy confirmed histological evidence of microgranulomatous inflammation. polymorphic ebv-positive lymphoproliferation (formerly lymphomatoid granulomatosis) and lymphoma were excluded. a multigene panel identified a de novo pathogenic variant c.7411del p.(arg2471aspfs*14) in the kmt2d gene associated with kabuki syndrome. cerebral inflammatory lesions are a rare complication of kabuki syndrome and other genetic conditions associated with cvid, such as ctla-4 deficiency. although neuroimaging may suggest demyelinating lesions or neurosarcoidosis, the histologic pattern of t-cell dominant angiocentric infiltrates shares similarities with cerebral lymphocytic vasculitis.   free neuropathol 6:2:16 dna methylation profile in the human hypothalamus: comparison of the profiles of covid-19, alzheimer’s and control patients dewisme julie1,2, sreekala nampoothiri1, laurence stechly3, florent sauvé1, fabienne escande3, claude-alain maurage1,2, vincent prevot1 univ. lille, inserm, chu lille, lille neuroscience & cognition, umr-s 1172, lille, france pathology department, lille university, medical centre, lille, france molecular biology department, lille university, medical centre, lille, france introduction: sars-cov-2 could be associated with neurological and endocrine symptoms, linked to the infection of the central nervous system, particularly via circumventricular organs, including the median eminence of the hypothalamus. it may also interfere with the progression of alzheimer's disease. our objective was to compare the dna methylation profile of post-mortem hypothalamic tissue from covid-19 patients with that of controls and of patients who died in the context of alzheimer’s disease. methods: we studied the dna methylation profile of formalin-fixed and frozen hypothalamic tissue from 4 covid-19 patients, 6 controls, and 6 alzheimer's patients using illumina® infinium methylationepi ctechnology. results: the comparison of the cpg methylation profile of covid-19 patients to controls revealed only 4 differentially methylated sites. in contrast, we observed numerous dna methylation differences between alzheimer's patients and both covid-19 patients and controls. conclusion: our results suggest that hypothalamic epigenetic modifications are more pronounced in alzheimer's patients than in covid-19 patients compared to controls, highlighting numerous candidate cpg sites in alzheimer's patients. the study will continue with 1) the inclusion of new patients, 2) a more specific study of cpg islands in gene promoter regions.   free neuropathol 6:2:17 neuropathology of the first experimental transmission of the atypical prionopathy vpspr (variably protease sensitive) to non-human primate j. mikol1, j. delmotte1, w. zou2, j.p. deslys1, e. comoy1 cea/drf/jacol/sepia, 18 route du panorama, bp6 92265, fontenay aux roses, france the first affiliated hospital of nanchang university, 17 yongwaizheng street 330006, nanchang, jiangxi, china presumed to be the sporadic form of genetic cjd v180i, vpspr is classically considered as an atypical dementia, incompletely, if any-transmissible in experimental rodent models and often firstly misdiagnosed. we report here the first transmission of vpspr in a cynomolgus macaque, 8.5 years after intracerebral inoculation of a brain homogenate from a 75-years-old mv patient. neuropathology showed all the elements of the triad, with a specific profile. notably, immunostaining showed very thin synaptic and neocortical fuzzy deposits of prpd and biochemistry revealed lower amounts of prpres than in other prion strains, with a specific ladder-like glycophoretic profile. in addition, we observed unusual massive neocortical aβ deposits that are absent in mid-aged controls (13.5 years), and different from those observed in two-fold older animals. secondary transmission was observed in a limited number of transgenic mice. the transmission between vpspr and amyloidosis will be discussed. copyright: © 2025 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. 65th annual meeting of the canadian association of neuropathologists association canadienne des neuropathologistes (canp-acnp) feel free to add comments by clicking these icons on the sidebar free neuropathology 6:23(2025) meeting abstracts 65th annual meeting of the canadian association of neuropathologists association canadienne des neuropathologistes (canp-acnp) meeting abstracts october 23rd–25th, 2025 banff, alberta submitted: 28 november 2025 accepted: 29 november 2025 published: 04 december 2025   the canadian association of neuropathologists – association canadienne des neuropathologistes (canp-acnp) held their 65th annual meeting at the banff centre for arts & creativity – banff, alberta, from october 23rd–25th, 2025, under the leadership of dr. cynthia hawkins, president of the canp-acnp, dr. veronica hirsch-reinshagen, secretary treasurer of the canp-acnp, and with technical support from canp administrator colleen fifield. the academic program comprised 23 scientific abstracts, 13 unknown cases, the neuropathology in practice forum on immunohistochemical markers for inflammatory myopathies, and the presidential symposium. digital pathology images from the 13 unknown cases were available for online viewing prior to the conference (www.canp.ca). the david robertson lecture, pathomechanisms at the interface of neurological infectious and immune disorders, was delivered by dr. christopher power. the 2025 presidential symposium, advanced perspectives on neurodegenerative diseases, featured the gordon mathieson lecture by dr. ian mackenzie on new approaches to neurodegenerative disease diagnosis and research, and the jerzy olszewski guest lecture by dr. eric smith entitled vascular dementia. the symposium also included three invited presentations: dr. rosa rademakers discussed the role of genetics in understanding the pathophysiology of neurodegenerative diseases; dr. edward lee presented on the role of spatially defined omics in understanding the pathophysiology of neurodegenerative diseases; and dr. julie schneider spoke on insights from population-based studies into the pathological determinants of dementia. the mary tom award for best clinical science presentation by a trainee went to dr. erin stephenson (supervisor dr. kristopher d. langdon), and the morrison h. finlayson award for best basic science presentation by a trainee was won by dr. karina martin (supervisor harry v. vinters). the following abstracts were presented at the 65th annual meeting of the canadian association of neuropathologists – association candienne des neuropathologistes (canp-acnp) in october 2025.   https://doi.org/10.17879/freeneuropathology-2025-9165 keywords: canadian association of neuropathologists, canp, meeting abstracts, 65th meeting oct. 2025 contents abstract 1: decoding the diseased brain: using machine learning to gain comprehensive understanding of neurodegenerative diseases abstract 2: the relevance of identifying limbic neuronal synuclein pathology in multiple system atrophy abstract 3: spinal cord α-synuclein mapping in parkinson’s disease abstract 4: comparison of co-pathologies in cte-nc and ggt and case report of an ex-football player abstract 5: does the hla locus have relevance in progressing supranuclear palsy? abstract 6: cognitive subtyping in schizophrenia reveals distinct clinicopathologic signatures and a vascular-linked cognitive phenotype abstract 7: mapping the digital divide: underrepresentation of neuropathology in large language model-driven diagnostic literature abstract 8: onsight: a real-time computational pathology companion for histopathology abstract 9: validation framework for an ai decision support tool in intraoperative neuropathology: protocol for a multi-phase evaluation abstract 10: an internal audit of neuropathology consultation for autopsies at the london health sciences centre – a quality improvement project abstract 11: ischemic stroke in hypereosinophilic syndrome: a clinicopathologic study of two cases abstract 12: neuropathological correlates of serum biomarkers during the dying process in humans abstract 13: brainstem cap dysplasias: pathology and novel descriptions abstract 14: keratan sulfate proteoglycan is not expressed in the human fetal cerebellar system except in pontine nuclei and transitory vermal septa; not in corticospinal fasciculi caudal to internal capsule abstract 15: chromogenic in situ hybridization for detection of cdkn2a/b homozygous deletion abstract 16: routine immunohistochemical assessment of mismatch repair proteins in high-grade gliomas: retrospective analysis and clinical implications abstract 17: malignant transformation of craniopharyngioma with spinal metastasis: a case report and comprehensive literature review abstract 18: methylation classification in meningioma prognostication: strengths and caveats abstract 19: liquid biopsy for cns tumours abstract 20: pituitary inflammatory lesions including lymphocytic hypophysitis: a 30-year single centre experience abstract 21: additional presence of lztr1 mutation in a vgll-fused central nervous system schwannoma with neuroblastoma-like cell dense areas abstract 22: histopathological and clinical features can discriminate immune from non-immune statin-induced myotoxicity abstract 23: muscle biopsy findings in an infant with arthrogryposis associated with a thoc2 variant: a case report     abstract 1 free neuropathol 6:23:4 decoding the diseased brain: using machine learning to gain comprehensive understanding of neurodegenerative diseases ain kim1, shelley l. forrest1,2, gabor g. kovacs1–7 tanz center for research in neurodegenerative diseases, university of toronto, toronto, canada krembil brain institute, university health network, toronto, canada rossy centre for psp, toronto western hospital, toronto, canada edmond j. safra program in parkinson’s disease and the morton and gloria shulman movement disorders clinic, toronto western hospital, toronto, canada division of neurology, university of toronto, toronto, canada laboratory medicine program, university health network, toronto, canada department of laboratory medicine & pathobiology, university of toronto, toronto, canada neurodegenerative diseases are characterized by neuronal loss and deposition of misfolded proteins in the human brain. since neuropathological evaluation remains the gold standard for definitive diagnosis, many efforts have been made to understand the complex neuropathological and molecular changes underlying neurodegenerative diseases using fully characterized post-mortem tissues. these include evaluating cytopathologies, morphologies of pathological inclusions, biochemical properties of misfolded proteins using seed amplification assays (saa), and genotypic information. however, the results of these evaluations are not independent, but rather should be interpreted together. in order to integrate results from multiple disciplines and achieve a comprehensive understanding of the disease, a powerful and scalable tool is needed. we used machine learning, a branch of artificial intelligence, to learn latent patterns from high dimensional data and incorporate results from multiple disciplines. machine learning-based analysis has allowed: i) the discovery of novel subtypes of multiple system atrophy based on morphological variables, ii) the integration of alph α-synuclein cytopathology and seeding behavior to describe cell type-specific seeding in synucleinopathies, iii) the understanding of differences in in inflammatory markers of distinct inheritable dna sequence patterns, iv) the validation of subtype and staging in progressive supranuclear palsy, and v) the prediction of 4r-tauopathies using morphological differences in coiled bodies. machine learning can be applied not only for diagnostic predictions in clinical settings, but also as a research tool to understand the comprehensive biological underpinnings of neurodegenerative disease.   abstract 2 free neuropathol 6:23:5 the relevance of identifying limbic neuronal synuclein pathology in multiple system atrophy gabor g. kovacs1–6, ivan martinez-valbuena1, shelley l. forrest1, xiaoxiao xu6, renato p. munhoz5, jun li1, ekaterina rogaeva1, anthony e. lang5, masahiro enomoto7 tanz center for research in neurodegenerative diseases, university of toronto, toronto, canada krembil brain institute, university health network, toronto, canada department of laboratory medicine and pathobiology, university of toronto, toronto, canada laboratory medicine program, university health network, toronto, canada edmond j. safra program in parkinson’s disease and the morton and gloria shulman movement disorders clinic, toronto western hospital, toronto, canada department of medical biophysics, university of toronto, toronto, canada princess margaret cancer centre, university health network, toronto, canada two neuropathologically defined disorders that show consistently α-synuclein pathology include lewy body diseases (lbd) and multiple system atrophy (msa). msa is characterized by predominantly oligodendroglial cytoplasmic inclusions and distinct synuclein protofilament folds. to compare literature observations on hippocampal neuronal synuclein pathology in msa and to address the question whether brain regions showing unusual neuronal α-synuclein pathology are associated with distinct biochemical and structural profile of α-synuclein as a distinctive feature of atypical form of msa. literature overview of msa with limbic neuronal synuclein pathology (atypical msa). report of a case and comparison with typical cases of msa using α-synuclein seed amplification assay (α-syn saa), immunoblotting, proteinase k digestions, conformational stability assay (csa) and electron cryo-microscopy. neuronal cytoplasmic inclusions are thought to contribute to memory impairment when seen in the hippocampus in msa and additionally large argyrophilic neuronal inclusions are detected in atypical msa. in our case with atypical msa, we demonstrate distinct biochemical characteristics of α-synuclein linked to cytopathological differences (e.g. neuronal or oligodendroglial) and a new lewy-msa hybrid fold brain regions showing neuronal inclusions. we propose that cell-specific protein pathologies can be associated with distinct filament folds and expand the current structure-based classification of α-synucleinopathies.   abstract 3 free neuropathol 6:23:6 spinal cord α-synuclein mapping in parkinson’s disease pramath kakodkar1, javera tariq1, ali h. rajput2, alex rajput2, roland n. auer1 department pathology and lab medicine, university of saskatchewan, saskatoon, canada neurology division, university of saskatchewan, saskatoon, canada it has been hypothesized that the etiology of parkinson’s disease (pd) lies outside the body via an inhaled/swallowed agent entering the body to cause protein-catalyzed misfolding of proteins akin to prion diseases. early olfactory abnormalities and gut dysbiosis antedate a proposed march of α-synuclein misfolding through loci involving olfactory and autonomic systems in a body-first, not brain-first, sequence. using anti-phospho-ser129 α-synuclein antibodies and an immunohistochemical red readout, we mapped α-synuclein rostro-caudally, and at each level dorso-ventrally using rexed’s laminae. a semi-quantitative scale was used. lewy bodies, lewy neurites and granular α-synuclein in 35 spinal cords from a sequential series of patients deceased from 2016 to 2021 showed no caudo-rostral gradient along the length of the spinal cord. however, α-synucleinopathy was highest in the thoracic cord, lowest in the sacral cord. intermediate autonomic lamina vii of rexed showed the highest α-synuclein compared to dorsal sensory laminae i–iv (p = 0.02) and to ventral motor laminae viii–ix (p = 0.02). clinical stratification by braak stage showed only that the autopsy cases with later, more severe braak staging had earlier onset and longer disease duration. our results show that directionality of spread within the spinal cord of pd is heterogeneous and does not correlate with braak staging. the results lend no support for a gradient of α-synuclein within the spinal cord, neither along the neuraxis nor dorso-ventrally within any spinal level, while confirming the autonomic-predominant distribution of α-synuclein seen in the pd spinal cord.   abstract 4 free neuropathol 6:23:7 comparison of co-pathologies in cte-nc and ggt and case report of an ex-football player shelley l. forrest1,2, nusrat sadia3, mozhgan khodadadi3, charles tator3,4, robin green3,5, maria carmela tartaglia3,6, gabor g. kovacs1,2,7,8 tanz centre for research in neurodegenerative disease, university of toronto, toronto, canada laboratory medicine program & krembil brain institute, university health network, toronto, canada canadian concussion centre, krembil brain institute, university health network, and university of toronto, toronto, canada division of neurosurgery, toronto western hospital, university of toronto, toronto, canada kite-toronto rehab, university centre, toronto, canada university health network memory clinic & krembil brain institute, university health network, toronto, canada edmond j. safra program in parkinson's disease, rossy program for psp research and the morton and gloria shulman movement disorders clinic, toronto western hospital, toronto, canada department of laboratory medicine and pathobiology and department of medicine, university of toronto, toronto, canada to compare literature observations on the frequency and type of co-pathologies associated with chronic traumatic encephalopathy-neuropathological change (cte-nc) and globular glial tauopathy (ggt). to report an ex-football player with primary progressive aphasia, and an unprecedented combination of degenerative pathologies, including cte-nc. mixed neurodegenerative pathologies are common but can be overlooked when there is a more obvious pathology. literature review and summary of our observations in cte and ggt cases. an 86-year-old male former canadian football player was longitudinally followed in the cognitive neurology clinic at toronto western hospital and underwent clinical assessments and mri scans throughout the disease course. paraffin-embedded sections from all brain regions available were immunostained with histological and immunohistochemical stains. mixed pathologies are frequently associated with cte-nc associated also with ageing. in ggt the frequency is lower but unusual co-pathology constellations (e.g. tdp-43) may occur. in the case presented, multiple and rare constellations of degenerative pathologies observed: 1) cte-nc (high level); 2) high level of adnc); 3) lewy body disease (braak stage 4 or limbic type); 4) cerebral amyloid angiopathy; 5) argyrophilic grain disease; 6) globular glial tauopathy type ii (corticospinal tract involvement) with selective and asymmetric involvement of the corticospinal tract; and 7) neuronal intranuclear hyaline inclusion body disease. this study highlights the spectrum of proteinopathies that can be associated with multiple concussions and demonstrates that unusual constellations of rare neurodegenerative diseases beyond adnc and lewy body pathology can be observed as co-pathologies in the same individuals.   abstract 5 free neuropathol 6:23:8 does the hla locus have relevance in progressing supranuclear palsy? forrest sl1–3, zaheer ss3,4, kim a1, tanaka h1, chasiotis h1, li j1, dasari s1, kalyaanamoorthy s1, fox sh3,4,7, wang j8,9, rogaeva e1, fox s3,7, tartaglia mc1–3,7, lang ae1,3,4,7, kovacs gg1–7 tanz centre for research in neurodegenerative diseases, university of toronto, toronto, canada krembil brain institute, university health network, toronto, canada rossy centre for psp, toronto western hospital, toronto, canada edmond j. safra program in parkinson’s disease and the morton and gloria shulman movement disorders clinic, toronto western hospital, toronto, canada laboratory medicine program, university health network, toronto, canada; department of chemistry, faculty of science, university of waterloo, waterloo, canada department of chemistry, faculty of science, university of waterloo, waterloo, canada division of neurology, university of toronto, toronto, canada laboratory medicine program, university health network, toronto, canada department of laboratory medicine and pathobiology, university of toronto, toronto, canada to investigate the role of human leukocyte antigen (hla) haplotypes in progressive supranuclear palsy (psp) and evaluate its relation to clinical and pathological diversity. psp is a neurodegenerative 4-repeat tauopathy characterized by atypical parkinsonism and cognitive-behavioral changes. the hla locus has not been investigated in psp, but has been implicated in autoimmune and some neurological diseases. first, hla haplotyping was performed in 44 psp cases compared to a canadian deceased donor pool. binding predictions were used to explore relation of hla molecules and tau peptides. second, 32 autopsy-confirmed psp cases were grouped by hla haplotypes: and systematic analysis of inflammatory cells (t and b cells, microglia) and phosphorylated-tau was performed. statistical analysis considering confounding factors and machine learning was used evaluate the effect of hla haplotypes on pathology variables. the dqb106:01 allele showed an odds ratio of 2.94 (95 % ci 1.01–8.55, p = 0.047), and the narcolepsy-associated haplotype had an odds ratio of 2.59 (95 % ci 1.39–4.83, p = 0.0025). hla-tau peptide binding predictions confirmed strong tau peptide binding to alleles dqa101:02-dqb106:02 and dqa101:03-dqb1*06:01, but not to the psp-protofilament fold. we observed differences of the constellations of neuropathological variables between hla haplotype groups. machine learning identified inflammatory markers and neuropathological ratios as strong predictors of hla haplotypes (clustering accuracy: 86.96 % and 91.30 %), with clinical symptom sequences and neuropathological ratios achieving prediction accuracies of 80.00 % and 71.43 %, respectively. these findings highlight hla haplotype-dependent neuroinflammatory and pathological variations in psp, suggesting potential for patient stratification in immune-modulating therapy trials.   abstract 6 free neuropathol 6:23:9 cognitive subtyping in schizophrenia reveals distinct clinicopathologic signatures and a vascular-linked cognitive phenotype naomi c. futhey1–3, fidel vila-rodriguez4,5, shawn j. stochmanski4, elizabeth gregory4, william honer4, belen arranz6,7, belen ramos6–8, ana escanilla6, america vera-montecinos8,9, mark s. cembrowski1,2,5,10, veronica hirsch-reinshagen3 djavad mowafaghian centre for brain health, university of british columbia, vancouver, canada department of cellular; physiological sciences, faculty of medicine, university of british columbia, vancouver, canada department of pathology and laboratory medicine, faculty of medicine, university of british columbia, vancouver, canada department of psychiatry, university of british columbia, vancouver, canada school of biomedical engineering, university of british columbia, vancouver, canada parc sanitari sant joan de déu, dr. antoni pujadas, sant boi de llobregat, spain centro de investigación biomédica en red de salud mental, cibersam (biomedical network research center of mental health), ministry of economy, industry and competitiveness institute of health carlos iii, madrid, spain psiquiatria molecular, institut de recerca sant joan de déu, esplugues de llobregat, spain departamento de ciencias biológicas y químicas, facultad de ciencias, universidad san sebastián, sede tres pascualas lientur 1457, concepción, chile department of mathematics, university of british columbia, vancouver, canada cognitive impairment is a defining feature of chronic schizophrenia, but its underlying neuropathology remains unclear and treatment options are scarce. the contributions of alzheimer’s disease (ad) and cerebrovascular disease (cvd) pathology to cognitive decline in older patients are particularly understudied. this study examined the relationship between postmortem neuropathology and neuropsychological performance in 55 patients (mean age 78), representing, to our knowledge, the most detailed clinicopathologic investigation of late-life schizophrenia to date. although 70 % met criteria for cognitive impairment, the prevalence of ad pathology (35.1 %) was comparable to that reported in age-matched controls. in contrast, cvd pathology was more frequent (84.2 %) and significantly associated with lower mini-mental state examination (mmse) scores (b = –6.40, p    abstract 7 free neuropathol 6:23:11 mapping the digital divide: underrepresentation of neuropathology in large language model-driven diagnostic literature vladimir (vova) r. auer1, pramath kakodkar1, daniel markewich1, nooshin shekari2, bryan johnston1, viktor zherebitskiy1, roland n. auer1, jay kalra1 department pathology and lab medicine, university of saskatchewan, saskatoon, canada department of anatomy, physiology, pharmacology, university of saskatchewan, saskatoon, canada an llm (large language model) is an artificial intelligence (ai) trained on massive amounts of text to predict the next word, letting it generate and work with human‑like language. the objective of this study is to assess the representation and diagnostic utility of llms in clinical neuroscience with a focus on neuropathology compared to other subspecialties. we performed a prisma (preferred reporting items for systematic reviews and meta-analyses) compliant review of 539 articles (chatgpt (n = 508), google gemini (n = 14), perplexity (n = 10), deepseek (n = 7)) spanning the years 2022–2025. studies were categorized by subspecialty, input data type (admission note, radiological imaging, radiology report, pathology images, pathology report), and assessed llm outcomes (diagnosis, grading, prognosis). only 11.9 % (n = 64) of included studies focused on clinical neuroscience, and just 3 were dedicated to neuropathology. these studies targeted gliomas and neurodegenerative disorders. the other specialty that had the highest llm utilization was radiology (11.3 %, n = 61). neuropathology-dedicated studies lagged in sample size, external validation, and model robustness, especially compared with other pathology (n = 37) subspecialties such as head and neck (n = 9), general surgical pathology (n = 7), gastrointestinal (n = 5) and gynecological pathology (n = 4). neuropathology remains significantly underrepresented in the era of diagnostic ai. given the distinctive morphologic and molecular complexity of cns (central nervous system) lesions, there is an urgent need for dedicated model development and rigorous validation tailored to neuropathology.   abstract 8 free neuropathol 6:23:12 onsight: a real-time computational pathology companion for histopathology kevin faust1, jinzhen hu1, parsa babaei zadeh1, adrienn bourkas1, dimitrios oreopoulos1, phedias diamandis1,2 princess margaret cancer centre, toronto, canada department of laboratory medicine and pathobiology, university of toronto, toronto, canada precise microscopic examination of surgical tissue sections is a specialized skill critical for diagnosis and clinical care. while artificial intelligence (ai) shows promise for histological analysis, differences in slide digitization and proprietary software limit real-world deployment. we present onsight, a platform-agnostic computer vision software providing real-time ai inferences to pathologists during digital slide review. onsight is freely accessible as a single executable file, runs locally on consumer-grade computers, and requires no complex integration, enabling cost-effective and safe use in research and clinical workflows. onsight was benchmarked on classification, mitosis detection, and immunohistochemical quantification. for tumor classification (glioma, meningioma, schwannoma, epithelial metastasis), a vit-b/16 pretrained on the kaiko foundation model was fine-tuned on 80,000 h&e tiles from uhn at 20×. ki-67 nuclei were segmented with a yolo model trained on qupath-annotated patches from 10 cases. mitotic figures were detected with a retinanet fpn (resnet-50 backbone) trained on midog++ at 40×. validation used tcga and uhn cases. tumor classification accuracy was 95 % across public and institutional sets. mitosis detection showed high area under the curve and agreement with manual review. automated ki-67 indexing showed closer agreement with neuropathologist counts than qupath and is reported as a percentage. median per-field latency was 0.3 s, enabling real-time use. onsight provides accurate, si-unit-standardized, low-latency pathology slide analysis with potential to reduce inter-observer variability, improve accuracy, and increase efficiency. prospective multicenter evaluation is warranted for widespread use.   abstract 9 free neuropathol 6:23:13 validation framework for an ai decision support tool in intraoperative neuropathology: protocol for a multi-phase evaluation adrienn n. bourkas1, shane eaton1, phedias diamandis1–4 department of laboratory medicine and pathobiology, university of toronto, toronto, canada princess margaret cancer centre, toronto, canada department of medical biophysics, university of toronto, toronto, canada laboratory medicine program, department of pathology, university health network, toronto, canada intraoperative consultations (ioc) for neurosurgical cases rely on rapid assessment of h&e-stained frozen sections and cytologic preparations, often under time pressure and with variable tissue quality. we describe a validation framework for evaluating onsightpathology, an open and platform-agnostic artificial intelligence (ai) tool developed to classify intraoperative digital slides into five diagnostic categories: glioma, epithelioid, meningeal, schwannoma, and normal/reactive tissue. in the pilot phase, 10–20 representative cases from each class were selected from archival material, and 50–100 diagnostic tiles per case were extracted using an image feature-based clustering pipeline. groups of tiles were assigned coarse labels (e.g., glioma, artifact) via visual histologic inspection. ground-truth diagnoses were adjudicated on formalin-fixed paraffin-embedded sections through dual-review consensus. the annotated images will then be used to train a vision transformer model and will be deployed using onsightpathology to provide "real-time" inferences to help with intra-operative frozen section analysis. the tool is designed to assist pathologists by highlighting diagnostically relevant regions, providing class probabilities, and flagging low-confidence areas for review. this approach is intended to augment ioc workflows by improving confidence, reducing turnaround time, and standardizing interpretation in time-sensitive settings. subsequent phases will assess onsightpathology’s performance on external cohorts and in real-time clinical use.   abstract 10 free neuropathol 6:23:14 an internal audit of neuropathology consultation for autopsies at the london health sciences centre – a quality improvement project hao li1,2, qi zhang1,2 pathology and laboratory medicine, london health sciences centre, london, canada schulich school of medicine and dentistry, western university, london, canada in autopsy, neuropathological workups tend to be relatively extensive, requiring more tissue sampling, ancillary testing, and examination time compared to other body systems. as london health sciences centre (lhsc) is a high-volume autopsy institution, in this quality improvement project we present the utility of a centralized neuropathology medical (hospital) / biobank autopsy database as an internal audit for laboratory management purposes. the database is a simple digital spreadsheet recording retrospectively, on all lhsc medical and biobank neuropathological autopsy consultations from 2005 to 2023, the following parameters: case type (routine medical vs. biobank), in-house vs. externally referred, reason for neuropathology consultation, specimens examined (eg. brain, spinal cord, muscle), age at death, dates of autopsy / brain cut / sign-out, involvement of trainees, and final diagnosis. once established, the database is easy to utilize, and multiple laboratory management statistics can be mined using basic spreadsheet functions. these include volume trends of case types over time, proportions of different reasons for consultation, and turn-around-time as a function of other parameters such as diagnosis, deceased age, trainee involvement, among others. the practical applications of an institutional neuropathology autopsy database for internal audit and laboratory management are many, including guidance on resource management based on consultation patterns, technical expertise based on specimen types, analysis of factors influencing turn-around-time, and guiding curriculum design in neuropathology education. future directions include extending such strategy to forensic/medical-legal autopsies, which would also be useful in analyzing contributions of neuropathological consultations in addressing the forensic question.   abstract 11 free neuropathol 6:23:15 ischemic stroke in hypereosinophilic syndrome: a clinicopathologic study of two cases karina c. martin1, dan straathof2, chi lai3, harry v. vinters4 department of pathology and laboratory medicine, faculty of medicine, university of british columbia, vancouver, canada fraser health department of laboratory medicine & pathology, royal columbian hospital, new westminster, canada providence health, st. paul’s hospital, university of british columbia, department of pathology & laboratory medicine, vancouver, canada departments of pathology and laboratory medicine (neuropathology) and neurology, david geffen school of medicine, university of california, los angeles, usa ischemic stroke is a rare complication of hypereosinophilic syndrome (hes). manifestations of stroke in hes have been described mainly in the radiologic literature; however, the pathologic characterization of central nervous system (cns) involvement in hes is limited. we describe two patients who presented to hospital with systemic symptoms and were found to have peripheral blood hypereosinophilia. thorough investigations for primary and secondary etiologies of hypereosinophilia were negative. both patients subsequently suffered catastrophic strokes and died despite therapy and resuscitative efforts. pertinent general autopsy findings in both cases included the presence of intraventricular cardiac thrombi. post-mortem neuropathologic examination demonstrated a multi-territorial embolic infarct pattern in patient 1 and a watershed infarct pattern in patient 2. microscopic features included the presence of microabscesses consisting of a disproportionately large number of eosinophils and infarcts associated with blood vessels showing occlusive thrombi with surrounding eosinophils. in summary, we present two post-mortem neuropathologic examinations of patients exhibiting cns infarcts associated with hes, discuss potential pathomechanisms, and review the literature on this rare entity.   abstract 12 free neuropathol 6:23:16 neuropathological correlates of serum biomarkers during the dying process in humans chloe p. allen1, catie n. futhey2, aditya swaro3, jordan d. bird1, sophie stukas4, mark s. cembrowski3–6, cheryl l. wellington4,5,8, mypinder s. sekhon1,4,8, veronica hirsch‑reinshagen7,8 division of critical care medicine, department of medicine, vancouver general hospital and university of british columbia, vancouver, canada md/phd program, university of british columbia, vancouver, canada department of cellular and physiological sciences, life sciences institute, university of british columbia, vancouver, canada djavad mowafaghian centre for brain health, department of pathology and laboratory medicine, faculty of medicine, university of british columbia, vancouver, canada school of biomedical engineering, university of british columbia, vancouver, canada department of mathematics, university of british columbia, vancouver, canada division of neuropathology, department of pathology and laboratory medicine; general hospital and university of british columbia, vancouver, canada international collaborations on repair discoveries, university of british columbia, vancouver, canada neurocritical patients (ncps) admitted to the intensive care unit (icu) have severe neurological damage stemming from diverse causes including hypoxic ischemic brain injury (hibi), intracranial hemorrhage (ich), and traumatic brain injury (tbi), among others. recently, blood-based neurologic biomarkers have emerged as a promising tool for prognostication, however, their utility for the diagnosis of tissue injury severity and the insights they provide into the pathophysiology of this brain injury are limited. we aimed to investigate the relationship of 120 blood-based systemic and neurologic biomarkers prior to the withdrawal of life-sustaining therapies (wlst) in humans with pre-mortem clinical measures and post-mortem neuropathological features in different neurocritical conditions. clinical and demographic data, along with arterial biospecimens, were obtained from twenty-nine patients immediately prior to wlsm with subsequent post-mortem neuropathological evaluation. proteomic analysis of plasma samples was performed using the argo ht platform. a standard neuropathological evaluation was performed followed by exploratory and advanced data analyses. ten brain-enhanced markers were identified that exhibited significant positive associations with neuropathological features, most prominently selective neuronal death (snd) and reactive gliosis: eno2, mapt, nefh, nefl, nrgn, ptau-217, snap25, sncb, and uchl1. notably, these associations were also successful in striating patients by their respective cause of injury. no significant correlations were present with chronic vascular, neurodegenerative, or inflammatory neuropathology. collectively, these results provide neuropathological demonstration of the brain tissue changes in neurocritically-ill patients and their immediate correlation with serum biomarkers. they also demonstrate that select brain-enhanced biomarkers likely possess diagnostic and prognostic utility.   abstract 13 free neuropathol 6:23:18 brainstem cap dysplasias: pathology and novel descriptions stephanie r. beldick1, andrew gao2,3, yael fisher4, patrick shannon2,4 department of laboratory medicine and pathology, western university, london, canada laboratory medicine and pathobiology, university of toronto, toronto, canada university health network, toronto, canada mount sinai hospital, toronto, canada brainstem cap dysplasias are characterized by heterotopic axonal tracts coursing peripherally in the brainstem, identified by magnetic resonance imaging. however, there is a paucity of neuropathological descriptions of cap dysplasias. we therefore aimed to review the histopathology and expand the morphological spectrum of cap dysplasias. three forms are described, including pontine tegmental cap dysplasia (ptcd), medullary tegmental cap dysplasia (mtcd), and anterior mesencephalic cap dysplasia (amcd; a component of the joubert syndrome). we describe a series of seven brainstem dysplasias with "cap" features from our autopsy practice: two ptcd, one mtcd, two amcd, and two undescribed forms. both ptcd cases demonstrated hypoplasia of rhombic lip derivatives and a dorsal pontine tegmental tract. the mtcd case demonstrated hypoplastic rhombic lip derivatives, large axonal tracts in the medullary fourth ventricle and coursing along the ventrolateral medulla, post-necrotic tegmental calcifications, and spinal cord anomalies. the two amcd cases demonstrated joubert syndrome features with axonal bundles crossing the interpeduncular fossa as leptomeningeal heterotopias. the two additional cases of undescribed "cap" forms have a prominent tectal cap. in one case, this is formed by aberrancy in the pathway of the inferior colliculi brachia, and in the other it is formed by incomplete duplication and fusion of midbrain with cerebellar elements, and heterotopic dorsal and lateral medullary tracts, in the context of a complex supratentorial malformation. in summary, "cap dysplasia" should be considered a morphological descriptor rather than limited to axonal pathology or a particular disease entity, as a diversity of pathological developmental processes are evident.   abstract 14 free neuropathol 6:23:19 keratan sulfate proteoglycan is not expressed in the human fetal cerebellar system except in pontine nuclei and transitory vermal septa; not in corticospinal fasciculi caudal to internal capsule harvey b. sarnat1, weiming yu2, laura flores-sarnat3 departments of paediatrics, pathology (neuropathology), clinical neurosciences, university of calgary; alberta children’s hospital research institute (owerko centre), calgary, alberta, canada department of pathology (paediatric anatomical pathology), university of calgary and alberta children’s hospital, calgary, alberta, canada departments of paediatrics, clinical neurosciences, university of calgary and alberta children’s hospital research institute (owerko centre), calgary, alberta, canada we determined spatial and temporal expression of keratan sulfate (ks) proteoglycan in the cerebellar system, dysgeneses and in white matter fasciculi. ks proteoglycan is an extracellular matrix molecule secreted by astrocytes. ks forms an early template of neuroblastic migration to the cortical plate and some axonal fascicles before axons enter. ks immunoreactivity was studied in sections of cerebellar cortex and in deep cerebellar, inferior olivary and red nuclei and white matter in 18 human fetuses 14–41 weeks gestation at autopsy, 6 infants to 2 years, and 9 cerebellar dysgeneses. synaptophysin expression was compared. dentate, inferior olivary and red nuclei are nonreactive at all ages, but pontine nuclei are positive from early 2nd trimester. cerebellar white matter pathways remain non-reactive throughout fetal and postnatal life. bergmann glia are nonreactive at all gestational ages. transitory parasagittal ks septa demarcate vermis/hemispheric boundary. ks is not expressed in dandy-walker, joubert or chiari malformations, pontocerebellar hypoplasia or cerebellar heterotopia. axonal fascicles of the corticospinal tract caudal to the internal capsule do not exhibit ks reactivity. synaptophysin in cerebellar system was normal. absence of hindbrain ks contrasts with strong forebrain reactivity. lack of ks expression in normally developing cerebellum and associated brainstem nuclei except pontine, and its absence in cerebellar dysgeneses implies that ks does not contribute to pathogenesis in cerebellar system malformations. transitory ks septa define early vermal margins, similar to septa of neuromeric segmentation. the intense ks template of the internal capsule does not extend caudally to brainstem fascicles.   abstract 15 free neuropathol 6:23:20 chromogenic in situ hybridization for detection of cdkn2a/b homozygous deletion karina c. martin1,2, tara spence1,2, janine senz2, julia naso1,2, andrew churg1,2, stephen yip1,2 department of pathology, vancouver general hospital, vancouver, canada department of pathology and laboratory medicine, university of british columbia, vancouver, canada the presence of cdkn2a/b homozygous deletion (hd) denotes a higher who grade and more aggressive behavior in gliomas and meningiomas. the gold standard assays for detecting copy number variation (cnv) include chromosomal microarray, next generation sequencing, and fluorescence in situ hybridization (fish). however, these methods are costly, time consuming, and have limited availability in resource-sparse centers. thus, an inexpensive and time-efficient assay for detecting cdkn2a/b hd would be helpful for routine neuropathology cases. chromogenic in situ hybridization (cish) combines dna hybridization techniques utilized in fish with conventional peroxidase reactions utilized in immunohistochemistry to interrogate cnv with brightfield microscopy. we hypothesize that cish is a sensitive and specific method for detecting cdkn2a/b hd. we performed cdkn2a cish on a retrospective cohort of whole slide tissue sections of 5 gliomas and 10 meningiomas using the abnova cdkn2a/cep9 cish probes and implementation kit. two pathologists independently counted and scored 100 cells from each case showing any cep9 (red/control) signals and no cdkn2a (green) signals as hd, and cells showing both red and green signals as cdkn2a/b intact. the pathologists’ scores were averaged and compared to the cdkn2a/b status for each of the glioma and meningioma cases, acquired via fish testing and methylation profiling, respectively. our results demonstrate that detection of cdkn2a/b hd with cish showed high concordance with cdkn2a/b locus interrogation with orthogonal assays. cish is a sensitive and specific method for detecting cdkn2a/b hd in glioma and meningioma and is a more cost-effective and practical alternative to traditional cnv detection methods.   abstract 16 free neuropathol 6:23:21 routine immunohistochemical assessment of mismatch repair proteins in high-grade gliomas: retrospective analysis and clinical implications jacob a. houpt1, lee cyn ang1 department of pathology and laboratory medicine, london health sciences centre, london, canada testing for deficiencies in mismatch repair (mmr) proteins (mlh1, pms2, msh2, and msh6) via immunohistochemistry (ihc) has become a widely-adopted means of screening for tumour predisposition syndromes and for informing potential immunotherapeutic treatment options. while some centres rely on ihc for all 4 mmr proteins, others have relied on a more cost-effective means of screening only the secondary partner proteins pms2 and msh6 (as losses of mlh1/msh2 are expected to result in degradation of pms2/msh6s). just under 2 years ago, cancer care ontario (cco) began to fund routine mmr ihc testing on all high-grade gliomas (hggs). london health sciences centre has thus far tested 132 of these tumours for all 4 mmr proteins via ihc. of these, 4 (approximately 3 %) were found to have a loss of mmr immunopositivity (2 for msh6, 2 for pms2). these cases had particularly elevated nuclear pleomorphism when compared to most other hggs and involved sporadic loss of msh6 (with negative germline testing for lynch syndrome), loss of msh6 secondary to temozolomide chemotherapy (with intact mmr in the original tumour), and biallelic loss of pms2 in the context of 2 cases of congenital mismatch repair deficiency syndrome (cmmrd). this ihc approach has been efficacious in contributing towards the diagnosis of tumour predisposition syndromes, identifying monoallelic versus biallelic loss of mmr proteins, and providing potential targets for immunomodulatory therapies. furthermore, these results support the use of a two-antibody mmr ihc screening approach relying solely on msh6 and pms2.   abstract 17 free neuropathol 6:23:22 malignant transformation of craniopharyngioma with spinal metastasis: a case report and comprehensive literature review pramath kakodkar1, gina martin2, julia radic3, vijayanada kundapur4, adrian b. levine5, viktor zherebitskiy1, roland n. auer1 department of pathology and laboratory medicine university of saskatchewan and saskatchewan health authority, saskatoon, canada division of pediatric hematology-oncology, saskatoon cancer center, saskatoon, canada division of neurosurgery, dalhousie university, halifax, canada division of radiation oncology, saskatoon cancer center, saskatoon, canada department of laboratory medicine and pathobiology, university of toronto, toronto, canada malignant transformation of craniopharyngioma is exceedingly rare with few cases characterized by systemic spread or molecularly confirmed clonal progression. we present a pediatric case of adamantinomatous craniopharyngioma that evolved over 12 years into a high-grade epithelial neoplasm with clival extension and spinal metastases causing vertebral fracture and spinal cord compression; to our knowledge, this is the 3rd case of spinal dissemination in the literature. the transformed tumor demonstrated high-grade epithelial morphology (ki-67: 100 % vs 5 % initially), strong pancytokeratin and vimentin expression, partial h3 k27me3 loss, and absence of glial or neuroendocrine differentiation. molecular profiling confirmed the same ctnnb1 p.s33c mutation in both the initial and transformed tumor, along with a segmental 1p deletion acquired in the latter, supporting molecularly confirmed clonal evolution. a systematic review (n = 35) on craniopharyngioma cases with malignant transformation revealed consistent epithelial immunophenotype, frequent p53 overexpression (89 %, n = 17), high mean ki-67 (41 ± 21 %). more surgeries were significantly associated with longer time to transformation (r = 0.49, p = 0.005), suggesting that multiple surgical interventions delayed progression, while no significant correlation was observed with radiation dose (r = 0.01, p = 0.97) or tumor size (r = 0.40, p = 0.16). these findings support a clonal evolution model and highlight the importance of molecular surveillance in long-standing craniopharyngiomas.   abstract 18 free neuropathol 6:23:23 methylation classification in meningioma prognostication: strengths and caveats shane eaton1, robert siddaway2, anthony arnoldo3, arun vadivel3, adrian levine1,4, cynthia hawkins1,4 department of laboratory medicine and pathobiology, university of toronto, toronto, canada arthur and sonia labatt brain tumour research centre, the hospital for sick children, toronto, canada department of paediatric laboratory medicine, the hospital for sick children, toronto, canada department of pathology, the hospital for sick children, toronto, canada dna methylation patterns have emerged as a useful signature for classifying brain tumours. these patterns tend to be consistent within tumour entities and even within subtypes, and have proved to be a helpful tool in resolving diagnostically challenging cases. however, methylation classification is not always reliable. in particular, in light of the recent cimpact-now guidelines for meningioma prognostication, inconsistencies have appeared between the prognostic subtypes defined by methylation profiling and the grading recommended on the basis of copy number profiles. to explore this, we reviewed methylation results from cases at sickkids hospital, focusing on areas of concordance and discordance. from 841 total methylation cases, 46 were meningiomas. within this group 13 % showed discordant prognostication between methylation subtyping and the copy number-based stratification described in cimpact-now update 8. we present several illustrative cases to highlight when methylation was useful, when it led to conflicting results, and the factors contributing to the discordance. previous studies have suggested a multi-factorial approach to meningioma grading and prognostication underscored by our results. how to approach grading in the setting of discordant findings will require further study with well annotated clinical outcome data. while methylation can be a powerful diagnostic adjunct, these results caution against overreliance on this method alone for meningioma classification and prognostication.   abstract 19 free neuropathol 6:23:24 liquid biopsy for cns tumours yoshiko nakano1, robert siddaway2, liana nobre3, ian burns4, mansuba rana2, richard yuditskiy2, cyril li2, julie bennett1, anthony liu1, uri tabori1, cynthia hawkins2 division of haematology/oncology, the hospital for sick children, toronto, canada department of paediatric laboratory medicine, the hospital for sick children, toronto, canada department of paediatrics, university of alberta, edmonton, canada department of paediatrics, the hospital for sick children, toronto, canada liquid biopsy (lb) is an emerging diagnostic tool for detecting tumor-specific alterations in body fluids that has many potential advantages including: identifying tumors with higher sensitivity and specificity than imaging; replacing invasive surgical biopsy; and monitoring tumor evolution by serial assessments. we investigated the feasibility and utility of cerebrospinal fluid (csf)-based lb for patients with cns tumors. 475 csf samples were collected from patients with known or putative cns tumors. to detect circulating tumor dna (ctdna), cell-free dna was extracted and panel sequencing and/or low-pass whole genome sequencing were performed. at diagnosis/baseline assessment, ctdna was detected in 78 % of high-grade gliomas, 44 % of low-grade gliomas, 55 % of medulloblastomas and 93 % of germ cell tumor cases. in patients with non-biopsied cns lesions, ctdna was diagnostic in 14 patients including for glioma, neuroblastoma and leukemia. furthermore, in seven recurrent medulloblastoma cases where primary tumor copy number profiles were available, new copy number variants suggestive of tumor evolution/new subclones were seen. in patients for whom serial sampling was performed during and/or after completion of therapy, ctdna clearance was observed in response to therapy. however, persistent ctdna was observed in some patients despite imaging showing no residual tumor, highlighting the potential utility of ctdna as a molecular-based measurable residual disease assessment for better risk stratification. these findings pave the way for the clinical use of lb in cns tumors and provide proof of concept for future incorporation of lb into clinical trials.   abstract 20 free neuropathol 6:23:25 pituitary inflammatory lesions including lymphocytic hypophysitis: a 30-year single centre experience marc r. del bigio1 department of pathology, university of manitoba and shared health manitoba, winnipeg, canada in manitoba (population 1.4 million) all neurosurgical procedures and all autopsy neuropathology examinations are done at one center. in a retrospective review (1996–2025) i searched for cases with pituitary inflammation. among 678 surgical pituitary biopsies we encountered: 15 idiopathic lymphocytic hypophysitis; 18 pitnet with inflammation; 34 pitnet + apoplexy with inflammation; 13 inflammation associated with rathke cleft cyst; 3 bacterial infection. among 4768 pituitaries from autopsies we encountered: 16 idiopathic lymphocytic hypophysitis; 1 pitnet + apoplexy; 2 pituitary apoplexy without obvious cause; 3 inflammation associated with rathke cleft cyst; 19 pituitary inflammation and associated disseminated infection (2 herpes simplex; 5 suspected viral; 5 mycobacterial; 7 other bacterial sepsis / meningitis). in some autopsy cases lymphocytic hypophysitis was incidental, 1 had chronic demyelinating disease, 4 had prominent inflammation in the thyroid, and in 1 case it was considered to be a major contributor to death. four decedents had pre-mortem clinical endocrine syndrome (38-year female hypothyroidism; 49-year female hyperprolactinemia; 44-year male addison disease and hypothyroidism; 49-year female diabetes mellitus). conclusive proof of autoimmune lymphocytic hypophysitis is elusive. pitnet, apoplexy, and cyst-associated inflammation are common and of uncertain significance. in all specimens, complete history and imaging correlates are necessary. although rarely a contributor to death, examination of the pituitary gland is an important part of the autopsy in individuals who die unexpectedly, particularly if they have a history of endocrine disease or recent neurological symptoms.   abstract 21 free neuropathol 6:23:26 additional presence of lztr1 mutation in a vgll-fused central nervous system schwannoma with neuroblastoma-like cell dense areas david g. munoz1, sunit das2, ju-yoon yoon1, robert siddaway3, adrian levine3, kenneth d. aldape4 laboratory medicine and pathobiology, university of toronto; department of laboratory medicine, unity health toronto, toronto, canada department of surgery, division of neurosurgery, unity health toronto, toronto, canada department of laboratory medicine, hospital for sick children, toronto, canada laboratory of pathology, center for cancer research, national cancer institute, national institutes of health, bethesda, maryland, usa vestigial-like family (vgll) altered cns schwannoma (vgllacs) is a new tumor entity, recognizable by dna methylation approaches. the critical alteration is a fusion, involving either vgll1 or vgll3. the fusion partner can be ewsr1, chd7, and rarely ss18. a recent series of 20 vgllacs did not identify additional recurrent mutations. to add to the span of the condition, we report a previously healthy 29-year-old man who presented with a clonic seizure. head mri showed an avidly enhancing well-demarcated intra-axial nodule in the left superior frontal gyrus extending to cortex. at surgery the intra-axial firm mass, well-demarcated from the brain, was completely resected. histologically, most of the tumor consisted of spindle cells arranged in streams organized in rhythmic palisades in a collagenous background. in addition, there was a peripheral nodule made up of densely packed neuroblastoma-like cells. both areas showed gfap expression, and reticulin single cell wrapping. at the periphery the tumor was interspersed with synaptophysin-rich neuropil, and rosenthal fibers were present within the tumor. a methylation profile analysis carried out at the national cancer institute matched to cns schwannoma vgll-fused. trusight pancancer next generation sequencing (ngs) revealed a ewsr1::vgll1 fusion, specifically ewsr1 nm_013986 (exon 9, 331 aa) to vgll1 nm_016267 (5’ utr , exon 2). a tier ii mutation in lztr1 (p.trp265*nm_006767.4:c.794g>a) was identified by hybridization-capture ngs assay. our case represents another rare case of vgllacs with neuroblastoma-like areas in association with an additional mutation in lztr1, a gene mutated in both schwannosis and glioblastoma.   abstract 22 free neuropathol 6:23:27 histopathological and clinical features can discriminate immune from non-immune statin-induced myotoxicity benjamin ellezam1, yves troyanov2, océane landon-cardinal3 division of pathology, chu sainte-justine, montréal, canada division of rheumatology, hôpital du sacré-cœur, montréal, canada division of rheumatology, centre hospitalier de l'université de montréal; université de montréal, montréal, canda anti-hmgcr immune-mediated necrotizing myopathy (imnm) is a subacute to chronic progressive auto-immune myositis diagnosed on clinical phenotype, serology and muscle biopsy, and non-immune toxic rhabdomyolysis (tr) is an acute potentially lethal condition usually diagnosed on clinical phenotype alone. however, muscle biopsies are sometimes still performed in tr patients on statins to rapidly rule out imnm, particularly when there is no quick access to serology results. our objective was to describe histopathological and clinical features of tr patients and compare them with a group of imnm controls. out of 924 muscle biopsies received between 2019 and 2024 at our referral center, 36 biopsies from statin-exposed tr patients and 29 anti-hmgcr imnm controls were identified. histopathologic analysis revealed overlapping morphology in 85 % of cases. discriminating features highly suggestive of tr included predominance of pale acute necrotic fibers (p < 0.001), groups of 4+ adjacent necrotic fibers (p < 0.01), regenerative basophilic cuffs (p < 0.001), and lack of lc3+ granular staining in non-necrotic fibers (p < 0.001). review of clinical data revealed acute creatinine elevation in 94 % of tr patients and none of imnm controls (p < 0.001). peak creatine kinase levels (cks) > 25,000 iu/l were seen in 44 % of tr and none of imnm (p < 0.0001). cks normalized on average in 12 days (range 8–21) in tr and in >30 days in all imnm cases. although pathology can be discriminating, tr should be confirmed by following cks closely over a few days without immunosuppression and muscle biopsy only performed to confirm imnm in patients with persistently elevated cks.   abstract 23 free neuropathol 6:23:28 muscle biopsy findings in an infant with arthrogryposis associated with a thoc2 variant: a case report leslie e. hamilton1,2, priya bhola1,3, elizabeth nizalik1,2, brigitte bonin1,4, mark walker1,4, erika bariciak1,5, julie hurteau-miller1,6 faculty of medicine, university of ottawa, ottawa, canada department of pathology and laboratory medicine, children’s hospital of eastern ontario (cheo), ottawa, canada department of genetics, children’s hospital of eastern ontario (cheo), ottawa, canada division of maternal fetal medicine, department of obstetrics, gynecology and newborn care, the ottawa hospital, ottawa, canada division of neonatology, department of pediatrics, children’s hospital of eastern ontario (cheo) and the ottawa hospital, ottawa, canada department of medical imaging, children’s hospital of eastern ontario (cheo), ottawa, canada this case report provides the second reported description of muscle biopsy findings in an infant with arthrogryposis associated with a thoc2 variant. thoc2-related neurodevelopmental disorder is caused by deleterious variants in thoc2; this gene encodes a component of the trex (transcription-export) complex, which plays a critical role in mrna processing and export. thoc2-related disorder is characterized by intellectual disability, often associated with seizure disorders, infantile hypotonia, tremors, gait disturbance and growth impairment. more recently, thoc2 variants have been linked to x-linked recessive fetal arthrogryposis multiplex congenita (cureus 2021; 13(11):e19682; nmd 2023;33(12):978-982). we present a case of an infant who died shortly after birth at 33 weeks gestational age (ga). a fetal ultrasound at 30 weeks ga was significant for arthrogryposis, hydrops and massive polyhydramnios. genetics consultation and amniocentesis was pursued prior to delivery, and initial genetic testing showed a normal karyotype and rapid aneuploidy detection (rad). a limited postmortem examination was performed. biopsy of the quadriceps muscle was significant for moderate variation in myofiber size, and cytoplasmic bodies within several myofibers. trio whole exome sequencing detected a hemizygous, likely pathogenic variant in thoc2,c.2482-1_2482del . the results of muscle biopsy are identical to those described in a previously reported fetus with arthrogryposis and a thoc2 variant (nmd 2023;33(12):978-982), further validating this neuromuscular phenotype. this case report highlights the critical role that the correlation of whole exome sequencing and postmortem examinations play in expanding our clinical knowledge of rare genetic diseases and their phenotypes. copyright: © 2025 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. infantile pilocytic astrocytoma with persisting external granular layer of the cerebellum: a potential diagnostic pitfall feel free to add comments by clicking these icons on the sidebar free neuropathology 5:25 (2024) letter infantile pilocytic astrocytoma with persisting external granular layer of the cerebellum: a potential diagnostic pitfall arnault tauziède-espariat1,2, lauren hasty1, alice métais1,2, pascale varlet1,2 department of neuropathology, ghu paris psychiatry and neuroscience, sainte-anne hospital, 75014 paris, france université de paris, umr s1266, inserm, ima-brain, institute of psychiatry and neurosciences of paris, 75014 paris, france corresponding author: arnault tauziède-espariat · department of neuropathology · ghu paris psychiatry and neuroscience · sainte-anne hospital · 1 rue cabanis · 75014 paris · france a.tauziede-espariat@ghu-paris.fr submitted: 15 september 2024 accepted: 07 october 2024 copyedited by: werner paulus published: 21 october 2024 https://doi.org/10.17879/freeneuropathology-2024-5891 keywords: pilocytic astrocytoma, external granular layer, dysplasia introduction pilocytic astrocytomas (pa) are frequent astrocytic neoplasms that generally have a circumscribed architecture. they are molecularly characterized by mapk gene pathway alterations, mostly kiaa1549::braf fusions (1). pa affect children along the neuraxis, but are typically found in the posterior fossa (2). dysplastic modifications of the brain parenchyma have been described as being associated with epileptic supratentorial tumors (3,4). in the cerebellum, the gangliocytoma (known as “lhermitte-duclos disease”), which is generally a symptom of cowden syndrome, is defined by typical radiological and histopathological features (5,6). it is characterized by a distorted architecture with enlarged cerebellar folia containing dysplastic ganglion cells and a diffuse enlargement of the molecular and internal granular layers (6). whereas it is mainly reported in adults, it can be discovered during childhood (7). herein, we report two cases of young children presenting pa associated with major dysplastic modifications of the adjacent cerebellar parenchyma. case presentation the two cases concerned infants (two boys, 11 and 18 months respectively) who presented a cerebellar mass which was grossly resected. both tumors exhibited features of pa (figure 1 a-b, c and e). indeed, each tumor was biphasic with piloid and oligodendrocyte-like cells, had low to moderate cellularity and a significant amount of rosenthal fibers. there were no eosinophilic granular bodies, perivascular lymphocytic infiltrates, binucleated ganglion cells or neuropil-like islands. tumor cells did not present atypia and mitotic figures were scarce. glomeruloid microvascular proliferation was observed. the tumors were solid and diffuse, and peripherally infiltrated the adjacent parenchyma. on the surface of the cerebellar folia, clusters of small undifferentiated blue cells with round hyperchromatic nuclei and mitoses were present, and their cytoplasm was loosely abundant. using immunohistochemistry, the tumor cells of pa expressed olig2 (figure g and i) and gfap, and were not immunopositive for neuronal markers (such as chromogranin a, synaptophysin, neun, or insm1). the expression of h3k27me3 and in1i was retained. cd34 immunostaining highlighted the vascularization without extravascular expression. no necrosis was observed and the mib1 labeling index was low (1–2%) (figure 1k and m). the small cell component was only immunoreactive for insm1 (figure 1h, j, p and r) and the mib1 labeling index was high in these areas (figure 1l and n). the fish (fluorescent in situ hybridization) analysis of the braf locus identified a rearrangement of tumor cells in both cases (figure 1o and q) without abnormality in the superficial undifferentiated cells. fish did not reveal a 1p deletion for either case. a final diagnosis of pa of the posterior fossa, braf-rearranged associated with “dysplastic” modifications of the adjacent cerebellar parenchyma and the persistence of the external granular layer was suggested. fourteen and four years after the initial diagnoses, both patients were alive without tumor residue or recurrence. figure 1. histological and molecular features of the two cases case #1 (a, c, d, g, h, k, l, o and p) and case #2 (b, e, f, i, j, m, n, q and r). (a and b) atrophy of the folia of the cerebellum with a lowly cellular proliferation (hps, magnification x40). (c and e) the tumor was composed of astrocytic cells with a piloid appearance (hps, magnification x400). (d and f) at the surface, there was a subpial proliferation of small immature cells (hps, magnification x400 and 600 for inserts). (g and i) tumor cells diffusely expressed olig2 whereas superficial immature cells did not (h and j) (magnification x400). the proliferation index was low in both tumors (k and m) whereas it was high in the superficial immature component (l and n). (o and q) a rearrangement of the braf gene was evidenced in tumor cells of the pilocytic astrocytomas with a duplication of the 3’braf signal, and was absent in the immature component (data not shown) (magnification x800). (p and r) the superficial cell component diffusely expressed insm1, an immature neuronal marker (magnification x400). hps: hematoxylin phloxin saffron. discussion and conclusions the mature cerebellar cortex is composed of three layers: the upper layer (stratum moleculare) containing a small amount of neurons (basket neurons and small stellate neurons), a middle layer (stratum gangliosum) where purkinje cells are found, and the deepest layer (stratum granulosum) comprised of tiny neurons (granule cells) and golgi cells (8,9). during the development of the cerebellum, is has been shown that granule cell precursors proliferate in the external granular layer and migrate through the molecular layer to the internal granular layer (8). subsequently, during the late postnatal period (2–8 months), there is a slow decrease in the width of the external granular layer which disappears between the eighth and eleventh months (8). the two current cases illustrate the development of pa during the early life of two infants. the persistence of the external granular layer of the cerebellar parenchyma suggests that the formation of the tumors may have started before 2–8 months of life or before birth. these immature neurons, which normally disappear after childbirth, constitute a histopathological diagnostic pitfall, which could possibly be confused with an embryonal tumor or an undifferentiated tumor component. this is particularly confusing because these immature neurons present high mitotic and proliferative indexes. however, these cells have a peculiar position, aligned at the surface of the cerebellar parenchyma. using immunohistochemistry, they only expressed insm1, a marker of immature neurons (10). moreover, the absence of the braf rearrangement, which was identified in the adjacent pa, argues against a poorly differentiated component. in rare cases, pa can present a rhythmic palisaded pattern, previously referred to as spongioblastoma, in reference to polar spongioblasts (neuroectodermal cells known to initiate the differentiation of radial glial cells) (11), which was not observed in the current cases. pilocytic astrocytomas represent the most common form of pediatric glioma and may occur in rare infantile cases (12). the persistence of an external granular layer of the cerebellum differs from the features of lhermitte-duclos disease where the cerebellar folia are replaced by large ganglion cells and inner granule cells are absent (6). in conclusion, the persistence of an external granular layer of the cerebellum may occur in cases with antenatal/perinatal tumor development and is not to be confused with embryonal/poorly differentiated neoplastic cells. declarations ethics approval this study was approved by ghu paris psychiatry and neuroscience, sainte-anne hospital’s local ethics committee. consent for publication the legal guardians signed informed consent forms before treatment. conflicts of interest statement the authors declare that they have no conflicts of interest directly related to the topic of this article. funding statement the authors declare that they have not received any funding. acknowledgements we would like to thank the laboratory technicians at ghu paris neuro sainte-anne for their assistance. references 1. jones dtw, kocialkowski s, liu l, pearson dm, bäcklund lm, ichimura k, et al. tandem duplication producing a novel oncogenic braf fusion gene defines the majority of pilocytic astrocytomas. cancer res. 2008;68(21):8673–7. https://doi.org/10.1158/0008-5472.can-08-2097 2. collins vp, jones dtw, giannini c. pilocytic astrocytoma: pathology, molecular mechanisms and markers. acta neuropathol (berl). 2015;129(6):775–88. https://doi.org/10.1007/s00401-015-1410-7 3. blümcke i, thom m, aronica e, armstrong dd, vinters hv, palmini a, et al. the clinicopathologic spectrum of focal cortical dysplasias: a consensus classification proposed by an ad hoc task force of the ilae diagnostic methods commission. epilepsia. 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improve distinctive criteria for infant-type hemispheric glioma versus desmoplastic infantile ganglioglioma/astrocytoma. brain pathol. 2023;e13182. https://doi.org/10.1111/bpa.13182 copyright: © 2024 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. neurodevelopmental disorders: 2022 update feel free to add comments by clicking these icons on the sidebar free neuropathology 3:8 (2022) review neurodevelopmental disorders: 2022 update miguel sabariego-navarro1, álvaro fernández-blanco1, cesar sierra1, mara dierssen1,2,3,4 1 center for genomic regulation, the barcelona institute for science and technology, barcelona, spain 2 neurosciences research program, hospital del mar medical research institute, barcelona, spain 3 university pompeu fabra, barcelona, spain 4 biomedical research networking center for rare diseases (ciberer), barcelona, spain corresponding author: mara dierssen · systems biology program · crg-center for genomic regulation · c/ dr. aiguader, 88 · prbb building · 08003 barcelona · spain mara.dierssen@crg.eu submitted: 09 february 2022 accepted: 08 march 2022 copyedited by: christian thomas published: 21 march 2022 https://doi.org/10.17879/freeneuropathology-2022-3801 keywords: mitovesicles, repeatome, trnas methylation, chromatin remodeler, autism spectrum disorder, aicardi-goutières syndrome abstract with a prevalence of 2-4% of the worldwide population, neurodevelopmental disorders (ndds) comprise a heterogeneous group of disorders associated with neurodevelopmental dysfunction, including intellectual disability (id), autism spectrum disorder (asd), down syndrome (ds) and attention-deficit/hyperactivity disorder (adhd) among others. however, due to their heterogeneity and overlapping clinical features, ndds such as asd are often misdiagnosed, while for others with more distinct symptoms, such as rett syndrome or ds, the mechanisms underlying their pathogenesis remain elusive. last year, important steps in the mechanistic understanding of several ndds have been achieved. new preclinical models demonstrated causality between pak3 mutations and disorders associated with social deficiencies. arid1b mutations have been linked to neuroectoderm specification in coffin-siris syndrome and dna damage was established as an important pathologic mechanism in aicardi-goutières syndrome. moreover, alterations in basic molecular processes including translation and histone acetylation have been established as major traits in the pathology of x-linked id and rett syndrome, revealing new pathogenetic mechanisms. last year, advances in bioinformatics have begun to shed light on the human repeatome, a largely unexplored part of our genome, and how alterations in these sequences have a central role in asd. the role of mitochondria in neuropathology was clarified last year with the discovery of previously unknown vesicles derived from mitochondria with a putative role in ds. an interesting discovery in the field of basic neurodevelopment showed that during postnatal brain development, changes in genome architecture and transcriptional dynamics progress independently of sensory experience. finally, our neurocentric views of ndds are changing as new players such as astrocytes are revealed to be crucial in neuropathology. the role of astrocytes has been clarified for some pathologies such as asd and ds, linking well-known genetic mutations to impaired astrocyte function. abbreviations adar1 double-stranded rna-specific adenosine deaminase, ags aicardi-goutières syndrome, asd autism spectrum disorder, atp adenosine triphosphate, cgas gmp-amp synthase, cko conditional knockout, dip-c diploid chromatin conformation capture, dna deoxyribonucleic acid, ds down syndrome, ev extracellular vesicles, gaba gamma-aminobutyric acid, γh2ax: h2a histone family member x, hipsc human induced pluripotent stem cells, iba1 ionized calcium-binding adaptor molecule 1, id intellectual disability, ifn interferon, ip3r2 2 inositol 1,4,5-trisphosphate receptors, lc-ms liquid chromatography-mass spectrometry, mecp2 methyl cpg binding protein 2, ndd neurodevelopmental disorder, rna ribonucleic acid, rnaseh2 ribonuclease h2, nmda n-methyl-d-aspartic acid or n-methyl-d-aspartate, nova1 neuro-oncological ventral antigen 1,   psd95 postsynaptic density 95, rtt rett syndrome, rps4y1 40s ribosomal protein s4, tads topologically associated domains, trs tandem-repeat sequence, trex1 three prime repair exonuclease 1, trna transfer rnas, xlid x-linked intellectual disability. introduction single-term search in the pubmed database with the search term “neurodevelopmental disorders” from january 1st to december 31st 2021 retrieved more than 10,000 papers. in this review, we selected the 10 most interesting discoveries in the field during the last year. instead of centering our efforts around papers published in the highest impact factor journals, we decided to select the discoveries that open new research avenues and that can be expected to have substantial implications in the study of neurodevelopmental disorders paving the way to a change of paradigms. the major steps made during 2021 comprise very different topics, from the discovery of new shared molecular mechanisms involved in several neurodevelopmental syndromes such as aicardi-goutières syndrome or coffin-siris to disentangling the complexity of autism spectrum disorders by the identification of new players at the network, on the cellular and the molecular levels. advancement in modeling tools holds the promise to not only increase our understanding of the origin of neurodevelopmental disorders, including their evolutionary aspects, but also to start proving causality. in the last year, the role of several underexplored biological underpinnings such as trnas methylation or mitochondrial-derived extracellular vesicles were clarified in neurodevelopmental disorders, opening new perspectives for diagnosis and treatment. finally, new tools appeared to study human neurodevelopmental disorders, partially solving some of the associated problems with the use of animal models. the final list of papers and topics of 2021, selected as very interesting and/or important for the field of neurodevelopmental disorders, comprises: reconsidering the molecular neuropathological basis of aicardi-goutières syndrome (ags). understanding cortical neurodevelopment from evolution. new 3d in vitro models to unveil neuropathological mechanisms of human neurodevelopmental disorders. new insights and tools to explore the genome architecture and transcriptional dynamics in the mammalian brain development. new cellular mechanisms and new tools to establish causality in asd. new mechanism in asd pathology: astrocytes, atp and gabaergic signaling. disentangling the role of repeatome in asd. key switch between chromatin remodeler baf subunits explains coffin-siris syndrome neuropathology. trnas methylation and intellectual disability. mitovesicles: a new player in the neuropathology of neurodevelopmental disorders. reconsidering the molecular neuropathological basis of aicardi-goutières syndrome (ags) aicardi-goutières syndrome (ags) is a rare neurodevelopmental disorder. the main neuropathological signs are progressive microcephaly associated with basal ganglia and white matter calcifications, leukodystrophy, cerebral atrophy, and variable increase of lymphocyte count in the cerebrospinal fluid. it is caused by recessive mutations in nine known genes identified until now, namely trex1, rnaseh2a, rnaseh2b, rnaseh2c, samhd1, adar1, mda5, lsm11 and rnu7-1 that result in upregulation of type i interferon (ifn) signaling and is accompanied by neuroinflammation [1]. increased type i ifn activity is detected in cerebrospinal fluid and serum, and increased expression of ifn-stimulated gene transcripts in peripheral blood. moreover, a positive correlation between the level of cerebrospinal fluid ifn activity and the degree of motor and intellectual disability has been reported [2]. astrocytes are the main cell type that produce ifn-α in the brain and magnetic resonance image studies in patients suffering ags show calcifications in the white matter and white matter signal changes in the frontotemporal regions with associated astrocytosis [3]. interestingly, ags-related genes are critical in nucleic acid metabolism function [4, 5], and involved in the removal of redundant endogenous or exogenous dna, rna, or dna/rna hybrids. it is assumed that failure to remove intracellular remnants of nucleic acids leads to stimulation of the innate and adaptive immune responses, so that type i interferonopathies would represent a failure of selfversus non-self-discrimination, as these essential antiviral systems can also be triggered by host dna and rna. the ribonuclease h2 (rnaseh2), linked to ags, is a genome surveillance factor, which has the role of constantly inspecting dna integrity and removing ribonucleotides that incorporate by error into replicating dna [2]. however, the role of ifn hyperactivity and rnaseh2 mutations and their possible functional interaction on ags neuropathology is poorly understood. although the neurotoxic potential of type i ifn was highlighted by studies that recapitulated the neuropathological features of ags in transgenic mice chronically producing ifn-α from astrocytes [6], available genetic ags mouse models fail to show type i ifn response and inflammation in the brain [7, 8]. now, aditi et al. have provided evidence that dna damage-dependent signaling rather than type i ifn signaling underlies neurodegeneration, in this class of interferonopathy. they generated a murine model of neural rnaseh2b inactivation (rnaseh2bnes-cre) [9]. these mice showed smaller cerebellar volume and reduced granule cell and interneuron number. the authors found that reduced cerebellar volume was explained by the loss of proliferative neurons as a consequence of dna damage-induced cell death. they found increased dna double-strand breaks in cells using the h2a histone family member x (γh2ax) marker. the authors used rna-sequencing to compare cerebellar tissue of rnaseh2bnes-cre and controls. they found that genes involved in type i ifn-α response pathway were particularly enriched in rnaseh2bnes-cre cerebellar cells indicating that rnaseh2 deficiency leads to an increased type i ifn response. consistent with the upregulation of neuroinflammatory genes in rnaseh2bnes-cre cerebellum, they also found astroand microglial genes such as glial fibrillary acidic protein (gfap) and ionized calcium-binding adaptor molecule 1 (iba1) were particularly upregulated in rnaseh2bnes-cre cerebellum. these data suggest an ongoing astrocytosis and microglial activation that are hallmarks of neuroinflammation. remarkably, inactivation of tumor protein p53, which induces apoptosis in damaged cells, rescued dna damage-induced cell death and cerebellar atrophy but led to accumulation of cytoplasmic dna damage that resulted in the development of medulloblastoma. the authors also demonstrated that neurodegeneration of rnaseh2bnes-cre mice was not driven by type i ifn signaling since the deletion of cyclic gmp–amp synthase (cgas), a factor that is required for type i ifn activation, led to similar phenotypes in rnaseh2bnes-cre mice. all these data together provide new levels of understanding about ags neuropathology and suggest that neuroinflammation is not the main cause of ags phenotypes, but is a consequence derived from increased dna damage due to rnaseh2b mutations. in this regard, those individuals with additional mutations in dna repair genes might be more prone to present exaggerated immune responses as a consequence of increased dna damage. this discovery constitutes a paradigm-shift in interferonopathies and changes the assumptions about the genetic and molecular causes of ags. reconsidering the leading cause of ags neuropathology can help not only to disentangle the complex neuropathology of this neurodevelopmental disorder, but also to specifically design strategies that would help to ameliorate the symptoms associated with dna damage-driven neuroinflammatory response in ags. understanding cortical neurodevelopment from evolution genetic disease is a necessary product of evolution. in fact, evolutionary pressures have established the risk for many neuropsychiatric or neurodevelopmental diseases. nearly all genetic variants that influence disease risk have human-specific origins. fundamental biological systems, such as dna replication, transcription and translation, evolved very early in the history of life, to give rise to cellular life, but also created the potential for disease. among those, alternative splicing is a form of genetic regulation that enables the production of multiple proteins from a single gene. differences in splicing arose rapidly during a recent evolutionary transition and appear to contribute to adaptation but also to brain disorder. it is known that the transcriptional machinery is often mutated in neurodevelopmental disorders [10]. the brain displays the most complex pattern of alternative splicing of the body [10], with neuron-specific alternative exons such as dna-binding proteins and histone modifying enzymes that regulate evolutionarily conserved transcription. given that alternative splicing is necessary for the proper cortical development [11, 12], a better understanding of how mutations in genes associated with alternative splicing would help to unravel the enormous complexity of several ndds. one interesting example is the neuro-oncological ventral antigen 1 (nova1), which encodes a rna-binding protein that acts as a brain-specific splicing factor. it regulates neuronal alternative splicing of genes responsible for synapse formation in the developing nervous system [13]. increasing evidence has implicated nova1 in numerous pathological processes and neurodevelopmental disorders [14, 15]. mutations in nova1 have been associated with impairment in the development of the motor system [16] and severe neurodevelopmental delays [17]. last year, a comprehensive analysis of genetic variation between modern humans and neanderthals performed by trujillo et al. detected a specific nova1 genetic variation [18]. even though the genomes of humans and neanderthals are very similar, this archaic nova1 version could help to explain how nova1 variants can differentially regulate alternative splicing during human brain development. to study the functional relevance of nova1 mutations, authors used crispr-cas9 genome-editing technology to introduce the archaic variant into the genome of human induced pluripotent stem cells (hipscs). hipscs carrying the archaic mutation (nova1ar/ar) and control hipscs (nova1hu/hu) were used to generate cortical organoids to investigate the impact of the nova1ar/ar variant on the human brain. trujillo and colleagues found that nova1ar/ar cortical organoids were smaller than nova1hu/hu, a phenotype that was explained by a reduced number of rosettes and a higher number of apoptotic cells in nova1ar/ar along with a reduced number of proliferating cells compared to the nova1hu/hu organoids. the authors also found several genes differentially expressed in nova1ar/ar such as rps4y1 and nnat, which are involved in neuronal differentiation, tdgf1, involved in cell migration and proliferation, or pax6 and lhx5, transcription factors associated with cell differentiation during brain development. trujillo and colleagues investigated the gene-splicing variants of nova1ar/ar and nova1hu/hu using single-nuclei rna-seq and identified differences in alternative splicing events affecting 122 genes. remarkably, most of the differentially spliced genes in nova1ar/ar organoids are involved in synaptogenesis and neuronal connectivity. more in detail, nova1ar/ar cells had reduced levels of preand postsynaptic markers such as psd95 and nmda, closely related with activity-dependent plasticity. finally, they evaluated how these synaptic changes could impact neuronal network activity using a multielectrode array that measures several electrophysiological properties. nova1ar/ar cells showed increased number of bursts, lower synchrony and increased variability compared with nova1hu/hu organoids (fig. 1). overall, their results suggest that the archaic nova1 variant causes changes in alternative splicing of genes involved in brain development, proliferation and synaptic plasticity that might explain how mutations of the nova1 gene can contribute to different alterations in ndds. specifically, nova1 would negatively impact neuronal proliferation and survival, but also synaptic connectivity as a result of the reduced expression of proteins important for activity-dependent plasticity. this new study helps disentangle the evolutionary origins of ndds, also pointing to relevant biological processes. fig. 1. schematic representation of how the archaic nova1 variant affects cellular, molecular and neuronal network activity in cortical organoids. a) nova1 archaic version was introduced in ipscs derived from humans. nova1hu/hu and nova1ar/ar hipscs were differentiated into cortical organoids and their cellular, molecular and physiology were studied. b) size of nova1ar/ar organoids was smaller than nova1ar/ar organoids due to decreased neuronal proliferation and increased apoptosis. single-nuclei rna-seq revealed reduced levels of synaptic proteins such as psd95. the study of electrophysiological properties by means of a multielectrode array revealed an increased burst activity in nova1ar/ar cells along with higher variability and reduced levels of synchrony when compared to nova1hu/hu. new 3d in vitro models to unveil neuropathological mechanisms of human neurodevelopmental disorders until recently, the study of neurodevelopmental disorders (ndds) has mainly relied on in vivo animal models to characterize the changes occurring during early brain development. however, the establishment of in vitro models of brain development derived from hipscs represents an unprecedented opportunity to study key brain developmental processes, such as neuronal cell morphogenesis, migration and connectivity in human tissue. although this field is still in its infancy, an increasing number of studies demonstrate its potential to shed light on the mechanisms underlying ndds. this potential is illustrated by recent advances in the study of rett syndrome (rtt), a ndd characterized by cognitive, speech, behavioral and motor problems, that starts in the first year of life and that predominantly affects females. rett syndrome is a rare and complex x-linked neurodevelopmental disorder associated with mutations in methyl cpg binding protein 2 (mecp2) that cause its loss of function. mecp2 is an epigenetic reader that binds to methylated dna to promote transcriptional repression. male homozygous mecp2-/y mice are the most frequently used rtt model due to the early development of severe phenotypes, but even though the use of these models has significantly contributed to the understanding of the mechanisms underlying rtt, their clinical relevance might be limited. in fact, their face validity is constrained as male mice are used to model a disease mainly affecting females, and female patients have a milder phenotype than the one observed in mice. to overcome these limitations, several methodologies have been established to model rtt with hipscs from patients by developing 2d-based neuronal differentiation protocols. more recently, the generation of 3d human brain organoids, a more complex approach that better recapitulates human neurodevelopment in vitro, has been established. these culture platforms are increasingly contributing to a better understanding of the rtt etiology. the tremendous potential of such methodologies is illustrated by the recent work of samarasinghe et al. [19] who showed that brain organoids derived from induced pluripotent stem cells from individuals with rtt reproduce patterns of electrical brain activity that resembled seizures, a hallmark of the condition. using calcium sensor imaging and extracellular recording approaches, they demonstrated highly abnormal and epileptiform-like activity, accompanied by transcriptomic differences revealed by single-cell analyses. interestingly, trujillo et al. [20] used an innovative pipeline to model rtt neurodevelopment demonstrating that neurons differentiated from human mecp2-ko hipscs show altered expression of synapse-relevant genes, synaptic morphology, and decreased calcium and network activities compared to control neurons. an in silico neural network simulation affirmed that synaptic structural parameters link with neural network activity, supporting the approach of rescuing synaptic structure and increasing activity. they differentiated mecp2-ko hipsc into in vitro models of increasing complexity. first, 2d monolayer neuronal cultures revealed an impaired neurotransmission. the same hipsc were then used to produce a novel system of 3d brain neurospheres derived from a 50/50 mixture of healthy and mecp2-ko hipsc to better mimic the mosaicism observed in female patients, a phenomenon produced by random x-inactivation. this new model will be of great utility to investigate rtt and other x-linked genetic conditions. as a last step, complex 3d mecp2-ko cortical organoids were also produced. interestingly, this pipeline revealed important features of rtt pathology. the researchers found that rtt organoids displayed smaller diameters and both mecp-ko neuronal cultures and organoids showed that synaptic and neurotransmitter pathophysiology principally concentrated in glutamatergic and cholinergic dysregulation. furthermore, nefiracetam, a cholinergic, gabaergic, and glutamatergic agonist, and pha 543613, a α7-nachr agonist, had a rescuing effect supporting glutamatergic and cholinergic dysregulation. new insights and tools to explore the genome architecture and transcriptional dynamics in the mammalian brain development neurodevelopmental processes rely on the orchestration of finely tuned gene expression programs, which depend on the regulation of chromatin structure. over the last few years, mutations in genes encoding factors that can modify chromatin regulation and nuclear architecture have emerged as a frequent cause of ndds. post mortem studies on brains from patients with different ndds found chromatin and other epigenetic alterations linked with abnormalities in neural development. this is the case of opitz-kaveggia and fryns-lujan syndromes, caused by mutations in med12 [21], and cornelia de lange syndrome, which results from mutations in nipbl or in the cohesin subunits coding genes smc1 and smc3 [22]. mutations in the ctcf gene are also associated with intellectual disability [23, 24]. on the other hand, our knowledge of the critical role of the genome’s 3d organization in gene regulation is steadily increasing. this is important since the expression of a given gene in a specific time and cellular type is known to depend on its location within the nucleus and on its contacts with other loci, forming topologically associated domains (tads). however, although previous studies have contributed to characterize the transcriptome and 3d genome in the brain, they failed to answer this question mainly due to the use of bulk chromatin conformation capture techniques, which can only assess averaged maps of the 3d genome. this constitutes an important limitation in a highly heterogeneous tissue such as the brain. now, tan et al. [25] have overcome this limitation thanks to the use of a new diploid chromatin conformation capture (dip-c) method [26], a chromatin conformation capture method that allows to distinguish between the two haplotypes of each chromosome, which allowed them to create a 3d genome atlas of the developing mouse forebrain, including ~2,000 single-cell contact maps and ~800 3d structures from two brain regions and 6 time points. in parallel, they also produced a single-cell transcriptomic atlas to shed light on the “structure-function” relationship question during neurodevelopment. importantly, this approach revealed a major post-natal transcriptomic and 3d structure reorganization. in other words, the gene expression and chromatin architecture profiles of postmitotic neurons continue to mature during postnatal development. at the 3d genome architecture level, the radial positioning (preference for the nuclear periphery or nuclear core) of genes was the most prominent difference between neonatal and adult neurons. a similar phenomenon was previously thought to be unique for certain gene clusters, such as olfactory receptors (or), in olfactory receptor neurons [27] development. importantly, during developmental stages, ors genes move to the nuclear interior, detaching from the nuclear laminin, thereby allowing their activation, to ensure that each olfactory receptor neuron expresses one, and only one, or, a phenomenon called the “one neuron-one receptor expression” [27]. these results, however, show that this behavior might represent a generalizable principle of neuron development not restricted to olfactory receptor neurons. strikingly, tan et al. also showed that these transformations of the 3d genome are independent of sensory experience, suggesting that the 3d transformation is genetically predetermined. altogether, these results provide new tools and a conceptual framework to shed light on the molecular mechanisms governing neurodevelopmental processes at an unprecedented resolution. new cellular mechanisms and new tools to establish causality in asd throughout the last years, advances in the field of genetics have led to the discovery of hundreds of genes that contribute to serious deficits in communication, social cognition, and behavior of autism spectrum disorder (asd). however, these genes only account for 10–20% of cases, and similar pathogenic variants may lead to very different levels of affectation. in fact, understanding causal relationships between genes and phenotypes remains a challenge. recent evidence suggests that dysfunction of rho family guanosine triphosphatases (rho-gtpases) contributes substantially to the pathogenesis of ndds [28, 29], and as many as 20 genes encoding rho gtpase regulators and effectors are listed as asd risk genes [30, 31]. those genes are promising candidates to explain asd neuropathology. rho gtpase signaling is essential for cellular signaling and cytoskeleton dynamics [32, 33]. one interesting example is the p21-activated kinase 3 (pak3), which is a downstream component of the rho-gtpase cascade. genetic studies in humans revealed that mutations of pak3 are associated with moderate to severe intellectual disability (id) and with synaptic alterations in post mortem human tissues [34-36]. animal models with pak3 mutations (mpak3) also recapitulate these phenotypes and have shown that pak3 is important for recognition memory [37, 38]. in fact, pak activity, in combination with rac (a rho-gtpase), has been suggested to be responsible for the autistic cellular and clinical phenotypes in mouse models of asd, and may be related to synaptic plasticity alterations [39]. however, a causal link between these mutations and autistic phenotypes has not yet been directly demonstrated. one possible problem is the lack of spatial-temporal resolution of the investigations. for example, a gene mutation may have specific neuropathological impact on a subpopulation of cells, which cannot be selectively targeted. this is the case with memories, which are believed to be stored in a subpopulation of neurons that get activated and modify their functional properties during learning. those are called engram cells [40, 41], and might be specifically affected by pak3 mutations. for this reason, zhou et al. designed a tetracycline-inducible system (tta/teto) under the control of cfos, to express a mutant pak3 form tagged with green fluorescent protein (mpak3-gfp) specifically in neurons that were activated during social interaction [42]. cfos is an immediate early gene associated with neuronal activity and synaptic plasticity and is often used to identify engram cells [40, 41, 43]. the authors showed that disrupting pak3 signaling by the expression of mpak3 in cfos-expressing neurons during social interactions in fact was sufficient to impair social memory. contrarily, rescuing mpak3 levels in the same animals restored social memory deficits. the fact that pak3 disruption in just a subset of cfos positive cells is sufficient to produce social memory impairment underscores the critical importance of increasing the resolution of the techniques we use. in this regard, an increasing number of studies are using single-cell omics for resolving the cell-specific impact of specific mutations, and single-cell rna seq data are available from post mortem neurotypical human brain regions. even though the mechanisms by which mpak3 was altering social memory engrams were not covered in this study, to date this is the first experimental demonstration that establishes the necessity of pak-dependent molecular changes in engram cells underlying social memory pathology. certainly, this cell-specific system can be used to establish molecular causality of memory deficits in different mouse models of ndds given the ability to fine-tune the expression of particular genes that could be potentially implicated in the neuropathology of these disorders in an inducible manner. increasing the resolution and selectivity of our experimental approaches will certainly uncover new neuropathological mechanisms and possibly help to understand the actual contribution of risk genes to the pathogenesis of autism and other ndds. new mechanisms in asd pathology: astrocytes, atp and gabaergic signaling the research in asd has traditionally focused on neuronal dysfunction, as many asd genes encode for proteins that have a role in synaptic function, thus making the views of the pathological features of asd mainly “neurocentric”. however, selectively focusing on neuronal mechanisms has proven to be limited, and thus, alternative biological analyses may help to reveal previously unknown cellular and molecular mechanisms. astrocytes are newly identified players in several brain pathologies, and could play an important role in asd. in fact, rna sequencing revealed a close association between asd and the genes related to glial cell activation, immune and inflammatory categories [44]. studies in post mortem brain tissue from donors affected by asd have shown alterations in the expression of astrocyte markers, and ipscs derived from asd patients present alterations in astrocytic development [45, 46]. one recent preprint shows that astrocytes from asd patients cause autistic phenotypes when engrafted in mouse pups, establishing an important milestone in the asd field [47]. however, whether astrocytes alterations are the cause or the consequence of asd and how this glial cell type contributes to asd pathology remains unclear. interestingly, de novo mutations in type 2 inositol 1,4,5-trisphosphate receptors (ip3r2) gene have been identified in asd patients [48]. this receptor mediates the activation of astrocytes by increasing cytoplasmic calcium signaling [49], and previous studies in ip3r2 knockout (ip3r2 ko) mice showed selective dysfunction of astrocytes but not neurons leading to social behavior impairment, as revealed by increased repetitive behaviors and impaired social approach [50]. wang et al. [51] now show that astrocyte calcium signaling dysfunction can result in asd-like behavior in mice. the authors generated a conditional knockout (cko) mouse for ip3r2 using an astrocyte-specific promoter, aldh1l1, that induces the ip3r2 knockout during adulthood. this astrocyte specific ip3r2 ko in adult mice was sufficient to produce autism-related behaviors, suggesting that these phenotypes are not just a consequence of altered neurodevelopment. to further investigate the mechanism underlying social behavior alterations and astrocyte dysfunction in ip3r2 mutant mice, they analyzed the levels of different gliotransmitters. they found a strong reduction in atp levels, a known gliotransmitter, in ipr32 cko mice. this reduction in atp concentrations was only found in astrocytes but not in neurons isolated from ipr32 cko mice, indicating astrocytes-specific alterations. using chemogenetic techniques and an atp sensor under an astrocytic promoter, they demonstrated that atp release was significantly reduced in ip3r2 cko and that atp administration led to a total rescue of social interaction. this indicates the need of atp release by astrocytes for proper social interaction. this finding establishes a new pathogenic mechanism: lower release of atp after astrocyte activation leads to an increase of gabaergic inhibitory activity. however, the pathogenetic mechanisms are certainly more complicated, as shown by other recent studies reporting that astrocyte dysfunction could lead to an altered microglial state [52], and lead to increased neuroinflammatory processes. in conclusion, astrocyte activity, mediated by calcium signaling pathways, plays a pivotal role in asd pathology. disentangling the role of repeatome in asd approximately half of the human genome consists of repetitive dna sequences, and is called the repeatome. repetitive dna consists of more than one million tandem repeats, sections of dna in which a sequence is replicated many times in tandems, interspersed repeats and copy number variants, which biology remains under-studied and poorly annotated, being considered as “junk dna” compared to the protein coding dna [53]. however, more than 50 diseases such as huntington disease, fragile x, various ataxias and a major subset of amyotrophic lateral sclerosis and frontotemporal dementia cases are caused by an expansion of a tandem-repeat sequence (trs), showing the importance of this non-coding dna in some pathologies [54]. repeat-associated non-atg translation (ran translation) of toxic peptides may contribute to the pathogenesis of those disorders and emerging evidence suggests that trs can also regulate gene expression in healthy individuals [54]. nonetheless, the role of trs in polygenic pathologies such as asd remains largely unexplored. even though changes in the number of copies of large segments of dna have been implicated in asd pathology [55], the capacity to analyze alterations in trs from a genome-wide point of view was, until now, unachievable. mitra et al. have now developed a new bioinformatic tool capable of analyzing trs in people with asd and their family members [56]. this new bioinformatic tool that the authors named monstr, allowed the researchers to discover that trs expansions are more common in people with asd than in their unaffected siblings. the expansions occurred in genes involved in brain development such as foxp1. some genes previously associated with asd, e.g. pdcd1 or kcnb1 present some tandem-repeat mutations, increasing the possibility that these repeats are involved in asd pathology. in a recent paper, trost et al. [57] also analyzed the role of trs in asd but with a different approach, not taking into account the appearance of de novo mutations. this study used three times more genomes as compared to the analysis of mitra et al., thus substantially increasing the statistical power. although the studies identified different mutations, probably due to the differences in their methodological approach, they were more complementary than controversial. interestingly, the study from trost et al. relates several trs expansions with particular clinical features like cognitive disturbances. the findings open a new research line that could contribute to disentangle the contribution of trs to other neurodevelopmental disorders and pathologies. however, the most critical question now is how these alterations in trs generate disturbances in neurodevelopmental processes, accounting for the multifactorial nature of asd with the environment playing a pivotal role, thus opening the question of how the environment interacts with these alterations in trs. key switch between chromatin remodeler baf subunits explains coffin-siris syndrome neuropathology the brain exhibits an extraordinarily precise morphogenesis that leads to the correct cell types and proportions at the appropriate sites. during the neurodevelopmental processes that lead to the formation of the central nervous system, cell fate is acquired by the decline in neural stem cell self-renewal and amplification potential to give rise to fate-committed progenitors that will ultimately undergo differentiation into neurons (neurogenic) or glial cells (gliogenic) [58]. the establishment of these cell fates requires the orchestration of timely regulated gene expression programs which, in turn, depend upon epigenetic and chromatin regulators. atp-dependent chromatin remodeling factors have been described to be vigorous regulators of chromatin state and dna accessibility, both of which have an important impact on the coordination of gene expression. interestingly, a substantial proportion of ndd associated genes are involved in chromatin and/or transcriptional regulation including the broad family of atp-dependent chromatin remodelers including the multi-subunit brg1/brm associated factor (baf) chromatin modifier family, also known as switch/sucrose non-fermentable (mswi/snf) chromatin remodeling complex. a dna-binding domain targets the swi/snf complex to specific genes and facilitates dna access for transcription factors. these complexes play a central role in different developmental processes. all described canonical configurations of baf require the presence of a subunit containing an at-rich dna binding domain (arid). namely, arid1b and its paralog arid1a encode for the two largest, mutually exclusive, subunits of the complex: arid1a and arid1b. interestingly, the arid1b gene, but not arid1a, is among the most commonly mutated genes in asd and in syndromic and non-syndromic ids [59]. mutations observed include deletions and truncations due to de novo nonsense or frameshift mutations as well as translocations leading to arid1b haploinsufficiency in the individuals with intellectual disability. furthermore, de novo haploinsufficient mutations in this gene are found in ~75% of coffin-siris syndrome patients, a neurodevelopmental condition characterized by learning disability and craniofacial features. despite the biochemical knowledge on their mechanisms of action, the roles of chromatin remodeling factors during brain development have not been identified. furthermore, the brain defects that arise as a consequence of changes in chromatin structure, dynamics and function remain largely unexplored. paglaroli et al. [60] have studied the molecular role of arid1b in cell fate commitment during neurodevelopment. using arid1b+/– coffin-siris patient-derived hipscs, they described for the first time that a switching mechanism between arid1b containing baf (arid1b-baf) and arid1a-baf is required for lineage specification and for exiting the pluripotent state. in pscs, pluripotency is conveyed via binding of an arid1a-containing baf to pluripotency-associated enhancers of the sox2 and nanog networks. in order to perform transition to a multipotent state, arid1b-baf is transiently activated to replace arid1a-baf at the sox2/nanog enhancers and elicits their repression (see fig. 2). the haploinsufficient mutation of arid1b in coffin-siris syndrome impairs the switch from arid1a-baf to arid1b-baf, thus maintaining arid1a-baf at pluripotency enhancers. this leads to aberrant binding of sox2 that interferes with the gene expression patterns required for neurodevelopment. altogether, these alterations result in an impaired cranial neural crest differentiation that accounts for the phenotypes observed in coffin-siris patients. these findings provide the molecular mechanisms underlying not only coffin-siris syndrome but also in some non-syndromic ids and asd in which arid1b is frequently mutated [59]. fig. 2. schematic representation of arid1a-baf switch to arid1b-baf during psc to multipotent state transition (top). in arid1b+/mutations, arid1a-baf is maintained at pluripotent enhancers, leading to an impaired neurodevelopment (bottom). trnas methylation and intellectual disability transfer rnas (trna) are small and highly abundant rna molecules in the cell, representing around 10% of the total cell’s rna pool [61]. trna help decode a messenger rna (mrna) sequence into a protein, coupling transcriptional and translational processes. trnas are highly structured and contain a high number of post-translational modifications such as methylation and acylation. these modifications contribute to the stability and integrity of trnas, to the codon-anticodon interaction, which contributes to translation efficiency and to the rna quality control and regulation of trna localization in the cell. neuronal function requires high translation efficiency due to the high translation rate and alterations in translation are now considered a pivotal pathogenic mechanism in several neurologic disorders, especially in ndds [62]. one of the most common trna modifications is methylation, with more than 30 methylated nucleotides found at different positions in trnas. trnas methylation is fundamental for the normal function of different organs, and the disruption of this process is associated with cancer, neurodegeneration and intellectual disability (id) and neurological phenotypes are often the primary manifestation of mutations affecting the trna regulome [63, 64]. trna methyltransferase genes such as trmt10a, trmt1, ftsj1, elp1, wdr4 and nsun2 have been linked to different forms of id [65-67]. the human ftsj1rna 2’-o-methyltransferase 1 (ftsj1) gene is located on the x chromosome and several mutations in this gene have been associated with the development of x-linked intellectual disability (xlid) in several families [68]. nagayoshi et al. have analyzed why the loss of ftsj1 and its methyltransferase activity would lead to the development of id despite its ubiquitous expression [69]. using ftsj1 ko mice and xlid patient-derived cells, they observed a reduction in 2’o-methylated nucleotides in 11 species of trna including trnaphe, a ftsj1 substrate. among all trna species, trnaphe is the most reduced in ftsj1 ko mouse brain compared to the subtle reduction of the other trnas. trna-sequencing demonstrated an accumulation of fragments of trnaphe in the ftsj1 ko mouse brains. these findings revealed that the absence of the ftsj1 methylation leads to a cleavage in specific positions, and, ultimately, to perturbed decoding at phenylalanine (phe) codons. the authors hypothesized that alterations in phe codon translation leads to dysfunction in the translation of fundamental proteins for neuron function as they found dysregulation in genes involved in synapse organization and cell projection, including wnt7b and ctnnb1 as two interesting candidates related to the wnt signaling pathway [70]. ftsj1 ko showed an increase of thin dendritic spines indicating immaturity of synapses, and a decrease in the length and width of the postsynaptic density indicating alterations in connectivity. finally, the authors demonstrate that ftsj1 ko mice show altered ltp and ltd, and behavioral impairment with increased anxiety, slower spatial learning and poor associative learning. these results establish for the first time how alterations in the normal physiology of trna methyltransferases lead to aberrant translation, altered cell biology, impaired electrophysiological properties and, ultimately, behavioral deterioration. mitovesicles: a new player in the neuropathology of neurodevelopmental disorders mitochondria, the organelles classically seen as the powerhouse of the cell, also play a role in a plethora of crucial cellular functions, from regulation of calcium homeostasis, initiation of apoptosis, thermoregulation, red-ox balance, to epigenetic regulation by means of methylation, acetylation, or phosphorylation. genes encoding mitochondrial proteins are increasingly associated with a wide variety of ndds such as epileptic encephalopathy, id, or asd and recent reports show that proteins associated with intellectual disability are linked to mitochondrial function. interestingly, mitochondrial components can be mediators for intraand inter-cellular communication under physiological and pathological conditions, being transferred through extracellular vesicles (evs). the study of evs, defined as heterogeneous nanoscale vesicles secreted into the extracellular space, is a growing field in biomedicine and particularly in neuroscience due to its potential as a biomarker. microvesicles and exosomes are the main types of ev, with subtle differences in composition [71]. in a recent study, the ability of evs to transfer mitochondrial components was examined in fmr1 knockout mice, a model of fxs [72]. the authors found reduced mitochondrial components in mitochondrial fractions from cortical tissues and astrocytes of fmr1 ko mouse brains, and were able to monitor mitochondrial dysfunction in evs. in fact, mitochondrial components are the least studied components in evs [73]. during years, there was a controversy about the nature of this secreted mitochondrial material with evidences suggesting the secretion of exosomes packed with mitochondrial material or the secretion of whole mitochondria, leading to exchange of these organelles between cells [74-76]. however, the lack of standardized methodologies to accurately separate these different components impeded the proper study of the biological mechanisms behind the different subtypes of evs [77]. d’acunzo et al. now developed a new method based on density gradients to achieve high resolution isolation of different ev subpopulations. this new method led to the finding of a new class of previously unknown, mitochondria-derived evs [78]. first, by using a high-resolution step gradient, the authors purified different ev fractions from mouse brains and demonstrated that the content and composition of evs was different between the isolated fractions. using cryogenic electron microscopy, they identified three brain ev subtypes based on protein content, morphology, electron density and size. then, they analyzed the protein composition of these different fractions, and identified a new class of vesicles with high abundance in mitochondrial markers, “mitovesicles”, of mitochondrial origin. using liquid chromatography-mass spectrometry (lc-ms) on the fraction enriched with mitovesicles, the authors provide data about their protein composition, with 279 mitochondria-specific proteins of 673 proteins identified in this fraction. alterations in mitochondria, from the fetal to the adult stage, are a pathological signature of patients with down syndrome (ds) [79]. the authors hypothesized that the newly described mitovesicles were aberrant in ds. in a trisomic mouse model of ds, ts[rb(12.17)]2cje (ts2), the authors found alterations in mitochondrial proteins in ev fractions and a higher number of mitovesicles in the parenchyma. they replicated these results in humans using post mortem brains of individuals with ds. although the pathogenic role of these alterations in number and in protein composition of mitovesicles remains unknown, ds cells are deficient in the elimination of mitochondria, a process called mitophagy, and thus, the excess of mitovesicles could contribute to the removal of damaged mitochondria, working as a homeostatic process to reduce cellular stress. another possibility is that mitovesicles may activate microglia through acting as proinflammatory agents, which would explain the over-activated microglia detected in several ds mouse models [80]. this study describes and characterizes for the first time a new type of ev derived from mitochondria and also describes a potential role in an ndd such as ds. discussion the complexity and heterogeneity of the myriad of ndds makes it difficult to decipher the ultimate mechanisms of ndd neuropathology. accumulating evidence suggests that their multidimensional nature can be explained by the interaction of numerous genetic and environmental factors. luckily, we are now facing exciting times in which we can study neurodevelopmental disorder from a holistic perspective using advanced omics and cell engineering techniques that are increasing the temporo-spatial resolution of our mechanistic understanding. techniques such as single cell omics or spatial transcriptomics and proteomics help us to unravel the enormous diversity and complexity of both cell composition and cell type-specific molecular signatures throughout the brain. the number of publications associating new cellular players such as the astrocytes with brain alterations have increased exponentially in the last three decades suggesting an important role of this cell type in the onset and progression of several ndds. every year, selected discoveries open new avenues to investigate ndds from completely unexplored perspectives and serve as building blocks to better comprehend the enormous complexity of ndds, hopefully leading to better diagnostics and new therapeutic avenues. acknowledgements the lab of md is supported by the secretaria d’universitats i recerca del departament d’economia i coneixement de la generalitat de catalunya (grups consolidats 2017 sgr 926). we also acknowledge the support of the agencia estatal de investigación (pid2019-110755rb-i00/aei / 10.13039/501100011033), h2020 sc1 go-ds21-848077 and 101057454-psych-strata, jerôme lejeune foundation #2002, nih (grant number: 1r01eb 028159-01), fundació la marató-tv3 (#2016/20-30), ministerio de ciencia innovación y universidades (rtc2019-007230-1 and rtc2019-007329-1). cs received an fi grant (2020fi_b2 00143) from the agència de gestió d’ajuts universitaris i de recerca (agaur) de la generalitat de catalunya; ms received an fpu fellowship (fpu19/04789) and afb received an fpi-so fellowship (pre2018-084504) of the ministerio de universidades, spain. we acknowledge support of the spanish ministry of science and innovation to the embl partnership, the centro de excelencia severo ochoa and the cerca programme /generalitat de catalunya. the ciber of rare diseases is an initiative of the isciii. references crow, y.j. and n. manel, aicardi-goutieres syndrome and the type i interferonopathies. nat rev immunol, 2015. 15(7): p. 429-40. crow, y.j., et al., characterization of human disease phenotypes associated with mutations in trex1, rnaseh2a, rnaseh2b, rnaseh2c, samhd1, adar, and ifih1. am j med genet a, 2015. 167a(2): p. 296-312. 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down syndrome: molecular mechanisms and therapeutic targets. mol med, 2018. 24(1): p. 2. pinto, b., et al., rescuing over-activated microglia restores cognitive performance in juvenile animals of the dp(16) mouse model of down syndrome. neuron, 2020. 108(5): p. 887-904 e12. copyright: © 2022 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. mesial temporal lobe epilepsy and hippocampal sclerosis associated with brafv600e mutant neurons in the cornu ammonis: an uncertain pathogenesis and a diagnostic challenge feel free to add comments by clicking these icons on the sidebar free neuropathology 5:18 (2024) case report mesial temporal lobe epilepsy and hippocampal sclerosis associated with brafv600e mutant neurons in the cornu ammonis: an uncertain pathogenesis and a diagnostic challenge samir alsalek1*, alexander s. himstead2,3*, scott self2, gianna m. fote2, sumeet vadera2, edwin s. monuki3, mari perez-rosendahl3, william h. yong3 kaiser permanente bernard j. tyson school of medicine, 98 s los robles ave, pasadena, ca 91101, usa department of neurosurgery, university of california, irvine, 200 south manchester ave, suite 210, orange, ca 92868, usa department of pathology and laboratory medicine, university of california, irvine, 101 the city drive south, orange, ca 92868, usa * the authors contributed equally to this manuscript corresponding author: william yong · department of pathology and laboratory medicine · uc irvine health, school of medicine · 101 the city drive south · orange, ca 92868 · usa yongwh@hs.uci.edu additional resources and electronic supplementary material: supplementary material submitted: 03 february 2024 accepted: 13 august 2024 copyedited by: félicia jeannelle published: 21 august 2024 https://doi.org/10.17879/freeneuropathology-2024-5269 keywords: hippocampal sclerosis, epilepsy, braf, fancl, cd34, mtle, brain tumor abstract mesial temporal lobe epilepsy (mtle) is a common cause of seizures, and hippocampal sclerosis (hs) is the predominant subtype. brafv600e mutations in mtle-hs have only been reported infrequently. herein, we illustrate the neurologic, radiological, and histopathological details of a patient with mtle-hs and brafv600e mutant neurons. a 31-year-old male with medically refractory epilepsy presented with magnetic resonance imaging (mri) and electroencephalography (eeg) findings typical of mesial temporal sclerosis without a mass lesion. the surgical specimens showed ilae type 1 hs with neurons immunopositive for brafv600e mutant protein distributed along the cornu ammonis (ca) curvature. instead of the normal mostly perpendicular orientation of pyramidal neurons relative to the hippocampal surface, the braf mutant neurons were often oriented in a parallel manner. on cd34 immunostaining, sparse clusters or nodules of cd34+ stellate cells and single immunopositive stellate cells were identified. brafv600e or cd34 immunopositive cells were less than 1 % of total cells. the patient responded well to surgery with no further seizures after 2 years and occasional auras. hippocampal braf mutant non-expansive lesion (hbnl) has been used to describe such lesions with preserved cytoarchitecture and without overt tumor mass. others may argue for the dual pathology of hs with early ganglioglioma. whether pre-neoplastic lesions or early tumors, these cases are important for understanding early glioneuronal tumorigenesis and suggest that brafv600e studies should be routinely performed on mtle-hs cases in the setting of clinical trials. with next-generation sequencing, a fancl deletion was detected in almost half of the alleles in our case, suggesting that many of the histologically normal-appearing cells of the hippocampus contain this alteration. fancl mutations can result in cytogenetic anomalies and defective dna repair and therefore may underlie the development of a low frequency braf alteration. abbreviations ca cornu ammonis; eeg electroencephalography; fancl fanconi anemia complementation group l; gtc generalized tonic-clonic; hbnl hippocampal braf mutant non-expansive lesion; hs hippocampal sclerosis; ihc immunohistochemistry; ilae international league against epilepsy; ipi inciting precipitating injury; leats low-grade epilepsy-associated tumors; mri magnetic resonance imaging; mtle mesial temporal lobe epilepsy; pcr polymerase chain reaction; plnty polymorphous low-grade neuroepithelial tumor of the young; seeg stereoelectroencephalography; t1 mp-rage t1-weighted magnetization prepared rapid gradient echo; t2/flair t2-weighted-fluid-attenuated inversion recovery. introduction among the various causes of epilepsy, mesial temporal lobe epilepsy (mtle) is noteworthy for its variability in presentation and resistance to anti-epileptic drug treatment.1–3 while mtle has myriad etiologies, including focal cortical dysplasia, vascular lesions, tumors, and ischemia, hippocampal sclerosis (hs) remains the most common.4 the natural history of this entity classically involves an inciting precipitating injury (ipi) and subsequent development of epilepsy after a variable latency period.1 the disease is heterogeneous in clinical presentation, response to therapy, ictal semiology, and ictal electroencephalography (eeg).5 similarly, the histopathological findings are diverse and include different patterns of granule cell layer alterations, neuronal loss, and gliosis in different regions of the hippocampus and amygdala.6 in 2013, the international league against epilepsy (ilae) defined several patterns of hs: type 1 involves severe neuronal loss and gliosis in the cornu ammonis (ca) 1 and 4 regions; type 2 involves neuronal loss and gliosis predominantly in ca1; and type 3 involves loss mainly in ca4.7,8 the fourth category, no-hs, shows reactive gliosis without neuron loss.8 immunohistochemistry (ihc) for cd34 may highlight multipolar or stellate cells. in a very small fraction of hs cases, brafv600e immunostaining of neurons with cytoarchitectural preservation of the hippocampus has been reported and named braf+ hs or hippocampal braf mutant nonexpansive lesion (hbnl).6,9 however, this entity is controversial, as some consider such lesions to represent dual pathology (hippocampal sclerosis with ganglioglioma) and requires further study. prior studies have searched for associations between histopathology, ihc, clinical features, and outcomes in patients with mtle-hs; these suggest that different histopathological patterns may be linked to outcomes after surgery.7,10 based on limited early data, patients with braf mutant hs may have good seizure control with surgery, and diagnostic criteria to distinguish hbnl from existing braf mutant brain tumors have been proposed.9 in this report, we discuss the clinical and histopathologic findings in the case of a young male patient with mtle-hs, whose resected tissue demonstrates ilae type 1 hs with neurons that are immunopositive for brafv600e (brafv600e+). we also argue that there is value in routine brafv600e testing of tissues in mtle-hs clinical trials. case presentation a 31-year-old right-handed male with a five-year history of focal epilepsy refractory to medical management presented to the comprehensive epilepsy center for advanced monitoring and surgical planning. the patient’s seizures developed shortly after undergoing surgery for the removal of a bone spur that was complicated by a deep vein thrombosis requiring anticoagulation. the initial semiology included focal seizures with impaired awareness and generalized tonic-clonic (gtc) seizures with rightward head turn. although the gtc seizures were controlled with valproic acid and lacosamide, he continued to have focal seizures without impaired awareness and failed a trial of levetiracetam. these seizures began with a sensation of déjà vu, followed by hyperventilation and oral automatisms. magnetic resonance imaging (mri) showed left hippocampal hyperintensity and mild volume loss, consistent with left mesial temporal lobe sclerosis, without evidence of mass lesion (figure 1). after two unsuccessful attempts at electrographically characterizing seizures with noninvasive eeg, the patient underwent stereoelectroencephalography (seeg) implantation, which localized the seizures to the left temporal lobe. a wada test demonstrated the dominance of the left hemisphere for language and the right for memory. the case was discussed at the multidisciplinary epilepsy clinic, and after patient consent was obtained for surgery and involvement in research, he underwent left craniotomy for temporal lobectomy and amygdalohippocampectomy. the procedure was uncomplicated, and the patient went home on postoperative day 1. a neuropsychological evaluation performed 11 months after surgery showed improvement in measures of verbal memory and a cognitive profile essentially within normal limits. two years after surgery, the patient has had occasional episodes of dysmnesic phenomena (déjà vu auras) but has not had any seizures, corresponding to engel class ib (supplementary figure 1). figure 1. preoperative coronal mri at the level of the hippocampal body (a) t1 mp-rage demonstrating left hippocampal atrophy and architectural distortion, and (b) t2/flair showing abnormal hyperintense signal with atrophy and associated widening of the temporal horn (right arrowhead) and choroid fissure (left arrowhead). postoperative axial t2 (c) mri at the level of the pons demonstrating postoperative changes following left anterior temporal lobectomy and (d) t2/flair at the level of the mesencephalon showing abnormal hyperintense signal in the residual left hippocampal tail. pathology surgical specimens of the temporal lobe, amygdala, and hippocampus were submitted. immunostaining for neun highlighted the relative paucity of neurons in the ca1 and ca4 regions consistent with hs ilae type 1 (figure 2a). granule cell dispersion was present. chromogranin immunostaining showed a similar pattern of regional neuronal loss. in the patient’s specimen, neurons of the ca1 and ca2 regions were often oriented parallel to the ventricular surface (figure 2b–g) instead of the normally predominant perpendicular orientation, giving a streaming appearance. occasional scattered brafv600e+ cells that have the morphology of neurons were present in the ca, most prominently in the ca4 and ca3 regions but represented less than 1 % of cells. in ca2/ca3, braf+ neurons also tended to have a parallel streaming orientation (figure 2d). there were far more neun immunopositive neurons than braf mutant neurons. hypertrophic and dysmorphic neurons were identified (figure 2e–f). a curvilinear distribution of braf mutant neurons mimics the usual neuroanatomic architecture of the ca (figure 2g). in the amygdala specimen that also had dentate gyrus of the hippocampus, there were a few scattered brafv600e+ neurons. a section of the temporal lobe did not show any brafv600e immunoreactivity. in the dentate gyrus, the brafv600e+ neurons were sparse and retained the perpendicular orientation of their many immunonegative peers (figure 2h). brafv600e+ threads, probably reflecting neurites, were few, but in some areas were abundant and excessive for what might be anticipated relative to the observed neuronal soma (figure 2g, 2i). cd34 immunostaining of the hippocampus-stained blood vessels was otherwise largely negative except for sparse foci of cd34 immunopositive (cd34+) stellate cells present as loose or nodular aggregates, or single cells (figure 2j–l). cd34+ cells are less than 1 % of cells. gfap immunostaining showed extensive reactive astrocytosis (figure 2m). neurofilament highlighted occasional pyramidal neurons and abundant axonal injury characterized by variably thickened axons with spindled expansions, torpedoes or spheroids in the hippocampus (figure 2n). synaptophysin immunostaining demonstrated strong staining of the brain neuropil with scattered immunopositive neurons. the ki-67 proliferation index was very low with only rare scattered immunoreactive nuclei. an idh1 r132h immunostain was negative. we attempted two brafv600e polymerase chain reaction (pcr) assays which did not detect a braf alteration. we then attempted next-generation sequencing that also did not detect a braf alteration but did detect a fanconi anemia complementation group l (fancl) deletion in 48.5 % of alleles. the frequency of the braf v600e+ neurons in our case is very low estimated at less than 1 % and our pcr testing can only detect reliably at the 20–40 % or above mutant allele level, while our next-generation sequencing detects down to ∼5 % mutant alleles. the brafv600e immunohistochemistry is robust as routine use produces clean staining and, in the setting of tumors with abundant brafv600e+ tumor cells, the immunostaining is corroborated by positive pcr findings. figure 2. a. neun immunostaining highlights the loss of neurons in the ca1 and ca4 regions of the hippocampus. (20x). b. in the ca1 and subiculum, residual pyramidal neurons often stream in parallel to the ventricular surface rather than being mostly oriented in the typical perpendicular orientation. (h&e 200x). c. neun immunostained ca1 neurons are oriented parallel to the ventricular surface lined by ependymal cells. e = ependymal lining of ventricle. (200x). d. brafv600e+ neurons in the stratum radiatum of the ca3 region. (200x). e. occasional neurons may be large and, in this case, dysmorphic with an ovoid or swollen appearing soma and abnormal distribution of basophilic nissl substance. (h&e 400x). f. two pyramidal neurons show lumpy cytoplasmic contours, reminiscent of popcorn. a variably thickened neuritic process (asterisk) is also present. (neun 400x). g. brafv600e+ neurons in the ca3 region stream in a curvilinear fashion around a terminus of the dentate (d) gyrus. many slender, delicate immunopositive “threads” – presumably neuritic processes – are in the background, including areas without obvious neuronal soma. (200x). h. only sparse brafv600e+ neurons are identified in the dentate gyrus. (200x). i. numerous braf immunopositive “threads” are seen in this area of ca4. an immunopositive neuron is in the upper image. immunonegative neurons are marked by an asterisk (200x). j. aggregates of cd34+ stellate cells (a) form irregular plaques (200x). k. cd34+ stellate cells may have a “nodular” pattern (n) or may be single (asterisk). (200x). l. the cd34+ stellate cell at higher magnification. (400x). m. gfap immunopositive hypertrophic astrocytes in the dentate gyrus and ca4 region are illustrated. (100x). n. neurofilament stain shows axonal injury characterized by thickened axons including spheroidal, spindled, or torpedo-shaped expansions. (400x). discussion mtle-hs is a heterogeneous syndrome characterized by intractable epilepsy, progressive neurological changes, and variable histopathology. although mtle-hs is thought to have a genetic component, it classically arises after an ipi, such as trauma, hypoxia, or febrile seizures, followed by a latent period and eventual seizure onset.11 when refractory to medical management, surgical resection of the seizure focus can yield favorable shortand long-term outcomes, including seizure control, seizure freedom, and improved quality of life.12,13 nonetheless, positive response to surgery is not uniform, and prior studies have reported association of febrile seizures, hippocampal sclerosis, and certain histopathological patterns with surgical outcomes.14 the ilae classification system defines types of hs in temporal lobe epilepsy, according to regions of hippocampal neuronal loss and gliosis. type 1 hs is the most common, features the most severe neuronal loss, and is frequently associated with an ipi before the age of 5. histologically, neuronal loss occurs mostly in the ca1 and ca4 regions.7,8 types 2 and 3 are less common and involve predominantly ca1 and ca4 neuronal loss (with gliosis), respectively. of all types, type 2 is most closely associated with an ipi.6,15 lastly, a no-hs pattern involves gliosis only with no neuronal loss.8 significant interest and research have been dedicated to describing the clinical correlates and surgical outcomes of different ilae subtypes.16–18 a retrospective cohort study of 213 patients found that those with type 1 hs had a longer duration of epilepsy and older age at the time of surgical treatment, compared to patients with type 2 hs.16 independent of hs type, greater than 80 % of patients had engel class i (free of disabling seizures) outcomes in the shortand long-term. however, the type 2 hs patients had better engel class ia (complete seizure freedom) outcomes than the type 1 hs patients.16 in another cohort of 389 patients, there was no detectable correlation between ilae hs type and surgical outcome.19 a more recent series of 247 patients reported that type 1 hs with hypertrophic neurons in the ca4 was associated with worse outcomes in both engel i (freedom from disabling seizures) and ia (complete seizure freedom) scores.6 while our case is type 1 hs and also contains hypertrophic neurons, our patient remains seizure-free at the latest follow-up. in addition to the pattern of hs, expression of cd34, a stem-cell marker of hematopoietic cells involved in early neurulation and cell function,20 has recently been recognized as a marker that varies among histological subtypes of mtle-hs.6 cd34 was previously shown to be useful in the prognostication of a range of low-grade epilepsy-associated tumors (leats), including gangliogliomas, dysembryoplastic neuroepithelial tumors, and multinodular and vacuolating neuronal tumors.15,21 a retrospective series of 187 patients identified cd34 expression in up to half of patients with leats.22 in those patients, cd34 positivity was associated with longer duration of epilepsy, older age at the time of surgical treatment, and a higher likelihood of achieving complete tumor resection. the authors also noted that 89 % of patients with cd34+ histology had drug-resistant epilepsy and that a slightly higher proportion of patients with unfavorable seizure outcomes (engel class ii–iv) were cd34+.22 cd34 immunoreactivity is not restricted to glioneuronal neoplasms. a study by calderon-garciduenas et al.6 described the distribution and clinical correlates of cd34 in a large series of surgically treated mtle-hs patients. they reported that 17 % (40/236) of their mtle-hs cases had cd34+ stellate cells. these cd34+ cells could form a “nodule” or exist as scattered single cells. type 1 hs was least likely to express cd34 compared to other types. cd34+ stellate cells were more frequently found in type 2 hs and non-hs cases (i.e., gliosis only), while a cd34+ nodular pattern was more frequently found in type 3 hs and non-hs cases and was associated with dysmnesic auras. nodular aggregates were noted in our case and the patient does have dysmnesic auras. the authors found that cd34+ stellate cells were associated with a trend towards better postsurgical outcomes and speculate that cd34+ cells may represent epileptogenic seizure foci curable by surgery.6 interestingly, our patient presented with the less common combination of type 1 mtle-hs with a few nodules of cd34+ stellate cells. he responded favorably to surgical treatment and, while he still has occasional auras, he remains seizure-free after 2 years. of particular interest in our patient is the detection of phenotypic neurons with brafv600e immunopositivity. braf is a serine/threonine kinase involved in the ras/mapk signaling pathway, necessary for cellular division and differentiation. brafv600e mutations are among the most common ras/mapk pathway abnormalities driving human neoplasms.23 brafv600e mutations have been identified in the histopathology of many tumors, including leats,24,25 but have also been reported in a significant subset of mtle-hs.6 determination of braf mutation status has been made easier by the advent of mutation-specific monoclonal antibodies, replacing the timeand resource-intensive genetic sequencing.26 neither pcr nor next-generation sequencing detected brafv600e alterations in our case, but the frequency of braf mutant neurons was below the threshold of detection by these modalities. with next-generation sequencing of our case, a fancl deletion was detected in almost half of the alleles. fancl mutations can lead to cytogenetic instability, increased chromosomal breakage and defective dna repair among other effects. this high percentage of mutant allele frequency suggests that many of the histologically normal/braf-wildtype cells in the hippocampus have the deletion. we speculate that a fancl alteration during development may have contributed to acquiring a later braf alteration given the very low percentage (less than 1 %) of braf immunopositive cells. gwas studies have previously shown a linkage between the fancl locus and epilepsy.27 brafv600e is associated with a variety of tumors including brain tumors, but it also has been reported in pre-neoplastic or dysplastic processes such as cutaneous nevi, serrated polyps, and cortical dysplasia.23,28,29 a variety of braf alterations, including several in the kinase domain region that codon 600 lies within, are associated with epilepsy and cortical dysplasia in patients with cardiofaciocutaneous syndrome.30 cd34+ cells are well described in glioneuronal tumors but also in cortical dysplasia,20,23 and in about 17 % of hippocampal sclerosis cases.6 accordingly, the brafv600e and cd34 immunopositivity in our case should not be considered specific for tumor, but can also be seen in dysplastic or pre-neoplastic processes. in their cohort, calderon-garciduenas et al. identified 5 out of 236 cases (2 %) of mtle-hs with cd34+ cells and brafv600e+ neurons.6 three of these cases exhibited the nodular cd34+ pattern, and 2 were cd34+ scarce. there were no neoplasms seen in these 5 patients, suggesting that brafv600e mutations may predispose to epilepsy in the absence of overt tumorigenesis.6 in that report, braf+ hs was the proposed nomenclature. in a follow-up study from the same group, lerond et al.9 used the designation of hippocampal braf-mutant non-expansive lesion (hbnl) and described similar findings in the neocortex. in 28 cd34 negative cases of hs, no braf mutant neurons were identified. in contrast, 20 % of 25 cd34+ mtle-hs cases were braf mutant and therefore designated as hbnl. lerond et al.9 raised the question of other mapk alterations besides braf in the cd34+ braf wildtype cases. research in mouse models has implicated brafv600e mutations in neuronal hyperexcitability31 and epileptogenic activity.32 in 2018, koh et al.32 showed that somatic brafv600e mutations led to intrinsic epileptogenic activity in mice neurons and concluded that braf mutations in progenitor neurons during embryonic development predispose to epilepsy-associated diseases. moreover, a case series of 20 patients with leats found that braf mutations were not only present in tumor cells but also in the dysplastic neurons associated with these tumors, further supporting the hypothesis that common braf-mutated progenitors may produce dysplastic neurons that accompany the neoplastic dysplasia.23 lerond et al showed that interictal-like discharges can be detected in hbnl hippocampal slices but that the spatial and discharge patterns differed from braf wildtype mtle-hs cases.9 in our patient with ilae type 1 mtle-hs and immune expression of cd34 and brafv600e, it is reasonably likely that these histopathological findings correlate with hyperexcitable neurons promoting epileptogenesis, and their resection may be associated with long-term seizure freedom. in addition to its utility as a predictor of disease pathogenesis and clinical presentation, brafv600e may represent a potential target for emerging pharmacologic agents.23,33 braf inhibitors, such as vemurafenib and dabrafenib, are commonly and successfully used to treat brafv600e+ melanoma.34 remarkably, emerging rodent data has applied braf inhibitors to brafv600e+ epileptic disease with promising results. in their study, koh et al.32 noted that intraventricular injections of vemurafenib in mice with brafv600e mutations alleviated epileptic seizures. oral administration of the drug, however, yielded no benefit, perhaps due to its poor blood-brain barrier penetrance. trials are currently underway to explore the efficacy of these drugs in treating brafv600e-associated gliomas in human subjects. there remains the unsettled question of whether the braf mutant cells in mtle-hs reflect a benign dysplastic state that has the potential to be converted by additional alterations into a neoplasm, an early stage of a developing neoplasm that would have continued to progress without surgical intervention or a forme fruste wherein a developing neoplasm is effectively frozen before forming a radiologically detectable mass, for example by oncogenic senescence. lerond et al.32 suggested the presence of somatic mosaicism given the absence of detectable brafv600e alterations in the temporal lobe of some of their hippocampus mutant cases. a brafv600e immunostaining of the temporal lobe in our cases is negative but this is a limited assessment of the temporal lobe specimen, which consisted of multiple blocks of tissue. at a practical level, we were initially uncertain as to whether classify the lesion as a braf mutant hs or as hs associated with a braf mutant neoplasm. the lack of a radiological mass, mild hippocampal volume loss by mri, localization of braf mutant cells in a curvilinear distribution that approximated the usual ca cytoarchitecture, and the low ki-67 index led us to favor the designation of a brafv600e+ hs consonant with the preliminary nomenclature at that time.6 the parallel streaming pattern raises the question of aberrant neuronal migration akin to classical cortical dysplasia. given the unclear nature of our case, the most recently proposed nosology of hbnl, which is a descriptive, neutral term that does not ascribe a non-neoplastic or neoplastic etiology may be appropriate.9 our case could reflect dual pathology (hippocampal sclerosis and a ganglion cell neoplasm) though that may require postulating migration of mutant neoplastic neurons along the curvature of the ca. we did not identify diagnostic features of a variety of tumor entities that may be cd34+ and braf+ such as polymorphous low-grade neuroepithelial tumor of the young (plnty), pleomorphic xanthoastrocytoma, multinodular and vacuolated neuronal tumor, and dysembryoplastic neuroepithelial tumor. the major concern for our case was to exclude a gangliocytoma or a ganglioglioma. based on a limited number of cases, it has been proposed that diffuse sheets of cd34+ cells are more typical of plnty and ganglioglioma than for hbnl wherein single scattered cd34+ cells are common9. the nodular cd34+ pattern was not included as a distinguishing feature.9 in addition to the aforementioned radiologic, cytoarchitectural and other histopathologic features, our case only had sparse cd34+ nodules and scattered cells and did not have sheets of such cells, overall favoring an hbnl by the published preliminary criteria. conclusion we provide details of a case of ilae type 1 mtle-hs with brafv600e+ neurons and cd34+ stellate cells. we describe a general parallel orientation of many ca neurons relative to the ventricular surface including braf mutant ones. braf mutant neurons are not restricted to the stratum pyramidale but may be more prominent in the stratum radiatum and strata lucidum. whether this parallel streaming pattern is typical of hbnl, or an inconstant coincidental dysplastic feature or migration of neoplastic cells requires further research. additional study is required to better understand the nature of this finding as to whether it is a pre-neoplastic or early neoplastic state or even a forme fruste of a glioneuronal tumor. nevertheless, these hbnl cases are important as they likely provide a very important glimpse of the early genesis of glioneuronal tumors. such cases may be missed as brafv600e studies are not routinely obtained on hs cases and tumor may be radiologically and histologically inapparent as in our case. since only approximately 2 % of mtle-hs cases will have this alteration, it may not be cost-effective to routinely test for brafv600e alterations in the community. however, major epilepsy centers that perform relevant mtle research as well as clinical trials involving mtle-hs patents should consider doing so routinely. acknowledgments we thank dr. jefferson chan of uc irvine for assistance with pcr and sequencing of the tissue 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https://doi.org/10.1152/jn.00523.2019. 32. koh hy, kim sh, jang j, et al. braf somatic mutation contributes to intrinsic epileptogenicity in pediatric brain tumors. nat med. 2018;24(11):1662-1668. https://doi.org/10.1038/s41591-018-0172-x. 33. bollag g, hirth p, tsai j, et al. clinical efficacy of a raf inhibitor needs broad target blockade in braf-mutant melanoma. nature. 2010;467(7315):596-599. https://doi.org/10.1038/nature09454. 34. gutzmer r, stroyakovskiy d, gogas h, et al. atezolizumab, vemurafenib, and cobimetinib as first-line treatment for unresectable advanced brafv600 mutation-positive melanoma (imspire150): primary analysis of the randomised, double-blind, placebo-controlled, phase 3 trial. lancet. 2020;395(10240):1835-1844. https://doi.org/10.1016/s0140-6736(20)30934-x. copyright: © 2024 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. acox1 gain-of-function post-mortem neuropathology is distinct from acox1 loss-of-function: case report and literature review feel free to add comments by clicking these icons on the sidebar free neuropathology 6:19 (2025) letter acox1 gain-of-function post-mortem neuropathology is distinct from acox1 loss-of-function: case report and literature review zita hubler1 *, kaleigh filisa roberts1 *, nima sharifai2, julia sim3, sophia a. hung4, grace e. robvais4, alan pestronk3, robert e. schmidt1, sonika dahiya1, robert c. bucelli3 washington university in st. louis,department of pathology and immunology, usa university of maryland, school of medicine, department of pathology, usa washington university in st. louis, department of neurology, usa washington university in st. louis, department of biology, usa * these authors contributed equally to this manuscript corresponding author: robert c. bucelli · department of neurology · washington university in st. louis · 517 s euclid ave · st. louis · usa bucellir@wustl.edu submitted: 18 august 2025 accepted: 29 september 2025 copyedited by: alessia sciortino published: 10 october 2025 https://doi.org/10.17879/freeneuropathology-2025-8894 keywords: mitchell syndrome, acox1, pseudo-neonatal adrenoleukodystrophy, case report introduction mitchell syndrome is a newly described progressive childhood neurodegenerative disorder characterized by myeloneuropathy, hearing loss, and skin changes, progressing to encephalopathy, paralysis, and death before the third decade [1]. mitchell syndrome is caused by a gain-of-function (gof) missense variant in peroxisomal acyl-coa oxidase 1 (acox1) [1]. acox1 is the rate-limiting enzyme of peroxisomal β-oxidation, including very long-chain fatty acids (vlcfas). acox1 loss-of-function (lof), known as pseudo-neonatal adrenoleukodystrophy, is associated with a spectrum of genetic variants, the accumulation of vlcfas in plasma, hypotonia, seizure, early-onset leukodystrophy, and death in early childhood [2]. both gainand loss-of-function results in myelin loss based on imaging and animal studies [1,3]. we report, for the first time, the postmortem neuropathologic findings in mitchell syndrome (acox1 gof), compare them to acox1 lof, and discuss the implications for future studies. case presentation an 11-year-old boy presented with ocular keratopathy, clumsiness, sensory ataxia, bilateral hearing loss, and hyporeflexia. he developed progressive myeloneuropathy; imaging showed a longitudinally extensive lesion preferentially involving the dorsal columns (fig. 1a–b). the patient was extensively investigated, and other known causes of longitudinally extensive transverse myelopathy were ruled out, including neoplastic/paraneoplastic, infectious, mitochondrial and other known metabolic disorders, neurodegenerative, toxic exposures, and autoimmune conditions. for additional details on clinical course please see paper chung et al., 2020 supplemental document methods s1 [1]. initially, the brain was relatively unaffected on imaging. the spinal cord disease progressed to involve the anterolateral cord. at age 13 a peripheral nerve biopsy showed mildly abnormal myelin profiles and ongoing and chronic axon loss [1]. an undiagnosed diseases network evaluation uncovered a novel de novo heterozygous acox1 n237s variant. he suffered a relapsing and remitting course with overall progressive decline despite treatment with n-acetyl cysteine, plasmapheresis, intravenous immunoglobulin, corticosteroids, rituximab, cyclophosphamide, and tocilizumab [1]. at 19, he was admitted for a urinary tract infection and developed encephalopathy and neuromuscular respiratory failure requiring mechanical ventilation. brain mri showed new, diffuse, bilateral subcortical fluid-attenuated inversion recovery (flair) and t2-weighted turbo spin echo (tse) hyperintensities with u-fiber sparing, consistent with disease progression (fig. 1c). the patient passed away after elective extubation and underwent a complete autopsy within 24 hours after death. to date, an additional 30 cases have been identified. figure 1: perimortem imaging and gross pathology t2-weighted turbo spin echo (tse) sequence in the transverse (tra) plane of a) sagittal cervical cord, b) transverse cervical cord showing hyperintensity of the dorsal column (arrow), and c) axial brain with artifact due to cochlear implants. d) post fixation cross-section of the cervical cord with translucency of the dorsal column (arrow). pathology the weight of the unfixed brain was 1510 grams (reference range 1250–1400 grams), possibly increased due to edema. the intact brain was grossly unremarkable. microscopic sections of the cortex revealed variable scattered white matter pallor and focally increased rarefaction. the cingulate gyrus showed white matter pallor with sparing of the u-fibers, confirmed by luxol fast blue and periodic acid-schiff (lfb-pas) stain. a neurofilament immunostain showed these areas of white matter to have a disrupted axonal array with numerous axonal swellings. mildly increased numbers of oligodendrocytes were noted in the demyelinated and adjacent areas. a cd68 immunostain of the cingulate gyrus highlighted increased microglia and perivascular macrophages in the white matter. the occipital lobe showed modest white matter pallor, without neuronal dropout, while the superior frontal lobe shows no obvious white matter pathology. the cortical gray matter in the affected lobes showed a normal number of neurons and intact laminar architecture. sections of the basal ganglia showed relative neuronal preservation of the caudate and putamen without significant astrocytosis. sections of the thalamus showed a normal neuronal density and glial component. the hippocampus showed neuronal dropout in the ca4 region, with hirano bodies in ca1 and the subiculum. the midbrain and pons were unremarkable. the medulla showed white matter pallor within the medullary pyramids, mild neuronal dropout, and associated gliosis in the inferior olivary nucleus. the cerebellum was unremarkable with a well-preserved complement of cortical purkinje, granule, and dentate nucleus neurons. grossly, the spinal cord showed translucency and discoloration of the dorsal column throughout, but particularly in the cervical cord (fig. 1d). microscopically there was total destruction of the dorsal columns with loss of myelin, loss of axons, and reactive gliosis (fig. 2a–b, d–e, g–h, j–k). areas near the dorsal column showed relative preservation of the axonal array with gliosis (fig. 2c, f, i, l) and active macrophage infiltrate showed with a cd68 stain (fig. 3a). in addition, there was pallor and vacuolation in the anterior and lateral corticospinal tracts. cd68 immunostain highlighted macrophages most prominent in the corticospinal tract in a perivascular distribution with pas-positive intracytoplasmic material corresponding to digested myelin. there were few scattered oligodendrocyte transcription factor 2 (olig2) positive oligodendrocytes. the cervical and thoracic cord showed neuron loss and atrophy with associated gliosis, greater in the dorsal horn than the ventral horn. sections of the lumbosacral cord showed similar pathology that was less severe. frozen sections of sciatic nerve were immunostained for neurofilament (nf, axon marker), neural cell adhesion molecule (ncam, non-myelinating schwann cell marker), and myelin basic protein (mbp, myelinating schwann cell marker) (fig. 3c–d, control nerve fig. 3 e–f) [4]. consistent with staining from a prior biopsy [1], the staining showed a severe loss of large (white arrow) greater than small (purple arrow), myelinated axons and a moderate loss of small, non-myelinated axons. the dorsal root ganglia showed frequent nageotte nodules, indicating ganglion cell loss (fig. 3b). the ventral roots were unremarkable. figure 2: spinal cord histopathology h&e at a) low power cross sections (1.25x) of cervical spinal cord and b) high power section of the dorsal column shows destruction, while c) adjacent areas show relative preservation. loss of myelin is seen on lfb-pas d–f), loss of axons by neurofilament g–i), and reactive gliosis by glial fibrillary acidic protein (gfap) j–l) in the dorsal column. clicking into the picture will lead you to the full virtual slide https://doi.org/10.57860/min_dts_000024 figure 3: macrophage staining, dorsal root ganglion and peripheral nerve pathology macrophage staining, dorsal root ganglion and peripheral nerve pathology. a) cd68 stain of spinal cord (4x) showing active macrophage infiltration. b) dorsal root ganglia (10x) showing frequent nageotte nodules (arrow). frozen sections of sciatic nerve were immunostained for neural cell adhesion molecule (ncam), neurofilament (nf), and myelin basic protein (mbp). overlay images of b) ncam (red)/nf (green) (200x) show a severe loss of large greater than small, myelinated axons (very few large green dots, example white arrow) and a moderate loss of small, non-myelinated axons (red staining relative to yellow staining). overlay images of c) mbp (red)/nf (green) (200x) again show the loss of large (white arrow) greater than small (purple arrow), myelinated axons, empty mbp sheaths (evidence of ongoing axon loss) and small axons with associated mbp staining (immature/regenerating axons). for additional details on the pathologic correlates of this pattern of staining findings, see pestronk et al., 2023 [4]. for a comparison to these stains on the patient’s sural nerve from six years prior see chung et al., 2020 supplemental figure 6 [1]. for comparison, control frozen sural nerve at 200x stained with e) overlay images of ncam (red)/nf (green) showing green dots with white arrow, and f) mbp (red)/nf (green) showing a large myelinated fiber with white arrow. (used with permission from https://neuromuscular.wustl.edu/pathol/nervenl.htm#axsur) discussion in comparison to acox lof, mitchell syndrome has notable differences in the gross presentation and distribution of pathology. lof is associated with prominent brain involvement [5], while gof has early and persistent burden of disease in the spinal cord that ultimately involves the brain. however, acox1 lof has limited reports evaluating the peripheral nervous system components; therefore, it cannot be ruled out that both conditions affect the peripheral nervous system and spinal cord. in lof the cerebrum and cerebellum show prominent atrophy [5], while gof results in a grossly normal brain. in lof, the cerebral, cerebellar, and brainstem white matter were severely affected; these regions were less affected in gof. both conditions have macrophage infiltration in affected areas. under electron microscopy, macrophages in lof samples show spiculated inclusions in membrane-bound aggregates [5]. however, these aggregates were not seen on peripheral nerve electron microscopy in the gof case [1]. differences in disease distribution between these conditions suggest selective vulnerability of specific cell types. for example, peripheral axonal loss with dorsal root neuronal dropout and destruction of the dorsal columns suggest selective vulnerability of primary sensory afferent neurons. afferent sensory neuron vulnerability is supported by the relative preservation of the ventral root and the presenting symptom of sensory ataxia. preferential early involvement for afferent neurons/axons could also underlie the early hearing loss in these patients and both peripheral and central nervous system involvement. to better understand this selective vulnerability of the primary sensory afferent neurons, animal models with dorsal root ganglia may be beneficial. however, sensory neurons are not the only cell type affected, as demonstrated by the early t2-hypertensity and obvious loss of myelin. in addition, the neuron loss in the ventral horn and corticospinal tract degeneration indicates eventual motor neuron vulnerability, findings compatible with the later development, clinically, of quadriparesis and neuromuscular respiratory failure. while both gainand loss-of-function of acox1 affect myelin and neurons, mitchell syndrome provides stronger support for a neuronal contribution to the pathology rather than acox1 lof. the findings of axon degeneration in the peripheral nervous system on electron microscopy [1], without a preponderance of naked axons, suggest that this is not a myelin-driven process early in disease in the peripheral nervous system. however, based on the distinct t2-hyperintensity in the dorsal column, myelin involvement cannot be ruled out. the areas of pallor identified in this autopsy show relatively proportional disruptions to myelin and axons based on lfb-pas and nf respectively, making it difficult to infer chronology of the injury. therefore, both neurons and myelinating cells likely contribute to disease progression. several overexpression acox1 mitchell syndrome non-human models exist that show different phenotypes and do not fully align in their proposed mechanisms. these phenotypes partially align with what we report here in the human pathology. studies overexpressing drosophila gof acox1 in drosophila and human acox1 in rat schwann cells highlight increased hydrogen peroxide, reactive oxygen species, and ensheathing glial cell death as a likely mechanism [1]. while overexpression of human acox1 in zebrafish showed no difference in oligodendrocyte precursor cell number, nor were differences detected in catalase or nitric oxide synthase 2a (nos2a) (markers of oxidative stress), it instead showed increased activating transcription factor 4 (atf4) expression, decreased peroxisomes, and decreased swimming [3]. further studies are needed to evaluate the neuronal contribution to the zebrafish phenotype. a consistent feature among these distinct models with differing phenotypes is the rescue with n-acetylcysteine (nac) amide or dendrimer-nac, which can function as an antioxidant and cytoprotectant [1,3]. nac amide allows for better blood brain barrier penetration but is limited to non-human models. instead, nac is available for human use and has been used clinically for numerous conditions, including mitchell syndrome. nac may have partially improved symptoms, but has been insufficient to prevent disease progression, as evident in this patient [1]. while nac/nac amide are pervasive in the literature, the mechanisms of action are not agreed upon [6]. other currently used treatments focus on neuroprotection and immunomodulation/immunosuppression [1]. there is no unifying model that reproduces the phenotypes seen in humans. while acox1 lossand gain-of-function are rare diseases, their pathology presents a unique opportunity to understand the contribution of critical metabolic pathways to the nervous system. the differences and similarities between these conditions highlight cell-specific vulnerabilities that can help us develop more accurate disease models and possibly provide clues to disease mechanisms. acknowledgements we thank the mitchell and friends foundation for their ongoing advocacy. funding statement this work was supported by the mitchell and friends foundation gift fund. this research was supported by the grant t32gm139799, which supports dr. zita hubler, and was awarded to washington university in st. louis by the national institute of general medical sciences. the content is solely the responsibility of the authors and does not necessarily represent the official views of the national institutes of health. conflicts of interest statement robert bucelli is a medical advisor for the mitchell and friends foundation and is the principal investigator on the mitchell and friends foundation gift fund. references 1. chung, h.l., et al., lossor gain-of-function mutations in acox1 cause axonal loss via different mechanisms. neuron, 2020. 106(4): p. 589-606 e6. https://doi.org/10.1016/j.neuron.2020.02.021 2. ferdinandusse, s., et al., clinical, biochemical, and mutational spectrum of peroxisomal acyl-coenzyme a oxidase deficiency. hum mutat, 2007. 28(9): p. 904-12. https://doi.org/10.1002/humu.20535 3. raas, q., et al., generation and characterization of a zebrafish gain-of-function acox1 mitchell disease model. front pediatr, 2024. 12: p. 1326886. https://doi.org/10.3389/fped.2024.1326886 4. pestronk, a., et al., schwann cells and myelin in human peripheral nerve: major protein components vary with age, axon size and pathology. neuropathol appl neurobiol, 2023. 49(2): p. e12898. https://doi.org/10.1111/nan.12898 5. wang, r.y., et al., effects of hematopoietic stem cell transplantation on acyl-coa oxidase deficiency: a sibling comparison study. j inherit metab dis, 2014. 37(5): p. 791-9. https://doi.org/10.1007/s10545-014-9698-3 6. pedre, b., et al., the mechanism of action of n-acetylcysteine (nac): the emerging role of h(2)s and sulfane sulfur species. pharmacol ther, 2021. 228: p. 107916. https://doi.org/10.1016/j.pharmthera.2021.107916 copyright: © 2025 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. a novel kdm2a::yap1 fusion in a pediatric supratentorial cns neoplasm resembling a tumor with bcor internal tandem duplication feel free to add comments by clicking these icons on the sidebar free neuropathology 6:5 (2025) letter a novel kdm2a::yap1 fusion in a pediatric supratentorial cns neoplasm resembling a tumor with bcor internal tandem duplication arnault tauziède-espariat 1,2 , alice métais 1,2 , dorian bochaton 1 , euphrasie servant 1 , guillaume chotard 3 , mégane le quang 3 , benjamin bonhomme 4 , nathalène truffaux 4 , volodia dangouloff-ros 5 , nathalie boddaert 5 , lauren hasty 1 , edouard gimbert 6 , pascale varlet 1,2 on behalf of the renoclip-loc department of neuropathology, ghu paris psychiatry and neuroscience, sainte-anne hospital, f-75014 paris, france université de paris, umr s1266, inserm, ima-brain, institute of psychiatry and neurosciences of paris, f-75014 paris, france department of pathology, pellegrin hospital, bordeaux, france department of biopathology, institut bergonié, bordeaux, france pediatric radiology department, hôpital necker enfants malades, ap-hp, university de paris, paris, france department of pediatric neurosurgery, pellegrin hospital, bordeaux, france corresponding author: arnault tauziède-espariat · department of neuropathology · ghu paris psychiatry and neuroscience · sainte-anne hospital · 1, rue cabanis, 75014 · paris · france a.tauziede-espariat@ghu-paris.fr additional resources and electronic supplementary material: supplementary material submitted: 12 december 2024 accepted: 16 february 2025 copyedited by: monica miranda published: 18 february 2025 https://doi.org/10.17879/freeneuropathology-2025-6152 keywords: yap1, kdm2a, bcor, tumor of the central nervous system introduction the central nervous system (cns) tumor with bcor internal tandem duplication (itd), initially named high-grade neuroepithelial tumor (hgnet), was introduced as a novel embryonal neoplasm in the last world health organization (who) classification of cns tumors and in the german cancer research center (deutsches krebsforschungszentrum, dkfz) classifier as a distinct methylation class (1,2). since its initial description, other alterations of bcor and its ligand bcorl1 have been reported in cns tumors having similar histopathological features. among them, the fusion ep300::bcor was evidenced in several cases with a distinct methylation class (3,4). moreover, cns tumors harboring fusions implicating other genes of the complex polycomb repressive complex 1.1 (prc1.1) (kdm2b and nutm2a/b genes) have been described (5). herein, we report a brain neoplasm presenting the histopathological features of a cns tumor with bcor itd, but harboring a kdm2a::yap1 fusion. case presentation a 10-year-old boy presented with intracranial hypertension, found to be caused by a supratentorial (st) intraventricular neoplasm. magnetic resonance imaging (mri) revealed a large mass within the left lateral ventricle having solid and cystic content (mri performed after cyst aspiration) (figure 1b–f). computerized tomography (ct) showed the solid component to have intermediate density and calcification (figure 1a), intense contrast enhancement (figure 1d), and no diffusion restriction (figure 1e). the tumor was subtotally resected. morphologically, this tumor was mainly well-circumscribed from the brain parenchyma (with few infiltrating isolated cells around the periphery of the tumor). pseudorosettes, microcysts and calcifications were observed (figure 2a–b). there was no necrosis or microvascular proliferation, but the tumor presented a high mitotic count (6 mitoses per 5 high-power fields representing 1.6 mm2) and proliferation index (10 %). immunohistochemistry (ihc) confirmed the preserved expression of h3k27me3, ini1 and atrx. there was no immunopositivity for gfap, olig2 or mn1, and the expression of ema was cytoplasmic without dot-like or microlumen staining. neun and neurofilament were expressed by a subset of tumor cells, and nuclear translocation of β-catenin was present. there was a weak, but diffuse, immunoreactivity for bcor (figure 2c) and a focal positivity for satb2 (figure 2d). rna sequencing evidenced a kdm2a::yap1 gene fusion (figure 2e). next-generation sequencing failed to reveal any other alteration. the tumor was not classifiable using the heidelberg brain tumor and sarcoma (v12.8) or bethesda classifiers, nor by t-distributed stochastic neighbor embedding (t-sne) or uniform manifold approximation and projection (umap) analyses (supplementary figure 1), which included hgnet-bcor and sarcomas with bcor alterations and cns and soft tissue tumors with ywhae:nutm2 or kdm2b fusions from our in-house database and previously reported (5). forty-two months later, the patient presented a local recurrence of the tumor and a second surgery was performed. the recurrence histopathology was identical to the initial resection. the patient is alive without residue 60 months after the initial diagnosis and without any adjuvant treatment. figure 1: radiological features axial unenhanced ct(a), t1-weighted (b), flair (c), post-contrast t1-weighted (d), diffusion (e), and apparent diffusion coefficient (adc) map (f) images of the patient, showing a large mass within the left lateral ventricle having solid and cystic content (mri performed after cyst aspiration). ct showed the solid component to have intermediate density and calcification, intense contrast enhancement, and no diffusion restriction. figure 2: histological and molecular features the tumor was solid and composed of uniform oval or spindle-shaped cells with round to oval nuclei, a dense capillary network (a,hematoxylin phloxin saffron (hps), magnification x400), pseudorosettes and microcystic formations (b, hps, magnification x400). bcor protein immunoreactivity was weak in the tumor cells (c, magnification x400). expression of satb2 in a subset of tumor cells (d, magnification x400). e rna-sequencing analysis evidenced a kdm2a::yap1 fusion. discussion and conclusions in the heidelberg classifier (v12.8), two distinct methylation classes of cns tumors characterized by different bcor alterations exist: cns tumors with bcor itd and those with ep300::bcor fusion, with only the first being part of the current who classification (2). regarding data from the literature (n = 82), cns tumors with bcor itd affect mainly children (95 % of reported cases are pediatric with a median age of 3.5 years-old) and are distributed supratentorially and infratentorially (58 % and 42 % of reported cases, respectively) with a poor outcome (37 % patients were dead with a median overall survival of 20 months) (6–20). contrarily, cns tumors with an ep300:bcor fusion (n = 24) concern young adults (median age of 28 years-old), are mostly supratentorial (83 % of reported cases) and seem to be associated with a better prognosis (no death reported to date in the literature and a median overall survival of 16 months) (3,4,16). whereas cns tumors with a ep300::bcor fusion can present a broad histological spectrum (4), these two molecular entities may share similar histopathological features: a predominantly solid growth pattern, uniform oval or spindle-shaped cells with round to oval nuclei, a dense capillary network, and pseudorosette formations (3). because of the nature of the antibody, only tumors with bcor itd showed a constant overexpression of the bcor protein using ihc (4,21). in the literature, other alterations implicating the bcor gene (crebbp::bcor fusions) or its ligand bcorl1 (ep300::bcorl1, crebbp::bcorl1 fusions) have been reported (16,22,23), for which satb2 ihc may constitute a helpful diagnostic tool, being positive in cns tumors with bcor alterations (regardless of their molecular abnormality), and in sarcoma harboring kdm2b or ywhae fusions (1,2). the current case showed a focal positivity for this antibody. interestingly, this case along with other rare cns tumors and soft tissue/visceral sarcomas which have been found to harbor classically bcor alterations, have been shown to have kdm2a or kdm2b fusions (5,24,25). associated with bcor in the prc1.1 complex, kdm2a and b (lysine demethylase 2a and b) proteins mediate the transcriptional repression of tumor suppressors through the post-translational modifications of histones. the predicted fusion protein of the current case maintained major functional domains, such as the jumonji c and cxxc dna-binding domains of the kdm2a gene and the ww domain of the yap1 gene which is a transcriptional co-activator as previously described (2,5). yap1 fusions are well known in the tumorigenesis of ependymomas (epn) and it has been evidenced that they are sufficient to form tumors in the developing mouse brain (3). moreover, the overexpression of the yap1 protein in cortical progenitor cells induces an activation of the hippo pathway and is implicated in cell proliferation (3). in contrast to previously reported cns cases harboring an alternative gene fusion of the prc1.1 complex, the current tumor presented histopathological similarities to cns tumors with bcor alterations but not sarcomas (5). however, the dna-methylation analysis did not permit its classification. this is potentially due to the fact that only cns tumors with bcor alterations are present in the current classifier and that the boundaries of the current methylation classes are not clearly defined, with a subset of cases being misclassified (4,22). other samples having these rare alterations are necessary to determine if they cluster together and form a distinct methylation class from those described. in closing, we report herein a cns tumor having histopathological similarities to cns tumors with bcor alterations, but having a fusion implicating the gene kdm2a, encoding a protein of the prc1.1 complex. in cases of a suspected cns tumor with bcor alteration without a strong and diffuse immunopositivity for the bcor protein, rna-sequencing analysis may help to diagnose alternative fusions. ethics approval this study was approved by ghu paris psychiatry and neuroscience, sainte-anne hospital’s local ethics committee. consent for publication the patients signed informed consent forms before treatment began. conflict of interest statement the authors declare that they have no conflicts of interest directly related to the topic of this article. funding statement the authors declare that they have not received any funding. acknowledgements we would like to thank the laboratory technicians at ghu paris psychiatry and neuroscience, sainte-anne hospital for their assistance. references 1. capper d, jones dtw, sill m, hovestadt v, schrimpf d, sturm d, et al. dna methylation-based classification of central nervous system tumours. nature. 22 mars 2018;555(7697):469‑74. https://doi.org/10.1038/nature26000 2. louis dn, perry a, wesseling p, brat dj, cree ia, figarella-branger d, et al. the 2021 who classification of tumors of the central nervous system: a summary. neuro-oncol. 2 août 2021;23(8):1231‑51. https://doi.org/10.1093/neuonc/noab106 3. tauziède-espariat a, pierron g, siegfried a, guillemot d, uro-coste e, nicaise y, et al. the ep300:bcor 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https://doi.org/10.1097/pgp.0000000000001026 25. kao yc, sung ys, zhang l, chen cl, huang sc, antonescu cr. expanding the molecular signature of ossifying fibromyxoid tumors with 2 novel gene fusions: crebbp-bcorl1 and kdm2a-wwtr1. genes chromosomes cancer. janv 2017;56(1):42‑50. https://doi.org/10.1002/gcc.22400 copyright: © 2025 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license ( https://creativecommons.org/licenses/by/4.0/ ), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver ( https://creativecommons.org/publicdomain/zero/1.0/ ) applies to the data made available in this article, unless otherwise stated. adult glioblastoma with lynch syndrome-associated mismatch repair deficiency forms a distinct high-risk molecular subgroup feel free to add comments by clicking these icons on the sidebar free neuropathology 5:32 (2024) original paper adult glioblastoma with lynch syndrome-associated mismatch repair deficiency forms a distinct high-risk molecular subgroup maria-magdalena georgescu neuromarkers, houston, texas, usa corresponding author: maria-magdalena georgescu · neuromarkers · 6907 academy street, 77025 · houston · texas · usa mmgeorgescu@yahoo.com additional resources and electronic supplementary material: supplementary material submitted: 17 september 2024 accepted: 17 november 2024 copyedited by: joão gama published: 10 december 2024 https://doi.org/10.17879/freeneuropathology-2024-5892 keywords: glioblastoma, mmr deficiency, lynch syndrome, tp53, pik3ca, chromatin remodeling genes, multinucleated giant cells, p53 immuno-histochemistry, immune checkpoint inhibitors, ipilimumab, nivolumab abstract glioblastoma is the most frequent and malignant primary brain tumor. although the survival is generally dismal for glioblastoma patients, risk stratification and the identification of high-risk subgroups is important for prompt and aggressive management. the g1–g7 molecular subgroup classification based on the mapk pathway activation has offered for the first time a non-redundant, all-inclusive classification of adult glioblastoma. five patients from the large, 218-patient, prospective cohort showed germline mutations in mismatch repair (mmr) genes (lynch syndrome) and a significantly worse median survival of 3.25 months post-surgery than those from the g1/egfr and g3/nf1 major subgroups, or from the rest of the cohort adjusted for age. these rare tumors were assigned to a new subgroup, g3/mmr, a g3/nf1 subgroup spin-off, as they generally show genomic alterations leading to ras activation, such as nf1 and ptpn11 mutations. an integrated clinical, histologic and molecular analysis of the g3/mmr tumors showed distinct characteristics as compared to other glioblastomas, including those with iatrogenic high tumor mutation burden (tmb), warranting a separate subgroup. prior history of cancer, midline location or multifocality, presence of multinucleated giant cells (mgcs), positive p53 and mmr immunohistochemistry, and specific molecular characteristics, including high tmb, msh2/msh6 alterations, biallelic tp53 arg mutations and co-occurring pik3ca p.r88q and pten alterations, alert to this high-risk g3/mmr subgroup. the mgcs and p53 immunohistochemistry analysis in g1–g7 subgroups showed that one in 7 tumors with these characteristics is a g3/mmr glioblastoma. the fda-approved first-line therapy for many advanced solid tumors consists of nivolumab-ipilimumab immune checkpoint inhibitors. one g3/mmr patient received this regimen and survived much longer than the rest, setting a proof-of-principle example for the treatment of these very aggressive g3/mmr glioblastomas. introduction glioblastoma is the most frequent and deadly primary brain tumor [1], assigned by the world health organization (who) classification of central nervous system tumors the highest tumor grade, who grade 4 [2]. its dismal prognosis is mainly due to diffuse brain invasion, tumor heterogeneity, but also activation of pathways that impart resistance to therapy. virtually all glioblastomas show activation of the extracellular signal-regulated kinase / mitogen-activated protein kinase (erk/mapk) and phosphatidyl-inositol 3-oh kinase (pi3k) canonical growth pathways [3–5]. non-redundant molecular alterations in the upstream mediators of the erk/mapk pathway, including the receptor tyrosine kinases (rtks) triggering pathway activation, allowed an all-inclusive molecular classification of glioblastoma into 7 subgroups [4,5]. these subgroups, termed after the most upstream gene showing activating rtk or mapk effector alterations are: g1/egfr, g2/fgfr3, g3/nf1, g4/raf, g5/pdgfra, g6/multi-rtk, and g7/other, the latter showing dominant pi3k pathway activation [4,5]. of these, the g1/egfr, g3/nf1 and g7/other are major subgroups, containing together approximately three quarters of all glioblastoma cases, and showing distinct risk stratification, with overall poor prognosis for the g7/other subgroup [5]. the treatment of glioblastoma relies on surgical resection, the extent of which correlates to the survival benefit [6]. subsequent radiation and temozolomide (tmz) chemotherapy represent the standard adjuvant regimen for the past 20 years [7], with few recent trials proven beneficial for the younger glioblastoma patient population [8]. beside risk stratification, the aim of the g1–g7 molecular subgroup classification is to identify subgroups benefiting of specific targeted therapy [5]. for example, the g6/multi-rtk has been analyzed in detail and shows rtk gene fusions for which addition of specific rtk inhibitors improves survival [5,9,10]. a very small but significant subgroup of glioblastomas that harbor germline mismatch repair (mmr) gene deficiency consistent with lynch syndrome has been identified in a previous study [5]. this subgroup has been termed g3/mmr, as ras activation appears to be dominant in these tumors, similarly to the g3/nf1 tumors. lynch syndrome is a cancer predisposition syndrome caused by germline pathogenic alterations in the mmr genes mlh1, pms2, msh2 and msh6, in which various types of tumors, the most prevalent being colorectal carcinoma (crc), arise by inactivation of the second mmr allele and a subsequent high rate of repair errors during dna replication [11]. this mmr deficiency is reflected in a high tumor mutation burden (tmb) that has been shown to benefit from immune checkpoint inhibitors [12]. the current study focuses on the clinical, histologic and molecular characterization of the g3/mmr subgroup in comparison to other glioblastoma cases from the same cohort, including cases with high tmb due to tmz therapy. it shows the distinct characteristics of g3/mmr tumors occurring in older adults, similar to most glioblastoma cases, and reviews few additional adult cases from the literature [13,14], delineating diagnostic guidelines for the identification of these rare cases. importantly, this is the first study showing survival advantage in adult g3/mmr glioblastoma by addition of the dual nivolumab-ipilimumab regimen of immune checkpoint inhibitors to the standard adjuvant regimen. methods tumor specimens, histology and immunohistochemistry (ihc). surgical specimens were obtained from patients, as previously described [15–17]. formalin-fixed paraffin-embedded (ffpe) sections from glioblastoma biopsies or surgical resections were stained with hematoxylin and eosin (h&e) or various antibodies for protein expression by ihc: p53 (do-7), p16 (e6h4), ki-67 (30-9), mlh1 (m1), pms2 (a16-4), msh2 (g219) and msh6 (sp93) (roche / ventana medical systems inc., tucson, az), and gfap (ep672y) (ventana / cell marque, rocklin, ca), as described [5,16]. images were acquired with nikon eclipse ci microscope equipped with nikon digital sight ds-fi2 camera (nikon instruments inc., melville, ny) [4]. an integrated histologic and molecular diagnosis, according to the most recent who guidelines [2] was obtained for all cases, prior to classifying them in the g1–g7 molecular subgroups [4,5]. next generation sequencing (ngs), copy number variation (cnv), and transcriptomics. the dna and rna ngs analyses were performed from ffpe samples by using the xt-648-gene panel (tempus labs, chicago, il), as previously described [15,17,18]. for each case, the same ffpe block was used for dna and rna extraction to allow direct comparison of results. variant and cnv assessment and interpretation, and the rna expression analysis were performed as extensively described elsewhere [15,18]. assessment of mutation germline origin. genetic testing from blood was performed for patient f70. for patients m62, m68 and m72, the germline origin was inferred by comparing the variant allele fraction (vaf) for the mmr mutations and the vaf for somatic heterozygous mutations (e.g. pten, pik3ca, tp53), in parallel with cnv analysis. a clear distinction between the vaf in the germline versus somatic range was present in each of these cases (see suppl. table s1). when possible, this method was extended to the rest of mmr mutations from tumors not mapping to the g3/mmr subgroup (see suppl. table s2). this assessment based on vaf showed reliable somatic versus germline prediction on the ngs platform used in the cases for which genetic testing was performed in parallel [15,17–19]. statistical analysis. survival kaplan-meyer curves were analyzed by log-rank (mantel-cox) and gehan-breslow-wilcoxon tests for statistical significance, as described [4,5]. parametric unpaired t-test with welch’s correction was performed by using graphpad prism (version 10.2.3, graphpad software, la jolla, ca). statistical significance was considered for p < 0.05, and confidence intervals were 95 % for all tests. data were analyzed and plotted by using microsoft excel (microsoft corp., redmond, wa), and graphpad prism. results dismal prognosis of g3/mmr glioblastoma patients: treatment of one patient with nivolumab-ipilimumab immune checkpoint inhibitors improves outcome. in a prospectively assembled cohort of 218 glioblastoma patients where all the tumors were subjected to dna and rna ngs, and classified in molecular subgroups according to the mapk-based g1–g7 subgroup classification [4,5], five cases with germline mutations in mmr genes stood apart as a separate subset, g3/mmr (fig. 1a). the g3/mmr subgroup represented approximately 2 % of the glioblastoma cohort, and showed distinctive clinical, histologic and molecular characteristics, summarized in table 1. the g3/mmr patients frequently had a clinical history of prior cancers, sometimes with multiple different malignancies, the most commonly reported being crc in two of the five patients. skin cancer, either melanoma or basal cell carcinoma, was also relatively common in these patients, and lymphoma and thyroid carcinoma less common (table 1). figure 1: location of g3/mmr molecular subgroup tumors a. pie chart distribution (%) of 218 prospective glioblastoma cases in g1–g7 molecular subgroups. purple arrow shows the g3/mmr subgroup. gbm, glioblastoma; egfr↑, egfr-amplified; egfrm, egfr-mutated. b. regional distribution of the glioblastomas from the g3/mmr, as compared to the rest of the cohort (gbm). cc, corpus callosum. c. selected mri images showing multifocality (red arrows) and brainstem infiltration (blue arrow) in three g3/mmr cases. *gbm-mmr n=213 1(192) g3/mmr n=5 m62 m68 f70 f72 f79 median age: years 65 1 (66) 70 62 68 70 72 79 sex m:f ratio 1.5 : 1 4 : 1 male male female male male race w:h ratio 53 : 1 4 : 1 white hispanic white white white history of prior cancer nd 60 % no no crc bcc, lymphoma melanoma, crc, thyroid 2location 22 % midline 40 % midline r bg l frontal r temporal l bg r frontal 2midline/crosses midline 22 % 60 % yes no no yes yes 2multifocal nd 60 % yes yes no yes no 2tumor size: cm variable large 4.9 x 4.3 x 4.2 4.3 x 4.1 x 3.1 7.7 2.8 x 1.8 7 x 5 3survival: months 91 (8) 3.25 2 3.25 10 1.7 4.5 surgery 24 % biopsy 40 % biopsy biopsy str str biopsy gtr adjuvant treatment none rt/icpi4 rt/tmz/icpi none rt/tmz mgmt promoter methylation negative positive nd positive nd 5histologic clusters all 5 #3 and #5 mix #3 + #5 #3/ana mix #3 + #5 #5/epi mix #3 + #5 multinucleated giant cells 18 % 100 % yes yes yes giant cell gbm yes mmr ihc retained loss msh2/msh6 msh6 msh2/msh6 nd msh2/msh6 germline mutation no msh2/6 msh2/msh6 msh6 msh6 msh2 msh6 msh2/msh6 detected at gbm workup na 80 % yes yes prior yes yes tmb: mutations/mb 4.3 ± 0.1 24.3 ± 5.1 9 29.5 38.9 17.6 26.3 msi status stable mixed stable stable high nd stable 6dna mutation signature none mmr mmr mmr mmr mmr mmr table 1: demographic, clinical, radiologic, histologic and molecular characteristics of g3/mmr patients. gbm, glioblastoma; m, male; f, female; w, white / caucasian; h, hispanic; r, right; l, left; bg, basal ganglia; crc, colorectal cancer; bcc, basal cell carcinoma; gtr, gross total resection; str, subtotal resection; rt/tmz, radiation / temozolomide adjuvant therapy; icpi, immune checkpoint inhibitors; nd, not determined; na, not applicable. *gbm cohort without the g3/mmr cases. 1numbers in parenthesis apply to the age-corrected cohort excluding less-than-50 year-old patients. 2parameters determined by pre-operative mri. size of enhancement measurements: m62, the whole enhancing area; m68 and m72, the largest focus. 3median survival is indicated for the g3/mmr subgroup and for the rest of the cohort (gbm–mmr) with or without age correction. 4rt/icpi for patient m68 was started 1.5 months post-surgery and stopped 3 weeks later as the tumor progressed during therapy. 5histologic clusters include the #3/ana (anaplastic) and #5/epi (epithelioid) clusters. 6the mmr-type dna mutation signature consists in a predominance of c:g to t:a transitions. the survival in glioblastoma depends on many factors, and these include tumor location and subsequent extent of surgical resection, age and comorbidities, accessibility to and effectiveness of adjuvant treatment [20,21]. the intrinsic aggressivity of the tumor may also influence survival [5,21]. the location of the tumors varied, with a high percentage of tumors showing midline location and multifocality, the latter often interpreted as metastatic disease (table 1, fig. 1b–c). the two multifocal tumors located in the midline that were only biopsied associated with the worst survival in patients m62 and m72 (table 1, fig. 1c). patient m79, with peripherally located tumor crossing the midline, and receiving gross total resection and standard radiation-tmz adjuvant therapy, marginally survived longer. patient m68 underwent subtotal resection of the larger right frontal focus (fig. 1c), and had slow post-surgical recovery, during which the residual tumor focus progressed. the radiation and immune checkpoint inhibitor therapy with pembrolizumab was initiated 1.5 months post-surgery and did not stop tumor growth, being aborted after 3 weeks. the median survival calculated from the date of the surgery was 3.25 months for the g3/mmr subgroup patients, and 9 months for the rest of the cohort (table 1). the survival was equally dismal for four out of the 5 g3/mmr patients, within a range between 1.7 and 4.5 months, with only f70 surviving to 10 months. the age range between 62 and 79 years within the g3/mmr subgroup did not appear to influence the survival, as patients in their 60s or 70s performed the same in this subgroup (table 1). however, age-adjusted survival showed a highly significant difference between patients older or younger than 50 years in the rest of the cohort, with 8 versus 20 months median survival values, respectively (fig. 2a). the median age of the g3/mmr patients was comparable to the ≥  50-years age-adjusted cohort, but the median survival was significantly lower, at 3.25 months versus 8 months, respectively (fig. 2a). compared to the three major glioblastoma subgroups, a statistically significant difference was apparent between g3/mmr patients and the g1/egfr and g3/nf1 subgroup patients that showed 11.3and 8.5-month median survival, respectively, but not the g7/other subgroup patients that showed a lower median survival of 7 months (fig. 2b). figure 2: survival of g3/mmr subgroup patients a-b. survival curves for g3/mmr patients in comparison to patients from the rest of the cohort: older than 50 years (≥50 yo) or younger than 50 years (< 50 yo) (a), and from other g1–g7 subgroups (b). statistical significance: *, p < 0.05; **, p < 0.01; ***, p < 0.001. n, number of deaths; n, number of patients in follow-up. the median survival in months, and the median age in years, are indicated. c. timeline of disease progression and treatment for the g3/mmr patient f70. gbm, glioblastoma; str, subtotal resection; icpi, immune checkpoint inhibitors. d. expression of the ctla4, pd-l1, pd-1 and ido1 immune therapy response targets in the 5 g3/mmr tumors. levels were normalized to the lower expression values for the respective immune target. note very high and high expression levels for ido1 and ctla4, respectively, in m62 and m72 tumors. f70 was the only patient from the g3/mmr subgroup with significantly longer survival to 10 months (table 1, fig. 2c). she had family history of first-degree cousin with glioblastoma, and history of colorectal cancer 10 years prior, status post resection and 5-flurouracil chemotherapy. she presented with dizziness, unsteady gait, sleepiness and confusion and received subtotal resection of the 7.7 cm, right temporal lobe glioblastoma, leaving residual enhancing and non-enhancing tumor in the anterior mesial temporal region. surgery was followed by the standard radiation-tmz regimen consisting of radiation with concurrent tmz. a regimen of immune checkpoint inhibitors consisting of combination nivolumab-ipilimumab (opdivo-yervoy), in the dosing and schedule as for metastatic non-small cell lung cancer (nsclc) [22], was then added for the following 4 months and interrupted when the patient showed decline of cognitive skills. the patient survived an additional 4 months post-therapy. the gene expression for various immune checkpoint inhibitor targets is one of the modalities used for assessing immune checkpoint inhibitor therapies [23]. the expression of ctla4, the cd274/pd-l1 and pdcd1/pd-1 interactors, and ido1, a negative immunomodulator in glioblastoma [24,25], showed variable levels in the g3/mmr tumors (fig. 2d). all markers were low in the m68 tumor. ido1 showed very high levels in the m62 and m72 tumors and low levels in the rest. the ctla4 levels were also slightly elevated in the m62 and m72 tumors. the pd-l1 and pd-1 levels were variable, with at least one slightly elevated in the m62, m72 and m79 cases. mmr glioblastoma shows multinucleated giant cells (mgcs) and ihc compatible with p53 mutation and mmr deficiency. histologic examination of the g3/mmr tumors showed presence of mgcs in all cases (table 1, fig. 3). however, except for case m72 that was diagnosed as giant cell glioblastoma, the mgcs were either scattered or focal in the other cases and admixed with neoplastic cells with high-grade neuroendocrine (hgne) or gemistocytic morphology, which were the predominant histologic patterns. moreover, myxoid extracellular matrix, a feature present in many tumors with hgne morphology from the g7 / other molecular subgroup [4], was present in three cases, m62, m68 and f70. the gfap expression was variable, being lost in subsets of neoplastic cells, in correlation with the hgne morphology [4]. figure 3: histology and ihc of g3/mmr molecular subgroup tumors h&e panels showing tumor morphology with presence of mgcs for all 5 cases, as indicated in the upper left corner. ihc with color-coded antibodies: yellow for mmr, green for gfap, blue for p53, and pink for p16. the mmr ihc is shown only for the f70 case and is representative for the other g3/mmr cases. all panels are shown at 200x magnification, and the scale bars are all the same, at 100 μm. note gigantic cells in the m72 tumor, the only of the 5 g3/mmr tumors diagnosed as giant cell glioblastoma. ihc for the mmr proteins mlh1, pms2, msh2 and msh6 consistently showed preserved nuclear staining for the mlh1-pms2 pair and loss of nuclear staining for the msh2-msh6 pair, or just msh6 for m68 (table 1, fig. 3). ngs confirmed germline msh2 or msh6 alterations, showing perfect correlation with mmr ihc. importantly, the combined ihc and ngs workup revealed the mmr deficiency for the first time in 4 of 5 patients, including in patients with prior history of cancer (table 1), underscoring the importance of combined ihc and ngs workup for glioblastoma. all the tumors showed diffuse nuclear p53 immunostaining, suggestive of tp53 mutation. the ki-67 proliferation index was variable, showing moderate elevation of approximately 20 % in m62 and m79 tumors and high rate of approximately 50 % in the m68 and f70 tumors. ihc with p16 antibody, performed in the m62, m68 and m79 tumors, showed a different expression pattern for each case (fig. 3). this ranged between strong expression for m79, lack of expression for m68, and mixed expression for m62, correlating with the intact, deleted, and mutant status of the cdkn2a gene, respectively (suppl table s1). mgc histology associates with tp53 mutation in multiple glioblastoma subgroups. the morphologic classification of glioblastoma in 12 patterns assigned to 5 histologic clusters, #1/astrocytic (previously called egfr-like), #2/small neuronal, #3/anaplastic, #4/spindle and #5/epithelioid, has been previously described [4]. histologic examination of the entire cohort showed that mgcs were also present in tumors from other molecular subgroups, making up approximately one fifth (18 %) of the total glioblastoma cases other than g3/mmr ones (fig. 4a). tumors containing scattered mgcs resembling the g3/mmr tumors were especially enriched in the g1/egfr-mutant subgroup (50 %), and less in the g6/multi-rtk (25 %), g7/other (24 %), g3/nf1 (20 %) and g5/pdgfra (20 %) subgroups, with the major g1/egfr-amplified subgroup showing only few cases (8 %). in total, 43 primary glioblastoma cases featured mgcs, with the g7/other and g3/nf1 subgroups contributing 10 cases each, and the g3/mmr subgroup, g1/egfr-amplified, g1/egfr-mutant and g6/multi-rtk subgroups contributing 5 cases each (fig. 4b). these data indicated that the presence mgcs in a tumor is not specific for the g3/mmr subgroup. figure 4: mgcs in glioblastoma: distribution in g1–g7 molecular subgroups, histologic profiles, and p53 pathway mutations a. incidence of cases with mgcs in the g1–g7 glioblastoma subgroups. gbm, glioblastoma; egfr↑, egfr-amplified; egfrm, egfr-mutated; total gbm-mmr, gbm cases without the g3/mmr cases. b. pie-chart distribution (%) of the 43 cases with mgcs in g1–g7 molecular subgroups. c. distribution of the 43 cases with mgcs into histologic clusters in the g1–g7 molecular subgroups (upper bar graph), or as % (lower pie chart). #, number of cases; ana, anaplastic; epi, epithelioid. d. distribution of p53 pathway alterations in the 43 tumors containing mgcs. tp53/ihc+ and tp53/ihc-, mgc cases with tp53 mutations and positive or negative p53 ihc, respectively; other (than tp53 or mdm2/4) alterations include one case each with rpl5 and ebf1 mutations. e. bar graph showing the % of cases with or without mgcs from the total gbm cases with tp53 mutations (n) and per g1–g7 molecular subgroup. total gmb-mmr, tp53-mutant cases without the g3/mmr cases. note that overall, only 45 % of the tp53-mutant tumors contain mgcs. f. bar graph showing the number of allelic tp53 mutations. gbm, tp53-mutant glioblastoma cases without the g3/mmr cases. examination of the associated morphologies in these cases revealed that they either fell into the #3/anaplastic, #5/epithelioid, mixed anaplastic/epithelioid or mixed spindle/epithelioid clusters, indicating that anaplastic and epithelioid morphologies are dominant in the cases with mgcs (fig. 4c). moreover, the mixed spindle/epithelioid morphology was limited to g3/nf1 subgroup tumors that may show a sega-like histologic profile [16]. other than this sega-like morphology that was noted only in the g3/nf1 subgroup, and the giant cell glioblastoma case m72 that was unique to the cohort, the other cases showed scattered mgcs, significantly smaller than in the m72 case (fig. 3), regardless of the molecular subgroup. no giant cells were recorded in cases from the minor subgroups g2/fgfr3 and g4/raf, and only 5 cases mapped to the largest molecular subgroup g1/egfr-amplified, as these three subgroups predominantly display morphologies in the #1/astrocytic or #2/small neuronal clusters that were virtually exclusive of mgcs. all the cases in the g3/mmr subgroup showed ihc positive for p53 (fig. 3 and 4d), and carried tp53 mutations (suppl. table s1). analysis of the other cases with mgcs showed that the large majority also showed p53 ihc positivity and carried tp53 mutations, regardless of the molecular subgroup (fig. 4d). one tumor occurring in a patient with li-fraumeni syndrome showed frameshift mutation in the beginning of tp53 gene with lack of p53 protein expression, and another 12 % of cases with mgcs showed alterations in mediators of the p53 pathway, such as mdm2, mdm4, rpl5 or ebf1 [5,26,27] (fig. 4d). only 7 % of the cases with mgcs did not show alterations in the p53 pathway, and they all clustered in the g3/nf1 subgroup. in total, approximately one fifth (21.3 %) of the cases with mgcs were negative for p53 ihc (fig. 4d, pie chart). examination of the tp53 mutations in the glioblastoma cohort showed some distinctive features of the g3/mmr cases. whereas all the cases in the g3/mmr subgroup showed tp53 mutations and mgcs, only approximately half of the cases with tp53 mutations from other molecular subgroups showed an mgc phenotype, except for the g1/egfr-mutant subgroup that showed 83 % of tp53 mutant cases displaying an mgcs (fig. 4e). overall, the g3/mmr cases represented one seventh (14.3 %) of the cases showing both mgcs and p53 immunopositivity. a major difference also consisted in the number of the tp53 mutations: whereas in g3/mmr cases, tp53 was inactivated by different missense mutations most likely targeting both alleles (suppl. table 1), 83 % of the tumors in the rest of the cohort showed only one tp53 mutation, with the second allele inactivated by loss or neutral loss of heterozygosity (fig. 4f). molecular landscape of the g3/mmr glioblastoma subgroup tumors. the tmb of the g3/mmr cases was significantly higher than that of the other glioblastoma cases, with or without high msi (table 1 and fig. 5a). for comparison, three additional cases from the glioblastoma cohort showed very high tmb (fig. 5a; suppl. table s2). one was a tumor at initial presentation in a 53-years-old male (m53/sc) that showed clonal vaf in few mutations common to g1/egfr-amplified tumors, and subclonal vaf in a multitude of less common mutations indicative of a subclonal population of cells with very high tmb. two other cases were recurrent, g1/egfr-amplified subgroup tumors in male and female patients aged 66 and 58 years, respectively (m66/tmz and f58/tmz), previously treated with tmz, a known hypermutator phenotype inducer [28]. interestingly, of the 20 post-tmz, recurrent tumors subjected to ngs in this cohort, of which only 5 mapped to the g1/egfr-amplified subgroup, both tumors with hypermutator phenotype belonged to the g1/egfr-amplified subgroup. all these three g1/egfr-amplified tumors harbored very high tmb, stable msi, but lacked mgc histology. their immunohistochemical profiles were undistinguishable from those of g1/egfr-amplified tumors, showing negative p53 and p16 ihc. figure 5: molecular profiling of the g3/mmr subgroup a. tmb mean±sem graph of individual case values from indicated cases. post-tmz, post tmz chemotherapy; total gbm-mmr, gbm cases without the g3/mmr cases. statistical significance: *, p <  0.05. b. genomic alterations in effectors of the pathways indicated on the left are shown by color-coded squares. telom, telomere maintenance; cc g1, cell cycle g1 phase; ddr, dna damage response; chrm, chromatin remodeling; chrom, chromosomes; expr, rna expression. c–d. line graphs tracing the incidence of the indicated alterations in the g3/mmr subgroup in comparison with the rest of the g1–g7 molecular subgroups. e. rna expression heatmaps for the indicated rtks, transcription factor ap-1 complex and mapk pathway feedback effectors. a comparison of the molecular landscape of the g3/mmr cases with the 3 cases with very high tmb and, in general, with the distribution of mutations from the rest of the g1–g7 molecular subgroups (suppl. tables s2-s3), showed distinctive features for the g3/mmr subgroup (table 1; fig. 5b–d; suppl. table s1). germline pathogenic alterations in msh2 or msh6 mmr genes were present in all g3/mmr cases but absent in the rest of the cohort. these were either splice site or frameshift mutations, resulting in truncation of the protein, or loss of the msh2/msh6 locus on chromosome 2p16.3, an exceedingly rare event in glioblastoma present only in the m79 g3/mmr case from the entire cohort. interestingly, the msh6 p.f1088fs mutation was recurrent, either as germline or as second hit somatic mutation (suppl. table s1). in comparison, the 3 cases with very high tmb showed pathogenic or likely pathogenic msh2 or msh6 mutations with vaf in the somatic range, of which only one resulted in protein truncation, in m66/tmz, the other being missense mutations, with subclonal vaf in the m53/sc tumor (suppl. table s2). in comparison, a paucity of pathogenic mutations in mmr, pole and mutyh genes, the latter two also known to induce hypermutator phenotype [29,30], was apparent in the cohort, without high tmb in any of these mutant cases (suppl. table s2). in particular, the g2/fgfr3 molecular subgroup displayed higher incidence of mmr/mutyh mutations, with the m60 case showing the only msh6 truncating mutation with vaf in the somatic range from the cohort. as previously described, this tumor showed a typical g2/fgfr3 molecular profile and lacked mgcs [15] (suppl. table s2). a truncating mutation in mlh3, a paralog of mlh1, with vaf in the germline range, in the g1/egfr-amplified m31 tumor, was the only other germline mmr truncating mutation from the cohort, and did not show high tmb, most likely due to compensation by the mlh1-pms2 mmr pair. the only two tumors that showed concomitant mgcs, tp53 mutations and mutations in either mutyh or msh3 but without hypermutator phenotype belonged to the g5/pdgfra subgroup (suppl. table s2). it has been shown that deficiency in dna repair results in a specific dna mutation signature in tumors, represented by predominance of c:g to t:a transitions in mmr-deficient tumors, and c:g to a:t transversions in pole, pold1 and mutyh mutant tumors [31,32]. all g3/mmr tumors, as well as the m66/tmz and f58/tmz tumors, showed an mmr-type dna mutation signature (table 1). surprisingly, the m53/sc tumor showed a pole-type dna mutation signature, and mutation analysis revealed two subclonal polh mutations, including one pathogenic (fig. 5b, suppl. table s2). since no report is available in the literature for association of polh mutations with high tmb, the latter may be due to the msh2 subclonal mutations but possibly fine-tuned by the polh deficiency. the g3/mmr subgroup tumors lacked or showed low incidence for many of the common glioblastoma molecular alterations, such as simultaneous chromosome 7 gain and 10 loss (see also [5]), tert promoter mutations and cdkn2a homozygous loss (fig. 5b–c, suppl. table s3). conversely, they showed 100 % tp53 mutation rate and high incidence of mutations in genes from dna damage response (ddr) and chromatin remodeling pathways, especially from the swi/snf complex (fig 5b–c, suppl. tables s1, s3). interestingly, likely pathogenic notch1 missense mutations were detected in three of the five g3/mmr cases. 100 % of g3/mmr tp53 mutations were biallelic (fig. 4f) and targeted arg residues (suppl. table s3). in the rest of the cohort, tp53 arg mutations were seen in a quarter of cases, the highest rate of 40 % being noted in the g7 / other subgroup. the pi3k pathway was strongly activated by mutations in pik3ca and pten upstream effectors, but also by tsc2 mutations (fig. 5b–d, suppl. table s1). the 80 % mutation rates for pik3ca and pten were the highest in the cohort, the pik3ca mutation rate being three-fold higher than the next highest rate from the g5/pdgfra subgroup (suppl. table s3). three g3/mmr cases showed overlapping pik3ca and pten mutations, and one of these showed additional pik3r1 subclonal mutation (fig. 5b). the g3/mmr 60 % rate of overlapping mutations in pi3k pathway upstream effectors was 10-fold higher than the 5.6 % rate in the rest of the cohort (fig. 5d). strikingly, pik3ca p.r88q was the activating mutation in all four g3/mmr mutant cases, whereas it was a single event in the 34 pik3ca-mutant cases from the rest of the cohort (fig. 5b, d; suppl. table s3). similar to tp53, pik3ca mutations on arg residues were scarce in the rest of the cohort, numbering one p.r88q and one p.r38l in the g7 / other and g3/nf1 subgroups, respectively. pten mutations were biallelic in two g3/mmr cases (40 %), whereas two concomitant pten mutations were noted only in 3 cases in the rest of the cohort (2.5 %), all showing subclonal vaf, suggestive of heterogeneity rather than of biallelic hit. double hit mutations were also detected in tsc2 tumor suppressor gene, in two out of three mutant g3/mmr cases (fig. 5b, suppl. table s1). tsc1/2 mutations are relatively uncommon in glioblastoma, numbering 8 cases in the rest of the cohort (3.8 %), without double hit (fig. 5d). the mapk pathways was activated by mutations in many effectors, some overlapping, such as ptpn11 and nf1 or kras (fig. 5b). none of the major rtks defining the g1/egfr, g2/fgfr3, g5/pdgfra or g6/multi-rtk subgroups was amplified or mutated. likely pathogenic missense mutations in ret in two cases, and flt1 and csf1r in one case each, were detected (suppl. table s1), of which the ret mutation in case f70 was accompanied by overexpression (fig. 5e, suppl. table s4). virtually all the g3/mmr cases showed moderate overexpression of at least one rtk except for egfr, and the m72 tumor showed high met overexpression close to the range of the met-amplified tumors from g6/multi-rtk subgroup (fig. 5e, suppl. table s4). fos and jun families of transcription factors heterodimerize to form the activator protein-1 (ap-1) complex that is phosphorylated and activated by mapk [33]. the four members of the fos family are transcription activators, whereas jund from the jun family acts mostly as repressor. the expression of the ap-1 complex transcription factors in g3/mmr cases showed overexpression of one or more activating transcription factors, with the m72 and m79 tumors highly overexpressing fosb, and additionally fosl1 for m72 (fig. 5e, suppl. table s5). consistent with its role as suppressor, jund showed no overexpression and was decreased in the m68 and f70 tumors that showed lesser overexpression of the transcription activator family members (suppl. table s5). the activation of the mapk and pi3k canonical growth pathways has been shown to result in an inhibitory feedback response in glioblastoma [15]. the dual-specificity phosphatases (dusp) directly dephosphorylate and inactivate mapk, with dusp5 and dusp1/6 specifically dephosphorylating erk1 and erk2, respectively, and dusp4 dephosphorylating erk1, erk2 and junk [34]. of these, dusp5 was upregulated in all five g3/mmr tumors, and dusp1 in three tumors (fig. 5e and suppl. table s6), suggesting mapk activation in all tumors. moreover, the more specific inhibitors errfi1 and spry1/4 targeting downregulation of growth signaling from the egfr and fgfr rtk families, respectively [35,36], were strongly upregulated in m72 and f70 tumors, respectively (fig. 5e and suppl. table s6). discussion the identification of molecular subgroups susceptible to targeted therapies is the ultimate goal of tumor classifications. the mapk pathway-based g1–g7 molecular classification of glioblastoma allowed subclassification of the small number of cases with genetic mmr deficiency in the g3/mmr molecular subgroup [5]. the incidence of these tumors in the prospective cohort of 218 glioblastoma cases is low, approximately 2 %. unlike three recent studies that have characterized few glioblastoma cases with mmr deficiency mainly in younger adults [13,14,37], this is the first study assembling five cases of glioblastoma with germline mmr deficiency in adults over 60 years of age, who represent the large majority of patients with glioblastoma. of the studies in younger mmr patients, the only one showing survival data concluded that these mmr patients exhibit better survival by comparing them to a standard glioblastoma cohort significantly much older, with a difference of 13 years in median age [13]. studies, including the current one, have clearly shown significant age-dependent differences for glioblastoma patient survival [20,21], and therefore the conclusion of a longer survival even for younger adult mmr patients is inaccurate methodologically in the afore mentioned study [13]. the current study unequivocally showed dismal survival for the adult g3/mmr glioblastoma patients, significantly shorter than for the rest of the glioblastoma cohort adjusted for age. this finding stratifies the g3/mmr as a high-risk glioblastoma subgroup and aligns it with a recent study reporting poor prognosis for lynch-syndrome patients with idh-mutant astrocytoma [38]. the speedy identification of the g3/mmr patients is peremptory for prompt therapy with immune checkpoint inhibitors. the only controlled study to date of immune checkpoint therapy for recurrent mmr-deficient glioblastoma has not found a significant difference to control when using pembrolizumab, a pd-1 inhibitor [39]. in addition, a recent case report has shown lack of effectiveness of pembrolizumab for controlling lynch-syndrome-associated glioblastoma in a young adult patient [40]. in agreement with these data, the m68 patient from the g3/mmr subgroup was briefly treated with pembrolizumab without response, although it is not clear if the lack of immune checkpoint marker expression may have contributed to the inhibitor resistance. in contrast, the f70 patient who was treated with the dual regimen of nivolumab-ipilimumab after standard concurrent radiation-tmz adjuvant therapy, showed much better survival than the other patients from the g3/mmr subgroup. this result, even if unique to this adult g3/mmr patient, shows that the combination nivolumab-ipilimumab targeting both pd-1 and ctla-4, respectively, may be effective on adult g3/mmr glioblastoma. few pediatric examples of mmr/lynch syndrome glioblastoma were successfully treated with pd-1 inhibitors [41,42], but in one case, a sustained response could only be achieved after addition of anti-ctla-4 ipilimumab to the initial anti-pd-1 nivolumab regimen [43]. this dual regimen is also used in metastatic crc associated to lynch syndrome [44], and as first-line therapy for metastatic or advanced nsclc with tmb ≥  10 mutations / mb, regardless of the pd-l1 expression levels [22,45,46]. the identification of the g3/mmr patients relies on clinical, histologic and molecular clues. a review of the literature identified 5 additional adult (≥ 50-years) g3/mmr cases with clinical, histologic and molecular data [13,14], and the integrated findings from the total of 10 g3/mmr adult glioblastoma patients is summarized in table 2. clinical history of prior cancers pertains to half of the patients, with highest frequency of crc, in 4 of the 10 cases. older g3/mmr patients appear to be prone to multifocal or deep-seated tumors that warrant subtotal resections or only biopsies. histologically, 100 % of these tumors contain mgcs. as shown in this study, mgcs are not specific for g3/mmr tumors, and other g1–g7 subgroups, especially the ones with tumors displaying morphology classified in the anaplastic and epithelioid clusters, may show a variable number of tumors with mgcs (see fig. 4). in addition to mgcs, all the g3/mmr tumors also show positive p53 ihc. however, as shown in this study, nearly 80 % of the tumors with mgcs showed p53 immunopositivity, with the g1/egfr-mutant molecular subgroup mimicking the closest the g3/mmr subgroup in terms of morphology and p53 immunopositivity (see fig. 4). overall, the g3/mmr subgroup cases represented 14.3 % (one seventh) of the total glioblastoma cases showing both mgcs and p53 immunopositivity. the ihc with the mmr panel appears to be specific for g3/mmr cases (table 2), but will only be yielding positive results in 1 out of 7 glioblastoma cases with concomitant mgcs and p53 immunopositivity. current study kim et al. [14] hadad et al. [13] total 3 studies number of patients 5 3 2 10 median age (range) in years 70 (62 to 79) 69 (50, 69 and 75) 53.5 (50 and 57) 68.5 (50 to 79) sex m:f ratio 4 : 1 1 : 2 1 : 1 1.5 : 1 history of prior cancer 60 % 67 % 0 % 50 % location: multifocal or midline 60 % 67 % 0 % 50 % survival: months 3.25 (median) nd 6.4 and 50.5 4.5 (median) surgery 40 % biopsy 100 % gtr 100 % gtr 60 % gtr adjuvant treatment (rt/tmz) 67 % 100 % 100 % 80 % multinucleated giant cells 100 % 100 % 100 % 100 % ihc msh2/msh6 100 % 100 % 100 % 100 % mmr mutation frequency msh6 > msh2 msh6 > msh2 msh2 msh6 ≥ msh2 ihc p53 100 % 100 % 100 % 100 % tp53 mutations (biallelic/r mutations %) 100 % (100/100) 100 % (67/100) 100 %(50/100) 100 % (80/100) mapk pathway (ptpn11/ras/nf1 %) 60 % (40/20/40) 100 % (67/0//67) 100 % (0/0/100) 80 % (40/10/60) pi3k pathway (pik3ca/pik3ca r88q/pten %) 100 % (80/100/80) 100 % (67/0/33) 100 % (50/100/100) 100 % (70/71/70) ddr pathway 80 % 67 % 50 % 70 % chromatin remodeling (dnmt3a/ setd2/ swi/snf %) 100 % (40/20/80) 100 % (0/33/100) 100 % (100/100/100) 100 % (40/40/90) common gbm alterations (tert; chr 7gain&10 loss) 0 % 0 % 0 % 0 % average tmb (mutations/mb) 24.3 23 35 27.3 msi-high 25 % 100 % 100 % 67 % table 2: review: integrated characteristics of g3/mmr subgroup glioblastoma in ≥ 50-year-old patients. gbm, glioblastoma; m, male; f, female; gtr, gross total resection; str, subtotal resection; rt/tmz, radiation/temozolomide; nd, not determined; chr, chromosome. the ngs molecular analysis with tmb inclusion is necessary for detecting and/or confirming the presence of g3/mmr cases. in general, high tmb is a relatively rare occurrence in untreated, first-presentation glioblastoma, and is more often seen in recurrences with tmz-related hypermutator phenotype [28]. noteworthy, both post-tmz recurrences described here mapped to the g1/egfr-amplified subgroup, suggesting an association worth looking into. a hypermutator phenotype in untreated tumors may be the result of germline and/or somatic mutations in mmr, pole or mutyh genes. germline mmr mutations were found only in msh2 or msh6, with the msh6 p.f1088fs mutation recurrent in 2 studies, the current one and that by kim et al. [14]. in the current study, somatic mmr mutations did not result in hypermutator phenotype in untreated, first-presentation glioblastomas (see suppl. table s2). however, at least three ≥ 50-year-old patients in the studies by hadad et al. and kim et al. presented with hypermutator phenotype in sporadic glioblastomas with somatic mmr alterations [13,14]. the question arises if these tumors should be included in the g3/mmr high-risk subgroup where mmr mutations are causative, or if their tumors, like the m53/sc case, show only added hypermutator phenotype to a baseline mutation core that fits another g1–g7 subgroup and show therefore better prognosis. more studies are necessary to clarify this issue, and treatment with immune checkpoint inhibitors may prove beneficial in glioblastomas with both germline and somatic mmr mutations. for mutyh, a pan-cancer recent study has shown that biallelic mutations and not heterozygous germline variants result in high tmb [30]. glioblastoma cases with high tmb due to mutyh mutations have not been reported in the literature, and in our series, the two tumors with heterozygous germline mutyh mutations did not show high tmb (see suppl. table s2). pole mutations in glioblastoma are equally rare, with only one in this cohort, without high tmb. however, pole somatic mutations with hypermutator phenotype and usually accompanied by germline or somatic mmr mutations have been reported mainly in young glioblastoma patients [13,29]. beside high tmb, the g3/mmr subgroup tumors showed a common signature represented by mutations resulting in ras activation, with high incidence of ptpn11 and nf1 mutations (table 2), an association also noted in the g3/nf1 subgroup [4]. the resulting mapk activation was accompanied by high levels of the transcription factors from the ap-1 effector complex and by upregulation of a negative feedback, of which dusp5 was the most consistently upregulated. the activation of the pi3k pathway was present in all the cases, with high incidence of pik3ca mutations, especially of p.r88q, of overlapping pik3ca and pten mutations, and of tsc2 mutations (table 2 and fig. 5d). as shown, tp53 was mutant in all the cases, with predominance of biallelic arg mutations. very high incidence of mutations in the ddr and chromatin remodeling pathways, particularly in dnmt3a, setd2 and the swi/snf complex, coupled to absence of the characteristic tert promoter mutations or concomitant chromosome 7 gain and 10 loss, distinguished the g3/mmr subgroup from the other g1–g7 subgroups (table 2 and fig. 5c) [5]. mutations in apc and notch1/2 genes were also overrepresented in g3/mmr patients from this study and that by kim et al. [14]. in general, the g3/mmr molecular profile resembled closer the profiles of other solid cancers and was highly divergent from that of non-mmr glioblastoma [47]. conclusions in conclusion, this study characterizes rare cases of adult glioblastoma associated to lynch syndrome that appear to represent a high-risk g3/mmr subgroup showing distinctive clinical, histologic and molecular characteristics. it reviews the available cases in the literature, and proposes immune checkpoint inhibitors as adjuvant treatment, particularly the dual nivolumab-ipilimumab regimen that showed net survival benefit in the g3/mmr patient receiving it. more studies are necessary to validate this regimen and correlate it with the molecular parameters usually used in crc associated to lynch syndrome, such as tmb, msi status and immune checkpoint markers expression levels. ethics, consent and permissions this study was conducted in accordance to the declaration of helsinki, and approved by the ethics committee of neuromarkers (protocol code nm-rec-2 on 9 january 2020 and 30 january 2023) for studies involving human subjects. informed consent was obtained for all subjects. consent for publication patient consent for publication was waived due to the study not identifying the patients. availability of data and materials the genomic and transcriptomic datasets supporting the conclusions of this article are de-posited in the public repository neuromarkers open research database https://neuromarkers.org/database under the accession numbers g-nm/24-1 and t-nm/24-1, respectively. conflicts of interest statement the author declares no conflicts of interest. funding statement this work was supported by an award from neuromarkers [nm2024-1] to m.-m.g. acknowledgements this paper is dedicated to the patients and their families. references 1. ostrom, q.t.; truitt, g.; gittleman, h.; brat, d.j.; kruchko, c.; wilson, r.; barnholtz-sloan, j.s. relative survival after diagnosis with a primary brain or other central nervous system tumor in the national program of cancer registries, 2004 to 2014. neurooncol pract 2020, 7, 306-312. https://doi.org/10.1093/nop/npz059 2. who classification of tumors editorial board. central nervous system tumors, 5th ed., international agency for research on cancer, lyon (france), 2021. https://doi.org/10.1093/neuonc/noab106 3. georgescu, m.m. pten tumor suppressor network in pi3k-akt pathway control. genes & cancer 2010, 1, 1170-1177. https://doi.org/10.1177/1947601911407325 4. georgescu, m.m. multi-platform classification of idh-wild-type glioblastoma based on erk/mapk pathway: diagnostic, prognostic and therapeutic implications. cancers (basel) 2021, 13. https://doi.org/10.3390/cancers13184532 5. georgescu, m.m. translation into clinical practice of the g1–g7 molecular subgroup classification of glioblastoma: comprehensive demographic and molecular pathway profiling. cancers (basel) 2024, 16. https://doi.org/10.3390/cancers16020361 6. li, y.m.; suki, d.; hess, k.; sawaya, r. the influence of maximum safe resection of glioblastoma on survival in 1229 patients: can we do better than gross-total resection? j neurosurg 2016, 124, 977-988. https://doi.org/10.3171/2015.5.jns142087 7. stupp, r.; mason, w.p.; van den bent, m.j.; weller, m.; fisher, b.; taphoorn, m.j.; belanger, k.; brandes, a.a.; marosi, c.; bogdahn, u., et al. radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma. n engl j med 2005, 352, 987-996. https://doi.org/10.1056/nejmoa043330 8. oster, c.; schmidt, t.; agkatsev, s.; lazaridis, l.; kleinschnitz, c.; sure, u.; scheffler, b.; kebir, s.; glas, m. are we providing best-available care to newly diagnosed glioblastoma patients? systematic review of phase iii trials in newly diagnosed glioblastoma 2005-2022. neurooncol adv 2023, 5, vdad105. https://doi.org/10.1093/noajnl/vdad105 9. cruz da silva, e.; mercier, m.c.; etienne-selloum, n.; dontenwill, m.; choulier, l. a systematic review of glioblastoma-targeted therapies in phases ii, iii, iv clinical trials. cancers (basel) 2021, 13. https://doi.org/10.3390/cancers13081795 10. dunn, d.b. larotrectinib and entrectinib: trk inhibitors for the treatment of pediatric and adult patients with ntrk gene fusion. j adv pract oncol 2020, 11, 418-423. https://doi.org/10.6004/jadpro.2020.11.4.9 11. lynch, h.t.; snyder, c.l.; shaw, t.g.; heinen, c.d.; hitchins, m.p. milestones of lynch syndrome: 1895-2015. nat rev cancer 2015, 15, 181-194. https://doi.org/10.1038/nrc3878 12. therkildsen, c.; jensen, l.h.; rasmussen, m.; bernstein, i. an update on immune checkpoint therapy for the treatment of lynch syndrome. clin exp gastroenterol 2021, 14, 181-197. https://doi.org/10.2147/ceg.s278054 13. hadad, s.; gupta, r.; oberheim bush, n.a.; taylor, j.w.; villanueva-meyer, j.e.; young, j.s.; wu, j.; ravindranathan, a.; zhang, y.; warrier, g., et al. 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creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. a malignant choroid plexus tumor with heterologous differentiation and bap1 deletion suggesting choroid plexus blastoma feel free to add comments by clicking these icons on the sidebar free neuropathology 6:18 (2025) letter a malignant choroid plexus tumor with heterologous differentiation and bap1 deletion suggesting choroid plexus blastoma arnault tauziède-espariat1,2, alice métais1,2, léa guerrini-rousseau3,4, farah sassi1, lauren hasty1, raphaël saffroy5, volodia dangouloff-ros6, kévin beccaria7, euphrasie servant1,2, pascale varlet1,2 on behalf of the renoclip-loc department of neuropathology, ghu paris psychiatry and neuroscience, sainte-anne hospital, paris, france université de paris, umr s1266, inserm, ima-brain, institute of psychiatry and neurosciences of paris, paris, france department of children and adolescents oncology, gustave roussy, université paris-saclay, villejuif, france molecular predictors and new targets in oncology, inserm u981, gustave roussy, université paris-saclay, villejuif, france department of biochemistry and oncogenetic, paul brousse hospital, villejuif, france department of pediatric radiology, aphp, hôpital universitaire necker enfants malades; inserm u1299 and umr 1163, institut imagine, université paris-cité, paris, france department of neurosurgery, paris hospitals public assistance, hôpital necker-university paris cité, paris, france department of pediatric neurosurgery, aphp hopital universitaire necker enfants malades, université paris-cité, paris, france corresponding author: arnault tauziède-espariat · department of neuropathology · ghu paris -psychiatry and neuroscience·sainte-anne hospital · 1, rue cabanis, 75014 · paris · france a.tauziede-espariat@ghu-paris.fr submitted: 25 august 2025 accepted: 29 september 2025 copyedited by: georg haase published: 02 october 2025 https://doi.org/10.17879/freeneuropathology-2025-8899 keywords: choroid plexus, malignant, heterologous components, bap1, tp53 pediatric intraventricular tumors are mainly represented by choroid plexus neoplasms. among them, the choroid plexus carcinoma (cpc) represents the malignant form. this tumor type is characterized by frequent tp53 alterations, two methylation classes (adult and pediatric subtypes) (1,2), and can sometimes occur in the hereditary context of syndrome. previously, our team reported a malignant plexus tumor presenting heterologous elements such as a pleomorphic spindle cell component and a polyphenotypic profile, which classified it as a choroid plexus tumor, subclass pediatric b by dna-methylation profiling analysis (3). herein, we present a morphologically similar choroid plexus tumor, this time harboring bap1 and tp53 alterations. a three-year-old girl, whose sister had ewing sarcoma, presented with signs of intracranial hypertension. magnetic resonance imaging (mri) revealed the presence of a large tumor located in the posterior part of the left lateral ventricle. cranial computed tomography (ct) scans revealed heterogeneous enhancement and intracranial calcifications (figure 1a–g). ct scan of the chest, abdomen, and pelvis did not find other abnormalities. germ cell markers were negative in the blood and in the cerebrospinal fluid. gross total resection was achieved. histopathologically, the tumor was pleomorphic with epithelial (arranged in solid sheets and papillary structures), mesenchymal (fascicles of spindle cells in a myxoid stroma and osteoid formation), and melanocytic elements (figure 2a–d). there was no myogenic differentiation. the cellular density was high, with nuclear pleomorphism, necrosis, and frequent mitotic figures (> 20/2.3 mm2). using immunohistochemistry, tumor cells were found to variably expressed cytokeratins ae1/ae3, ck18 (figure 2e), gfap, ps100, mitf, hmb45 (figure 2f), desmin and smooth muscle actin. smarcb1/ini1 (figure 2g), brg1/smarca4 (figure 2h) and h3k27me3 expressions were maintained. there was no immunoreactivity for lin28a, olig2, ema, sall4, myogenin, nut, etv4, bcor or germinal markers (oct3/4, plap, beta-hcg, and alpha-fetoprotein). there was no expression of p53, and ki67/mib1 was expressed in 70 % of cells (figure 2i). next-generation sequencing analyses of tumor cells evidenced a homozygous deletion of 3p21.1, including the bap1 gene and a loss of function tp53 mutation (p.y220c). rna-sequencing analysis did not identify any fusion transcript. dna-methylation profiling analysis classified the tumor as a cpc, pediatric subtype (calibrated scores > 0.9 in both classifiers dkfz v12.8 and bethesda nih v2.0). using t-distributed stochastic neighbor embedding analysis, the tumor clustered with a case previously reported (3), in close vicinity to cpc, pediatric subtype (figure 3a–c). whole exome sequencing analysis failed to reveal a bap1 or tp53 germline alteration. considering all these results, a complementary immunostaining of bap1 was performed and evidenced a loss of protein expression in tumor cells (figure 3b). the patient received conventional and high-dose chemotherapy, followed by local radiation therapy. one year post treatment, a distant recurrence in the anterior right lateral ventricle was detected on imaging and confirmed by repeat resection. histopathology was consistent with the initial tumor. at present, 21 months after the initial diagnosis, the patient is alive. figure 1. radiological features. a. an intraventricular mass in contact with the choroid plexus showing heterogeneous enhancement after injection of gadolinium on t1-weighted computed tomography (ct) images. b. intermediate t2 signal. c calcifications and hemorrhagic changes on susceptibility weighted imaging. d–e. diffusion restriction and low apparent diffusion coefficient. f. elevated perfusion on arterial spin labeling perfusion imaging. g. ct scans showing intracranial calcifications. figure 2. histopathological features a–d. histopathologically, the tumor was pleomorphous, and composed of an epithelial component with tubular structures, a mesenchymal component with spindle cells arranged in a myxoid stroma, or with an osteoid or melanin component (hps, magnification x400). e expression of ck18 (magnification x400) and hmb45 for a subset of tumor cells (f. magnification x400). maintained expression of smarcb1/ini1 and brg1/smarca4 (g–h. magnification x400). high mib1 labeling index (i, magnification x400).hps: hematoxylin phloxin saffron. scale bars represent 60 μm. clicking into the respective picture will lead you to the full virtual slide https://doi.org/10.57860/min_dts_000023 figure 3. epigenetic features. a. copy number variation analysis of the tumor showed several aneuploidies without any amplification but a deletion of chromosome 13. b. absence of protein bap1 immunolabeling in the tumor cells (magnification x400). c. t-distributed stochastic neighbor embedding analysis which included all dna-methylation reference classes, showed that the current tumor clustered alongside the previously reported case of choroid plexus blastoma (3) and within the molecular subgroup "choroid plexus tumor, subclass paediatric b". the scale bar represents 60 μm. in conclusion, we present the case of a pediatric malignant choroid plexus tumor with heterologous components and a bap1 gene deletion. the current observation illustrates the diagnostic difficulties in pediatric tumors showing polyphenotypic histopathology. in the cns, embryonal tumors, teratomas, and glioneuronal tumors can present heterologous differentiations. rare observations of metaplasia have been reported in low-grade choroid plexus papillomas (7,8). the current intraventricular tumor contained a component resembling that of choroid plexus carcinoma. the differential diagnosis of a germ cell tumor was ruled out because of the negativity of germ cell markers, the absence of a mature teratomous component, and the epigenetic results classifying the tumor as a cpc. the dna-methylation profiling was in line with this diagnosis, and the case was very similar to a previous observation in terms of clinics (age of onset, location), radiology, histopathology (presence of blastematous elements), and epigenetics (3). the current and the previous observations also share a familial context of tumors but without evidence of a germline alteration (particularly tp53 and dicer1). in contrast to the previous observation where immunohistochemical analysis retrospectively confirmed the maintained expression and no variant in bap1 gene (3), the current case harboured a bap1 deletion and a loss of protein expression, but no germline variant was evidenced. moreover, no bap1 deletion has been reported in cpc (1,2). different tumor types have been identified as having somatic bap1 alterations, and a subset belong to bap1-tumor predisposition syndrome. in the cns, meningiomas of various histologies (9) belong to this spectrum but no choroid plexus tumor has been reported to date. here, there was no evidence for li-fraumeni-syndrome and bap1-tumor predisposition syndrome. indeed, the tumor does not resemble other neoplasms with bap1 deletions reported in extra-cns locations, and the patient’s whole body imaging did not reveal any other tumor. while bap1 is generally not associated with ewing sarcoma, clinvar does report a single case with a likely pathogenic bap1 variant in the germline (see https://www.ncbi.nlm.nih.gov/clinvar/rcv000722041.1/). our patient did not harbor any germline bap1 variants, suggesting that the occurrence of ewing sarcoma in the sibling is coincidental. we could like to point out that this case represents the second observation of a malignant choroid plexus tumor showing heterologous elements, thus highlighting the value of dna-methylation profiling in the diagnosis of choroid plexus tumors with unusual histopathological features. additional observations are needed to confirm if this neoplasm represents a novel tumor type (blastoma) or a subtype of choroid plexus carcinoma. ethics approval this study was approved by ghu paris psychiatry and neuroscience, sainte-anne hospital’s local ethics committee. consent for publication the patients signed informed consent forms before treatment began. conflict of interest statement the authors declare that they have no conflict of interest directly related to the topic of this article. funding statement the authors declare that they have not received any funding. acknowledgements we would like to thank the laboratory technicians at ghu paris neuro sainte-anne for their assistance. references 1. thomas c, soschinski p, zwaig m, oikonomopoulos s, okonechnikov k, pajtler kw, et al. the genetic landscape of choroid plexus tumors in children and adults. neuro-oncol. 12 avr 2021;23( 4 ):650‑60. https://doi.org/10.1093/neuonc/noaa267 2. thomas c, metrock k, kordes u, hasselblatt m, dhall g. epigenetics impacts upon prognosis and clinical management of choroid plexus tumors. j neurooncol. mai 2020;148( 1 ):39‑45. https://doi.org/10.1007/s11060-020-03509-5 3. tauziède-espariat a, pagès m, masliah-planchon j, bourdeaut f, doz f, beccaria k, et al. a malignant choroid plexus tumour with prevailing immature blastematous elements. neuropathol appl neurobiol. févr 2022;48( 2 ):e12764. https://doi.org/10.1111/nan.12764 4. tauziède-espariat a, dufour c, zerah m, gareton a, dangouloff-ros v, lechapt e, et al. embryonal tumor with multilayered rosettes, c19mc-altered with sarcomatous differentiation: histopathologic and molecular characterization of one case. clin neuropathol. 2021;40( 1 ):11‑6. https://doi.org/10.5414/np301274 5. tauziède-espariat a, siegfried a, nicaise y, kergrohen t, sievers p, vasiljevic a, et al. supratentorial non-rela, zfta-fused ependymomas: a comprehensive phenotype genotype correlation highlighting the number of zinc fingers in zfta-ncoa1/2 fusions. acta neuropathol commun. 13 août 2021;9( 1 ):135. https://doi.org/10.1186/s40478-021-01238-y 6. tauziède-espariat a, dangouloff-ros v, sievers p, duchesne m, siegfried a, nicaise y, et al. glioneuronal tumors patz1-fused: clinico-molecular and dna methylation signatures for a variety of morphological and radiological profiles. acta neuropathol commun. 24 mai 2025;13( 1 ):114. https://doi.org/10.1186/s40478-025-02037-5 7. corcoran gm, frazier sr, prayson ra. choroid plexus papilloma with osseous and adipose metaplasia. ann diagn pathol. févr 2001;5( 1 ):43‑7. https://doi.org/10.1053/adpa.2001.21478 8. yap wm, chuah kl, tan ph. choroid plexus papilloma with chondroid metaplasia. histopathology. oct 1997;31( 4 ):386‑7. pmid: 9363459 9. sievers p, arora s, hielscher t, savran d, schrimpf d, banan r, et al. molecular signatures define bap1-altered meningioma as a distinct cns tumor with deregulation of polycomb repressive complex target genes. neuro-oncol. 18 avr 2025;noaf105. https://doi.org/10.1093/neuonc/noaf105 copyright: © 2025 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. neurooncology: 2024 update feel free to add comments by clicking these icons on the sidebar free neuropathology 5:21 (2024) review neurooncology: 2024 update michel mittelbronn1–6 national center of pathology (ncp), laboratoire national de santé (lns), dudelange, luxembourg luxembourg centre of neuropathology (lcnp), luxembourg department of oncology (donc), luxembourg institute of health (lih), luxembourg, luxembourg department of life sciences and medicine, university of luxembourg, esch sur alzette, luxembourg luxembourg centre for systems biomedicine (lcsb), university of luxembourg, esch-sur-alzette, luxembourg faculty of science, technology and medicine (fstm), university of luxembourg, esch-sur-alzette, luxembourg corresponding author: michel mittelbronn · national center of pathology (ncp) and luxembourg center of neuropathology (lcnp) · laboratoire national de santé (lns) · 1, rue louis rech · 3555 dudelange · luxembourg michel.mittelbronn@uni.lu submitted: 14 august 2024 accepted: 08 september 2024 copyedited by: joão gama published: 27 september 2024 https://doi.org/10.17879/freeneuropathology-2024-5809 keywords: neurooncology, neuropathology, brain tumors, glioblastoma, brain metastasis abstract as in previous years, including 2023, a major focus in the neurooncological area of neuropathology was put on more precise and constantly faster diagnostic procedures, even reaching the level of ultra-fast intraoperative diagnostics based on methylation profiling. neuropathological diagnostic precision and clinical follow-up treatment has been further increased by combining dna methylation profiling with targeted panel sequencing. a few new, molecularly defined tumor subtypes have been proposed, among others, a glioneuronal tumor with atrx alteration, kinase fusion and anaplastic features (in its abbreviated form named gtaka) and the de novo replication repair deficient glioblastoma, idh-wildtype both having either distinct prognostic or therapeutic implications. regarding the understanding of brain tumor development and progression, several novel mechanisms have been presented which might also be considered as treatment targets in the future, such as a) autonomous rhythmical ca2+ oscillations in interconnected glioma cell networks driving tumor growth; b) transfer of mitochondria from normal astrocytes to glioma cells enhancing proliferation and self-renewal; c) brain endothelial cell remodeling upon matrix-metalloprotease 9 secretion by tumor cells metastasizing into the cns and d) anti-tumor activity of microglia in cns metastasis of breast cancer. finally, in contrast to previous years, several very promising neurooncological treatment studies have been conducted, focusing on specific targets such as h3k27m or idh1/2 mutations for which a proper neuropathological assessment is key. the continuous translation of potential new treatment targets using faster and precise diagnostic procedures will further pave the way for better individualized clinical care of neurooncological patients. introduction as in the previous year, the author’s aim for the selection of the “top ten” paper series in the field of neurooncology was to select an equilibrated mix of high-quality manuscripts that provide new tumor biological concepts, new tumor entities, new diagnostic tools or approaches, and clinical studies with specific targeted approaches, all touching the field of neuropathology. this should not at all devalue excellent papers in the field of neurooncology with a different focus, such as neuroradiology, neurophysiology or neuropsychology, but rather focus on the central aims and scope of free neuropathology, namely morphological or molecular techniques based on biospecimens such as tissues, cells of fluids. the search strategy for this year’s “neurooncology update” series was similar as previously described [mittelbronn, 2023]. whereas in 2022, a major focus was put on studies focusing on profiling, “omics” and new diagnostic applications, many more cutting-edge studies presenting new tumor biological mechanisms in the field of neurooncology were published in 2023. one point that negatively struck the author last year was the fact that despite the large amount of profiling data, very few studies aimed at translating those findings into clinical applications. it is a central paradigm of our field that an excellent clinical treatment necessitates precise and fast diagnosis as well as identification of specific targets, allowing for the best individualized clinical care. it is gratifying to see the development and implementation of new treatment approaches to address molecular targets that have been introduced in daily neuropathological routine over recent years. although this year’s series also presents some new tumor subtypes and further improvements in diagnostic procedures, a larger part focuses on novel, more basic tumor biological mechanisms, as well as very promising clinical studies that aim to treat brain tumors in a more specific and individualized manner to complement or even replace the classic neurooncological treatment triad of surgery, radioand chemotherapy. with this, the “top ten” series in neurooncology for 2023 reads as follows: frequency-dependent mapk and nfkb pathway-activation by rhythmic ca2+ oscillations as potential treatment target in glioma [hausmann et al., 2023]. gap43-dependent transfer of mitochondria from astrocytes to glioma cells increasing cellular proliferation and self-renewal [watson et al., 2023]. brain endothelial cell remodeling by cns metastatic cell-derived matrix-metalloprotease 9 (mmp9) as mechanism for extravasation [karreman et al., 2023]. microglia suppresses brain metastasis formation in breast cancer [evans et al., 2023]. proposal of a new tumor entity: glioneuronal tumor with atrx alteration, ntrk gene fusion and anaplastic features (gtaka) [bogumil et al., 2023]. “de novo replication repair deficient glioblastoma, idh wildtype”: a new glioblastoma subtype with implications for targeted treatment [hadad et al., 2023]. ultra-fast, machine-learning based intraoperative methylation profiling [vermeulen et al., 2023]. multiomics neuropathology improving diagnostic precision, targeted therapy and detection of cancer predisposition syndromes in pediatric neurooncology [sturm et al., 2023]. clinical vaccination study targeting h3k27m in adult diffuse midline glioma [grassl et al., 2023]. clinical study in patients with idh mutant low-grade glioma using an oral, brain-penetrant inhibitor of mutant idh1 and idh2 enzymes [mellinghoff et al., 2023]. interdisciplinary discussions are important as the key role of neuropathological shifts from being purely diagnostic to prognostic to more and more theranostic. an increased number of neurooncological patients are currently treated based on biomarker-guided decisions jointly made in multidisciplinary molecular tumor boards [renovanz et al., 2023]. as in many neurooncological centers the immunohistochemical, dna/rna in-situ, genetic and/or epigenetic biomarker analyses are performed by neuropathologists, it is imperative that neuropathologists help to rapidly translate unequivocal findings from both basic tumor biological and clinico-oncological studies into diagnostic or theranostic tests to facilitate improved patient care. the author strongly hopes that the selected papers will both fascinate the readers and further stimulate discussions around neurooncological topics in neuropathology. 1. frequency-dependent mapk and nfkb pathway-activation by rhythmic ca2+ oscillations as potential treatment target in glioma [hausmann et al., 2023]. it has been previously reported that glioma cells are not only interconnected between each other but also to neurons via microtubes enabling intercellular communication via calcium signaling [venkataramani et al., 2019]. calcium signaling induced by neuronal activity increased the formation of glioma microtubes and cell invasion [venkataramani et al., 2022]. however, the exact mechanisms how the intercellular communication between neurons and glioma cells or uniquely amongst glioma cells were still not fully deciphered. in the present study, hausmann et al. showed that a minor glioma cell fraction, accounting for only 1–5 % of all tumor cells is able to rhythmically trigger ca2+ oscillations [hausmann et al., 2023]. those cells belong to the cellular fraction showing a considerably higher number of tumor microtube connections to other glioma cells. as the ca2+ oscillations in glioma cells were not only observed in vivo in the presence of neurons but also in neuron-free cell culture conditions, one can assume that this periodic activity is a glioma-cell autonomous mechanism. this hypothesis was further corroborated by the finding that blocking gap junction and/or tumor microtube formation did not reduce the periodic ca2+ activity in this small glioma subpopulation, but considerably reduced the related ca2+ activity in the intertumoral network. a pharmacological screen revealed that specific inhibition of the calcium-activated potassium channel kca3.1 reduced ca2+ activity also in those glioma cells. the respective potassium channel appears to be of interest in glioma biology as implications for enhanced malignant behavior, in particular enhanced glioma cell invasion in the presence of kca3.1, have been reported [d'alessandro et al., 2013; turner et al., 2014]. kca3.1-positive cells rhythmically stimulated ca2+ signaling at a mean frequency of 12.5–12.7 mhz (in vivo) and 9.7–11.3 mhz (in vitro) within the glioma network, apparently a range in which also the mapk and nfkb pathways increase cell proliferation and survival. along this line, the inhibition or knockout of kca3.1 led to reduced mapk and nfkb pathway activity while its stimulation enhanced the activity of both pathways. the knockdown of kca3.1 was associated with significantly reduced glioma growth in animal models and survival analyses of an openly accessible patient cohort (the cancer genome atlas tcga) revealed that glioblastoma patients with high kca3.1 expression showed significantly worse survival as compared to their counterpart with low kca3.1 expression. in summary, those findings point to a new treatment option for glioblastoma patients by targeting more specifically kca3.1 thereby focusing on the cell population that seems to negatively orchestrate the glioma cell network. although it was demonstrated that the cell state of rhythmically performing ca2+ signaling is hierarchical, but highly plastic, it still remains to be determined why only a very small subpopulation of glioma cells acquire this state at any given time (summary of the proposed mechanism in figure 1). figure 1: rhythmic ca2+ oscillation mechanism in glioma cell networks leading to enhanced malignancy via nf-kb and mapk pathway activation. 2. gap43-dependent transfer of mitochondria from astrocytes to glioma cells increases cellular proliferation and self-renewal [watson et al., 2023]. it has previously been shown that brain cells interact via tunneling nanotubes (tnt), in neoplastic conditions also known as tumor microtubules (mts), by which larger cellular organelles such as mitochondria can be transferred. in particular, glioblastoma cell-derived mitochondria can be shuttled into non-neoplastic astrocytes that subsequently adapted to tumor-like metabolism and hypoxia conditions [valdebenito et al., 2021]. the study of watson et al. analyzed the reverse, namely how mitochondria are transported from residual non-neoplastic astrocytes to glioma cells and how this impacted glioma cell function [watson et al., 2023]. to elucidate this question, the authors used different mouse and rat glioma models with mitochondria and glioma cells fluorescing in different colors. by doing so, they could demonstrate that between 15 to 60 % of glioma cells incorporated mitochondria of non-neoplastic astrocytic, and to a lesser extent also microglial origin. it could further be shown that physical contact between non-neoplastic astrocytes and glioma cells is necessary for mitochondrial transfer. inhibition of actin polymerization by cytochalasin b reduced the mitochondrial transfer rate, indicating an important role of actin cytoskeleton remodeling as an underlying mechanism. hypothesizing that the actin-associated protein gap43 that is involved in neurite outgrowth could be implicated in this mechanism, the authors knocked down gap43 and observed significantly reduced mitochondrial transfer. importantly, rna sequencing showed an increase of gene activity related to electron transport and mitochondrial organization therefore suggesting a functional implication of mitochondrial transfer. this was corroborated by metabolic analyses showing a higher basal and maximal respiration rate in glioma cells having incorporated a higher number of astrocyte-derived mitochondria. this glioma fraction was also more aerobic and energetic as compared to their counterpart with lower mitochondria incorporation rate. in addition, amino acid and nucleotide metabolism were significantly altered in glioma cells with a higher transfer rate of mitochondria indicating a metabolic reprogramming that might be important for pro-proliferative, antioxidative and various cell signaling processes. the first evidence that this mechanism might play an important clinical role in neurooncology was observed in a mouse glioma model in which animals with glioma cells that incorporated astrocyte-derived mitochondria showed significantly shorter survival rates as compared to glioma cells without mitochondria transfer (summary of the proposed mechanism in figure 2). figure 2: mitochondrial transfer from residual non-neoplastic astrocytes to glioma cells thereby conferring respiratory and metabolic functions linked to glioma cell proliferation and tumorigenicity. 3. brain endothelial cell remodeling by cns metastatic cell-derived matrix-metalloprotease 9 (mmp9) as a mechanism for extravasation [karreman et al., 2023]. brain metastases still constitute the most frequent malignant cns tumors, being much more frequent than their primary cns tumor counterparts. there is a broad agreement on the key steps of cancer cell extravasation from the bloodstream to the cns similarly to leukocyte extravasation including a) rolling/adhesion of cancer cells to brain endothelia, b) alteration of the blood-brain barrier (bbb) permeability and c) trans-endothelial migration, however, the exact underlying mechanisms are still not fully understood [alsabbagh et al., 2023]. previous observations, that primary cancer cells are able to considerably change the structure of brain vessels [haskó et al., 2019], prompted the authors to decipher more precisely the potential underlying mechanism. by combining fluorescent and electron microscopy on murine brain metastasis models, the authors were able to trace the moment of interaction between cancer and endothelial cells and study those events at high resolution. the observed vascular arrest of cancer cells was mainly seen at vessel branch points where cancer cells acquired an elongated shape and surrounding vessels a significantly dilated state with disorganized basement membranes. furthermore, the moment of extravasation could also be captured by showing tumor cell nuclei both partly inside and outside of brain capillaries with a nuclear diameter of less than 1 µm. the most prominent disruption of the basement membranes was observed during the extravasation process of cancer cells, without surpassing the astrocytic or microglial cell layers. brain capillaries affected by cancer cell extravasation showed three major, reproducible findings, namely a) the formation of neo-lumina parallel to the pre-existing ones, b) formation of endothelial cell projections and c) obstruction of brain capillary lumina by swollen endothelial cells (figure 3). of note, those changes were reversible within 4 days if cancer cells left the respective blood vessel without performing extravasation. furthermore, the faster cancer cells were able to extravasate, the more successful the survival rate of those cells was in the perivascular niche. once cancer cells successfully extravasated and started perivascular metastatic growth, prominent endothelial cell remodeling led to the formation of capillary loops that followed the localization of tumor cells. the formation of capillary loops was even a prerequisite for the formation of macrometastases. this mechanism was mediated by cancer cell derived matrix metalloprotease 9 (mmp9). pharmacological or genetic inhibition of mmp9 expression led to significantly reduced endothelial cell remodeling, extravasation of cancer cells, and the formation of metastases. these findings contribute to a better understanding of brain metastasis formation and may pave the way for new prevention or treatment strategies. figure 3: alteration of brain endothelium upon interaction with mmp9 producing cancer cells. 4. microglia suppress breast cancer brain metastasis formation [evans et al., 2023]. multiple studies have suggested a detrimental role of microglia in the development and/or progression of brain metastases [izraely s et al., 2023; pukrop et al., 2010]. to overcome the issue of distinguishing the contribution of primary cerebral microglia from secondarily invaded cells of the myeloid lineage (most importantly macrophages), the authors chose a complex study design including single cell rna sequencing (scrnaseq) as well as genetic and humanized mouse models approaches [evans et al., 2023]. in a murine breast carcinoma brain metastasis model in which intracardiac injection of tumor cells led to brain metastasis formation, scrnaseq revealed stable pro-inflammatory expression signatures related to cytokine production, antigen processing and presentation, and interferon-beta responses. as this signature was microglia-specific and only encountered upon brain metastasis formation, the authors hypothesized that these transcriptional changes were related to tumor cell sensing and pathological tissue alterations. an experimental depletion of microglial cells using a genetic model with deletion of a key super-enhancer in the csf1r locus was associated with enhanced metastasis formation and reduced survival rates. mechanistically, the authors showed that in the context of brain metastasis formation, microglial cells activate nk/nkt and cd8+ t cells, while attracting relatively less immunosuppressive t regulatory cells. of note, experimental depletion of effector t cells led to reduced antigen presentation and ifn response in microglial cells, suggesting that only the orchestrated interaction between both cell types enables their full antitumoral capacity (figure 4). the antitumoral responses were conserved in an additional experimental mouse model with human microglial cells. the authors reanalyzed data from an rna sequencing study from human breast cancer brain metastasis patients [varešlija et al., 2019]. in line with their findings and hypotheses, this patient cohort demonstrated significantly longer survival rates if breast cancer brain metastases were associated with a high expression of canonical microglial markers indicating a higher tumor infiltration by microglial cells. although still many scientific papers stress a pro-tumorigenic role of microglial cells in primary and secondary brain tumors, the findings of the present study are also in line with some human tissue-based studies describing a beneficial contribution of microglial cells to prolonged patient survival in glioblastoma [zeiner et al., 2019]. figure 4: proinflammatory microglial signature associated with activation of cytotoxic t and nk cells and inhibition of regulatory t cells inhibiting brain metastasis growth. 5. proposal of a new tumor entity: glioneuronal tumor with atrx alteration, ntrk gene fusion and anaplastic features (gtaka) [bogumil et al., 2023]. although glioneuronal neoplasms constitute a fairly rare group of brain tumors, new entities belonging to this group are constantly emerging, mainly due to the increasing use of molecular methods and respective analyses in larger, centralized databases that allow for a faster collection of isolated rare cases. dna methylation profiling revealed a distinct new group of 20 cases (median age: 19 years) of mainly supratentorial (84 %) location with histologically glioneuronal features as compared to a reference cohort of more than 100,000 patients. in a more fine-tuned approach comparing those cases with a reference cohort of 718 neuroepithelial tumors, unsupervised, non-linear t-distributed stochastic neighbor embedding (t-sne) projection, this distinct subgroup could be confirmed. further detailed neuropathological and molecular pathological analyses could be performed in 16 out of those 20 tumors. all tumors showed alterations of atrx, assessed by dna sequencing and/or immunohistochemistry. in addition, all cases showed a potentially druggable gene fusion involving members of the receptor tyrosine-kinase family, mainly ntrk1-3. in slightly more than half of the patients, homozygous deletion of cdkn2a/b was observed. the neuropathological assessment of those tumors showed isomorphic, round, often condensed nuclei with perinuclear clearing, prominent mitotic indices and vascular proliferations. for this new entity, the proposed acronym “gtaka” is composed of its central neuropathological and molecular features, namely glioneuronal tumor with atrx alteration, kinase fusion and anaplastic features (table 1). at the current stage, it is too early to provide precise prognostic estimations, however this new subgroup of glioneuronal tumors with a median progression-free survival of only 12.5 months seems to be clearly more aggressive than many other, already established glioneuronal tumor entities. table 1: proposition of diagnostic criteria for glioneuronal tumor with atrx alterations, kinase fusion and anaplastic features (“gtaka”). 6. “de novo replication repair deficient glioblastoma, idh wildtype”: a new glioblastoma subtype with implications for targeted treatment [hadad et al., 2023]. over decades, the treatment of glioblastoma patients consisted of neurosurgical resection followed by radio-chemotherapy and is, however, still associated with a detrimental prognosis. despite a multitude of clinical studies, valid biomarkers for potential targeted therapies are largely lacking [rodgers et al., 2024]. therefore, it is highly important to define glioblastoma subtypes, even if they only occur at low percentages, that might benefit from new treatment approaches. in a recent larger series of 459 consecutive glioblastoma patients, 9 patients (2 %) with a biallelic inactivation of a canonical mismatch repair gene (msh2, msh6 or mlh1) were identified, partly with a heterozygous germline mutation (indicating an underlying lynch syndrome) in a part of the glioblastoma patients [hadad et al., 2023]. while overall mutation rate was significantly increased in this tumor group, they showed lower rates of classic genetic glioblastoma alterations such as trisomy of chromosome 7, monosomy of chromosome 10, tert promoter mutation, egfr amplification or homozygous cdkn2a deletion. in 3 out of those 9 patients, additional mutations in the proofreading domain of the dna polymerase pole were detected being associated with an ultrahypermutational state of more than 100 mutations per megabase. of note, the mismatch repair deficient (mrd) glioblastomas presented as a giant cell variant of glioblastoma and dna methylation profile either matched with “diffuse pediatric-type hgg, rtk1 subtype, subclass a”, “adult-type diffuse hgg, idh-wt, subtype e” or did not match at all. clinically, the median age at tumor occurrence was significantly lower (50 years) in the mrd group as compared to non-mrd glioblastoma patients (63 years). in addition, median overall survival was more than twice as long (36.8 months) in mrd glioblastoma as compared to non-mrd patients (15.5 months). microscopically, mrd glioblastoma were associated with significantly more cd8+ cytotoxic t cells and microglia cells indicating a stronger immune activation in the context of a hypermutational state. as smaller studies demonstrated that some mrd glioblastoma patients radiologically and clinically profited from immune checkpoint inhibition treatment, the identification of such patients (table 2) is important [bouffet et al., 2016]. with this, a better clinical patient stratification regarding improved survival times and suitability for treatments with immune checkpoint inhibitors is possible. table 2: clinical and neuropathological parameters that should trigger mmr and tmb testing in patients with high-grade gliomas. 7. ultra-fast, machine-learning based intraoperative methylation profiling [vermeulen et al., 2023]. methylation profiling considerably improved the unbiased and precise classification of brain tumors and was rapidly acknowledged as an essential criterium for diagnostics of most brain tumor entities by the who guidelines [capper et al., 2018; who classification of brain tumors, 2021]. most institutions performing methylation profiling still use an array-based approach for which the entire laboratory process before bioinformatic analyses takes approximately one week. however, for the intraoperative surgical guidance and potentially upcoming new treatment approaches with application of substances or cells directly in the tumor cavity, it might be important to have a precise, rapid on-site diagnosis. as brain tumors are more and more stratified according to molecular classes, an intraoperative histological assessment for primary cerebral neoplasms is of limited interest in many such cases. to overcome those obstacles, a fast genomic and epigenomic assessment using nanopore sequencing had been previously developed [euskirchen et al., 2017]. the authors of the current study applied nanopore sequencing in combination with a neural network analysis (named “sturgeon”) to molecularly classify cns tumors intraoperatively [vermeulen et al., 2023]. in 45 out of 50 samples, an accurate diagnosis was already obtained 40 min after initiation of the sequencing procedure in a retrospective cohort. after this highly promising proof of concept results, the authors were able to achieve intraoperative molecular diagnoses within a turnaround time of 90 min. a major advantage of the sturgeon model is that it can be universally used without retraining (see https://github.com/marcpaga/sturgeon) on several different flow cell types (e.g. minion or promethion using r9 and r10 chemistry). however, the method is still strongly dependent on the tumor cell content. therefore, a concomitant mirroring histological assessment of the tissue assessed by the sturgeon model could be beneficial. 8. multiomics neuropathology improving diagnostic precision, targeted therapy and detection of cancer predisposition syndromes in pediatric neurooncology [sturm et al., 2023]. over the past decade, diagnostic improvement in neuropathology was strongly linked to constantly improved methylome profiling [https:// www.molecularneuropathology.org/mnp based on capper et al. 2018]. however, this approach has not yet contributed significantly to improved treatment approaches, partially due to a lack of related prognostic data or information about underlying genetic predisposition for tumor development that might be additionally important for further clinical follow-up [farouk et al., 2021]. in their study, sturm et al. combined methylation profiling with targeted panel sequencing in a cohort of more than 1,200 newly diagnosed pediatric brain tumors [sturm et al., 2023]. with this approach, they were able to achieve a refined diagnosis in almost every second case as compared to current who classification alone. moreover, in almost every second case, a potential therapeutically relevant alteration was detected and 15 % of all tumors revealed a directly druggable genetic alteration with braf-v600e being the most frequent (7.4 %), followed by fgfr1/3 (4.0 %), alk (0.8 %), ntrk2/3 (0.4 %), met (0.1 %) and ret (0.1 %). for 10 % of all cases an underlying cancer predisposition syndrome was found, amongst those, most frequently neurofibromatosis type 1 (1.5 %), followed by tp53-related li-fraumeni syndrome (1.2 %), constitutional mismatch repair gene deficiency or lynch syndrome including mlh1, msh2 and msh6 genes (1.1 %), ataxia-telangiectasia and atm heterozygous carriers (0.9 %), neurofibromatosis type 2 (0.8 %), dicer syndrome (0.6 %) and rhabdoid tumor predisposition syndrome 1 related to smarcb1 (0.4 %) were observed. compared to the initial cohort [capper et al., 2018], 3 % of pediatric neurooncological cases in the present study belong to potentially new tumor classes. of note, in up to one third of this pediatric patient cohort, discordant results were obtained when comparing standard whoand methylation-based analyses, particularly for the children histologically displaying tumors with characteristics of a high-grade glioma. taken into account that clinical follow-up revealed that molecular profiling provides a better clinical prediction for patient prognosis than morphological assessment, it was highly recommended to implement multiomics neuropathological assessment in the diagnostic routine. the present study still highlights the importance of methylation profiling, as methylation data being discordant to the initial neuropathological diagnosis was considered as relevant in up to 70 % of the cases discussed in interdisciplinary tumor boards. a very useful tool of the study is the proposed molecular risk stratification of pediatric high-grade gliomas that have been classified according to cns who grade 3–4 (table 3). although the who classification would put such tumors in a very similar biological class, it has to be taken into account that their clinical behavior might be very different and that multiomics, in particular methylation profiling, provides a much more precise prediction in those cases. of note, the highly successful results of the present study further had a strong impact on health politics. german national health insurances now provide financing for dna methylation profiling and gene panel sequencing (for the latter in both tumor and blood leukocyte samples) in parallel for every newly diagnosed brain tumor case in children and adolescents, thereby changing the standard-of-care. table 3: molecular risk stratification classes for of pediatric high-grade gliomas otherwise classified according to cns who grade 3–4. the molecular risk remains unknown for cases revealing high score for non-neoplastic control tissue or being unclassifiable in the dna methylation classifier. as the biological behavior is still unclear for pediatric brain tumor with patz1 or plagl1 fusion, those tumors were excluded from the risk stratification. 9. clinical vaccination study targeting h3k27m in adult diffuse midline glioma [grassl et al., 2023]. while mutations in histone h3 have been detected in several human tumors, the h3k27m mutation in diffuse gliomas of mainly younger patients constitutes a specific brain tumor entity usually showing a detrimental clinical prognosis [who classification of brain tumors, 2021]. although, h3k27m mutant diffuse midline gliomas are incurable, the first promising results were obtained from mhc-humanized mice showing a mutation specific immune response to a h3k27m long peptide vaccine [ochs et al., 2017]. this prompted the authors to start a phase i clinical trial with 8 patients suffering from a h3k27m-positive diffuse midline glioma [grassl et al., 2023]. all patients received a 27-mer peptide (p14-40) including the mutant h3k27m via bi-weekly subcutaneous injection over 6 weeks followed by monthly administration for 4 months and quarterly afterwards until disease progression. the vaccine was well tolerated and without any relevant higher-grade side effects. after 2 injections, h3k27m-vaccine related neoepitope-specific immune responses (in blood and csf) were detected in 5 out of 8 patients and were associated with promising neuroradiological findings showing reduced axial contrast enhancement. for patients from which previous tumor material was accessible, a colocalization of mutant h3k27m and hla class-ii-dr was detected, indicating that the neoepitope is probably presented by antigen-presenting cells which might lead to a stimulation of h3k27m-specific t cells. of note, the two patients of this phase i study with the best clinical outcome after detection of peripheral immune response also showed highest numbers of hla class-ii-dr-positive cells associated with mutant h3k27m which may indicate that enhanced immune cell responses associated with mutant neoepitope presentation are responsible for better patient prognosis. with this first human vaccination trial for h3k27m mutant diffuse midline glioma, the authors demonstrated patient safety and neoepitope immune responses. as a most promising finding, complete remission for more than 31 months was achieved in one patient. however, all other patients died within the study period. to further improve the h3k27m vaccine-based effects, the authors propose to administer the vaccine concomitantly with first line treatment to increase the time for t cell mediated antitumoral effects. 10. clinical study in patients with idh mutant low-grade glioma using an oral, brain-penetrant inhibitor of mutant idh1 and idh2 enzymes [mellinghoff et al., 2023]. in 2009, the detection of specific point mutations in idh1 and idh2 genes in distinct brain tumor entities, in particular diffuse astrocytoma and oligodendroglioma, considerably changed the neuropathological diagnostic approach paving the way from a mainly histological to a more and more molecular classification [yan et al., 2009]. the most frequently altered amino acid is 132 in idh1 and 172 in idh2. thus, those alterations make them a promising treatment target. the application of the dual idh1 and idh2 inhibitor vorasidenib in an orthotopic murine glioma model showed considerably reduced 2-hydroxyglutarate levels that acts as an oncometabolite [konteatis et al., 2020]. in addition, it was demonstrated that vorasidenib also penetrated the brain of several non-human species therefore showing its cns bioavailability in preclinical models. this prompted several neurooncological teams to test idh1/idh2 inhibitors in the clinical context of which the double-blind, phase 3 trial of mellinghoff and colleagues is one of the most advanced studies for residual or recurrent cns who grade 2 glioma comprising 331 patients in total [mellinghoff et al., 2023]. the patients orally received vorasidenib or placebo daily until disease progression or toxic treatment side effects occurred. vorasidenib treatment was mainly associated with low-grade toxic side effects; however grade 3 or higher side effects were observed in 22.8 % of the patients in the vorasidenib as compared to 13.5 % in the placebo control group. of note, only 1.8 % of the patients treated with vorasidenib showed serious side effects, therefore showing a clinically acceptable safety profile. the median progression-free survival was significantly improved upon vorasidenib treatment, reaching 27.7 months compared to only 11.1 months in the placebo control group. furthermore, time to next follow-up treatment was significantly longer in the vorasidenib treated patients for which the median had not been reached by the time of publication of the study while the placebo group reached the median at 17.8 months. this study is another milestone on the way to more specific, efficacious and safe brain tumor treatments. the us food and drug administration has now approved vorasidenib as a systemic therapy for patients with grade 2 astrocytoma or oligodendroglioma with a susceptible idh1 or idh2 mutation, based largely on the result of this study (see https://www.fda.gov/drugs/resources-information-approved-drugs/fda-approves-vorasidenib-grade-2-astrocytoma-or-oligodendroglioma-susceptible-idh1-or-idh2-mutation). discussion in 2023, several novel pathogenic mechanisms were published that might serve as potential targets for oncological treatment approaches in the future. however, it still remains to be determined if those mechanisms can be targeted with high specificity without having negative side effects. for example, spontaneous ca2+ oscillations are key in the modulation of glioneuronal synaptic information [goenaga et al., 2023]. therefore, it is unclear if rhythmic ca2+ oscillations that activate frequency-dependent mapk and nfkb pathway in the context of brain tumors may be selectively targeted without negatively impacting the normal neurophysiological circuits [hausmann et al., 2023]. to which degree a potential inhibition of gap43-dependent transfer of mitochondria from normal astrocytes to glioma cells may slow down tumor growth also in the human context has to be clarified [watson et al., 2023]. although it seems obvious that fresh and healthy mitochondria transferred to potentially exhausted glioma cells may serve as a powerhouse, a high metabolic plasticity has been reported for glioma cells allowing for sustainable tumor propagation via glycolytic energy production if mitochondrial-type energy supply is impaired [shibao et al., 2018]. this glioma cell feature may limit a treatment approach targeting the intercellular transfer of mitochondria between normal and neoplastic glial cells. the findings regarding the involvement of matrix-metalloprotease (mmp) in the process of brain metastasis formation may serve as another promising therapeutic target in the prevention and treatment of brain metastasis, especially as several synthetic mmp inhibitors have been produced [karreman et al., 2023]. unfortunately, to date, many oncological trials applying mmp inhibitors failed because of either lack of efficacy or severe adverse side effects [lopez-navarro and gutierrez, 2022]. finally, the tumor-suppressive effects of microglia cells demonstrated in the context of brain metastasis formation need a careful reassessment in humans as to date still many studies claim a promoting microglial effect for brain tumor formation [evans et al., 2023]. as previous studies in glioma have shown, the role of microglial cells in brain tumors is probably less black and white and rather reflects a high cellular plasticity that is related to intratumoral microenvironmental changes resulting in both classic proand anti-tumoral features within the same tumor [zeiner et al., 2019]. in summary, although the aforementioned studies provide fascinating findings about brain tumor pathomechanisms, it seems to be too early to concretely judge the related diagnostic and, most importantly, therapeutic potential. in contrast, the diagnostic and prognostic potential for the proposed new tumor entities seem to be clear. for the glioneuronal tumor with atrx alteration, ntrk gene fusion and anaplastic features (gtaka), the detrimental clinical course that is rather unusual for many glioneuronal tumors has to be stressed [bogumil et al., 2023]. as gtaka could be unequivocally defined as a new class using methylation classification, its diagnosis also seems to be straightforward. the proposed new entity of “de novo replication repair deficient glioblastoma, idh wildtype” harbors a better prognosis as compared to other glioblastoma variants [hadad et al., 2023], and may also profit from checkpoint inhibitors. both diagnosis and treatment rely on a neuropathological diagnostic approach requiring mmr and tmb testing in diffuse high-grade gliomas: a) presenting in patients with a younger median age (50 years) as compared to conventional glioblastoma, b) showing a giant cell morphology, and c) lacking classic methylation or mutational features usually observed in conventional glioblastoma (table 2). the diagnostics of such new entities should also be taken into account when developing new diagnostic tools. the ultra-fast, machine-learning based intraoperative methylation profiling can be applied without retraining but it is still based on methylation data that has been published a few years ago [vermeulen et al., 2023]. it is therefore essential that the neuropathological community aligns on joint diagnostic standards, techniques and platforms so that molecular profiling can achieve similar results world-wide. this is even more important if different layers of omics approaches are combined [sturm et al., 2023]. over the recent years, neuropathology was always at the forefront regarding technical development (e.g. methylation profiling) and diagnostic precision in oncology (e.g. entity specific mutations such as idh1/idh2, h3k27m, atrx only to name a few), subsequently also impacting other oncological domains. these developments prompted an extremely rapid clinical translation focusing on specific drug targets such as h3k27m, idh1/idh2 or mrd tumors [hadad et al., 2023; grassl et al., 2023; mellinghoff et al., 2023]. in conclusion, it is fascinating to see how fast the improvements in diagnostic neuropathology with the definition of specific treatment targets reached the bedside. after having shown drug safety and initial clinical efficacy, larger clinical trials are now necessary to define best treatment schemes. acknowledgements the figures were prepared using https://biorender.com figure creation tool. the author would like to thank prof. patrick harter, prof. david capper, dr. ann-christin hau, prof. joachim steinbach, pd dr. michael ronellenfitsch and prof. werner stenzel for one or the other discussion around neurooncological topics discussed in this paper. 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multiomic neuropathology improves diagnostic accuracy in pediatric neuro-oncology. nat med. 2023 apr;29(4):917-926. https://doi.org/10.1038/s41591-023-02255-1. pmid: 36928815. 26. turner kl, honasoge a, robert sm, mcferrin mm, sontheimer h. a proinvasive role for the ca(2+) -activated k(+) channel kca3.1 in malignant glioma. glia. 2014 jun;62(6):971-81. https://doi.org/10.1002/glia.22655. pmid: 24585442. 27. valdebenito s, malik s, luu r, loudig o, mitchell m, okafo g, bhat k, prideaux b, eugenin ea. tunneling nanotubes, tnt, communicate glioblastoma with surrounding non-tumor astrocytes to adapt them to hypoxic and metabolic tumor conditions. sci rep. 2021 jul 15;11(1):14556. https://doi.org/10.1038/s41598-021-93775-8. pmid: 34267246. 28. varešlija d, priedigkeit n, fagan a, purcell s, cosgrove n, o'halloran pj, ward e, cocchiglia s, hartmaier r, castro ca, zhu l, tseng gc, lucas pc, puhalla sl, brufsky am, hamilton rl, mathew a, leone jp, basudan a, hudson l, dwyer r, das s, o'connor dp, buckley pg, farrell m, hill adk, oesterreich s, lee av, young ls. transcriptome characterization of matched primary breast and brain metastatic tumors to detect novel actionable targets. j natl cancer inst. 2019 apr 1;111(4):388-398. https://doi.org/10.1093/jnci/djy110. pmid: 29961873. 29. venkataramani v, tanev di, strahle c, studier-fischer a, fankhauser l, kessler t, körber c, kardorff m, ratliff m, xie r, horstmann h, messer m, paik sp, knabbe j, sahm f, kurz ft, acikgöz aa, herrmannsdörfer f, agarwal a, bergles de, chalmers a, miletic h, turcan s, mawrin c, hänggi d, liu hk, wick w, winkler f, kuner t. glutamatergic synaptic input to glioma cells drives brain tumour progression. nature. 2019 sep;573(7775):532-538. https://doi.org/10.1038/s41586-019-1564-x. pmid: 31534219. 30. venkataramani v, yang y, schubert mc, reyhan e, tetzlaff sk, wißmann n, botz m, soyka sj, beretta ca, pramatarov rl, fankhauser l, garofano l, freudenberg a, wagner j, tanev di, ratliff m, xie r, kessler t, hoffmann dc, hai l, dörflinger y, hoppe s, yabo ya, golebiewska a, niclou sp, sahm f, lasorella a, slowik m, döring l, iavarone a, wick w, kuner t, winkler f. glioblastoma hijacks neuronal mechanisms for brain invasion. cell. 2022 aug 4;185(16):2899-2917.e31. https://doi.org/10.1016/j.cell.2022.06.054. pmid: 35914528. 31. vermeulen c, pagès-gallego m, kester l, kranendonk meg, wesseling p, verburg n, de witt hamer p, kooi ej, dankmeijer l, van der lugt j, van baarsen k, hoving ew, tops bbj, de ridder j. ultra-fast deep-learned cns tumour classification during surgery. nature. 2023 oct;622(7984):842-849. https://doi.org/10.1038/s41586-023-06615-2. pmid: 37821699. 32. watson dc, bayik d, storevik s, moreino ss, sprowls sa, han j, augustsson mt, lauko a, sravya p, røsland gv, troike k, tronstad kj, wang s, sarnow k, kay k, lunavat tr, silver dj, dayal s, joseph jv, mulkearns-hubert e, ystaas lar, deshpande g, guyon j, zhou y, magaut cr, seder j, neises l, williford se, meiser j, scott aj, sajjakulnukit p, mears ja, bjerkvig r, chakraborty a, daubon t, cheng f, lyssiotis ca, wahl dr, hjelmeland ab, hossain ja, miletic h, lathia jd. gap43-dependent mitochondria transfer from astrocytes enhances glioblastoma tumorigenicity. nat cancer. 2023 may;4(5):648-664. https://doi.org/10.1038/s43018-023-00556-5. pmid: 37169842. 33. world health organization classification of tumours of the central nervous system. 5th ed. lyon: international agency for research on cancer; 2021. 34. yan h, parsons dw, jin g, mclendon r, rasheed ba, yuan w, kos i, batinic-haberle i, jones s, riggins gj, friedman h, friedman a, reardon d, herndon j, kinzler kw, velculescu ve, vogelstein b, bigner dd. idh1 and idh2 mutations in gliomas. n engl j med. 2009 feb 19;360(8):765-73. https://doi.org/10.1056/nejmoa0808710. pmid: 19228619. 35. zeiner ps, preusse c, golebiewska a, zinke j, iriondo a, muller a, kaoma t, filipski k, müller-eschner m, bernatz s, blank ae, baumgarten p, ilina e, grote a, hansmann ml, verhoff ma, franz k, feuerhake f, steinbach jp, wischhusen j, stenzel w, niclou sp, harter pn, mittelbronn m. distribution and prognostic impact of microglia/macrophage subpopulations in gliomas. brain pathol. 2019 jul;29(4):513-529. https://doi.org/10.1111/bpa.12690. pmid: 30506802. copyright: © 2024 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. title feel free to add comments by clicking these icons on the sidebar free neuropathology 5:28 (2024) letter glioneuronal heterotopia in the right middle cranial fossa emilie russler-germain1, shamaita majumdar2, theresa nguyen2, keiko hirose3, peter h. yang4, ali mian2, sonika dahiya1 department of pathology & immunology, washington university school of medicine, st. louis, usa mallinckrodt institute of radiology, washington university school of medicine, st. louis, usa department of otolaryngology, washington university school of medicine, st. louis, usa department of neurosurgery, washington university school of medicine, st. louis, usa corresponding author: sonika dahiya · department of pathology & immunology · washington university school of medicine · 660. s euclid ave. · st. louis, mo 63110 · usa sdahiya@wustl.edu submitted: 05 september 2024 accepted: 19 october 2024 copyedited by: monica miranda published: 12 november 2024 https://doi.org/10.17879/freeneuropathology-2024-5845 keywords: glioneuronal heterotopia, intracranial extracerebral, pigmented ocular epithelium, choroid plexus a newborn boy, delivered vaginally at 40 weeks and 2 days gestation at home under the care of a midwife to a 28-year-old gravida 2 para 2 mother, was noted to have bleeding from his oral cavity at birth. prenatal care was adequate and the pregnancy was uncomplicated, with no concerning findings noted on 20-week ultrasound. the patient was brought to the hospital for evaluation and was admitted to the neonatal intensive care unit. the pediatric otolaryngology service was consulted, and examination revealed fullness of the right temporal fossa with absence of a portion of the squamous temporal bone. a mass of the right oropharynx was noted directly posterior to the tonsil, and the soft palate and uvula were asymmetric, resembling a shallow cleft. despite this, the patient was breathing comfortably without evidence of respiratory distress. magnetic resonance imaging (mri) of the skull base revealed a large cystic and solid extra-axial mass in the right middle cranial fossa, displacing the right temporal lobe and extending into the right infratemporal fossa through a large skull base defect, as well as cleft palate. furthermore, the mass protruded into the oropharynx (figure 1a). the adjacent brain parenchyma appeared radiologically separate from the mass. the mass had signal intensity resembling brain parenchyma except for faint enhancement at the center and diffusion restriction in the infratemporal component (figure 1b–d). the vascular supply of this mass appeared separate from the adjacent brain with its own feeding vessel and distal perfusion (figure 1e–f). mri sequences demonstrating diffusion restriction suggested this lesion could represent a malignancy. however, the lesion did not invade adjacent structures and appeared to induce bony erosion not by infiltration but by resorption and remodeling (figure 1g). therefore, a benign diagnosis was favored. figure 1. head and neck imaging. coronal t2 mri (a) shows a mass extending from the middle cranial fossa through a skull base defect into the right infratemporal fossa. the adjacent normal brain parenchyma is displaced but not involved by the mass, with a clear cerebrospinal fluid (csf) cleft separating the two. axial pre-contrast (b) and post-contrast (c) t1 mprage images show minimal enhancement at the center, but the majority of the mass is non-enhancing. axial diffusion-weighted imaging (d) shows diffusion restriction in the infratemporal mass, suggestive of hypercellularity, a feature often concerning for an aggressive tumor, though this can also be seen in benign lesions. sagittal t1 space mri (e) and post-contrast mra (f) show a prominent vascular pedicle (red arrows) in the center of the mass and the displaced right middle cerebral artery (yellow arrow). 3d renderings of the skull (g) show budging of the squamosal temporal bone and widening of the suture rather than osseous destruction. on day 4 of life, the patient underwent transoral biopsy of the oropharyngeal portion of the mass. on days 18 and 33 of life, the patient underwent staged debulking of the intracranial and right orbital components with reservoir placement and covering of the skull base defect due to concern for progressive intracranial mass effect. intraoperatively, the mass was noted to respond to surgical manipulation with consistency akin to normal brain parenchyma. the patient was discharged home on day 44 of life in good condition. histopathological analysis of the mass revealed fragments of malformed, disorganized glioneuronal tissue with calcospherites and perivascular dystrophic calcifications, as well as, reactive changes including gliosis. in addition, few detached fragments of unremarkable choroid plexus and foci of pigmented ocular epithelium were notable (figure 2). minuscule fragments were suggestive of putative ependymal lining and meningeal tissue. the final histological diagnosis was consistent with intracranial extracerebral glioneuronal heterotopia (iegh) with oropharyngeal extension. developing brain tissue and robust reactive changes resulted in high proliferation indices, a potential diagnostic pitfall. the differential possibility of teratoma was ruled out due to lack of endodermal components. figure 2. histopathological analysis. oropharyngeal biopsy (a–c): low(a) and high(b) power hematoxylin and eosin-stained sections demonstrate widespread ulceration (*) of the lining squamous epithelium (inset), hyalinized ectatic vessels, and reactive glioneuronal tissue (arrows indicate calcospherites). glial fibrillary acidic protein (gfap) confirms a dominant glial component (c). resection of the intracranial mass (d-f) further substantiates the malformed nature of the glioneuronal tissue (d, arrows indicate calcospherites; inset highlights the disorganized arrangement of neurons by neun immunostaining), as well as, pigmented ocular epithelium (arrow, e) and unremarkable fragments of choroid plexus (f). whole exome sequencing and rna-sequencing of the oropharyngeal biopsy were performed by caris life sciences. whole exome sequencing revealed no pathogenic gene alterations or whole arm chromosomal alterations. a number of variants of uncertain significance and unclassified gene variants with variant allele frequencies approximating 50 % were detected, suggestive of germline alterations. additionally, uncharacterized fusions of umad1::glcci1 and kansl1::arl17a were detected through rna-sequencing, the significances of which are unclear but which are possibly germline1. karyotype analysis, performed using whole exome sequencing, identified duplications of the centromeric portion of chromosome 22, as well as the distal portion of chromosome 22q. no chromosomal losses were detected. microsatellite status was stable, genomic loss of heterozygosity was 1 %, and tumor mutational burden was 2 mutations / megabase (mb). overall, the genetic analyses were consistent with a non-neoplastic process. the term glioneuronal heterotopia / ectopia encompasses microscopic, small nodular or large mass-like ectopic neuroectodermal tissue, both within or outside of the neuraxis2. a well-recognized example of ectopic glioneuronal tissue is the intranasal or extranasal “nasal glioma”2–5. intraparenchymal, dural / leptomeningeal, intracranial extracerebral, and “distal” (e.g. orbit, palate, middle ear, internal auditory canal, oral cavity, oropharynx, and lung) locations are also possible2,3,6–8. mass-like glioneuronal heterotopias are uncommon developmental malformations and can be difficult to distinguish from primary central nervous system (cns) neoplasms on radiologic and pathologic evaluation. while microscopic glioneuronal heterotopia likely result from aberrant glial and neuronal migration9, the pathogenesis of large mass-like glioneuronal heterotopia is less well understood. possible etiologies include aberrant sequestration of embryonic tissue during development with subsequent dysregulated growth versus encephalocele with obliteration of the connecting stalk5. iegh most commonly occurs in the middle cranial fossa, with fewer than 20 cases reported in the literature and even fewer reports of extracranial extension2,3,6,10–23. literature review of published iegh cases has been previously performed2,3,6,10,23. the association of iegh with craniofacial anomalies, such as cleft palate, implies an origin during early embryogenesis, likely during the 5th to 6th week of gestation, possibly via evagination of a third inferior telencephalic vesicle, in the case of middle cranial fossa lesions24. patients typically present within the first six months of life, and many are identified via prenatal ultrasonography, although cases in older children have been documented2,3,6,17,24,25. the recommended management for iegh is staged resections, beginning with the intracranial component3,25. it is important to recognize these lesions as they are benign. grossly, the lesions are often large and cystic2,3,6,10. histological examination of previous cases has yielded similar findings as the case presented here; namely, disorganized neuroglial tissue (variably mature neurons, astrocytes, and oligodendrocytes) with scattered calcospherites, and the presence of other cns tissue elements including leptomeninges, ependyma, choroid plexus, and ocular pigmented epithelium2,3,6,10,11. in some cases, cortical or cerebellar architectural patterns are evident2,10,22. to our knowledge, this is the first example of iegh with genetic / molecular analysis performed. in contrast to a recent case series of nasal glioma5, this case showed no whole arm chromosomal gains or losses. overall, the condition appears to have excellent prognosis after resection, including normal neurologic development3,13,25. however, complications related to airway obstruction are possible7,15,16. the infant in this case is doing well at 10 months of age, with normal development and without evidence of growth of the residual oropharyngeal mass. conflicts of interest statement the authors have no conflicts of interest to report. funding statement the authors have no relevant funding sources to disclose. references 1 .zhou jx, yang x, ning s, et al. identification of kansarl as the first cancer predisposition fusion gene specific to the population of european ancestry origin. oncotarget. aug 01 2017;8(31):50594-50607. https://doi.org/10.18632/oncotarget.16385 2 .oya s, kawahara n, aoki s, et al. intracranial extracerebral glioneuronal heterotopia. case report and review of the literature. j neurosurg. jan 2005;102(1 suppl):105-12. https://doi.org/10.3171/ped.2005.102.1.0105 3 .abel tj, chowdhary a, thapa m, et al. ectopic glioneuronal tissue in the middle cranial fossa region. report of four cases. j neurosurg pediatr. mar 2009;3(3):188-96. https://doi.org/10.3171/2008.12.peds0892 4 .husein of, collins m, kang dr. neuroglial heterotopia causing neonatal airway obstruction: presentation, management, and literature review. eur j 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2008;44(4):318-23. https://doi.org/10.1159/000134924 24 .harris cp, townsend jj, klatt ec. accessory brains (extracerebral heterotopias): unusual prenatal intracranial mass lesions. j child neurol. oct 1994;9(4):386-9. https://doi.org/10.1177/088307389400900410 25 .muzumdar d, michaud j, ventureyra ec. anterior cranial base glioneuronal heterotopia. childs nerv syst. mar 2006;22(3):227-33. https://doi.org/10.1007/s00381-005-1222-5 copyright: © 2024 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. 6th annual conference of the neuropathology society of india (npsicon) meeting abstracts feel free to add comments by clicking these icons on the sidebar free neuropathology 4:9 (2023) meeting abstracts 6th annual conference of the neuropathology society of india (npsicon) virtual conference meeting abstracts february 23–25, 2023 submitted: 12 may 2023 accepted: 12 may 2023 published: 06 june 2023   the 6th annual conference of the neuropathology society of india, (npsicon 2023) was held in a virtual mode, from 23rd to 25th february 2023, hosted by the division of neuropathology, department of pathology, christian medical college vellore. dr geeta chacko was the organizing chairperson. the pre-conference workshop was on fluorescence in situ hybridization with nearly 90 registrants. the workshop was conducted by nimhans bangalore with dr shilpa rao, dr vaishali suri, dr vani santosh and dr geeta chacko as resource persons. there were 253 national registrants and 7 international registrations. the conference had participation from reputed national (34) and international (8) faculty and covered a wide range of topics in both neoplastic and non-neoplastic neuropathology including sessions on perinatal pathology, epilepsy, neuromuscular pathology and several sessions in neurooncology. there were five symposiums with several sessions of cases-based discussion. the first prof ashru k banerjee oration was delivered by dr kenneth aldape, from nih bethesda on: "practical aspects of methylation-based diagnostics in central nervous system tumors”. the first prof sk shankar oration was delivered by dr avindra nath, nih bethesda on "neuropathology of two pandemics: aids and covid". the presidential oration was delivered by dr chitra sarkar. the key note speakers included dr monika hofer, dr david capper, dr takashi komori and dr maysa husseini. there were 28 papers for oral presentation and 38 posters. a highlight of the conference was a lively quiz competition, which was held as the penultimate session. despite being a virtual conference, there was active participation from all the delegates and the conference was very well received.   https://doi.org/10.17879/freeneuropathology-2023-4898 keywords: neuropathology society of india, npsi, meeting abstracts table of contents free papers abstract 1 insulin-like growth factor 2 mrna binding protein 3 (imp3) immunoreactivity in pilocytic astrocytoma and pilomyxoid astrocytomadoes it help to differentiate? abstract 2 series of 4 cases of subependymal giant cell astrocytoma abstract 3 diffuse meningeal melanocytic neoplasms of the brainmorphological and molecular analysis of braf and kras mutations abstract 4 histopathological spectrum of astrocytoma in tertiary care hospital abstract 5 study of zfta and yap-1 fusion in supratentorial ependymomas abstract 6 the many facets of astrocytes and blood-brain-barrier alterations in fungal infections of central nervous system abstract 7 spectrum of skeletal muscle disorders -an audit of data over four decades from a tertiary referral center abstract 8 does the macrophage subtype drive the immunological subtypes of leprous neuropathy? abstract 9 study of ki67 pi amongst meningiomas of different who grades & its association with os & recurrence abstract 10 utility of immunohistochemistry in subtyping posterior fossa group a ependymoma abstract 11 posterior fossa ependymomas a clinicopathological study abstract 12 clinicopathological study of ependymomas with special reference to l1cam expression in prognosis and survival abstract 13 gfap and sox10 expression in neonatal cerebral white matter injury: a postmortem minimally invasive tissue sampling study abstract 14 olig2-positive cell density in moghe versus other epilepsy surgical samples: a morphometric study abstract 15 significance of emerging prognostic markers in meningiomas   posters abstract 1 glioblastoma with predominant sarcomatoid differentiation: a rare case report abstract 2 does mib-1 labelling index correlate with who grade and outcome in spinal meningiomas? – a clinicopathological study abstract 3 non-hodgkins lymphoma nk/t cell type of nasal cavity : a rare case report abstract 4 a case of papillary tumor of pineal region – a rare entity abstract 5 a rare case of atypical teratoid/rhabdoid tumour: case report abstract 6 smoke and mirrors: a tale of two unusual cerebellopontine angle tumors abstract 7 all proliferating vessels are not haemangiomas a case report of rare entity of sporadic meningioangiomatosis abstract 8 extensive spherical amyloid deposition in lactotroph pituitary neuroendocrine tumour masquerading as a primary bone tumour abstract 9 late onset pompe disease: role of fluorescence microscopy abstract 10 nemaline myopathy: a case report abstract 11 cutaneous clue to amoebic encephalitis: a case report abstract 12 malignant transformation of intracranial epidermoid cyst: a rare case report abstract 13 neuropathological findings in a 17-month-old boy with delayed milestones and kinkyhair abstract 14 pituitary macroadenoma with apoplexy presented as sudden bilateral vision loss abstract 15 polymorphous low-grade neuroepithelial tumor of the young (plnty): rare but curable cause of refractory epilepsy abstract 16 a rare case of cerebral cavernous malformation with concomittant intracanial mucormycosis infection abstract 17 suprasellar masquerader: chordoid glioma abstract 18 paediatric high-grade glioma’ in association with constitutional mismatch repair deficiency syndrome (cmmrd) abstract 19 dumbell spinal tumor with ini-1 loss in an infanta potential diagnostic pitfall e-posters abstract 1 braf and kras mutations analysis in rosai-dorfman disease involving central nervous system: an observational study abstract 2 role of inflammation in muscle atrophy of limb girdle muscular dystrophy 2a/r1 (lgmd 2a/r1) abstract 3 gene expression signature of meningioma identifies potential prognostic biomarker for tumor progression abstract 4 detection of braf fusion expression patterns in pilocytic astrocytoma of the paediatric age group abstract 5 comparative differential gene analysis in supratentorial ependymoma abstract 6 surgical spectrum of parasitic infections of nervous system an audit from neuropathology archives abstract 7 o6 methylguanine-dna methyltransferase (mgmt) promoter methylation in adult type diffuse high grade gliomas, cns who grade 4 by droplet digital pcr   free papers   free papers abstract 1 free neuropathol 4:9:5 insulin-like growth factor 2 mrna binding protein 3 (imp3) immunoreactivity in pilocytic astrocytoma and pilomyxoid astrocytomadoes it help to differentiate? sridevi koduru1, shilpa rao1, vani santosh1 department of neuropathology, nimhans, bangalore background: pilomyxoid astrocytoma (pma), located mainly in the hypothalamic/chiasmatic region, is a subtype of pilocytic astrocytoma (pa) with subtle histomorphological differences. pma also harbors similar molecular alterations of pa including mapk pathway alterations. however, pmas are known for higher rates of local recurrences. few studies have shown a difference in their gene expression profiles and from these studies, insulin-like growth factor 2 mrna binding protein 3 (imp3), has been identified as a gene helpful in differentiating the two tumors. objectives: to assess the value of imp3 in differentiating pma from pa. materials and methods: retrospective study was carried out on cases histopathologically diagnosed as pa and pma between years 2015 and 2021, including review of clinical, histological and ihc features. all tumors were stained for olig2, sox10, p16, brafv600e, ki67 and imp3 expressions. imp3 immunoreactivity was scored as 0, +1 and +2, based on intensity. labeling index was calculated only with 2+ intensity. results: nine cases of pma were diagnosed during the study period, and nine age and site matched pa were included. the mean age at diagnosis was 7.3 years (1-17), with male preponderance (m: f – 2.6:1). most common site was hypothalamic/chiasmatic region. pma and pa showed the well-described histomorphological features and both showed similar diffuse immunoreactivity for olig2, sox10, p16 and imp3 with a low ki67 labeling index. brafv600e ihc was positive in one case of pa. imp3 immunoreactivity was noted in both pma and pa. median labeling index for imp3 in pma was 30% (10%-70%) and 50% (10%-70%) in pa which was not statistically significant (p=0.283). conclusion: although there are reports that suggest imp3 to be a useful maker of pma, our study does not support this observation. there are no specific ihc markers to distinguish pma from pa on a routine basis and the diagnosis predominantly depends solely on histomorphology. our observation also points to the fact that pma could just represent a less mature from of pa.   free papers abstract 2 free neuropathol 4:9:6 series of 4 cases of subependymal giant cell astrocytoma vrushti devendrakumar patel1, hansa goswami1, urvi parikh1 department of pathology, b.j. medical college, ahmedabad, gujrat background: subependymal giant cell astrocytoma (sega) is a slowly growing tumor of unknown histogenesis with incidence of 0.027 per 100,000 person-years1. it mainly arises in the periventricular regions adjacent to the foramen of monro, which causes increased intracranial pressure, seizures, and focal neurologic signs. objectives: to study the age-distribution, clinical features and microscopy of sega in patient having clinical symptoms of tuberous sclerosis. material and methods: all four-biopsy received as multiple brown soft tissue structure aggregate each measuring 2.1x1.9x0.6 cm3, 1.5x1x0.2cm3, 3.7x0.8x0.3xcm3, 2.4x1.9x0.2cm3. sections were fixed with 10% neutral buffered formalin solution and processed by routine tissue processing and paraffin embedding. haematoxylin & eosin-stained sections were studied microscopically. results: we reported four cases of sega over a period of two years with all having the clinical symptoms of tuberous sclerosis. out of four cases two were male and two were female. out of four cases three were adult and one was adolescent. on microscopic findings sections show fascicle of spindle shaped cells with admixture of large gemistocyte like cells with homogenous eosinophilic cytoplasm and prominent nucleoli with eccentric nucleus. histomorphological findings are suggestive of subependymal giant cell astrocytoma who grade-1. conclusion: sega is a rare tumour of the central nervous system and diagnosis is based on clinical, radiological, histolopathology findings. it should be included in the differential diagnosis of mass near the foramen of monro even if there are features of tuberous sclerosis present or not.   free papers abstract 3 free neuropathol 4:9:7 diffuse meningeal melanocytic neoplasms of the brain morphological and molecular analysis of braf and kras mutations neha bhardwaj1, debajyoti chatterjee1, renu madan2, navneet singla3, bishan radotra1 department of histopathology, post graduate institute of medical education and research, chandigarh department of radiotherapy post graduate institute of medical education and research, chandigarh department of neurosurgery, post graduate institute of medical education and research, chandigarh background: primary meningeal melanocytic tumors (mmt) of the central nervous system (cns) can be circumscribed or diffuse. the latter arise from leptomeningeal melanocytes with predilection for spinal canal and posterior fossa. their sub-categorization into melanocytoma and melanoma is based purely on histomorphology. due to their rarity, not much prognostic information or molecular data is available at which this study is aimed. material and methods: the cases of mmt were retrieved from surgical database over a period of 9 years, and re-evaluated for histomorphology and immunohistochemistry (hmb-45, melan a, s100 and ki67). dna was extracted from formalin-fixed, paraffin-embedded tissue and real time polymerase chain reaction was performed using entrogen (krbrrt-50) kit for braf v600e and kras (exon 2, 3 and 4) hotspot mutations. results: six cases of melanocytomas and nine cases of melanomas were included with mean age of 35.3 years and 46.6 years, respectively. melanocytomas were identified in spinal, posterior fossa, and basitemporal locations in 2 cases each. melanomas were identified in spinal and cerebral location in 2 and 7 cases, respectively. no case of melanoma showed any other primary on further workup. melanomas showed nuclear pleomorphism, mitotic activity, and coagulative necrosis in contrast to melanocytoma. no case showed braf v600e or kras mutation. conclusion: kras and braf mutations are uncommon in mmts, hence suggesting that their mutational profile is different as compared to systemic melanomas. meticulous histological examination along with ki-67 help to differentiate meningeal melanoma from melanocytoma.   free papers abstract 4 free neuropathol 4:9:8 histopathological spectrum of astrocytoma in tertiary care hospital nidhi thaker1, smita shah1, hansa goswami1 department of pathology, b.j. medical college, ahmedabad, gujrat background: astrocytoma is most common primary central nervous system (cns) tumor that arise from the cells that form supportive tissue of the brain. astrocytoma is a subset of glial tumors, usually affecting the brain and sometimes the spinal cord. the two major categories of astrocytic tumors are the diffusely infiltrating astrocytoma (who grade ii to iv) and the more localized astrocytoma which is pilocytic astrocytoma (who grade i). objectives: to identify frequency of various histopathological types of astrocytoma. to study age, sex, site wise distribution and clinical presentation. materials and methods: a retrospective study is conducted from january 2022 to december 2022. for histopathological study, the specimens were fixed in 10% formalin, subsequently dehydration, clearing, embedding in paraffin wax were carried out. the sections were cut and stained using routine hematoxylin and eosin stain. results: in present study, most common astrocytic tumor was diffuse astrocytoma (42%). pilocytic astrocytoma comprises 20% of cases and anaplastic astrocytoma comprises of 14% cases. most cases belonged to age group between 31-40 years followed by 21-30. 58% of the cases were males and the rest were females. 82% of astrocytic tumors were supratentorial origin and 18% were infratentorial origin. conclusion: most common astrocytic tumor was diffuse astrocytoma followed by pilocytic astrocytoma. most common symptom was headache followed by convulsion, weakness, giddiness and vomiting. combining histopathological and molecular features helps in the definitive diagnosis and management of astrocytic tumors.   free papers abstract 5 free neuropathol 4:9:9 study of zfta and yap-1 fusion in supratentorial ependymomas vijayanirmala1, vaishali suri1, manmohan singh2, amandeep k. jadgevan2, ajay garg3, mehar chand sharma1 department of pathology, all india institute of medical sciences, new delhi department of neurosurgery, all india institute of medical sciences, new delhi department of neuroradiology, all india institute of medical sciences, new delhi background: ependymomas are classified according to a combination of histopathological and molecular features along with anatomical sites. they occur in three anatomical compartments of the central nervous systemsupratentorium, posterior fossa and spinal region. supratentorial ependymomas are relatively rare, shows considerable genetic heterogeneity and are molecularly stratified into 2 subtypeszfta fusion and yap-1 fusion positive supratentorial ependymomas. objectives: to perform molecular characterization of st-epn according to who cns 2021 classification. material and methods: a total of 50 cases of supratentorial ependymomas were retrieved from departmental archives between 2018-2022. histopathological examination and immunohistochemistry for l1cam, p65 were done. l1cam immunopositive tumors were evaluated for zfta fusion. tumors which were negative for zfta fusion and l1cam negative tumors were subjected for yap-1 rearrangement by florescence in situ hybridization. results: our study included 26 female and 24 males, and the tumors were commonly located in frontal and parietal lobes in the paraventricular region. on immunohistochemistry, 35 cases were l1cam positive and 25 cases were p65 positive. out of these 35 cases, 34 were positive for zfta fusion. one zfta fusion negative and 15 l1cam negative cases were tested for yap-1 rearrangement and 2 cases showed yap-1 rearrangement. who grade was assigned for cases that are not molecularly defined. conclusion: supratentorial ependymomas have distinct molecular profiles and an integrated histopathological and genetic workup is needed for precise diagnosis. yap-1 fusion tumors are rare. some cases cannot be diagnosed based on this classification system and further research is needed to find out other molecular alterations to understand their oncogenesis.   free papers abstract 6 free neuropathol 4:9:10 the many facets of astrocytes and blood-brain-barrier alterations in fungal infections of central nervous system aditi goyal1, shilpa rao1, m netravathi2, arivazhagan a3, jitender saini4, anita mahadevan1 departments of neuropathology, nimhans, bangalore departments of neurology, nimhans, bangalore departments of neurosurgery, nimhans, bangalore departments of neuroimaging, nimhans, bangalore background: astrocytes, most abundant cells in cns, are diverse in morphology and function. alterations following cellular injury, leads to dysfunction and blood-brain-barrier (bbb) permeability. astrocytic response in cns infections has not been systematically evaluated. we sought to study astrocytic and bbb alterations in fungal infections of cns, incidence of which is rising due to increasing immunosuppression, ecological changes etc. objectives: to evaluate morphological patterns of astrocytic response and changes in bbb in cns fungal infections. material and methods: clinico-demographic and neuropathological changes of twenty-nine patients with cns fungal infections were reviewed. immunohistochemistry for gfap and aquaporin-4 (aqp4) was performed for astrocytic and bbb alterations. results: pathological patterns of 29 cases [mucormycosis-11, aspergilloma-7, invasive aspergillosis-2, cryptococcoma-2, cryptococcal meningitis-2 and chromoblastomycosis-4, and candida-1 infection] were classified as granulomatous [aspergilloma and cryptococcoma], abscess [mucormycosis and dematiaceous fungi], diffuse cerebritis [invasive aspergillosis and mucormycosis], microabscesses [candida], and minimal inflammation [cryptococcal meningitis (cm)]. astrocytes’ phenotype was dictated by pathology pattern. granulomatous inflammation and abscess were similar with loss of astrocytes and aqp4 within necrotic centres, dysmorphic astrocytes with fragmented and beaded processes with perivascular accentuation of aqp4 in periphery of lesion and hypertrophic astrocytes in adjoining parenchyma with diffuse aqp4 distribution. in diffuse cerebritis, stellate astrocytes with marked loss of aqp4 expression was seen, whereas in cm, stellate astrocytosis with increased perivascular aqp4 was seen. conclusion: tissue patterns were associated with distinct astrocytic phenotypes and bbb integrity. perivascular accentuation of aqp4 in perilesional zone in granulomas and cm correlate with milder perilesional edema in contrast to loss of aqp4 in suppurative inflammation with extensive perilesional edema. understanding glial pathophysiological changes may help abrogate neurological sequelae in survivors.   free papers abstract 7 free neuropathol 4:9:11 spectrum of skeletal muscle disorders -an audit of data over four decades from a tertiary referral center rima s1, nalini a2, madhu n2, madalambika nm1, vani santosh1, anita mahadevan1, yasha tc1, nandeesh bn1, gayathri n1 departments of neuropathology, nimhans departments of neurology, nimhans background: diseases of skeletal muscle constitute a significant proportion of neurological disorders. the developments in investigative procedures and the advent of molecular diagnostic techniques have improved our understanding, clinical and diagnostic approach. objectives: to describe the spectrum of neuromuscular disorders (nmds) over four decades in a tertiary neuropathology referral center, and the changing trend if any. material and methods: review of muscle biopsies from patients of nmds received in the department of neuropathology. these biopsies were evaluated by histology and special tests (enzymehistochemistry, electron microscopy, immunohistochemistry and immunoblot, wherever indicated). the data is collated from nimhans myobank data base from 1983-2022. results: during the period 1983-2022, a total of 28,506 muscle biopsies were received. the major categories of muscle disorders diagnosed include muscular dystrophies(n=9027;31%), neurogenic atrophy(n=6111;21%), inflammatory disorder(n=3836;13%), congenital myopathies(n=388;1.3%), mitochondrial disorders(n=556;1.9%), miscellaneous (non-mitochondrial metabolic, myofibrillar and others). a notable change observed was a decline in the number of biopsied muscular dystrophy cases over the past two years (2021&2022), which formed 14% of biopsies, in contrast to 33% (2000-2020) in the previous years. contrastingly, an increase from 12% to 20%, in inflammatory myopathies was observed. conclusion: the present study observed reduction in the biopsies for conventional inherited myopathies and an increase in acquired myopathies, probably reflecting the advancement and application of genetic tests. review and regular audits of the biopsies are required to study the spectrum of disease prevalence, pattern and the associated changes(trend). this will facilitate our understanding, preparation and management. hence there is requirement for adopting new testing platforms in the diagnosis of nmds for optimal patient care.   free papers abstract 8 free neuropathol 4:9:12 does the macrophage subtype drive the immunological subtypes of leprous neuropathy? hema av1, madhu nagappa2, mahadevan a1 departments of neuropathology, nimhans, bangalore departments of neurology, nimhans, bangalore background: leprosy caused by mycobacterium leprae, is a common cause for infective neuropathies in india. the host’s immune response determines the pattern and progression of disease. macrophages are the main cell types directly infected by the bacillus. recently, macrophage subsets with distinct functions have been described. m1 macrophages are proinflammatory, m2 anti-inflammatory and m4 are related to the formation of foam cells described in atherosclerosis. no studies have evaluated macrophages subsets in leprous neuropathy. objectives: to determine macrophage subtypes in immunological subgroups of leprous neuritis (ln) material and methods: leprous neuritis (n= 47) diagnosed between january 2021 to may 2022 were reviewed and subtyped using ridley jopling criteria. immunohistochemistry for subtyping tlymphocytes (cd3, cd4, cd8), blymphocytes (cd20) and macrophages (m1=cd68+cd1630 ifn+; m2=cd68+cd163+ tgf-beta+; m4 = cd68+cd1630, mrp8+0) was performed and correlated with ln subgroup. results: tuberculoid tuberculoid (tt) and chronic leprous neuritis (cln) showed predominance of m1 macrophages with tnfα and small proportion of m2 > m4 macrophages. bcells were absent in endoneurium but found around epineurial vessels with cd4 & cd8+tcells. borderline tuberculoid (bt) was similar to tt with m1 macrophages, but admixed m4 > m2 macrophages. in borderline borderline (bb), borderline lepromatous (bl) and lepromatous leprosy (ll), there was equal proportion of m1 and m4 macrophages. conclusion: macrophage subtype dictates ln subgroup. the paucibacillary forms (tt, bt and cln) with florid inflammation and nerve destruction have pro-inflammatory phenotype (m1), multibacillary forms (bb, bl, ll) with scant inflammation have anti-inflammatory macrophages (m2) with m4. deciphering macrophage subtype in ln immunobiology has therapeutic implications to develop immunomodulatory therapies to limit nerve damage.   free papers abstract 9 free neuropathol 4:9:13 study of ki67 pi amongst meningiomas of different who grades & its association with os & recurrence ishita das1, dr vandita singh1 department of pathology & lab medicine aiims raipur background: meningiomas comprise 39 % of cns tumours and all grades are known to recur. who histopathological grading of meningiomas does not always correlate with, did not always correlate with chances of recurrence or progression and molecular diagnostics are not available everywhere. we studied the ki67 pi in different grades of meningiomas. material and methods: this was a retrospective study of ki67 pi in 100 meningiomas received at a tertiary hospital in central india, over 4-year period and its correlation with histopathological parameters of grading (as per who 2021 criteria), overall survival (os in months) and recurrence where data was available. results and conclusions: amongst 100 meningiomas studied m:f was 1:2. mean age of presentation was 45 years. the different who grade 1/2/3 meningiomas and their median mitoses, ki-67 indices, os (available in 68 cases) and recurrence (data available 70 cases) rates were noted: grade 1(n=65) mitoses median 1/10 hpf, ki-67 pi median 3.5%, os median 32 months, recurrence 10.76% (n=7); grade 2 (n=28) grade 2a n=12 (mitoses <4/10 hpf) median 1/10hpf, ki67 pi median 6.5% , os median 28 months, recurrence 16.7% (n=2) ; grade 2b n=15 cases (mitoses >4-19/10hpf) median mitoses 5/10hpf, ki-67 pi median 10%, os median 19 months, no recurrence, grade 3 (n=7): median mitotic count 21/10 hpf, ki-67 pi median 20%, os median 10 months, recurrence 29% (n=2). mitotic count was not an independent predictor of recurrence in our cohort while ki67 pi was. ki-67 was significantly associated with os in our cohort   free papers abstract 10 free neuropathol 4:9:14 utility of immunohistochemistry in subtyping posterior fossa group a ependymoma abhishek chowdhury1, shilpa rao1, nandeesh bn1, vani santosh1 department of neuropathology, national institute of mental health and neurosciences background: posterior fossa ependymomas (pf-epn) have been classified into posterior fossa group a (pfa) and posterior fossa group b (pfb) ependymomas. pfa-epn show loss of h3 p.k27me3 expression. further subdivided into molecular subgroup 1(pfa-1) and subgroup 2 (pfa-2), they encompass 9 subtypes (pfa 1a to 1f and 2a to 2c). otx2 and h3 p.k27m immunopositivity is noted in pfa-2c and 1f respectively and have prognostic implications. objectives: to assess the frequency of otx2 and h3 p.k27m immunopositivity in pfa-epn. material and methods: this retrospective study included pfa-epn diagnosed at our institute from 2020 to 2022. they were diagnosed based on loss of expression of h3p.k27me3 by immunohistochemistry. in these cases, immunohistochemistry for otx2 and h3 p.k27m was carried out. results: a total of 28 cases of pfa-epn were encountered. age range was 10 months to 23 years, with median age being 3.5 years. otx2 immunopositivity was seen in 3 tumors (10.7%). only one tumor was positive for h3 p.k27m (3.6%). the youngest patient (10-month-old girl) had the h3 p.k27m positive tumor. two otx2 positive tumors were seen in two 2 year old and one in a 12 year-old patient. conclusion: pfa-epn are most common in younger children. occasional tumors are otx2 and h3 p.k27m immuno-positive, conforming to pfa 2c and 1f. whilst pfa 2c is known to be associated with a better prognosis, h3 p.k27m positive tumors are associated with a worse outcome. assessment of frequency of otx2 and h3 p.k27m immunopositivity with data on survival can help in prognostication and exploring therapeutic avenues in pfa ependymomas.   free papers abstract 11 free neuropathol 4:9:15 posterior fossa ependymomas a clinicopathological study s. amritha¹, geeta chacko1 prabhu k2, chacko ag2, rajshekhar v2, moorthy r2, dr joseph bv2, john rr3, dr rajesh b4 and reka k5 departments of pathology1, neurosurgery2, paediatric oncology3, radiation oncology4 and biostatistics 5 christian medical college, vellore background: the 5th edition of who cns 5 has classified ependymomas into ten subgroups based on the anatomical site together with their molecular signature, of which supratentorial ependymomas with zfta fusion, posterior fossa ependymomas group a and spinal ependymomas with mycn amplification are found to have a dismal outcome. this study aimed to subgroup posterior fossa ependymomas (pfe) into posterior fossa ependymoma group a (pfa) and posterior fossa ependymoma group b (pfb) as it was of prognostic significance. the objective was to assess the expression of h3k27me3 in pfe and correlate it with the clinicopathological profile. objectives: to access the expression of h3k27me3 (histone h3 lysine 27 trimethylation) and to access the clinicopathological profile of posterior dosa ependymomas. materials and methods: a twelve-year study including 77 cases of posterior fossa ependymomas were subjected to immunohistochemical marker h3k27me3 and were sub-grouped as pfa and pfbs. further, all pfas were subjected to immunohistochemistry using the ezhip antibody. demographic details, site of the tumour, extent of surgery, and adjuvant therapy were obtained from the medical records. results: the prevalence of the pfa and pfb subgroup in this study population was 36.4% and 63.6% respectively. pfa tumours were more common in the younger age group, whereas pfb tumours were more common in the adult age group. pfa tumours were predominantly high-grade who cns grade 3 tumours; in contrast, pfb tumours were predominantly who cns grade 2 tumours. it was found that age (<10 years), who cns grade 3, mib1 labelling index >10% and the pfa subgroup had significantly poorer progression-free survival. conclusion: it is important to subgroup pfe based on their molecular signature as it is an independent indicator of outcome.   free papers abstract 12 free neuropathol 4:9:16 clinicopathological study of ependymomas with special reference to l1cam expression in prognosis and survival tista basu1,mou das1, uttara chatterjee1 department of pathology, ipgme&r and sskm hospital, kolkata background: ependymomas exhibit heterogeneity across age, location, histology, molecular nature and survival. the cns who classification (2021), identifies nine different molecular sub-groups of ependymomas based on dna methylation studies thus suggesting an epigenetic component its pathogenesis. studies suggests that these molecular sub-types remain stable throughout the course of disease. immnunohistochemical expression of l1cam, has been identified as a surrogate marker for zfta/c11orf95-rela fusion in supratentorial ependymomas. objectives: this study aims at realising the utility of l1cam as a surrogate marker for zfta/c11orf95-rela fused supratentorial ependymomas specially in resource-poor setups. materials and methods: forty-three histopathologically-proven cases of ependymoma under treatment over the period of two and half years from december 2019 to june, 2022 were selected. histopathological examination followed by ihc staining for gfap, s-100, ema and ki-67 in all cases and l1cam in the supratentorial ependymomas. we have followed-up most cases during our study period. ihc expression was correlated with various clinico-pathological parameters, including survival. results: in our study the commonest location for ependymomas was the spine in adults and posterior fossa in the pediatric age group. majority cases belonged to cns who grade 2 both in adults and in the paediatric age group. supratentorial location of ependymomas with positive immuno-reactivity for l1cam and a higher ki-67 labelling index were associated with poor survival. conclusion: the study revealed that l1cam was an effective surrogate marker for supratentorial ependymomas possibly carrying the zfta fusion gene product. the l1cam immuno-reactivity also corresponded with the survival data. however, larger population-based studies amongst the indian population are required to validate these results further.   free papers abstract 13 free neuropathol 4:9:17 gfap and sox10 expression in neonatal cerebral white matter injury: a postmortem minimally invasive tissue sampling study mary mathew1, athira sreenivas1, jayashree p1, leslie lewis2 department of pathology, centre for foetal and perinatal medicine, and neonatal unit, kasturba medical college department of pediatrics, kasturba medical college background: cerebral white matter injury (wmi) due to hypoxia-ischemia is the most type of neurological injury seen in term and preterm infants resulting in cerebral palsy. two pathogenetic pathways have been described that include the upper pathway where severe hypoxia-ischemia results in pancellular death of neurons as evidenced by severe white matter necrosis, microcysts and loss of glia and axons and the lower pathway where mild hypoxia-ischemia causes selective death of the preoligodendrocyte progenitors (preol) resulting in reactive gliosis. objectives: to study the utility of gfap and sox10 in cerebral white matter injury. material and methods: 50 postmortem brain cores of neonates with suspected neurological injuries were obtained following parental consent. the brain cores were studied for evidence and patterns of neurological injury. immunohistochemical markers gfap and sox 10 were used as surrogate markers to demonstrate loss glial cells and preoligodendrocytes or reactive gliosis respectively results: the various changes observed in the biopsies included anoxic neurons, reactive gliosis, cystic change, microgliosis, microglial nodules, meningitis, germinal matrix, intraventricular and white matter hemorrhage and subarchnoid hemorrhage. gfap was increased in 31 preterm and 8 term neonates and reduced or absent in 6 preterm and 1 term baby. sox 10 was absent in 35 preterm and 5 term infants and retained in 4 preterm and 6 term infants. conclusion: the above findings demonstrate that both pathways are involved in preterm and term infants with wmi and identifying the patterns of injury would aid in early therapeutic interventions and prediction of outcomes.   free papers abstract 14 free neuropathol 4:9:18 olig2-positive cell density in moghe versus other epilepsy surgical samples: a morphometric study deepti narasimhaiah1, george c vilanilam2, ramshekhar n menon3, ajith cherian3, bejoy thomas4, kesavadas c4, rajalakshmi poyuran1 department of pathology, sree chitra tirunal institute for medical sciences and technology, trivandrum department of neurosurgery, sree chitra tirunal institute for medical sciences and technology, trivandrum department of neurology, sree chitra tirunal institute for medical sciences and technology, trivandrum department of imaging sciences and interventional radiology, sree chitra tirunal institute for medical sciences and technology, trivandrum background: abnormalities in oligodendroglia are reported in surgical samples of patients with drugresistant epilepsy. “mild malformation of cortical development with oligodendroglial hyperplasia (moghe)” described mostly in patients with frontal lobe epilepsy is characterized by significantly higher degree of oligodendroglial hyperplasia. objectives: to compare the density of olig2-positive cells in white matter between moghe and other types of epilepsy specimens. material and methods: 59 epilepsy surgical samples comprising 6 groups (10: focal cortical dysplasia, 7: gliosis, 19: hippocampal sclerosis, 4: moghe, 10: oligodendrogliomas, 9: others) were included in the study. olig2-positive cell density/mm2 area in the white matter was determined on olig2-immunostained sections using image j software. one-way anova test was used to compare the olig2-positive cell densities between different groups (p <0.05 significant). results: the mean density of olig2-positive cells in the white matter in the six groups was: focal cortical dysplasia: 1094/mm2, gliosis: 1080/mm2, hippocampal sclerosis: 1426/mm2, moghe: 2138/mm2, oligodendroglioma: 2300/mm2 and others: 1198/mm2. the p-value (one-way anova) between groups was 0.002. to further understand which groups were different, multiple comparisons test was performed. as the group of interest was moghe, the mean olig2-positive cell density of each group was compared with the mean olig2-positive cell density of moghe group. the mean olig2-positive cell density/mm2 was significantly different between moghe and all other groups (p: <0.05), except oligodendroglioma (p: 0.71). conclusion: the mean density of olig2-positive cells/mm2 in white matter in moghe was significantly higher than in other epilepsy samples and was like that of oligodendroglioma.   free papers abstract 15 free neuropathol 4:9:19 significance of emerging prognostic markers in meningiomas trishala mohan1, jyotsna singh1, ravi sharma3, ashish suri2, mehar c sharma1, vaishali suri1 department of neuropathologya, all india institute of medical sciences, new delhi department of neurosurgeryb, all india institute of medical sciences, new delhi department of neurosurgery, all india institute of medical sciences, jhajjar background: meningiomas are the most common primary intracranial tumours. who cns5 classification updates has incorporated prognostic biomarkers like ptert promoter mutation and cdkn2a/b homozygous deletion. expression of p16 is being correlated with cdkn2a gene alterations in gliomas and can be detected via immunohistochemistry. objectives: to assess the significance of prognostic markers (ptert, cdkn2a homozygous deletion, and p16 expression) in meningioma to identify their biological and clinical utility. material and methods: 200 cases (93 males and 107 females) of grade 1 (n=107), 2 (n=78), and 3 (n=15) were included. they were assessed for ptert mutation (c228t/c250t) using sanger sequencing; cdkn2a deletion by fish, and p16 expression by ihc. results: there were 192 adult and 8 paediatric cases. only 5% (10/200) of meningiomas showed ptert c228t mutation. all were histopathologically grade 2. histopathological grade 2 meningiomas with ptert mutation survival varied from 7 to 64 months with mean survival of 26 months while without ptert alteration grade 2 meningiomas varied from 10 to 91 months with mean survival of 36 months. 62% (124/200) of cases showed total and 29.5% (59/200) showed partial loss of p16 expression. only a small fraction of meningiomas (2.5%, 5/200) showed cdkn2a homozygous deletion including histologically grade 3 (4/5) and 2 (1/5) tumours. conclusion: integrated diagnostic protocol simplifies meningioma categorization and provides improved accuracy in predicting outcome and recurrence. there is no association between cdkn2a deletion and p16 expression. further, the predictive power of ptert and cdkn2a status identifies aggressive meningiomas and provides biomarker for new therapeutic interventions.   posters   posters abstract 1 free neuropathol 4:9:20 glioblastoma with predominant sarcomatoid differentiation: a rare case report saloni parikh1, hansa goswami1, urvi parikh1 department of pathology, b.j medical college, ahmedabad, gujarat background: is a rare primary malignant tumor of central nervous system with biphasic morphological pattern, glial and malignant mesenchymal components and is considered as variant of glioblastoma multiforme. located in the cerebrum, involving the temporal, parietal, frontal, and occipital lobes in decreasing order of frequency. objectives: to correlate frozen section findings with histopathological examination and immunohistochemistry for exact histogenetic typing. material and methods: received one reddish brown soft tissue measuring 2.5x2x0.3cm3 in normal saline during ongoing surgery, processed as frozen section with cryostat machine. another three greyish brown soft tissue were received in 10% neutral buffered formalin total measuring 2.8x2.6x1.5 cm3 which was processed by routine tissue processing. after paraffin embedding, hematoxylin and eosinstained section were studied microscopically. ihc for gfap, vimentin, ema and p53 was performed. results: frozen section showed histology of high grade glioma -glioblastoma with small cell component and pleomorphic spindle cell component who grade-iv. on biopsy, microscopy showed glial component with pleomorphic astrocytic cells showing nuclear atypia, mitosis, pseudopalisading necrosis and microvascular proliferation. sarcomatous component shows spindle shaped cells with nuclear atypia. this showed impression of glioblastoma with predominant sarcomatoid differentiation-who grade iv. ihc was positive for p53, vimentin and negative for gfap in areas with sarcomatoid differentiation, ema negative which confirmed the case as gliosarcoma. conclusion: frozen section plays crucial role in deciding staging of tumor and determining margin for resection. biopsy and ihc has important role in confirmation of diagnosis and prognosis.   posters abstract 2 free neuropathol 4:9:21 does mib-1 labelling index correlate with who grade and outcome in spinal meningiomas? – a clinicopathological study hemanth kumar r1, afshan fhs1, chacko g1, moorthy r2, joseph bv2, chacko ag2 department of pathology, christian medical college, vellore neurosurgery, department of neurological sciences, christian medical college, vellore background: spinal meningiomas are slow growing indolent tumours. the histological grade and extent of resection do not reliably predict prognosis. the mib-1 labelling index is known to be a useful prognostic marker. objectives: to assess mib-1 labelling index (mib-1 li) in spinal meningiomas, and to correlate it with who grade, histological parameters, and outcome. material and methods: this was an ambispective study on 115 cases of spinal meningiomas diagnosed between january 2012 and june 2021. based on who (2021) criteria, these tumours were assigned into benign, atypical, and anaplastic categories. mib-1 immunostaining was performed on all these cases. outcome (recurrence) was correlated with the who grade, histological parameters, and mib-1 li. results: of the 115 cases, 65 were cns who grade 1, and 50 were cns who grade 2. there were no cns who grade 3 tumours. cns who grade 2 meningiomas had a higher mib-1 li compared to grade i tumours, with a threshold index of 5% exhibiting highest diagnostic validity at a sensitivity/specificity of 98% / 41.5% respectively. this cut-off also had significant correlation with certain ‘atypical’ features, namely, sheeting, small cell change, prominent nucleoli, and necrosis. follow-up was available for 66.9% of cases, with a mean duration of 26 months. only one patient with a cns who grade 2 meningioma had recurrence. conclusion: mib-1 li threshold of 5% distinguished grade 2 from grade 1 meningiomas and had a significant correlation with certain individual ‘atypical’ features. association with recurrence could not be ascertained due to limited follow-up, and a low incidence of recurrence.   posters abstract 3 free neuropathol 4:9:22 non-hodgkins lymphoma nk/t cell type of nasal cavity : a rare case report dharti mukeshbhai ramani1, urvi parikh1, hansa goswami1 neuropathology laboratory, neurosciences centre, all india institute of medical sciences, new delhi, india background: rare, aggressive and predominantly extranodal lymphoma of nk / t cell origin characterized by angiotropism, angiodestruction , necrosis and association with epstein-barr virus (ebv). also known as midline malignant reticulosis, polymorphic reticulosis, nasal t-cell lymphoma, lymphomatoid granulomatosis and angiocentric t cell lymphoma. objectives: to study a rare case for its clinical features, microscopic findings and immunohistochemistry for exact histogenetic typing and to rule out remote possibility of embryonal rhabdomyosarcoma-small cell type and high-grade neuroblastoma. material and methods: received multiple greyish white soft tissue structure aggregate total measuring 0.8x0.6x0.2 cm3 in histopathology section of pathology department, b j medical college. sections were fixed with 10% neutral buffered formalin and processed by routine tissue processing. after paraffin embedding, haematoxylin & eosin-stained sections were studied microscopically. results: serial sections from received small, fragmented biopsy specimen shows markedly cellular and necrotic tumor tissue with diffusely arranged monomorphic population of poorly differentiated round to oval cells with dense hyperchromatic nuclei, scant cytoplasm and extensive areas of necrosis. histopathological findings are suggestive of undifferentiated malignant round cell tumor –possibly non-hodgkins lymphoma-nk/t cell type, with remote possibility of other round cell tumorembryonal rms. immunohistochemistry done for further confirmation and rule out other possibilities. conclusion: histopathological findings give clue for diagnosis of nk /t cell non-hodgkins lymphoma and immunohistochemistry is confirmatory.   posters abstract 4 free neuropathol 4:9:23 a case of papillary tumor of pineal region – a rare entity roseline rekha hebziba1 mathew suresh kumar1, monika nanavati1 , hansa goswami1 department of pathology, civil hospital, ahmedabad background: tumors of the pineal region comprises 1% or less of all intracranial neoplasms. papillary tumor of pineal region (ptpr) is a very rare entity, arising from specialized secretory ependymocytes of the subcommissural organ. headache is the common presenting symptom occurring due to increased intracranial tension as a result of compression of the aqueduct. this is a case report of a 17-year-old male, presenting with headache and altered sensorium, subsequently diagnosed with papillary tumor of pineal region. objectives: to study about the cause, symptoms, histological pattern and its differentiating feature from other tumors of pineal region. materials and methods: excised specimen was received in histopathology department. specimen was then preserved in 10% neutral buffered formalin, processed in automated tissue processor, embedded in paraffin blocks, stained using h&e stain following which microscopic examination was performed. results: histopathology revealed oval to columnar tumor cells arranged in pseudorosettes formation and papillary architecture with fibro-vascular core and small foci of tumor cells arranged in group. immunohistochemistry study showed strong reactivity for vimentin and s 100 protein. ema and gfap were negative. overall histomorphological appearance was suggestive of papillary tumor of pineal region who grade 2. conclusion: papillary tumor of the pineal region is a neuroepithelial tumor with distinct morphological and immunohistochemical features. ihc is used to differentiate it from other primary and metastatic tumors in this region. it’s potential for disease progression, frequent local recurrence and csf dissemination warrants early diagnosis and early aggressive therapy in these patients.   posters abstract 5 free neuropathol 4:9:24 a rare case of atypical teratoid/rhabdoid tumour: case report keta patel1, hetal jani1, hansa goswami1 department of pathology, civil hospital, ahmedabad background: atypical teratoid rhabdoid tumour is a rare, malignant, highly aggressive paediatric intracranial neoplasm. it comprises 1-2% of all paediatric brain tumours and 10–20% of cns tumours up to 3 years of age, with male preponderance. it originates mostly from cerebral and cerebellar hemispheres, cerebellopontine angle and brain stem, and is easily misdiagnosed as medulloblastoma on ct/mri. the tumour progression is associated with loss of ini-1 expression in the neoplastic cells and immunohistochemistry helps confirming the diagnosis. this is a case report of a 4-year-old male presented with complain of vomiting, lethargy and macrocephaly. ct/mri suggestive of trigeminal schwannoma and surgical excision was performed. objectives: to study the histopathology and immunohistochemistry in a case of atypical teratoid rhabdoid tumour. material and methods: specimen of the excised greyish-white soft tissue aggregates was received and fixed in 10% neutral formalin. after gross examination, sections were taken and processed by automated tissue processing. slides were stained using h&e stain, and microscopic examination was performed. results: serial sections showed highly cellular embryonal neoplasm composed of sheets histology of markedly cellular, mitotically active high grade cns tumour-atypical teratoid/rhabdoid tumour, who grade-iv. immunohistochemistry was positive for vimentin, ema, gfap, but negative for ini-1 which confirmed the diagnosis. conclusion: this highly aggressive tumour can metastasize rapidly via the cerebrospinal pathway, resulting in poor prognosis. earlier detection and adjuvant therapy can improve the prognosis. reporting such rare tumours is of utmost importance as it provides information on clinicopathological patterns and factors that impact prognosis.   posters abstract 6 free neuropathol 4:9:25 smoke and mirrors: a tale of two unusual cerebellopontine angle tumors deepak nayak m1, geetha v1, bhavna nayal1, girish menon2 department of pathology, kasturba medical college, manipal, mahe department of neurosurgery, kasturba medical college, manipal, mahe background: tumors of the cerebellopontine angle (cpa) are frequent; with neural tumors and meningiomas predominating the scene. however, infrequently encountered tumors, derived from adjoining anatomical structures, can be confounding to a pathologist, especially under the duress of an intra-operative frozen section. we herein describe two such unusual neoplasms, having radiologic and clinical incongruity, which led to diagnostic difficulties and eventually, a learning experience. case reports: case-1: a 25-year-old lady with a history of headache and gait imbalance for 3 months. the radiology profile showed a left cpa tumor measuring 3.8x3.7cm., hinting at a possibility of a neural tumor. the on-table finding prompted a meningioma. the imprint and squash cytology & section suggested an epithelial neoplasm-? metastasis. the paraffin sections showed compact, organoid nests with a granular cytoplasm and intervening vasculature, positive for synaptophysin, chromogranin, s-100 & negative for cytokeratin, inhibin; a low ki67 index, compatible with a paraganglioma. case-2: a 48-year-old man with a history of gait imbalance and dizziness for 2 months. radiology suggested a mass lesion in the right cpa, compatible with either an epidermoid/ dermoid cyst or a schwannoma. the on-table diagnosis -? schwannoma or an epithelial cyst. the imprint and squash cytology & section showed acellular matrix with suspended epithelial cells and vacuolated cells, commensurate with the radiologic diagnosis. the paraffin sections showed lobules of large, vacuolated cells with a myxoid stroma with a pseudochondroid appearance. the ihc profile showed positivity for ck19, ema, s100 and negative for ck7; rendering a diagnosis of a conventional chordoma. conclusion: these cases intend to highlight the lesser encountered cpa neoplasms by revisiting the intra-op devices for diagnostic clues and a better precision.   posters abstract 7 free neuropathol 4:9:26 all proliferating vessels are not haemangiomas a case report of rare entity of sporadic meningioangiomatosis swetha narla1, annapurneswari s1 department of histopathology, apollo cancer centre, chennai background: meningoangiomatosis is a rare benign disorder which can occur sporadically or in association with neurofibromatosis. seizures are the first symptom in sporadic type whereas the one associated with neurofibromatosis is often asymptomatic. it is characterized by proliferation of fibroblasts with increased number of vessels surrounded by meningothelial cells. very few case reports have been reported out of which most of the cases were sporadic. we present the etiology, imaging and histopathological findings of a sporadic case of meningoangiomatosis. case report: we present a case of 8-year-old male child who was evaluated for complaints of multiple episodes of seizures, disorientation and speech disturbances. there was no significant past or family history. there was no association with neurofibromatosis. investigations revealed parietal space occupying lesion. the boy underwent craniotomy with surgical excision. histopathology and immunohistochemistry was consistent with meningoangiomatosis. within a short span, the boy came with early local recurrence. conclusion: meningoangiomatosis can occur sporadically or in association with neurofibromatosis and it must be considered in the differential diagnosis of vascular cortical lesion.   posters abstract 8 free neuropathol 4:9:27 extensive spherical amyloid deposition in lactotroph pituitary neuroendocrine tumour masquerading as a primary bone tumour abhilash krishnan.k1, ipsita panda1, aravind sekar1, debajyoti chatterjee1 sushant kumar sahoo2, pinaki dutta3 department of histopathology, pgimer, chandigarh department of neurosurgery, pgimer, chandigarh department of endocrinology, pgimer, chandigarh background: amyloid deposition is uncommon in pitnet. these deposits can be spherical, perivascular or stellate, commonly seen in prolactin and growth hormone-producing tumours. however, the spherical type of amyloid is rare and is exclusively described in prolactin-producing tumours. herein, we report a rare case of prolactin-producing pituitary pitnet with extensive spheroid amyloid deposits. case report: a 45-year-old male patient presented with a gradual diminution of vision in the left eye for three years associated with headache. mri showed the presence of a sellar mass measuring 2.4*2.3*1.9cm bulging inferiorly into the sphenoid sinus. biochemical investigation revealed elevated prolactin levels (34.9 microgram/l), for which he was treated with cabergoline for nine months. he subsequently underwent trans-sphenoidal resection of the mass. intra-operatively, the mass was firm to hard, and differentials of clival chordoma and pituitary adenoma were considered. on microscopic examination sections from the mass revealed pitnet showing therapy related changes, in the form of shrinkage of tumor cells. there was extensive large lamellated spheroidal acellular eosinophilic deposits masking the tumor cells. the lamellated deposits showed positivity for congo red and displayed apple green birefringence under polarised microscope, indicating amyloid in nature. on ihc, the tumour cells and spheroid collections showed exclusive positivity for prolactin. conclusion: prolactin producing pitnet may show extensive spherical amyloid deposits, masquerading as calcification in imaging or raise a suspicion of a primary bone tumor. microscopically, the extensive lamellated structure may deceptively camouflage the neoplastic tumour cells. hence, awareness of this rare histological finding is essential.   posters abstract 9 free neuropathol 4:9:28 late onset pompe disease: role of fluorescence microscopy divya aggarwal1, lokesh saini1 department of pathology, all india institute of medical sciences, jodhpur background: pompe disease is a type ii glycogen storage disorder (gsd), which is caused by acid alpha-glucosidase deficiency. onset usually occurs soon after birth, however, partial enzyme activity can result in late onset pompe disease (lopd) which can present as late as second decade of life. owing to presence of storage material in lysosomes, lopd is characterised by presence of intralysosomal lipopigment deposition, which can be depicted on immunofluorescence, aiding in diagnosis. case report: a 15-year-old boy presented with gradual onset weakness and progressive wasting for 3 years. he complained of difficulty in getting up from squatting position, climbing upstairs and lifting heavy objects. his elder sister had history of similar symptoms, before she expired at 18-years of age with sudden onset breathlessness. grade ii ejection systolic murmur was present in mitral area. ck-nac was 2347 u/l. muscle biopsy was done which revealed moth eaten appearance with marked cytoplasmic vacuolation in subsarcolemmal location. these showed periodic acid schiffdiastase (pas-d) positive material. lipopigment was seen as autofluorescent granules on fluorescent microscopy at 494 nm. this autofluorescence was quenched on sudan black b staining, hence confirming lipopigment deposition. combination of above features was useful for diagnosis of lopd which was subsequently confirmed on whole exome sequencing with homozygous mutation in gaa gene at exon 14 (c.2040g>a). conclusion: late onset pompe disease is a rare entity which is characterised by lipopigment accumulation in addition to other characteristic features of gsd. though the muscle biopsy findings of gsds show considerable overlap, knowledge of additional role of fluorescence microscopy can be helpful in arriving at correct diagnosis and in performing targeted genetic studies, if required.   posters abstract 10 free neuropathol 4:9:29 nemaline myopathy: a case report snehvarsha bhagat1, neha bhardwaj1, debajyoti chatterjee1, kirti gupta1, sucharita ray2 departments of histopathology, post graduate institute of medical education and research, chandigarh departments of neurology, post graduate institute of medical education and research, chandigarh background: nemaline myopathy is a heterogenous group of congenital myopathies caused by autosomal dominant or recessive mutations commonly involving acta1 and neb genes. the spectrum of clinical presentation is broad, ranging from congenital onset muscle weakness and hypotonia to adult forms which are mostly autoimmune related. ultrastructurally, these rod like or ovoid structures are derived from z lines and have a similar lattice structure and protein content. case report: we describe a case of an 18-year-old female presenting with very low body mass index since birth and proximal weakness since childhood. on examination, she had high arched palate, bilateral pedal edema, and ptosis associated with bulbar signs. gower’s sign was positive. biochemical analysis revealed elevated serum creatinine and uric acid. nerve conduction studies (ncs) showed sensori-motor axonal polyneuropathy.the electromyography (emg) performed from deltoid, vastus lateralis and tibialis anterior was suggestive of myopathic pattern. snap frozen left vastus lateralis muscle biopsy examined showed uniform myofibres with dominance of type 2 fibres. these fibres showed numerous subsarcolemmal rod shaped inclusions in the cytoplasm which stained red on modified gomori’s trichrome (mgt) stain. these regions stained negatively on nadh enzyme, thus confirming nemaline myopathy. conclusion: nemaline myopathy is a rare form of congenital myopathy. routine histology of muscle biopsy may be normal, hence, mgt and enzyme stains are essential for the diagnosis. a better recognition and understanding of the disease will pave the way for early diagnosis and development of potentially effective treatment modalities. genetic counselling is mandatory in the affected families.   posters abstract 11 free neuropathol 4:9:30 cutaneous clue to amoebic encephalitis: a case report divya aggarwal1, suryanarayanan bhaskar1, sarbesh tiwari1, anil budania1, deepak kumar1, vibhor tak1, sucharita anand1 department of pathology, all india institute of medical sciences, jodhpur background: pathogenic free living amoebae can cause fatal central nervous system infections in humans. these include acanthamoeba sp, naegleria fowleri and balamuthia mandrillaris. of these balamuthia is known to cause skin involvement with skin as one of the proposed entry routes. we present one such case report. case report: a 28-year-old male presented with generalised tonic clonic seizures for 1 month. mri brain was done which showed an ill-defined intra-axial cortical based sol in left superior and middle frontal gyri with marked perilesional edema, suggestive of infectious etiology. he also had well-defined erythematous plaques over back and left arm. biopsies were performed from both brain and skin lesions. histopathology of brain specimen revealed features of necrotising granulomatous inflammation with presence of trophozoites of balamuthia mandrillaris, confirmed on periodic acid schiff stain with diastase digestion. skin lesions showed pan dermal dense lymphohistiocytic infiltrate. patient was started on multi-drug regimen, however, he continues to deteriorate. multiple steps were included in species identification and confirmation, including pathology and microbiology consults, assessment of size of organism, presence of cutaneous lesions and pcr (which served as gold standard). conclusion: free living amoeba are neurotropic organisms which cause rapidly fatal meningoencephalitis. of the known species, balamuthia is known to cause skin lesions. thorough search for organisms must be performed in all cases of granulomatous inflammation of brain, as amoebic trophozoites are easily missed due to their resemblance to histiocytes. species identification is a key aspect in diagnosis and disease management which requires multi-speciality coordination.   posters abstract 12 free neuropathol 4:9:31 malignant transformation of intracranial epidermoid cyst: a rare case report farzana abdul majeed1, loretta ann pereira2 department of pathology, yenepoya medical college; mangalore department of oncopathology, yenepoya medical college, mangalore background: intracranial epidermoid cysts constitute approximately 0.2 – 1% of all brain tumours. malignant transformation of an intracranial epidermoid cyst to squamous cell carcinoma is extremely rare and very few cases have been reported in the world. case report: we hereby present a rare case of a 29 year old man who had presented with a new swelling on a previous surgical site and left paresis. the patient had presented three months prior with a dermoid cyst with a small focus of grade 1 scc, for which he underwent total excision. evaluation of the current lesion showed recurrence with significant interval growth, with imaging findings suggestive of residual lesion as well as pseudomeningocele. he underwent exploration and excision, resulting in improvement of paresis. histopathological examination revealed a moderately differentiated squamous cell carcinoma (grade 2) infiltrating adjacent glial parenchyma. conclusion: although malignant transformation of ec to scc is rare, possibility of such rare entities should be kept in mind. appropriate diagnosis with immunohistochemical and other ancillary techniques are mandatory for the treatment and management of the patient.   posters abstract 13 free neuropathol 4:9:32 neuropathological findings in a 17-month-old boy with delayed milestones and kinky hair yukim dong1, neha bhardwaj1, kirti gupta1, chaithanya reddy2, jitendra kumar sahu2 departments of histopathology, postgraduate institute of medical education and research, chandigarh, india departments of pediatrics, postgraduate institute of medical education and research, chandigarh, india background: menkes disease is rare x-linked recessive disorder reported in 1 in 300000 live births. it is caused by loss of function mutation in copper transport gene, atp7a gene located on chromosome x. patients classically present with neurodevelopmental delay, skin with hair changes and can be successfully treated with copper supplements. case report: we describe a case of 17-month-old boy presenting with cyanosis, breathing difficulty and seizures. there was loss of previously attained developmental milestones. on examination, he was lethargic with small head circumference. his hair was brittle with skin laxity, joint hypotonia and brisk deep tendon reflex. his elder sibling and maternal uncle died at young age with similar symptoms. investigations revealed low levels of serum copper and ceruloplasmin. magnetic resonance imaging revealed diffuse atrophy of the cerebrum, cerebellum along with tortuosity of intracranial vessels. due to worsening clinical condition, the child expired on day 32 of hospital stay. brain autopsy was performed and histopathological examination of cortex showed dense gliosis, myelin loss and significant loss of neurons. basal ganglia and other deep grey nuclei revealed variable loss of neurons in the background of gliosis. cerebellum showed aberrant dendritic arborization, somal sprouts and axonal torpedoes within the purkinje neurons. genomic dna extracted from blood revealed a hemizygous single base pair deletion in exon 17 of atp7a gene located on chromosome x hence confirming the diagnosis of menkes disease. conclusion: the index case illustrates the neuropathological features in a genetically proven case of menkes disease at autopsy.   posters abstract 14 free neuropathol 4:9:33 pituitary macroadenoma with apoplexy presented as sudden bilateral vision loss jyotsna naresh bharti1 department of pathology, aiims mangalagiri background: pituitary apoplexy is true when there is clinical and radiological evidence of pituitary hemorrhage. a true apoplexy is an uncommon event occurring in 2 to 3 % of pituitary adenoma. in most cases, the event is spontaneous; however, a precipitating cause is identified in 25–30% of patients. various precipitating causes reported in literature include hypertension, hypotension, major surgery, head trauma, radiation, drugs, endocrine anterior pituitary stimulation test, and thrombocytopenia. the two major mechanisms by which these factors cause apoplexy are either by causing acute fluctuations in blood pressure or by increasing the bleeding tendency. pituitary apoplexy with sudden vision loss requires urgent trans endoscopic transsphenoidal surgical resection. case report: a 35-year-old male presented with bilateral vision loss for 3 days and no other complaints. the patient was advised for mri head and hormone test. lh3.98 m iu/ml, fsh 12.8 m iu/ml, prolactin 2.36 ng/ml, testosterone 416.14 ng/dl. the mri head revealed a heterogenous sellar mass lesion with suprasellar extension measuring 45x32x22mm. the lesion uplifted the optic chiasma. the features were suggestive of pituitary macroadenoma with apoplexy. the patient was operated, and the tumor was sent for histopathological examination. the tumor cells were arranged in sheets with small round nuclei and moderate amounts of granular eosinophilic cytoplasm. there was no significant mitotic activity and atypia. many areas showed the presence of hemorrhagic infarction. tumor cells were immunoreactive to synaptophysin, and ki67 was 1%. reticulin stain showed a distorted and fragmented staining pattern. features were of pituitary macroadenoma with apoplexy. within a few days, the patient recovered from vision loss and now doing well on follow up. conclusion: early surgical intervention is associated with a good prognosis in such cases.   posters abstract 15 free neuropathol 4:9:34 polymorphous low-grade neuroepithelial tumor of the young (plnty): rare but curable cause of refractory epilepsy aishwarya karthikeyan1, debajyoti chatterjee1, navneet singla2 department of histopathology, postgraduate institute of medical education and research, chandigarh, india department of neurosurgery, postgraduate institute of medical education and research, chandigarh, india background: first described in 2016, polymorphous low grade neuroepithelial tumor of the young (plnty) is an epileptogenic tumour of children and young adults. they present with seizures resistant to anti-epileptic drugs. morphologically, they exhibit a diverse collection of neuro-epithelial neoplasms that, as a group, exhibit varying levels of glial with or without neuronal differentiation. case report: in our case report, a 16-year male presented with a history of seizures since 9 months of age. mri brain showed a well-defined expansile non enhancing multi-cystic cortex-based lesion involving the left medial temporal lobe with areas of mineralisation. intra-operatively, the lesion was yellowish partly calcified, firm, and non suckable. biopsy examined was in multiple fragments and showed grey and white matter fragments infiltrated by a moderately cellular tumour. the tumor was composed of uniform small round cells with bland vesicular chromatin and peri-nuclear halo having oligodendroglioma like appearance. some areas showed spindling. there were areas of extensive calcification. there were no rosenthal fibres or eosinophilic globular bodies. the tumor cells were positive for gfap, cd34 (diffuse strong) and negative for idh1, synaptophysin, p53 and olig2. neun highlighted entrapped neurons while tumour cells are negative. atrx show retained nuclear expression. the final impression was plnty. mri performed one year after surgery did not show any residual tumor, but he had intermittent episodes of seizures. conclusion: plnty is associated with drug resistant epilepsy. it can show extensive calcification and may resemble other low-grade gliomas. diffuse cd34 expression can clench the diagnosis.   posters abstract 16 free neuropathol 4:9:35 a rare case of cerebral cavernous malformation with concomittant intracanial mucormycosis infection pratishtha sengar1, vikas kailashiya1, nityanand pandey1 department of pathology, institute of medical sciences, banaras hindu university, varanasi background: cerebral cavernous malformation is angiographically detected occult solitary or multiple vascular anomalies. intracranial mucormycosis is rare and represents one of the most severe manifestations, often determining the survival and functional outcome of patient with few case reports in literature in immunocompetent individuals. we, hereby report a case of cavernous malformation made rarer with concomitant mucormycosis. case report: a 22-years old female presented with left sided facial seizures since age of 7 years and headache for past 3 years. frequency of seizure was 1-2 episodes per day. there was no history of fever, headache, vomiting or head trauma. past history was negative for tuberculosis, connective tissue disease or glucocorticoid use. neurological examination also did not reveal any cranial nerves, motor, sensory or cerebellar abnormality. magnetic resonance imaging revealed a contrast enhancing lesion with evidence of bleed in right posterior frontal lobe suggestive of cavernous malformation. right frontal craniotomy with excision of cavernoma was done. gross examination showed a 4x3 cm solid cystic mass with multiple mulberry protrusions. histopathological examination revealed features of cavernous malformation with evidence of broad aseptate branching fungal hyphae conforming with the morphology of mucormycosis. scant intervening brain parenchyma also revealed fungal infiltration along with reactive gliosis. few foci of angioinvasion were seen. final diagnosis of cavernous malformation with mucormycosis fungal infection was rendered and microbiological studies were advised. conclusion: to the best of our knowledge, this is the first case report of a cerebral cavernous malformation with mucormycosis in an immunocompetent patient without any risk factor.   posters abstract 17 free neuropathol 4:9:36 suprasellar masquerader: chordoid glioma neha bhardwaj1, chirag ahuja2 , navneet singla3, kirti gupta1 departments of pathology, postgraduate institute of medical education and research, chandigarh, india departments of radiology, postgraduate institute of medical education and research, chandigarh, india departments of neurosurgery, postgraduate institute of medical education and research, chandigarh, india background: chordoid glioma is a rare well-circumscribed glial neoplasm arising in adults and shows female predominance. tanycytes of third ventricle are proposed as the cell of origin owing to its location. it is characterized by chordoid features with myxoid and inflammatory stroma. the specific molecular hallmark is recurrent prkca p.d463h missense mutation. case report: we present 2 cases (30-year-old female and 45-year-old male) with similar complaints of behavioural change and headache. midline suprasellar homogenously enhancing mass was seen on contrast enhanced magnetic resonance imaging. both the patients underwent craniotomy and subtotal tumor resection. histopathology and immunohistochemistry was characteristic of chordoid glioma with cords and clusters of epithelioid cells arranged in a solid pattern. there was variable amount of myxoid stroma and lymphoplasmacytic infiltrate. no mitosis, necrosis or brain invasion was noted. the tumor cells expressed strong diffuse positivity for glial fibrillary acid protein (gfap) and weak nuclear thyroid transcription factor (ttf-1). ema and brachyury were negative. subsequently, the lady underwent gross total excision and demised soon in the post-operative period. the male patient received radiotherapy and is currently doing well post 6 months of follow-up. conclusions: the rare occurrence, radiological and morphological overlaps in chordoid gliomas make them a true masquerader. combination of gfap and ttf-1 in the immunohistochemical panel can be useful in differential diagnosis. mainstay of treatment is complete surgical excision, with evolving role of adjuvant radiotherapy.   posters abstract 18 free neuropathol 4:9:37 paediatric high-grade glioma’ in association with constitutional mismatch repair deficiency syndrome (cmmrd) shalini suman1, sumanta das1, kavneet kaur1, shweta kedia2, m c sharma3, vaishali suri3 department of pathology, all india institute of medical sciences, new delhi department of neurosurgery, all india institute of medical sciences, new delhi department of neuropathology lab (nsc), all india institute of medical sciences, new delhi background: paediatric high-grade gliomas (phggs), h3wildtype, idhwildtype are aggressive tumors considered as who grade 4. a small proportion of phggs are reported to be associated with cancer predisposition syndromes like li fraumeni syndrome, cmmrd and neurofbromatosis type 1 (nf1). glioblastoma arising in setting of cmmrd has unfavorable prognosis. case report: a 2.5-year-old female born of consanguineous marriage, presented with left parieto-occipital lobe sol with t2weighted mri suggestive of an enhancing tumor. the histomorphological, and immunohistochemical analysis showed features of paediatric type high-grade glioma with mib-1 labelling index of 35-40%, absence of idh1 r132h mutation, loss of atrx expression, p53 mutation, h3 wildtype, (absence of h3 k27 mutation; loss of nuclear expression of h3 k27 me3; absence of h3 g34 r and h3 g34 v mutation), loss of alk and n-myc expression. further immunohistochemistry for mismatch repair genes demonstrated loss of mlh1 and pms2 expression in tumour and vascular endothelial cells while msh2 and msh6 expression were retained. based on clinical, morphological and immunohistochemistry findings a final diagnosis of constitutional mismatch deficiency syndrome was rendered. conclusion: early diagnosis can guide genetic testing, family screening and surveillance, as well as directly impact treatment decisions like avoiding temozolomide and using pd-1 immune checkpoint inhibitors at progression.   posters abstract 19 free neuropathol 4:9:38 dumbell spinal tumor with ini-1 loss in an infant a potential diagnostic pitfall anjali chimnani 1, neha bhardwaj2, kirti gupta2, amita trehan3, prashant chhabra3, gargi kapatia4 departments of pathology, postgraduate institute of medical education and research, chandigarh departments of histopathology, postgraduate institute of medical education and research, chandigarh departments of pediatrics, postgraduate institute of medical education and research, chandigarh department of pathology, all india institute of medical sciences, bathinda, india background: atypical teratoid/rhabdoid tumour (at/rt) is a high-grade malignant neoplasm (cns who grade 4) composed of variable numbers of poorly differentiated and rhabdoid cells. spinal at/rt are extremely rare, belong to at/rt-myc molecular subgroup and carry a dismal prognosis. these are characterized by biallelic inactivation of smarcb1 corresponding to loss of nuclear expression of ini-1. at this location, while other small blue round cell tumors are commoner differentials, poorly differentiated chordoma is another rare possibility, which needs exclusion with appropriate immunohistochemistry. case report: a 11-month-old boy presented with quadriparesis. magnetic resonance imaging (mri) of the spine showed an intradural extramedullary dumbell tumor in the cervical region extending from c3-c5 causing cord compression. a radiological diagnosis of neurofibroma was kept. mri brain and ultrasonography abdomen revealed normal study. biopsy from the cervical mass revealed a tumor arranged in cords and singly scattered cells in a basophilic mucopolysaccharide-rich matrix. the tumor cells had eccentric nuclei with abundant homogenously eosinophilic cytoplasm giving a rhabdoid appearance. few cells showed cytoplasmic vacuolations. mitoses were frequent. immunohistochemistry performed showed loss of ini-1 expression along with negativity for synaptophysin, nkx2.2, desmin, wt1 and satb2 excluding neuroblastoma, ewing sarcoma, rhabdomyosarcoma, wilms tumor and osteosarcoma, respectively. poorly differentiated chordoma was also considered, as it features ini-1 loss similar to at/rt; however, immunonegativity for brachyury excluded the former. conclusion: at/rt demonstrates broad spectrum of morphology, and a panel of immunohistochemistry is mandated for an accurate diagnosis.   e-posters   e-posters abstract 1 free neuropathol 4:9:39 braf and kras mutations analysis in rosai-dorfman disease involving central nervous system: an observational study mayur parkhi1, debajyoti chatterjee1, dharambir kashyap1, ashish aggarwal2, bishan radotra1 department of histopathology, post graduate institute of medical education and research, chandigarh, india department of neurosurgery, post graduate institute of medical education and research, chandigarh, india background: rosai-dorfman disease (rdd) is characterized by clonal proliferation of s-100 positive histiocytes, variable emperipolesis, and commonly affecting cervical lymph nodes. the central nervous system (cns) involvement is extremely rare. objectives: we attempted to evaluate the cyclin d1 expression and frequency of kras and braf mutations in the rdd involving the cns. material and methods: all patients with histopathologically diagnosed rdd involving cns were recruited from year 2011 to 2022. all cases were subjected to immunohistochemistry for cd68, cd163, s100, cd1a, gfap, cd207, ema, igg4, and cyclind1. the real-time polymerase chain reaction (rt-pcr) for hotspot mutation analysis of kras (exons 2, 3 and 4) and braf (v600e) was done on formalin-fixed paraffin-embedded tissue using commercial kit (entrogen). results: a total of seven cases were included. the median age was 31 years with 6 male and 1 female. it showed spinal cord (n=4) and intracranial (n=3) involvement. histologically, all cases showed histiocyte rich inflammation with evidence of emperipolesis. these histiocytes are positive for s100, cd68, cd163, and cyclin d1, whereas negative for cd1a, cd207 and ema. none of the control cases with histiocytic infiltrate (5 cases each of demyelination and infarct) showed cyclind1 expression. four cases showed increase in igg4 positive plasma cells (>10/ hpf). braf v600e mutation was detected in one case (14.28%), while none showed kras mutation. conclusion: rdd involving cns is an extremely rare and diagnostically challenging. nuclear cyclin d1 expression is a strong diagnostic clue. braf and kras mutations are rare in cns rdd.   e-posters abstract 2 free neuropathol 4:9:40 role of inflammation in muscle atrophy of limb girdle muscular dystrophy 2a/r1 (lgmd 2a/r1) sukanya banerjee1, bishan dass radotra2, manni luthra-guptasarma3, manoj k goyal4 department of pathology, university of pittsburgh, pennsylvania, usa department of histopathology, pgimer, chandigarh, india department of immunopathology, pgimer, chandigarh, india department of neurology, pgimer, chandigarh, india background: muscle atrophy is one of the major characteristic features of limb girdle muscular dystrophy 2a/r1 (lgmd 2a/r1) patients. the ubiquitin proteasome system (ups) is known to play an important role in muscle protein degradation. inflammation is also observed in these patients, which may further activate the ups. objectives: the study aimed to explore the role of inflammation in muscle atrophy of lgmdr1 patients. materials and methods: expression of atrophy markers, murf-1 and atrogin-1 was analyzed in muscle biopsies of fifteen lgmdr1 patients by qrt-pcr and western blotting. expression of cytokines, tnf-α, il-1β, and il-6 was analyzed by qrt-pcr from muscle biopsies, and by elisa from serum samples. expression of nfkb, foxo1 and foxo3 genes was analyzed by qrt-pcr and western blotting from muscle biopsies. the expression of genes and proteins of the ups pathway under inflammatory conditions was studied in calpain-3 silenced c2c12 cell line (using calpain-3 sirna transfection) and further confirmed using a proteasome inhibitor (mg132). results: the present study showed significantly increased level of pro-inflammatory cytokine tnf-α in lgmdr1 patients, which activated the ups pathway by two transcription factors, i.e., nfkb and foxo. increased and decreased phosphorylation of nfkb and foxo respectively, resulted in activation of atrogin-1 and murf-1 proteins. the calpain-3 silencing of myoblasts simulated the patient data, and treatment with mg132 showed reversal of catabolic signaling of tnf-α by targeting the ups pathway mediators. conclusion: the study suggested that mg132 may prevent degradation of muscle protein breakdown by inhibiting the proteins related to the ups pathway.   e-posters abstract 3 free neuropathol 4:9:41 gene expression signature of meningioma identifies potential prognostic biomarker for tumor progression aanchal verma1, kirti gupta1, pravin salunke2, prateek bhatia3, bd radotra1 and renu madan4 department of histopathology, post graduate institute of medical education and research, chandigarh department of neurosurgery, post graduate institute of medical education and research, chandigarh department of pediatrics (hematology-oncology division), post graduate institute of medical education and research, chandigarh department of radiotherapy, post graduate institute of medical education and research, chandigarh background: meningioma are common intracranial tumors categorized into three who grades i, ii and iii based on histological characteristics. these who grades fail to reliably predict their biological behavior of frequent recurrences & progression. recently, immune checkpoint inhibitor, pd-l1 and nf-κb pathway have been implicated in the progression and recurrence of meningioma. also, pd-l1 expression is associated with up-regulation of genes of nf-kb pathway. objectives: we aimed to evaluate the expression of pd-l1 and pathways associated with it and genes related to tumor progression in 3 grades of meningioma. material and methods: transcriptomic sequencing was performed in 6 meningiomas (2 of each who grade). differential expression was assessed using deseq2 to shortlist differentially expressed genes. gene set enrichment analysis (gsea) was performed to identify their association with various pathways. results: transcriptomic profiling revealed gene expression signatures, which are distinct to each who grade. cd274 (pd-l1) immune check-point inhibitor, nfkb2 and relb genes belonging to nf-kb pathway were found to be significantly upregulated in grade ii and iii compared with grade i meningioma. while dsc2 and dsc3 genes belongs to cell-cell adhesion pathway were significantly downregulated in higher grades (ii and iii) compared to grade i meningioma. conclusions: genes related to pdl1 and nf-κb pathway are found to be upregulated while those of cell-cell adhesion pathway are downregulated in higher grades of meningioma, which can be used as potential therapeutic target to check tumor progression and recurrence.   e-posters abstract 4 free neuropathol 4:9:42 detection of braf fusion expression patterns in pilocytic astrocytoma of the paediatric age group jonali das1, mathew ss1, a. sharmila1, pai r1, john j3, p krishna2, chacko a.g2, v. rajshekhar2, joseph b.v2, moorthy r.k2, nancy r1 and chacko g1 department of pathology, christian medical college, vellore department of neurological sciences, christian medical college, vellore departments of community medicine, christian medical college, vellore background: pilocytic astrocytomas (pa) are among the most common primary tumors found in children and adolescents. majority (>70%) of pa cases demonstrate the presence of an oncogenic tandem duplication at 7q34. this duplication results in a kiaa1549::braf fusion gene with constitutive braf kinase activity and mapk pathway activation. the second most the braf v600e mutation. objectives: to determine the frequency of kiaa1549-braf fusions in posterior fossa pilocytic astrocytomas of the pediatric age group. material and methods: formalin-fixed paraffin-embedded tissues of 60 consecutive pilocytic astrocytomas (pas) from the posterior cranial fossa were evaluated for kiaa1549::braf fusion transcripts (kiaa1549::braf 16-9, kiaa1549::braf 15-9, and kiaa1549::braf 16-11) by reverse transcriptase polymerase chain reaction and 38 pas were evaluated for brafv600e. the sample was amplified by ddpcr using the qx100 ddpcr system (biorad, usa). results and conclusions: twenty two (22/60, 36.6%) cases showed fusion transcripts of which twenty (33.3%) showed kiaa1549-braf 16-9 transcripts and 2 (3.3%) the kiaa 1549::braf 15-9 fusion transcript. kiaa1549::braf 16-11 fusions were not detected in any of the cases. all the kiaa1549::braf fusion negative cases (38) were tested for brafv600e mutation via ddpcr and only 1 (1.6%) case was positive for braf v600e mutation. the frequency of molecular alterations (38%) seen in this cohort was lower than that reported in other parts of the world (60-70%), emphasizing the need to expand the panel of testing to make it more comprehensive. finally, the data will be more meaningful when these findings are correlated to outcome.   e-posters abstract 5 free neuropathol 4:9:43 comparative differential gene analysis in supratentorial ependymoma aastha saraswati1, kirti gupta1, prateek bhatia2, pravin salunke3 and bd radotra1 department of histopathology, post graduate institute of medical education and research, chandigarh department of pediatric (onco division), post graduate institute of medical education and research, chandigarh department of neurosurgery, post graduate institute of medical education and research, chandigarh background: supratentorial ependymoma (st-epn) is segregated into two molecular subgroups denoted as st-epn-rela and st-epn-yap1; the former subgroup accounts for 70% of st-epn. while activation of nf-kb pathway through c11orf95-rela fusion is known to drive the oncogenesis of rela fusion epn, not much is known about rela fusion negative epn. objective: transcriptomic profiling of fusion positive and negative epn for comparative analysis of gene expression and their association with cellular pathways. material and methods: transcriptomic sequencing was performed on 5 cases of st-epn negative, 3 cases of st-epn positive and 2 normal brain as control using ion ampliseq plateform. differential expression analysis between sample groups (positive and negative epn) was performed using module from bioconductor from r package. further analysis was carried out using kyoto encyclopedia of genes and genomes (kegg) pathways and gene ontology (go). results: transcriptomic sequencing identified 2366 significant genes that are abundantly expressed in st epn. genes (nfkbil1, nfkb2, il1b, ptgs, tnfrsf1b) of nf-κb and tnf signalling pathways were highly expressed in st positive and downreguated in st negative whereas genes (foxj1, pard6b, fgf19) of hippo signalling pathway and pathways of neurodegeneration were upregulated in st negative and downregulated in st positive. these genes play an important role in cell proliferation and distinguishing between st positive and negative epns. conclusion: our results revealed interim oral heterogeneity of epn seen on gene expression level. the prognostic significance and further validation of these gene expression signatures needs to be determined on in-vivo models.   e-posters abstract 6 free neuropathol 4:9:44 surgical spectrum of parasitic infections of nervous system an audit from neuropathology archives ranjitha nair1, aditi goyal1, abhishek chowdhury1, nandeesh bn1, shilpa rao1, yasha tc1, mahadevan a1 department of neuropathology, nimhans, bangalore background: parasitic infections of the nervous system can be caused by a wide array of organisms from protozoans to metazoa. they may affect both children and adults, and are more prevalent in immunosuppressed individuals. spectrum of parasitic infections involving the central and peripheral nervous system as well as neuromuscular system is not widely described, with no reports from india. objectives: to determine the spectrum of parasitic infections in the neuromuscular and central nervous system. material and methods: all cases histopathologically diagnosed as parasitic infections between 2017 to 2022 were reviewed. the clinical, demographic and neuropathological details were retrieved from records. results: over the past 6 years, parasitic infections were reported in 96/42,913(0.2%) of patients [age range: 6-61 yrs; m:f=47:49]. neurocysticercosis (ncc) was most frequent (53 cases, lobar-23, intraventricular-21, spinal-1) followed by cerebral toxoplasmosis (22 cases, multifocal lobar; hiv seropositivity status available in 68%), hydatidosis (14 cases; lobar-6, bone-1, spinal-5). less frequent were acanthamoeba meningoencephalitis (3 cases, 18-49 years; all lobar), toxocara (1 case, 64 yr/f, spinal), sparganosis (1 case, 40yr/m, multifocal lobar). in skeletal muscle, one case each of ncc, microsporidiosis in a hiv positive (40yr/f), and trichinosis (34yr/m) was diagnosed. conclusions: the most common infection amongst surgical biopsies was neurocysticercosis followed by toxoplasmosis. this spectrum pertains to surgical biopsies and does not reflect community prevalence. high index of clinical suspicion in appropriate clinical settings is essential for early diagnosis and optimum patient management.   e-posters abstract 7 free neuropathol 4:9:45 o6 methylguanine-dna methyltransferase (mgmt) promoter methylation in adult type diffuse high grade gliomas, cns who grade 4 by droplet digital pcr ranjani jayachandran1, abarna r1, rekha pai1, rajesh balakrishan2, grace rebecca3, baylis vivek joseph4, ranjith k moorthy4, krishna prabhu4, ari g chacko4, geeta chacko1 department of pathology, christian medical college, vellore department of radiotherapy, christian medical college, vellore department of statistics, christian medical college, vellore department of neurosurgery, christian medical college, vellore background alkylating chemotherapy for adult type diffuse high-grade gliomas, cns who grade 4 with epigenetic silencing of mgmt gene has been associated with a better prognosis. we designed a droplet digital pcr (ddpcr) assay, to determine mgmt promoter methylation and determined its utility in terms of ease and cost of analysis in comparison to methylation-specific pcr (ms pcr). objectives: to determine the o6-methylguanine-dna methyltransferase (mgmt) promoter methylation in adult type diffuse high grade gliomas using ddpcr. to ascertain if cg16 dinucleotide methylation can be a stand-alone marker for mgmt promoter methylation. to determine the ease of performance and cost in comparison with ms pcr. material and methods: to determine the o6-methylguanine-dna methyltransferase (mgmt) promoter methylation in adult type diffuse high grade gliomas using ddpcr. to ascertain if cg16 dinucleotide methylation can be a stand-alone marker for mgmt promoter methylation. to determine the ease of performance and cost in comparison with ms pcr. results: when compared with ms-pcr, mgmt gene promoter methylation by ddpcr had a sensitivity of 88.4% and a specificity of 81.3% and cg16 dinucleotide methylation by ddpcr had a sensitivity of 90.7% and specificity of 62.5%. discrepant cases were sequenced by bisulphite sequencing and sensitivity and specificity were reassessed. the cost per test for mgmt promoter methylation by ms-pcr was much higher than ddpcr. conclusion: although ddpcr and mspcr had equal sensitivity for detection of mgmt promoter methylation, ddpcr had a higher specificity. droplet digital pcr being an easier and cost-effective testing platform could replace the existing standard ms-pcr. cg16 dinucleotide methylation by ddpcr had a low specificity and hence cannot be used as a stand-alone marker to ascertain mgmt promoter methylation status.   copyright: © 2023 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. applying machine learning to assist in the morphometric assessment of brain arteriolosclerosis through automation feel free to add comments by clicking these icons on the sidebar free neuropathology 6:12 (2025) original paper applying machine learning to assist in the morphometric assessment of brain arteriolosclerosis through automation jerry j. lou1, peter chang2, kiana d. nava3, chanon chantaduly2, hsin-pei wang3, william h. yong1, viharkumar patel3, ajinkya j. chaudhari4, la rissa vasquez3, edwin monuki1, elizabeth head1, harry v. vinters5, shino magaki6, danielle j. harvey7, chen-nee chuah4, charles s. decarli8, christopher k. williams6, michael keiser9, brittany n. dugger3 department of pathology and laboratory medicine, school of medicine, university of california irvine, irvine, usa department of radiological sciences, center for artificial intelligence in diagnostic medicine, school of medicine, university of california irvine, orange, usa department of pathology and laboratory medicine, school of medicine, university of california davis, sacramento, usa department of electrical and computer engineering, university of california davis, davis, usa department of pathology and laboratory medicine and department of neurology, david geffen school of medicine, university of california los angeles, los angeles, usa department of pathology and laboratory medicine, david geffen school of medicine, university of california los angeles, los angeles, usa department of public health sciences, school of medicine, university of california davis, davis, usa department of neurology, school of medicine, university of california davis, sacramento, usa department of pharmaceutical chemistry, department of bioengineering and therapeutic sciences, institute for neurodegenerative diseases; bakar computational health sciences institute, university of california san francisco, san francisco, usa corresponding authors: jerry j. lou · department of pathology and laboratory medicine · school of medicine, university of california irvine · d440 medical sciences i, ca 92697-4800 · irvine · usa jerry.jierui.lou@gmail.com brittany n. dugger · department of pathology and laboratory medicine · school of medicine, university of california davis · 4400 v street, pathology building, ca 95817 · sacramento · usa bndugger@ucdavis.edu additional resources and electronic supplementary material: supplementary material submitted: 18 march 2025 accepted: 15 may 2025 copyedited by: cinthya agüero published: 02 june 2025 https://doi.org/10.17879/freeneuropathology-2025-6387 keywords: machine learning, artificial intelligence, neuropathology, arteriolosclerosis, blood vessel, morphometry abstract objective quantification of brain arteriolosclerosis remains an area of ongoing refinement in neuropathology, with current methods primarily utilizing semi-quantitative scales completed through manual histological examination. these approaches offer modest inter-rater reliability and do not provide precise quantitative metrics. to address this gap, we present a prototype end-to-end machine learning (ml)-based algorithm, arteriolosclerosis segmentation (artseg), followed by vascular morphometry (vasmorph) – to assist persons in the morphometric analysis of arteriolosclerotic vessels on whole slide images (wsis). we digitized hematoxylin and eosin-stained glass slides (13 participants, total 42 wsis) of human brain frontal or occipital lobe cortical and/or periventricular white matter collected from three brain banks (university of california, davis, irvine, and los angeles alzheimer’s disease research centers). artseg comprises three ml models for blood vessel detection, arteriolosclerosis classification, and segmentation of arteriolosclerotic vessel walls and lumens. for blood vessel detection, artseg achieved area under the receiver operating characteristic curve (auc-roc) values of 0.79 (internal hold-out testing) and 0.77 (external testing), dice scores of 0.56 (internal hold-out) and 0.74 (external), and hausdorff distances of 2.53 (internal hold-out) and 2.15 (external). arteriolosclerosis classification demonstrated accuracies of 0.94 (mean, 3-fold cross-validation), 0.86 (internal hold-out), and 0.77 (external), alongside auc-roc values of 0.69 (mean, 3-fold cross-validation), 0.87 (internal hold-out), and 0.83 (external). for arteriolosclerotic vessel segmentation, artseg yielded dice scores of 0.68 (mean, 3-fold cross-validation), 0.73 (internal hold-out), and 0.71 (external); hausdorff distances of 7.63 (mean, 3-fold cross-validation), 6.93 (internal hold-out), and 7.80 (external); and auc-roc values of 0.90 (mean, 3-fold cross-validation), 0.92 (internal hold-out), and 0.87 (external). vasmorph successfully derived sclerotic indices, vessel wall thicknesses, and vessel wall to lumen area ratios from artseg-segmented vessels, producing results comparable to expert assessment. this integrated approach shows promise as an assistive tool to enhance current neuropathological evaluation of brain arteriolosclerosis, offering potential for improved inter-rater reliability and quantification. introduction the study of vascular morphology pertains to several neurological disorders, including dementia, stroke, and cerebral vasculopathies. vascular contributions to cognitive impairment and dementia have emerged from decades of study, including the honolulu asia aging study,1 the rotterdam study,2 and the religious orders study and memory and aging project.3,4 vascular abnormalities can encompass numerous entities, including brain arteriolosclerosis. arteriolosclerosis can be associated with an increased likelihood of microinfarcts5 and subsequently dementia, including alzheimer disease (ad).6,7 currently, the guidelines used in vascular cognitive impairment neuropathology guidelines (vcing)8 and the national alzheimer’s coordinating center neuropathology data set9 for assessing the severity of vascular pathology such as brain arteriolosclerosis (b-asc) consists of a semi-quantitative scale that divides vascular pathologic change categorically into “none”, “mild”, “moderate”, and “severe”.10 according to vcing, occipital cortex white matter may be the optimal brain region for brain arteriolosclerosis assessment in terms of reliability and association with cognitive status.8 the semi-quantitative b-asc scale showed “moderate” inter-rater reliability (gwet’s ac2 coefficient 0.52) in the vcing study.8 alternative methods have been proposed for the analysis of arteriolosclerosis, including quantitative assessments such as the sclerotic index [1-(internal diameter / external diameter)].11,12 however, implementing these methods on a large scale may not be feasible. scalable means for deep phenotyping of vascular pathology to assess morphological features such as vessel shape, wall thickness, and/or degree of hyaline change are needed. the demand for reproducible and high-resolution quantitative metrics motivates the development of robust computational methods to segment vascular structures. machine learning (ml) is a promising computational paradigm capable of delivering expert-level performance in complex visual recognition tasks, including the classification of amyloid plaques,13 neurofibrillary tangles,14 and glial tauopathies.15 additional work has correlated machine learning generated quantitative findings with clinical, demographic, and pathological metrics.16 these ml models trained on whole slide image (wsi) datasets recognize features and patterns specific to target objects and demonstrate remarkable adaptability to variations generated by disparate institutional procedures in the production of wsis. in addition to classification, ml models can perform semantic segmentation by which a class label is assigned to each pixel in an image, generating collections of pixels called segmentation masks that form distinct objects within an image. these masks may be used for downstream analysis of the shape, diameter, and/or other features of the segmented object. generally, classification requires recognition of only a minimum number of object-specific features to categorize images into the correct class, whereas segmentation demands recognition of all object-specific features to classify all pixels belonging to the target. non-ml methods for segmentation and quantification of blood vessels have been explored, however, these methods were not extensively evaluated and compared to expert annotations.17 prior works have shown that ml models can segment non-arteriolosclerotic blood vessels in neoplastic tissue.18–22 we build upon these prior efforts and hypothesize that ml models could (1) detect and localize blood vessels in brain tissue, (2) classify arteriolosclerosis, (3) segment the walls and lumens of arteriolosclerotic vessels, and (4) facilitate morphometric analysis of vascular structure. as object occlusion has been shown to reduce model performance,23 we additionally hypothesized centering image patches onto the object of interest, the blood vessel in this study, mitigates occlusion and improves downstream segmentation performance. in this pilot study, we present a prototype end-to-end ml-based algorithm – arteriolosclerosis segmentation (artseg), followed by vascular morphometry (vasmorph) – to assist persons in the morphometric analysis of arteriolosclerotic vessels on wsis. while previous software for manual measurement of sclerotic index on digital histology images (vascalc)24 has been described, vasmorph represents an automated method to measure sclerotic index, vessel wall thickness, and vessel wall to lumen area ratio from artseg-segmented vessels. furthermore, we describe a novel custom recursive wrapper algorithm – object of interest recursive centering algorithm (orca) – that can flexibly interface with any segmentation ml model to recursively generate patches centered on an object of interest. we evaluated three ml models within artseg that show promising performance for the detection of blood vessels, recognition of arteriolosclerosis, and segmentation of arteriolosclerotic vessel walls and lumens. to aid in reproducibility and open science, we provide our code, training, and testing data, and image processing methodology (see data availability and code availability). to the best of our knowledge, this study constitutes the first demonstration of an open-source means for ml-based morphometric analysis of arteriolosclerotic blood vessels in digital histology images of human brain. methods participant consent and ethics compliance our investigation used de-identified human post-mortem tissues, which do not qualify as human subjects under federal law (45 cfr 46, protection of human subjects). the university of california, davis (ucd) alzheimer’s disease research center (adrc), university of california irvine (uci) adrc, and the former university of california los angeles (ucla) adrc programs obtained signed informed consent from all participants or legal representatives during the life of the participant. procedures were completed in accordance with the ethical standards of the helsinki declaration. operations of the ucd, uci, and former ucla adrc were approved by the institutional review board (irb) of ucd, uci, and ucla, respectively. all data were de-identified and shared through a randomly generated pseudo-identification number. the de-identified data does not contain personal health information such as addresses, phone numbers, names, date of birth, or social security numbers. participant selection and case cohort all brain samples were retrieved from archives of ucd adrc, uci adrc, and the former ucla adrc. thirteen participants and 42 wsis were included through three stages of selection. in the first stage, we chose 1 participant with mild arteriolosclerosis (global score) and 1 with severe arteriolosclerosis (global score) from uci adrc, each of whom had frontal and occipital lobe h&e-stained slides. we also chose 2 participants with mild arteriolosclerosis (global score) and 2 with severe arteriolosclerosis (global score) from ucd adrc, each of whom had frontal, frontal-periventricular white matter, occipital, and occipital-periventricular white matter h&e-stained slides. in the second stage, we added 6 additional participants from ucd adrc (2 with no arteriosclerosis, 1 with mild, 2 with moderate, and 1 with severe arteriosclerosis, global score) to expand the distribution of arteriosclerosis and increase the sample size. in the third stage, we added 1 participant from the former ucla adrc to create an external test set. cortical and subcortical (periventricular white matter) regions were chosen due to implications in possible vascular contributions to cognitive impairment and dementia.7 samples studied consisted of 5–7 μm formalin-fixed paraffin-embedded sections of the frontal and occipital lobes mounted on glass slides and stained with hematoxylin and eosin (figure 1). ucd adrc digitized slides with a zeiss axioscan scanning at 0.220 μm/pixel and x20 magnification. the former ucla adrc utilized an aperio cs2 with the same parameters as ucd adrc. uci adrc digitized slides with an aperio versa 200 scanner at 0.137 μm/pixel with a 40x objective. in total, our study included 8 male and 5 female participants from 52 to 89+ years of age (mean 81, median 86). see supplementary table 1 for detailed specifications and participant demographics of each wsi utilized and supplementary figure 1 for example wsis from respective brain regions. figure 1: overview of the process for sampling and digitization of slides. samples were retrieved from the cortical and/or periventricular white matter regions of the frontal and occipital lobes from 13 participants. these samples were formalin-fixed paraffin-embedded and processed into glass slides, which subsequently were digitized into wsis by a slide scanner. inclusion criteria for the selection of vessels only vessels (1) within white matter and (2) cut in cross-section with the entire circumference of the vessel wall within view were included for training and evaluation of the ml algorithm. because arteriolosclerosis is most common in cortical white matter, to optimize workflow, we chose to focus on only blood vessels located within white matter. we included the occipital lobe because the occipital cortex white matter may be the optimal brain region for brain arteriolosclerosis assessment in terms of reliability and association with cognitive status, according to vcing,8 cross-sectional views of vessels were selected as the purpose of the ml algorithm is to automate the calculation of the sclerotic index,11,12 vessel wall thickness, and vessel wall to lumen area ratio, which require measurements of the internal and external radius or diameter. dataset all uci and ucla wsis were stored in svs file format. all ucd wsis were stored in czi file format. due to the enormous gigapixel size of wsis, it is generally computationally inefficient or intractable to input the entire wsi directly into ml algorithms for training or inference, and wsis are typically divided into thousands of smaller image tiles. we performed wsi tiling utilizing the open-source libraries openslide25 and czifile,26 generating (512 x 512) pixel tiles. for arteriolosclerosis classification and segmentation, we divided our dataset (11 participants, 34 wsis) into three subsets: a training and validation subset, an internal hold-out test set, and an external test set (supplementary table 2). the training and validation set consisted of 28 wsis from 8 participants (26 wsis from 7 ucd participants and 2 wsis from 1 uci participant). we performed a 3-fold cross-validation procedure with on average 19 training and 9 validation wsis (supplementary tables 3 and 4) and reported the mean performance metrics of the three folds. the internal hold-out testing subset consisted of 4 wsis from 2 participants (2 wsis from 1 ucd participant and 2 wsis from 1 uci participant; supplementary table 5). the external testing subset consisted of 2 wsis from 1 ucla participant (supplementary table 5). for vessel detection, the training and validation subsets had an additional 8 wsis from an additional 2 ucd participants (supplementary table 6), who did not have arteriolosclerosis and therefore were not included in the dataset for arteriolosclerosis classification and segmentation. human annotations all annotators (vp, wy, jjl, hpw, kn) were blinded to wsi specifications and participant clinical information during annotation. vp and wy are attending neuropathologists; jjl is a neuropathology fellow; hpw was a junior specialist; and kn was an undergraduate student. all annotations for the training and validation subsets, as well as the internal hold-out test subset, were completed by jjl, hpw, and/or kn. all annotations for the external test subset were completed by vp. arteriolosclerosis was defined using criteria proposed by skrobot et al.8,10 segmentation masks for vessel walls and lumens were created by jjl, hpw, kn, and vp using imagej27 (detailed annotation protocol available in supplementary document 1). wy annotated vessel wall thicknesses (see section vascular morphometry or vasmorph) using imagej27 (detailed annotation protocol available in supplementary document 2). data augmentation for all training tiles, color augmentation developed by tellez et al.28 to simulate the spectrum of hues generated by different staining methods was performed through the open-source library histomicstk.29 tensorflow’s keras application programming interface30 produced morphological augmentations including random flip, random translation, and random rotation. gray scale augmentation through the open-source library opencv31 assisted further in reducing neural network dependence on color. for datasets with class imbalance, minority class oversampling was performed. the optimized color-to-gray augmentation ratio for arteriolosclerotic vessel segmentation was 5:1. for arteriolosclerosis classification, the optimized ratio was 2:1. overview of end-to-end machine learning-based pipeline our end-to-end ml-based pipeline consists of an ml component (phase 1) and a post-ml quantification module (phase 2). the ml component, arteriolosclerosis segmentation (artseg) receives wsis as input and outputs segmentations of arteriolosclerotic blood vessel walls and lumens, which are in turn input into the post-ml module vascular morphometry (vasmorph), which outputs quantitative metrics for the sclerotic index, vessel wall thickness, and vessel wall to lumen area ratio. phase 1: arteriolosclerosis segmentation (artseg) artseg comprises four algorithms that complete four sequential steps (figure 2). after wsi tiling, the first step (phase 1a) is to detect blood vessels and keep tiles that contain a blood vessel and discard those that do not. the second step (phase 1b) is to recursively shift tiles until the detected blood vessel appears at the center of the tile. the third step (phase 1c) is to keep tiles that contain a blood vessel with arteriolosclerosis and discard those that do not. the fourth step (phase 1d) is to segment the walls and lumens of blood vessels with arteriolosclerosis. figure 2: artseg overview.the ml pipeline received wsis of h&e-stained cortical and/or periventricular white matter brain tissue as input. each wsi was tiled into tens of thousands of (512 x 512) pixel image tiles. (phase 1a) the blood vessel detection ml model sorted tiles into those with blood vessels and those without. (phase 1b) object of interest: recursive centering algorithm (orca) (see figure 3) generated new tiles centered onto the detected blood vessels. (phase 1c) an arteriolosclerosis classification model separated tiles with centered blood vessels into those with arteriolosclerosis and those without. (phase 1d) a modified attention u-net segmented the arteriolosclerotic vessel walls and lumens to produce the final output. all models within artseg take advantage of fixed imagenet pretrained parameters from google’s efficientv2l to extract low-level features before learning vessel-specific features de novo. phase 1a: blood vessel detection the blood vessel detection neural network consisted of an attention u-net architecture32 with an encoder composed of an efficientnetv2l33 backbone with five semi-trainable convolution layers followed by two fully trainable convolution layers and a decoder composed of seven trainable convolution layers generated through the concatenation of a 2d transpose convolution of the prior layer and an attention gate32 that filters features propagated from the skip connections. to evaluate the classification performance of the vessel detection model for separating patches with blood vessels from those without, the segmentation output of the model was converted to confidence scores by: (1) obtaining the softmax probability per pixel for background (class = 0) and vessel (class = 1), (2) for each pixel where the softmax probability for the vessel class is greater than that for background, saving the vessel softmax probabilities into one set, and (3) calculating the mean of the set (supplementary figure 2). phase 1b: blood vessel centering blood vessel centering was achieved by a custom recursive algorithm – object of interest recursive centering algorithm (orca) – wrapping the blood vessel detection neural network (figure 3). the wrapper algorithm inputs raw (512 x 512) tiles into the blood vessel detection neural network, which segments blood vessels. subsequently, the wrapper algorithm generates a new (512 x 512) tile with shifted boundaries such that the detected blood vessel resides closer to the center of the tile. this process is repeated until the detected blood vessel lies in the center of the final output tile (figure 3). orca detects when the patch has been centered onto the blood vessel(s) by comparing the coordinates of the new patch with shifted boundaries to the original input patch; if the shift in boundaries is less than a preset threshold, then the patch is considered blood vessel(s) centered. the average runtime per wsi was approximately 37 minutes (see hardware section for further details). figure 3: object of interest recursive centering algorithm (orca). starting at step (1), the algorithm inputs a raw (512 x 512) image tile through the embedded blood vessel detection model, which produces an output segmentation (2). (3) orca creates a new patch from the input wsi using modified shifted coordinates based on the previous segmentation. (4–6) steps 1 through 3 are repeated until the vessel is centered. phase 1c: arteriolosclerosis classification the arteriolosclerosis classification neural network consisted of an efficientnetv2l33 backbone with five semi-trainable convolution layers topped by two fully trainable convolution layers, followed by three dense layers. phase 1d: arteriolosclerotic vessel segmentation the arteriolosclerotic vessel segmentation network used the same architecture as the model for blood vessel detection. implementation of ml models see supplementary figure 3 for detailed architecture and supplementary document 3 for detailed training hyperparameters of ml models in phases 1a, 1b, 1c, and 1d. phase 2: vascular morphometry (vasmorph) for each blood vessel analyzed, vasmorph outputs the sclerotic index, vessel wall thickness, and vessel wall to lumen area ratio, which have previously been used as an indicator of the degree of vascular stenosis.17 the final sclerotic index and vessel wall thickness outputs include the median, mean, standard deviation, minimum, and maximum of sclerotic indices and vessel wall thicknesses calculated in a 360-degree rotation around the center of the blood vessel lumen. sclerotic index to calculate the vessel sclerotic index, we first defined the sclerotic index at an angle theta (siθ), given the internal diameter (di(θ)) and external diameter (de(θ)) of the vessel at that angle, using the following formula: siθ = 1 – (di(θ) / de(θ)).11,12,24 vasmorph then measures the internal diameter (di(θ)) and external diameter (de(θ)) of the vessel to calculate the sclerotic index (siθ) at each degree angle over a 180-degree rotation in a half circle around the centroid34 of the lumen segmentation to obtain a set of sclerotic indices (figure 4). the output of vasmorph is the median, mean, standard deviation, minimum, and maximum of this set of vessel sclerotic indices. figure 4: sclerotic index calculation. an image patch (a) centered onto an arteriolosclerotic blood vessel is input into the segmentation model of artseg, which outputs the vessel wall and lumen segmentation (b). vasmorph measures the internal diameter (di(θ)) and external diameter (de(θ)) of the vessel to calculate the sclerotic index (siθ) at each degree angle over a 180-degree rotation in a half circle around the centroid34 of the lumen segmentation to obtain a set of sclerotic indices (b and c). the output of vasmorph is the median, mean, standard deviation, minimum, and maximum of this set of vessel wall thicknesses. vasmorph finds the center of the lumen segmentation. vessel wall thickness: tangent line-based method to calculate the vessel wall thickness, we first defined vessel wall thickness at point alpha (tα) on the curve lumen contour as the distance between a line tangent to point alpha (lα) and the external contour of the vessel (e): tα = e – lα (figure 5). vasmorph then measures the vessel wall thickness at 100 equally spaced points in the lumen contour to obtain a set of thicknesses (figure 5). the output of vasmorph is the median, mean, standard deviation, minimum, and maximum of this set of vessel wall thicknesses. figure 5: vessel wall thickness based on a line tangent to every point on the lumen contour. an image patch (a) centered onto an arteriolosclerotic blood vessel is input into the segmentation model of artseg, which outputs the vessel wall and lumen segmentation (b). vasmorph measures the vessel wall thickness at each point in the lumen contour to obtain a set of thicknesses, where thickness at point alpha (tα) is defined as the distance between a line tangent to point alpha (lα) on the curve of the lumen contour and the external contour of the vessel (e): tα = e – lα (b and c). vasmorph then outputs the median, mean, standard deviation, minimum, and maximum of this set of vessel wall thicknesses. vessel wall thickness: radii-based method we also considered an alternative definition of vessel wall thickness at angle theta (tθ) relative to the lumen center as the difference between the external radius at theta (re(θ)) and the internal radius at theta (ri(θ)): tθ = re(θ) – ri(θ) (figure 6). vasmorph then measures the internal radius (ri(θ)) and external radius (re(θ)) of the vessel to calculate the sclerotic index (siθ) at each degree angle over a full 360-degree rotation around the centroid34 of the lumen segmentation to obtain a set of thicknesses (figure 6). vasmorph outputs the median, mean, standard deviation, minimum, and maximum of this set of vessel wall thicknesses based on this alternative definition. figure 6: vessel wall thickness based on difference between internal and external radii. an image patch (a) centered onto an arteriolosclerotic blood vessel is input into the segmentation model of artseg, which outputs the vessel wall and lumen segmentation (b). vasmorph measures the internal radius (ri(θ)) and external radius (re(θ)) of the vessel to calculate the sclerotic index (siθ) at each degree angle over a full 360-degree rotation around the centroid34 of the lumen segmentation to obtain a set of thicknesses, where thickness at angle theta (tθ) relative to the lumen center is defined as the difference between the external radius at theta (re(θ)) and the internal radius at theta (ri(θ)): tθ = re(θ) – ri(θ) (b and c). vasmorph then outputs the median, mean, standard deviation, minimum, and maximum of this set of vessel wall thicknesses. vessel wall area to lumen area ratio we calculated the vessel wall area to lumen area ratio by counting the number of pixels in the vessel wall segmentation and the lumen segmentation, then converting from pixels to μm2 using the conversion factor 0.0484 μm2 / pixel, where the length and height of each pixel is 0.220 μm, and finally dividing to obtain the ratio. by coincidence, the 62 vessels that fulfilled inclusion criteria were derived from ucd wsis, which were scanned at 0.220 μm per pixel. the setting for the length and height of each pixel may be adjusted to match the wsi resolution. hardware all scripts and python 3 modules for this project were run on the compute environment provided by the center for artificial intelligence in diagnostic medicine at uci which includes a high-end cluster of 88 nvidia gpu hardware accelerators composed of a100 (80 gb x16), a40 (40 gb x8), rtx titan (24 gb x12), gtx titan (16 gb x4), and geforce rtx 2080 ti (11 gb x48) graphics cards. wsis and other data components were stored on a total of four cpu-optimized cluster nodes and three dedicated 0.24 pb file servers that are all interconnected on a high-speed 25 gbps local fiber optic network. results our internal hold-out test set contains wsis from the same institutions (uci and ucd) and annotators (jjl, hpw, and kn) as wsis used for model training and test internal deployment of artseg. the external test set contains wsis from a different institution (ucla) and annnotator (vp) from wsis used for model training and tests external deployment of artseg. hausdorff distance is converted from pixels to μm using a conversion factor of 0.220 μm / pixel (supplementary table 1). phase 1a: detection of blood vessels on wsis (artseg) the initial blood vessel detection model serves as a preprocessing step, providing coarse segmentation to localize and center patches containing blood vessels. the marginally lower dice score observed is consistent with the annotation quality at this stage and is adequate for the intended purpose of blood vessel detection. fine segmentation is achieved at a later stage (phase 1d) during the segmentation of arteriolosclerotic blood vessel walls and lumens. internal hold-out testing after converting the segmentation output of the model into classification confidence scores, the area under the receiver operating characteristic curve (auc-roc) of the model is 0.79 (figure 7). the vessel detection segmentation model achieved a dice score of 0.56 and a hausdorff distance of 0.56 μm on the hold-out test (table 1). external stress testing after converting the segmentation output of the model (obtained by running inferences on an external cohort) into classification confidence scores, the area under the receiver operating characteristic curve (auc-roc) of the model is 0.77 (figure 7). the vessel detection segmentation model achieved a dice score of 0.74 and a hausdorff distance of 0.47 μm on the external test (table 1). figure 7: roc curves of hold-out and external tests for the vessel detection. as expected, the model performs slightly worse for the external test. vessel detection model acts as a screening tool to quickly remove patches without vessels while retaining patches with vessels or objects suspicious of vessels. table 1: results of vessel detection segmentation internal hold-out and external testing dataset dice score hausdorff distance (µm) hold-out 0.56 0.56 external test 0.74 0.47 phase 1b: object of interest centering through recursive segmentation and boundary migration (orca) segmentation cnns such as u-net operate most effectively when the object of interest (oi) is fully visible and not cropped. the process of wsi tiling occurs irrespective of the positions of ois, frequently creating cropped ois that appear on the edge of the tile (figure 8). we hypothesized that cropping of blood vessels reduced segmentation performance and that centering tiles onto blood vessels would improve performance. to achieve oi centering, we designed a custom recursive algorithm – object of interest recursive centering algorithm (orca) – wrapping our blood vessel identification neural network. from 16 wsis, orca generated 401 tiles with arteriolosclerotic blood vessels and 7066 tiles with non-arteriolosclerotic blood vessels or without vessels. arteriolosclerosis was defined using criteria proposed by skrobot et al.8,10 visualization of tiles demonstrated centering onto blood vessels (figure 8). these vessel-centered tiles were utilized to train, validate, and test the arteriolosclerosis classification and arteriolosclerotic vessel segmentation ml models. figure 8: results of vessel centering by orca. (a) wsi tiling generates tiles that often contain blood vessel(s) at the tile edge, with some vessel cropped (arrow). (b) orca generates new tiles with the detected blood vessel(s) located in the center. phase 1c: arteriolosclerosis classification is feasible, albeit challenging (artseg) 3-fold cross validation for the classification of arteriolosclerosis, our model achieved a mean validation set performance of 0.94 accuracy and 0.68 auc-roc (table 2). table 2: results of arteriolosclerosis classification 3-fold cross-validation k specificity sensitivity (recall) precision accuracy f1 auc-roc 0 1.00 0.19 0.68 0.96 0.29 0.59 1 0.95 0.52 0.23 0.94 0.32 0.73 2 0.97 0.51 0.67 0.91 0.58 0.74 mean 0.97 0.37 0.47 0.94 0.38 0.68 internal hold-out testing our internal hold-out test set, which was not seen by the model during training, consisted of 4 cases from 2 participants, 1 each from ucd and uci, respectively, labeled by novice annotators jjl, hpw, and kn, who also labeled the training data set. for the classification of arteriolosclerosis, our model achieved an accuracy of 0.86 and an auc-roc of 0.87 (table 3). external stress testing our external stress test set, which was not seen by the model during training, consisted of 2 cases from 2 participants, both from ucla, labeled by expert annotator vp, who did not label the training data set. for the classification of arteriolosclerosis, our model achieved an accuracy of 0.77 and an auc-roc of 0.83 (table 3). table 3: results of arteriolosclerosis classification internal hold-out and external testing k specificity sensitivity (recall) precision accuracy f1 auc-roc hold-out 0.856 0.89 0.13 0.86 0.23 0.87 external test 0.752 0.92 0.26 0.77 0.41 0.83 phase 1d: attention u-net effectively segments the arteriolosclerotic vessel walls and lumen (artseg) 3-fold cross validation for the segmentation of vessel walls and lumens (figures 9–12), our model achieved a mean validation set performance of 0.68 dice score, 1.68 μm hausdorff distance, and 0.90 auc-roc (table 4). for the segmentation of vessel walls only, our model achieved validation set performance of 0.69 dice score, 1.41 μm hausdorff distance, and 0.86 auc-roc (table 5). for the segmentation of the vessel lumen only, our model achieved validation set performance of 0.63 dice score, 0.99 μm hausdorff distance, and 0.86 auc-roc (table 6). table 4: results of arteriolosclerotic vessel segmentation 3-fold cross-validation for vessel walls and lumens k dice score hausdorff distance (µm) 0 0.72 1.56 1 0.70 1.63 2 0.63 1.87 mean 0.68 1.68 table 5: results of arteriolosclerotic vessel segmentation 3-fold cross-validation for vessel walls only k dice score hausdorff distance (µm) 0 0.71 1.36 1 0.71 1.34 2 0.65 1.54 mean 0.69 1.41 table 6: results of arteriolosclerotic vessel segmentation 3-fold cross-validation for vessel lumen only k dice score hausdorff distance (µm) 0 0.72 0.97 1 0.63 0.95 2 0.54 1.06 mean 0.63 0.99 figure 9: example segmentation results for blood vessels with mild arteriolosclerosis as classified by a neuropathology fellow (jl) and annotated by non-experts (kn, hsw, jl). six example instances with input image, human annotation mask, model segmentation output, and an overlap image of input image and model segmentation are shown here. the blood vessels shown here were classified as having arteriolosclerosis by a human annotator. (a, b) example instances of good model performance. (c, d) example instances of intermediate model performance. (e, f) example instances of poor model performance. figure 10: example segmentation results for blood vessels with moderate arteriolosclerosis as classified by a neuropathology fellow (jl) and annotated by non-experts (kn, hsw, jl). six example instances with input image, human annotation mask, model segmentation output, and an overlap image of input image and model segmentation are shown here. the blood vessels shown here were classified as having arteriolosclerosis by a human annotator. (a, b) example instances of good model performance. (c, d) example instances of intermediate model performance. (e, f) example instances of poor model performance. figure 11: example segmentation results for blood vessels with severe arteriolosclerosis as classified by a neuropathology fellow (jl) and annotated by non-experts (kn, hsw, jl). six example instances with input image, human annotation mask, model segmentation output, and an overlap image of input image and model segmentation are shown here. the blood vessels shown here were classified as having arteriolosclerosis by a human annotator. (a, b) example instances of good model performance. (c, d) example instances of intermediate model performance. (e, f) example instances of poor model performance. figure 12: example segmentation results for challenging instances as classified by a neuropathology fellow (jl) and annotated by non-experts (kn, hsw, jl). six example instances with input image, human annotation mask, model segmentation output, and an overlap image of input image and model segmentation are shown here. the blood vessels shown here were classified as having arteriolosclerosis by a human annotator. (a) a corpora amylacea was mistaken for a vessel lumen by artseg. (b) artseg misidentified a non-arteriolosclerotic vessel as having arteriolosclerosis and a target for segmentation. (c) conversely, a vessel with arteriolosclerosis is misidentified as a non-arteriolosclerotic vessel and omitted for segmentation. (d) artseg fails to identify moderate hyaline vessel wall thickening and lumen stenosis. (e) image tiles with clusters of blood vessels present a particular challenge to artseg and vasmorph. (f) vessels within the leptomeninges are mistakenly segmented by artseg. internal hold-out testing our internal hold-out test set, which was not seen by the model during training, consisted of 4 cases from 2 participants, 1 each from ucd and uci, respectively, labeled by novice annotators jjl, hpw, and kn, who also labeled the training data set. for the segmentation of vessel walls and lumen, our model achieved a 0.73 dice score, 1.52 μm hausdorff distance, and 0.92 auc-roc (table 7). for the segmentation of vessel walls only, our model achieved a 0.73 dice score, 1.30 μm hausdorff distance, and 0.88 auc-roc (table 8). for the segmentation of the vessel lumen only, our model achieved a 0.71 dice score, 0.88 μm hausdorff distance, and 0.90 auc-roc (table 9). external stress testing our external stress test set, which was not seen by the model during training, consisted of 2 cases from 2 participants, both from ucla, labeled by expert annotator vp, who did not label the training data set. for the segmentation of vessel walls and lumen, our model achieved a 0.71 dice score, 1.72 μm hausdorff distance, and 0.87 auc-roc (table 7). for the segmentation of vessel walls only, our model achieved a 0.70 dice score, 1.36 hausdorff distance, and 0.83 auc-roc (table 8). for the segmentation of the vessel lumen only, our model achieved a 0.65 dice score, 1.03 μm hausdorff distance, and 0.80 auc-roc (table 9). table 7: results of arteriolosclerotic vessel segmentation internal hold-out and external testing for vessel walls and lumens dataset dice score hausdorff distance (µm) hold-out 0.73 1.52 external test 0.71 1.72 table 8: results of arteriolosclerotic vessel segmentation internal hold-out and external testing for vessel walls only dataset dice score hausdorff distance (µm) hold-out 0.73 1.30 external test 0.70 1.36 table 9: results of arteriolosclerotic vessel segmentation internal hold-out and external testing for vessels' lumens only dataset dice score hausdorff distance (µm) hold-out 0.71 0.88 external test 0.65 1.03 phase 2: vascular morphometry (vasmorph) sclerotic index calculation the mean and median of the sclerotic indices of 62 vessels in the test set calculated by vasmorph were 0.53 and 0.53, respectively. the standard deviation of the sclerotic indices was 0.05. the minimum was 0.43 and the maximum was 0.60. vessel wall thickness the vessel wall thickness metrics (mean, median, standard deviation, minimum, and maximum) for the manual measurements, tangent line-based method, and radii-based method are outlined in table 10. in this preliminary comparison of the two methods, the tangent line-based method appears to generate metrics with greater proximity to manual measurements. table 10: comparison of the tangent line-based method and the radii-based method to manual measurements vessel wall thickness metrics manual measurement tangent line-based method radii-based method mean 24.71 23.97 52.59 median 23.13 22.57 49.16 standard deviation 8.37 6.89 12.97 minimum 17.00 14.41 36.74 maximum 35.60 45.38 80.31 vessel wall to lumen area ratio the mean vessel wall area, lumen area, and vessel wall to lumen area ratio of 62 vessels in the test set, calculated by vasmorph, were 84.79 μm2, 286.8 μm2, 3.38 μm2, respectively. discussion in this study, we present a novel proof of concept ml pipeline (artseg) capable of automatically detecting blood vessels, classifying blood vessels by presence or absence of arteriolosclerosis, and segmenting arteriolosclerotic blood vessel walls and lumens. furthermore, we introduce a wrapper algorithm (orca) that centers tiles onto objects of interest; and a custom algorithm (vasmorph) that calculates the sclerotic index, vessel wall thickness, and vessel wall to lumen area ratio from arteriolosclerotic blood vessel segmentations. artseg performs four sequential steps: (1) blood vessel detection, (2) blood vessel centering, (3) arteriolosclerosis classification, and (4) arteriolosclerotic vessel segmentation (see figure 2 for overview). first, we trained an attention unet-based neural network to segment and identify blood vessels(s) within patches produced by wsi tiling. second, patches containing non-centered blood vessel(s) are then fed into orca to produce new patches centered onto the detected blood vessel(s). third, a binary classification model was trained to separate patches with arteriolosclerotic blood vessel(s) from those without. fourth, we trained an ml model to segment walls and lumens of arteriolosclerotic blood vessels. the segmentation outputs of artseg are input into vasmorph to obtain the sclerotic index, vessel wall thickness, and vessel wall to lumen area ratio of each arteriolosclerotic blood vessel. as a component of artseg, we developed a novel object of interest recursive centering algorithm (orca), which may be applied to any object of interest and not just blood vessels. orca wraps a segmentation model, which may be customized to fit the target task. orca first inputs raw image tiles generated by wsi tiling into the segmentation model, which segments the object of interest. based on this segmentation output, orca generates a new image patch with boundaries shifted such that the detected object of interest resides closer to the center of the tile. this process is repeated until the detected object of interest lies in the center of the final output tile (figure 3). when building vasmorph, we encountered a dearth of literature on the mathematical definition of blood vessel wall thickness. we utilized the following definition: the distance between the inner boundary of the vessel endothelium and the outer boundary of the tunica adventitia along a line perpendicular to the wall’s “backbone” or minimum skeleton – the arc equidistant from the outer and inner wall boundaries, which equates to the sum of the widths of the endothelium, tunica intima, tunica media, and tunica adventitia.35 because we cannot calculate the equation for the minimum skeleton, and local fluctuations make estimates of its slope unreliable, indirect methods to approximate the wall thickness have been described.35 while using the difference between the outer vessel radius and the inner vessel radius has been described, our method of finding the line perpendicular to a line tangent to each point along the lumen contour (tangent line-based method) has not been previously proposed to the best of our knowledge. in our preliminary study, we find that our tangent line-based method produces metrics comparable to expert annotations. there are several notable areas for improvement in vasmorph. first, the tangent line-based method for calculating vessel wall thickness mishandles lumens with highly convoluted and irregular contours (figure 13). second, the current version of vasmorph can only accept image tiles containing only one vessel. image tiles with more than one vessel cannot yet be analyzed by vasmorph (figure 14) because such image tiles often contain multiple vessels with contiguous vessel walls. finally, vasmorph currently does not correct for eccentricity of blood vessels due to the angle of sectioning through the vessel. the method to calculate vessel wall thickness using a tangent line tends to mishandle irregular lumens. rare blood vessels with lumens that contain an involution in the lumen contour (figure 13) pose a significant challenge to the current version of vasmorph. because the thickness measurement line is extrapolated from the lumen contour, these involutions cause the thickness measurement line to point towards the lumen center and to traverse across it (figure 13). the simplest solution for future implementations of vasmorph would be to exclude any thickness measurement line that points towards the center of the lumen. figure 13: involutions in the lumen contour produce inaccurate thickness measurement lines when using the tangent line-based method. because the thickness measurement line is extrapolated from the lumen contour, these involutions cause the thickness measurement line to point towards the lumen center and to traverse across it. vasmorph cannot process multi-vessel images because the segmentation output of artseg does not currently distinguish between two blood vessels with contiguous vessel walls (figure 14). artseg is unable to discern the border between the two blood vessels. the sclerotic index calculation depends upon having the complete circumference of the blood vessel's external contour to serve as an outer bound for the external diameter. similarly, both methods for calculating vessel wall thickness also require the complete circumference of the blood vessel external contour to serve as the outer bound for the external radius (radii-based method) and the thickness measurement line (tangent line-based method). several solutions are possible: (1) teach artseg to differentiate between two vessels with contiguous vessel walls by labeling each vessel as a separate object, or (2) exclude measurements that require external contour at the region of vessel wall contiguity. figure 14: images with multiple contiguous vessels pose a particular challenge to the current version of vasmorph. artseg is unable to discern the border between the two blood vessels. the sclerotic index calculation depends upon having the complete circumference of the blood vessel's external contour to serve as an outer bound for the external diameter. vasmorph may be improved by adding a correction for eccentric sectioning of blood vessels. a section through a cylindrical vessel at angle θ will result in an ellipsis with a short (ds) and long (dl) diameter. the ratio of ds and dl equals the cosine of θ, which can be used as a correction factor. the current version of vasmorph presented in this paper has yet to implement this type of correction. to the best of our knowledge, artseg is the first open-source ml-based pipeline for the classification of arteriolosclerosis in blood vessels and the segmentation of arteriolosclerotic blood vessel walls and lumens in wsis of the brain. orca is the first ml-based algorithm capable of generating image patches centered on objects of interest in human post-mortem brain tissue. and vasmorph is the first algorithm for automated calculation of sclerotic index, vessel wall thickness, and vessel wall to lumen area ratio using blood vessel segmentations. all three frameworks represent scalable prototype methodologies to achieve their respective goals and serve as blueprints for further refinement and improvement. notably, artseg is model-agnostic, meaning ml models contained within the algorithm may be replaced and updated as novel state-of-the-art architectures are discovered. several caveats merit mention. first is the small sample size of only 13 participants included in this proof-of-concept study. blood vessel morphology may vary from individual to individual, and scaling up artseg will likely require further training with wsis from hundreds to thousands of disparate participants. although small, the external validation cohort offers preliminary support for the algorithm’s performance. these promising pilot results warrant further validation with larger cohorts and additional wsis. we do want to acknowledge obtaining high-quality annotated datasets is labor-intensive and time-consuming. second, only three brain banks (uc davis, uci, and ucla) within california were involved in this study, which may not represent the diversity of cerebrovascular pathology, staining methods, and slide digitization protocols seen in brain banks across the united states. more diverse datasets spanning multiple institutions will provide a more robust and generalizable ml-based algorithm. third, all wsis were derived from only two brain regions, the frontal lobe or the occipital lobe. we only examined blood vessels within the white matter. a broader sampling of brain regions will provide more representative datasets spanning the entire brain. fourth, only 2 of the 13 participants in our cohort lacked arteriolosclerosis. the addition of more normal controls would strengthen the evaluation of the specificity and negative predictive value of the algorithm. fifth, our preliminary testing of the two methods for vessel wall thickness calculation includes measurements made by a single neuropathologist; a robust test set would include multiple experts. sixth, the current version of artseg does not differentiate and is unable to discern the border between multiple contiguous blood vessels within the same image tile. this presents a significant limitation when calculating the sclerotic index, vessel wall thickness, and vessel wall to lumen area ratio, which require accurate contours of each blood vessel wall and lumen within the analyzed image tile. seventh, we were only able to annotate each image tile once, and so we were unable to conduct a study on the inter-rater reliability of our annotations. eighth, we did not explicitly examine the internal elastic lamina to differentiate arterioles from venules, although we applied criteria from skrobot et al.8,10 to define arteriolosclerosis and referenced figure 3 in their paper as a guide for categorizing no, mild, moderate, and severe arteriolosclerosis. our prototype algorithm analyzes only h&e-stained sections; the addition of protein-specific stains such as cd31 immunohistochemistry or verhoeff–van gieson histochemistry may enhance visualization of specific vascular structures, including endothelial cells and the internal elastic lamina. and lastly, the ml models contained within the current version of artseg have not had hyperparameters exhaustively optimized for peak performance. the prototype artseg published here serves as a proof of concept to demonstrate the promising potential of our method, which provides a scalable blueprint for further refinement. furthermore, the blood vessel detection model currently uses an efficientnetv2l-based attention u-net, which may be computationally expensive in comparison to yolov7,36 one of the most computationally efficient and accurate object detection models for computer vision tasks. we specifically designed our training, internal hold-out, and external test sets to model real-world situations where the model may be deployed at an institution that did not contribute to its training dataset and evaluated by domain experts who did not participate in annotating the training dataset. the training and internal hold-out dataset consisted of wsis from uci and ucd labeled by annotators jjl, hpw, kn, whereas the external test set consisted of wsis from ucla annotated by attending neuropathologist vp, who did not see images in the training dataset. our study demonstrates that artseg performs slightly worse when applied to an external institution and evaluated by an external domain expert, but overall exhibits considerable resilience to these variables. in the future, we anticipate collecting datasets from multiple sources annotated by multiple brain arteriolosclerosis experts to build a more robust and reliable ml pipeline. another next step in algorithm validation would be to examine the correlation between the algorithm-derived sclerotic index and established semi-quantitative scales. learning from our pilot experience, we will ask annotators to differentiate between multiple blood vessels within an image tile, especially contiguous vessels. we may validate our tangent line-based method of calculating vessel wall thickness through a more rigorous test set annotated by multiple brain arteriolosclerosis experts. artseg may be further optimized by testing yolov7 for blood vessel detection as well as novel segmentation and classification architectures such as segment anything model,37 segformer,38 efficientvit,39 or coca.40 another consideration is to expand artseg to distinguish arteriolosclerosis from cerebral amyloid angiopathy on h&e-stained slides. to improve the ml-based tool, we plan to incorporate a quality assurance 'human-in-the-loop' step, allowing users to correct suboptimal segmentation outputs. we will assess its impact by comparing segmentation metrics before and after user supervision. during our labeling process, our annotators anecdotally observed subjectivity when applying the skrobot et al. arteriolosclerosis criteria to vessels with mild arteriolosclerosis from those without, which warrants a dedicated follow-up study on the interor intra-rater variability of arteriolosclerosis classification. future versions of artseg and vasmorph may potentially mitigate this variability by introducing quantitative thresholds – such as sclerotic index, vessel wall thickness, and vessel wall to lumen area ratio – to define arteriolosclerosis. finally, we plan to combine artseg with another ml pipeline that screens for microinfarcts41 to create a comprehensive ml-based tool capable of analyzing the relationship between vascular morphology and microinfarcts. conclusion taken together, the present study demonstrates a pilot ml approach to assist persons in the morphometrical analysis of blood vessels in histopathological images. our ml pipeline showed promising capabilities for inference on unseen wsis from a disparate institution and annotator, which would be encountered in real-world deployment of such a tool. within our pipeline, we present a generalizable novel algorithm capable of centering image tiles onto an object of interest. furthermore, we propose a novel method to calculate vessel wall thickness and present preliminary data showing this method agrees with human interpretations of vessel wall thickness. our pipeline is flexible as the ml models contained in the pipeline can be updated as novel state-of-the-art architectures are discovered by the artificial intelligence community. furthermore, our approach provides preliminary evidence that breaking a complex task into multiple steps, each addressed by a separate machine learning model, may be the optimal path for the segmentation of pathological features of interest. we hope this proof of concept inspires further work in this field. we provide the code and dataset for our pipeline openly available to the community (see data availability and code availability). data availability all available data are located in zenodo records listed within the github repository (https://github.com/jerryjlou/artseg-vasmorph). all wsis used in this study are available in their raw, de-identified form. preprocessing, training, and evaluation can be carried out using the codes listed in this manuscript. code availability all code for artseg and vasmorph can be found in the github repository (https://github.com/jerryjlou/artseg-vasmorph), which contains the full end-to-end pipeline. no outside code is necessary to reproduce the study results. funding statement this work was supported in part by national institutes of health (nih) r01ag062517, u24ns133949, and p30ag072972 as well as funding [2024-351073] from the chan zuckerberg initiative daf, an advised fund of the silicon valley community foundation and the california department of public health alzheimer’s disease program (grant # 19-10611) with partial funding from the 2019 california budget act. the views and opinions expressed in this manuscript are those of the author and do not necessarily reflect the official policy or position of any public health agency of california or of the united states government. this project is made possible in part by support from the mary s. easton center for alzheimer’s research and care at ucla. the uci alzheimer’s disease research center is supported by nih / national institute on aging (nia) p30ag066519. j.l. is supported by the uci institute for memory impairments and neurological disorders (mind) nia training in translational adrd neuroscience or titan t32ag073088. conflict of interest statement the authors declare no competing interests. supplementary material supplementary figures 1–3, table 3–6 (.pdf-file; 1139 kb) supplementary table 1 (.xlsx-file; 15 kb) supplementary table 2 (.xlsx-file; 19 kb) supplementary document 1 (.docx-file; 3510 kb) supplementary document 2 (sup fig 4,5) (.docx-file; 428 kb) supplementary document 3 (.docx-file; 108 kb) references 1. launer lj, hughes tm, white lr. microinfarcts, brain atrophy, and cognitive function: the honolulu asia aging study autopsy study. ann neurol. 2011;70(5):774-780. https://doi.org/10.1002/ana.22520 2. garcia-alloza m, gregory j, kuchibhotla k v., et al. cerebrovascular lesions induce transient β-amyloid deposition. brain. 2011;134(12):3697-3707. https://doi.org/10.1093/brain/awr300 3. schneider ja, arvanitakis z, leurgans se, bennett da. the neuropathology of probable alzheimer disease and mild cognitive impairment. ann neurol. 2009;66(2):200-208. https://doi.org/10.1002/ana.21706 4. schneider ja, wilson rs, bienias jl, evans da, bennett 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https://doi.org/10.1093/jnen/nlae120 copyright: © 2025 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. neurotrauma: 2024 update free neuropathology 5:26 (2024) priemer et al doi: https://doi.org/10.17879/freeneuropathology-2024-5849 page 1 of 15 copyright: © 2024 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. neurotrauma: 2024 update david s. priemer1,2, daniel p. perl1,2 1 the department of defense/uniformed services university brain tissue repository, bethesda, md, usa 2 department of pathology, f. edward hébert school of medicine, uniformed services university, bethesda, md, usa corresponding author: daniel p. perl · department of pathology · f. edward hébert school of medicine · uniformed services university (usu) · 4301 jones bridge rd · bethesda, md 20814 · usa daniel.perl@usuhs.edu submitted: 09 september 2024 · accepted: 03 october 2024 · copyedited by: joão gama · published: 23 october 2024 abstract 2023 was an important year for research in traumatic brain injury (tbi), particularly as it concerned interests in neuropathology. after reviewing the literature, we present the advancements that we felt were of particular importance to the neuropathology community. highlighted are articles that report upon: (1) the first large-cohort assessment for the neuropathology of intimate partner violence, (2) the assessment of chronic traumatic encephalopathy (cte) in young athletes, (3) the observation of cortical sulcal depth vascular changes in cte, (4) a proposal for a tau immunohistochemical panel to evaluate complex cases of cte in the context of multiple tauopathies, (5) the relationship of tbi and/or cte with tdp-43 pathology, (6) repetitive tbi inducing pathology in c9orf72-transgenic mice, (7) radiologic patterns of head and neck injury following vehicular underbody blast exposure, (8) chronic alterations in brain metal content following repetitive impact tbi, (9) neurovascular unit injury following low-level blast exposure, and finally (10) an assessment of muhammad ali’s clinical history leading to the conclusion that he suffered from young-onset, idiopathic parkinson disease. we close our writing with in memoriam to dr. byron a. kakulas, a renowned figure in the neuropathology of spinal cord injury who we lost in 2023. keywords: traumatic brain injury, chronic traumatic encephalopathy, tdp43, neurovascular, intimate partner violence, blast introduction the year 2023 was an especially fruitful year in the study of traumatic brain injury (tbi), particularly as it concerned aspects of neuropathology, demonstrating that interest in the field remains robust. many notable studies were published, providing many impactful advancements in knowledge. after conducting a thorough review of the literature regarding neurotrauma over the year of 2023, herein we present and summarize the discoveries that we felt were of particular importance and relevance to the neuropathology community. we conclude our review with memoriam to the life of dr. byron a. kakulas, a pioneer in the neuropathology of spinal cord injury, who died in 2023 at the age of 90. review https://doi.org/10.17879/freeneuropathology-2024-5849 https://creativecommons.org/licenses/by/4.0/ https://creativecommons.org/publicdomain/zero/1.0/ mailto:daniel.perl@usuhs.edu free neuropathology 5:26 (2024) priemer et al doi: https://doi.org/10.17879/freeneuropathology-2024-5849 page 2 of 15 the neuropathology of intimate partner violence while the large focus of research on the chronic neuropathologic effects of repetitive tbi has been dedicated to contact sports athletes and, to a lesser degree, military service, there are other circumstances in which repetitive tbi and its longterm sequelae must be investigated. one important setting is intimate partner violence (ipv), as it is a remarkably common global phenomenon of increasing medical research interest (figure 1) and as previous, highly-cited anecdotal reports have implicated this exposure with risk of chronic traumatic encephalopathy (cte).1-3 in 2023, authors damso’conner and folkerth et al. reported the comprehensive neuropathological examination and clinicopathological correlation of the first large case series of brains from victims of ipv.4 the authors used a multi-prong approach to address this question. first, they prospectively identified a total of 14 brains from documented ipv cases which had come to the new york city office of chief medical examiner over a 24-month period. these cases were comprehensively examined neuropathologically, and all available medical records, medicolegal records, and data available from postmortem interviews from next-of-kin were reviewed. the fourteen prospectively gathered cases were supplemented by the retrospective review of neuropathological material from 70 additional cases derived from additional victims of ipv. figure 1. total pubmed listings regarding intimate partner violence (ipv) and related terms, by year. medical research interest in ipv has grown substantially in recent years, particularly in the most recent decade, as is easily demonstrated by the dramatic increases in annual publications for ipv and related terms (domestic violence, domestic abuse) indexed by pubmed. within the box are the proportions of the total number of publications regarding a given term that have been published within the last decade, i.e. though the first publication listed in pubmed utilizing the term ipv appeared in 1976, 63.5 % of the total studies using this term have emerged only in the last decade. https://doi.org/10.17879/freeneuropathology-2024-5849 free neuropathology 5:26 (2024) priemer et al doi: https://doi.org/10.17879/freeneuropathology-2024-5849 page 3 of 15 the fourteen prospectively gathered cases, as could be expected, had complex histories associated with ipv, including known tbi in six cases, non-fatal strangulation in four, epilepsy in seven, and some history of cerebrovascular, neurological, or psychiatric condition (related to ipv or otherwise) in thirteen. in this group, evidence of prior tbi, such as old contusions or subdural membranes, was identified in eight cases, and evidence of more recent injuries was identified in six. amyloid precursor protein (app) immunohistochemistry demonstrated evidence of axonal injury in regions susceptible to diffuse traumatic axonal injury in nine cases; further, these regions were highlighted by perivascular or parenchymal microgliosis (cd68 immunohistochemistry) and/or iron deposition (perls stain) in twelve cases, many of which were in the absence of hypertensive arteriolosclerotic disease. this study illustrates that the complexity of ipv translates to the variable development of different corresponding neuropathologies, most of which are related to direct anatomic disruptions from trauma such as hemorrhage, contusion, and axonal injury. importantly, and despite high rates of neuropsychiatric symptomatology in the cohort, comprehensive examination did not reveal evidence of cte in any of the 14 cases. this finding was corroborated in the second portion of the study analyzing 70 archival cases of ipv, which similarly did not reveal a single case of cte. therefore, this study also suggests that tbi sustained in the context of ipv, similar to that sustained in other non-athletic contexts wherein impact tbi occurs but is less predictable and certainly less repetitive,5 is often of an insufficient dose and/or mechanism to produce cte neuropathology. moreover, this data further suggests that cte does not provide a “catch-all” or common denominator accounting for high proportions of chronic neuropsychiatric sequalae following tbi. as such, we also feel that this study serves as an important opportunity to urge caution regarding the overinterpretation of isolated cases reports in our research practices, particularly as it concerns diseases with high public interest such as cte. cte in young contact sports athletes the large majority of cte cases have been characterized in older age groups and/or in athletes with long careers, particularly those with an elite level of sport participation.6 however, a lingering question concerns development of cte at young ages and the risk of developing cte from lower level athletic exposures (e.g., high school sports). among the most impactful studies with respect to public awareness in 2023 was written by dr. ann mckee and colleagues, titled “neuropathologic and clinical findings in young contact sport athletes exposed to repetitive head impacts”.7 the study describes the neuropathologic evaluation for cte, with use of the “mckee” staging system to assess severity,8 in a convenience sample of 152 donated brains from former contact sports athletes and who were under the age of 30 years at death (range = 13–29 years). the majority of the series (60.5 %) had a history of participation in american football, with the average duration of football play being 10.29 years, and the average age at which football participation began being 9.25 years. of the 152 cases, 128 were classified as “amateur athletes” that did not play at a semiprofessional or professional level but rather played in youth, high school (most frequent), or up to collegiate levels. the authors report that cte was present in 63 of 152 cases (41.4 %), the large majority (95.2 %) of which had minimal/mild pathology (mckee stages i or ii). 45 of the 63 cte cases were from amateur athletes. consistent with what has been observed and reported in the literature,6 the brains with cte were from individuals who were significantly older, and had significantly longer durations of play than those without cte. however, the youngest individual reportedly diagnosed with cte in this study was 17 years old. the authors of the study have made an important contribution with the largest series of athletes in this young age group to be assessed for cte pathology, and identifying pathologic features at a relatively high frequency. however, apart from the largely unavoidable limitations of ascertainment bias in a convenience sample derived from a brain bank dedicated to tbi research, there are a number of concerns with this study that we care to highlight. first, though mckee et al state that evaluation for cte in this study was done in accordance with the most recent national institute of neurological disorders and stroke/national institute of biomedical https://doi.org/10.17879/freeneuropathology-2024-5849 free neuropathology 5:26 (2024) priemer et al doi: https://doi.org/10.17879/freeneuropathology-2024-5849 page 4 of 15 imaging and bioengineering (ninds/nibib) consensus criteria,9 there is no detail provided with respect to the extent or manner of sampling that was performed in these cases. in addition, the authors provide their interpretation of the pathognomonic lesion of cte as occurring “typically”, and “most frequently” at the depth of a cortical sulcus, which stands in notable contrast to the ninds/nibib consensus document which states that a pathognomonic lesion of cte must be at a sulcal depth by definition.9 this raises concern about how these cases were being diagnosed, as an especially liberal approach may very well have led to over-diagnosing cte in these mildly affected cases. accordingly, it will be hard to relate the data reported here to other published studies. it is also important to express concern with how the lay media has approached this study. indeed the publication garnered nationwide media attention, which involved the publication of several anecdotal stories involving very young athletes who had died by suicide, with the implication that contact sports participation and cte were contributory, if not causal.10-12 these media articles were written in this manner despite the fact that the study published by mckee et al identified no correlation with clinical symptoms (including neurocognitive and psychiatric symptoms) or cause of death (including suicide) with cte when compared to cases without cte, which itself is consistent with other recently published data.13 how media outlets may ultimately choose to interpret scientific articles is often out of our control. however, as neuropathologists who may concern ourselves with cte or other neurologic disorders that are within the public eye, we must be especially careful with how we communicate to the media and the lay public at large when given the opportunity, so as to avoid mischaracterization and/or unnecessary hyperbolizing of our findings. evidence of vascular injury and remodeling at sulcal depths in cte next, we would like to highlight the collective efforts of two separate studies, both largely focused out of the boston university cte center and the veterans administration healthcare system in boston, which have identified evidence of chronic vascular injury and remodeling concentrated at sulcal depths in cases of cte, demonstrating evidence that vascular insults may be intricately tied to the disease. first, in a study that investigated the theory that microvascular and associated blood brain barrier damage at sulcal depths may be involved in the pathogenesis of cte, authors kirsch et al. published a large immunohistochemical and immunoassaybased study using brain tissues from cohorts which included high stage cte, low stage cte, and cte-negative controls with and without history of repetitive tbi.14 most of those with a history of repetitive tbi were exposed via contact sports. a total of 156 cases were included in the immunoassay portion of the study, which quantified markers of vascular injury and inflammation, including intercellular adhesion molecule 1 (icam1), vascular cellular adhesion molecule 1 (vcam1), and c-reactive protein (crp) in samples from the dorsolateral frontal region; the authors proceeded to measure for associations between these quantifications and extent of repetitive tbi exposure (years of contact sports played), evidence of microgliosis, and p-tau pathology. a smaller sampling of 57 cases with high stage cte, low stage cte, and no cte were evaluated histologically via immunohistochemistry with antibodies including for icam1, albumin, p-tau (at8), and microglia (iba1, cd68). the authors found that quantified levels of icam1, vcam1, and crp were significantly increased in cte cases versus cte-negative cases, and generally increased with cte severity. further, these elevated levels were statistically associated with duration of repetitive tbi exposure, increased microglial density, and overall p-tau burden. in cte cases, particularly in high stage cases, histologic examination revealed dramatic co-localization of icam1 and at8 at sulcal depths. whether these increases relate to repetitive impact tbi exposure, p-tau aggregation, or a combination of both remains an important question. however, all cases wherein there was a history of repetitive tbi demonstrated extravascular albumin staining in both gray and white matter, in contrast to none of the of the cases without a history of repetitive tbi, suggesting chronic compromise to vascular integrity and the blood-brain barrier following this exposure. https://doi.org/10.17879/freeneuropathology-2024-5849 free neuropathology 5:26 (2024) priemer et al doi: https://doi.org/10.17879/freeneuropathology-2024-5849 page 5 of 15 in the second study, authors rosen et al. imaged vascular microstructure in passively cleared (shield tissue processing) and tomato lectinstained dorsolateral frontal samples from 41 total cases, including donors with high stage cte, low stage cte, and no cte (44 % of which had repetitive tbi history) using fluorescence microscopy.15 again, the majority of the cte cases corresponded to tbi from contact sports participation. among the factors evaluated in different cortical regions (e.g. sulcal depth versus gyral crest) included assessments for vessel branch density (the number of vessel branches per volume of the image stack), and vessel fraction volume (the volume of blood vessels per volume of the image stack). first, cte cases demonstrated evidence of vascular remodeling characterized by significantly increased vascular branch density at the sulcal depths compared to non-cte controls. further, there were statistically significant differences in both vessel branch density and vessel fraction volume at sulcal depths versus the gyral crest in cte cases that were not observed in controls. finally, at8 staining density correlated positively with vessel fraction volume at the sulcus, though this was independent of cte status. these two studies collectively demonstrate that chronic vascular injury is an important component of repetitive, mild tbi and its predilection to sulcal depths with remarkable correlation with p-tau deposition in cte offers a tantalizing theory for cte pathogenesis. indeed, vascular/blood-brain-barrier injury from tbi could induce insufficient delivery of nutrients (including oxygen) and removal of waste products, and promote a malnourished/hypoxic, pro-inflammatory, and even neurodegenerative environment in which p-tau can accumulate. further, while authors rosen et al. postulate that the changes in vascular branching and volume at sulcal depths in cte may be accounted for by atrophy in the sulcus relative to the gyral crest, it has also been demonstrated that chronic vascular insufficiency triggers pro-angiogenic proteins, such as vascular endothelial growth factor.16,17 nonetheless, it cannot be stated with certainty whether the processes of vascular injury and remodeling are pathogenically linked to cte rather than being co-occurring/parallel processes in locations susceptible to impact tbi.18 more research is needed. assessing multiple concurrent tauopathies in the setting of cte: a way forward? the second, and still the most recent, ninds/nibib consensus for the neuropathological diagnosis of cte made important strides regarding diagnostic approach, in that it refined the definition of the pathognomonic lesion for cte.9 specifically, the newest consensus definition of cte defines it as a disease of perivascular neuronal tau deposition at sulcal depths, with or without glial tau; the definition was adjusted in part to allow for a more careful delineation of cte from age-related tau astrogliopathy (artag), which as the name suggests is limited to tau accumulation in astrocytes but also tends to be limited to superficial cortex when it involves sulcal depths. beyond the discernment of artag, the consensus paper stated that the interpretation of cte in the context of other multiple, concurrent neurodegenerative diseases is a matter of future consideration, and specifically made “no assertion” in this regard. however, especially in brains from older individuals with cte, co-pathologies which include other tauopathies (e.g. alzheimer disease [ad], primary age-related tauopathy, progressive supranuclear palsy [psp], etc.) are common,8,19 and this creates a dilemma in neuropathology practice. though multiple co-occurring tauopathies can certainly be diagnosed (figure 2), given the degree of overlap between cte distribution and that of other tauopathies, distinguishing (i.e. determining which p-tau aggregates belong to which disease) and thus staging any individual tauopathy in this circumstance under current guidance can be extraordinarily difficult, if not impossible. importantly, this issue also precludes many aspects regarding the clinicopathological correlation of cte and the clinical validation of pathological staging criteria. while we hope for a third ninds/nibib consensus effort in the near future that could begin to address this unresolved issue, in 2023 authors sorrentino et al. investigated a solution and wrote a brief manuscript detailing a study in which they used an immunohistochemical panel of multiple antibodies for p-tau on diagnostic material from a small series of cases of ad, cte, psp, and controls.20 antibodies utilized in the panel included at8 (p-tau at https://doi.org/10.17879/freeneuropathology-2024-5849 free neuropathology 5:26 (2024) priemer et al doi: https://doi.org/10.17879/freeneuropathology-2024-5849 page 6 of 15 figure 2. chronic traumatic encephalopathy (cte) overlap with alzheimer disease. cte neuropathology overlapping with alzheimer neuropathologic change in the brain of a 72 year-old man with a history of repetitive impact-type traumatic brain injury. a: at8 immunohistochemical stain (scale bar = 4 mm) performed on a sample from the orbitofrontal cortex demonstrating phosphorylated tau aggregation throughout the cortex in a pattern consistent with alzheimer disease, overlapped by sulcal depth aggregations corresponding to cte. b: at8 immunohistochemical stain (scale bar = 200 µm) performed on a sample from the dorsolateral prefrontal cortex demonstrating extensive perivascular phosphorylated tau aggregation; this was identified at a sulcal depth. c: 4g8 immunohistochemical stain (scale bar = 4 mm) performed on a sample from the orbitofrontal cortex demonstrating florid involvement by aβ plaques. s202 and t205), at180 (p-tau at t231), phf1 (p-tau at s396 and s404), and mc1, which is an antibody specifically raised against p-tau in ad.21 the authors found little discernable difference in cases of ad, cte, and psp labeled with at8, at108, and phf1. however, interestingly, mc1 immunostaining was minimal to absent in cases of cte and psp in comparison to the strong staining identified in the ad case as rated subjectively and also calculated by assessment of p-tau inclusions per three 10x microscopic fields (p = ≤ 0.0002). in this study, the authors demonstrate evidence that the conformation of p-tau in ad is sufficiently different so as to potentially allow the differentiation of ad from other tauopathies, importantly cte, through the use of mc1 immunohistochemistry. this study, though small, therefore provides promise for a simple and practical means to potentially address major diagnostic issues concerning cte when concurrent with multiple tauopathies. more investigation with mixed cases is needed, particularly given that, at the time of this writing, the discussion regarding mixed neurodegenerative pathologies in brains from individuals exposed to repetitive tbi continues to grow.22 the relationship between tbi, cte, and tdp43 pathology tdp43 pathology has been described in a relatively small number of mostly advanced cte cases, including descriptions of neocortical deposits (sometimes at sulcal depths, coincident with cte pathognomonic lesions) as well as deposits in limbic structures, sometimes coincident with hippocampal sclerosis (hs).23-27 as it concerns cte with concurrent limbic tdp43 pathology and hs, the precise relationship between these entities has been uncertain, as has been how limbic tdp43 pathology and hs may differ in the context of cte versus that of aging alone (i.e., the impact of tbi on these lesions). in 2023, authors nicks et al. published a large cohort study to begin to address this gap in knowledge.28 the authors began with a 401-case cohort of cases with cte and known tbi history, predominantly from contact sports participation (mostly american football). the mean age of the cohort was 62.2 years, with a range of 20–100 years. the https://doi.org/10.17879/freeneuropathology-2024-5849 free neuropathology 5:26 (2024) priemer et al doi: https://doi.org/10.17879/freeneuropathology-2024-5849 page 7 of 15 authors found evidence of tdp43 pathology in 43.3 % of the cte cases, and hs in 23.4 % of cases. regardless of whether or not hs was identified, tdp43 pathology was most common in the limbic regions (93 % of tdp43-positive cases), and less so in the examined frontal cortex (47 % of tdp43positive cases, including 7 % which only had tdp43 accumulations in the frontal cortex in the absence of limbic pathology). perhaps expectedly, the most numerically significant factor between cte cases with and without hs was age; the average age in the cte group with hs was 77 years (range 52–100), and without hs was 57.7 (range: 20–91). interestingly, however, another statistically significant factor that emerged in the study was duration of contact sport participation, which was significantly longer in the cte group with hs as opposed to without (p=0.029). as a means of comparison, the group also assessed differences between the cte cohort and a 33-donor cohort diagnosed with hs related to aging, without a history of tbi and without cte (average age = 86.6 years). among the notable observations was that the cte group with hs had a substantially greater proportion of cases with tdp43 inclusions within both limbic and frontal regions (41.5 %) in comparison to the group with hs but no cte (18.2 %), which was particularly interesting given that the cte group had an average age of death that was 10 years younger than the hs group without cte. also interesting was that the distribution of tdp43 pathology in the hippocampus differed between the groups. in the cte group with hs, the ca1 region and dentate gyrus were statistically less likely to be involved by any tdp43 pathology, and the ca2 region was more severely involved tdp43 pathology. with this study, the authors demonstrate a high rate of tdp43 pathology in aged individuals with cte, and provide evidence that cte, and/or repetitive tbi, may exacerbate or accelerate age-related tdp43 pathology with or without hs, and/or may be a risk factor for the development of independent, and perhaps different, tdp43 pathology. the latter notion becomes particularly interesting when considering another 2023 article which identified tdp43 inclusions in the inner nuclear layer of 6/8 retinas from cases with cte, and in only 1/8 of the age-matched controls.29 we are excited to see what the future holds for these new developments. repetitive tbi elicits pathology in c9orf72 transgenic mice in our continued efforts to understand the link between amyotrophic lateral sclerosis (als) and frontotemporal dementia, the identification of c9orf72 hexanucleotide gene expansion as the most common genetic association between these disorders was a landmark discovery.30,31 interestingly, links between repetitive tbi and als have also been suggested, and bolstered by evidence epidemiologically, which itself includes demonstrations that a history of multiple tbis is associated with a threefold increased risk of als, and that incidence and mortality from als is nearly four times higher in professional american football players in comparison with the general population.32-34 questions surrounding how tbi may impact development of pathology in the setting of a known genetic risk for frontotemporal dementia/als should therefore be explored. in a 2023 article published in brain, authors kahriman et al. investigated the effect of repeated, mild tbi on producing pathology in a transgenic hemizygous and homozygous c9orf72 expansion mouse model.35 the authors experimented on 63 total mice, including transgenic and non-transgenic lines, 34 of which were subjected to repetitive, mild, closed-head tbi using a weight-drop impact model and 29 of which were sham. the tbi model delivered over 5 consecutive days, and the brains were removed for neuropathologic examination 52 weeks following exposure. in the interim period, mice were subjected to a variety of neurologic and behavioral testing. alterations in neurologic and behavioral testing during the long survival period were observed in the transgenic, tbi-subjected mice, which included but were not limited to significantly reduced grip strength, and behaviors that were similar to some phenotypes of human als. neuropathologic findings in these mice were also particularly striking. histological examination included chromogenic immunohistochemistry and immunofluorescent staining for https://doi.org/10.17879/freeneuropathology-2024-5849 free neuropathology 5:26 (2024) priemer et al doi: https://doi.org/10.17879/freeneuropathology-2024-5849 page 8 of 15 antibodies including tdp43, glial fibrillary acidic protein (gfap), iba1 and cd68, neurofilament (smi312), synaptophysin (a pre-synaptic marker) and psd-95 (a post-synaptic marker), and neuronal markers neun, tuj1 (beta-iii tubulin), and map2. examination revealed that brains from the tbi-exposed, transgenic mice suffered significantly more neuronal loss in the cortex along with axonal and synaptic marker loss and evidence of widespread microglial activation in comparison with controls. these findings were seen coincidently with dramatic cytoplasmic mislocalization of tdp43 in the remaining neurons of the cortex, which was not observed in either the non-transgenic mice which sustained tbi or the transgenic sham mice following the 52-week survival period. with these findings, the authors demonstrate evidence that repetitive tbi is sufficient to initiate, exacerbate, and accelerate the development of frontotemporal dementia/als-like pathology in the setting of genetic predisposition with c9orf72 expansion in a mouse model. the findings are most informative with regard the relationship of tbi and als, and also have implications with regard to development and implementation of therapy for tdp43 pathology and/or tbi. patterns of head and neck injury in fatal underbody blast exposures tbi is considered a “signature wound” of modern warfare, and blast exposure is acknowledged as the most common source of tbi in the military context.36,37 among the more common causes of military blast-related morbidity and mortality is so-called underbody, or “mounted” blast exposure. underbody blast exposure, which became notable due to the increased use of improvised explosive devices in the most recent wars in the middle east, is sustained when an explosive is detonated under a vehicle. this causes the vehicle, its contents/occupants to be propelled upwards in a rapid acceleration event that further includes impacts between vehicle occupants and other occupants, vehicle contents, and/or the inner walls of the vehicle. the precise mechanism(s) by which fatal central nervous system injuries occur in this context, and/or constellations that these injuries tend to occur in, have been poorly understood. ashworth et al. studied post-mortem computed tomography scans of 46 underbody blast fatalities which occurred from 2007–2013 among british military personnel.38 the authors identified that the most common pathology identified within or of the cranium was subarachnoid hemorrhage (80.4 % of total cases), followed by skull fracture (63 %, including 10.9 % of cases with an “eggshell” pattern of skull fracture reflecting direct head impact), 4th ventricular hemorrhage (63 %), and contusions (28.3 %). other injuries observed less frequently included subdural hematoma, epidural hematoma, brainstem hemorrhage, and pneumocephalus. radiologic evidence of diffuse axonal injury was uncommon (1 case). examination of the spine frequently revealed fractures, being most common in the lower spine (lumbar fractures in 52.2 % of cases, sacral in 50 %), followed by the thoracic and cervical. in examining the constellation of injuries to the central nervous system and its surroundings, together with secondary injuries to other regions (e.g. thorax, abdomen, limbs), the authors hypothesized five categories/patterns of potential head and neck injury in fatal underbody blasts. briefly, these constellations are published as follows: 1. multiple-level spinal injuries in addition to skull fracture: frequent association of fractures at the c0-c1 junction and three or more vertebral fractures at other spinal levels, along with base-of-skull and brainstem injuries. often coincident with injuries to the thorax and abdomen. the proposed mechanism is upward compression of the axial skeleton due to upward force of the blast. 2. peri-mesencephalic hemorrhage: peri-mesencephalic hemorrhage, as characterized by hemorrhage in the 4th ventricle, lateral ventricles, and/or posterior fossa. the proposed mechanism is increased jugular pressure and secondary non-aneurysmal, peri-mesencephalic bleeding. 3. spinal (cervical) fractures and brainstem injury: proposed tertiary blast injury, that is blunt force injuries occurring as a result of the person being accelerated by the blast and impacting objects, leading to flexion/extension https://doi.org/10.17879/freeneuropathology-2024-5849 free neuropathology 5:26 (2024) priemer et al doi: https://doi.org/10.17879/freeneuropathology-2024-5849 page 9 of 15 and rotational forces about the neck, and resulting c1 and condylar fractures. 4. contusions with injury to c0-c1: injury, including contusions, at c0-c1 combined with abdominal or thoracic injury. 5. “eggshell” skull fractures: eggshell pattern of skull fracture on the convexity of the skull, owing to direct impact, most likely with the vehicular cabin ceiling. the information in this study may be particularly important to medical examiners and/or neuropathologists who practice in a forensic setting. further, the article may also serve to emphasize the growing utility of post-mortem imaging in diagnostic practice. beyond the scope of neuropathology, understanding mechanisms by which the central nervous system can be injured in various settings of blast exposure is critical for diagnosis and care, including triage efforts, and also for the development of protective measures and mitigation. chronically altered brain metal homeostasis following repetitive impact tbi alterations in the content and metabolism of metals including iron, zinc, and copper have been identified in the context of aging and alzheimer disease, as well as parkinson’s disease, and have been implicated with misfolding of pathogenic proteins in neurodegeneration.39-43 while a small number of studies have reported iron accumulation following repeated traumatic brain injury,44-46 there has been little investigation with regard to alterations in iron regulatory proteins and the potential association of metal dysregulation with neurodegeneration in the context of tbi. paul adlard’s laboratory had previously shown altered brain metals following a single impact tbi, and demonstrated increased iron levels in the subacute period following repeated impact tbi, in mice.47,48 as a follow-up, they investigated chronic alterations in metal content, iron regulatory proteins, and neurodegenerative proteins in a mouse model of mild, repetitive impact tbi with prolonged survival within a study they published in 2023.49 the authors subjected groups of three-monthold mice to either single or repeated (five instances, each 48 hours apart) mild, closed-head impact tbi, and compared to corresponding sham controls. in order to demonstrate evidence of ongoing genetic changes following tbi, rna sequencing and differential gene expression analysis was performed on ipsilateral and contralateral cerebral hemispheres from mice sacrificed following 6 months of survival. in the mice with 12 months of survival, ipsilateral and contralateral cortical samples were subjected to metal analysis using inductively coupled plasmamass spectrometry (icp-ms) and western blot analysis using a list of antibodies related to iron regulatory proteins, to tau and tau regulatory proteins, and to amyloid precursor protein. the authors identified significant alterations in metal content and regulatory proteins in the mouse brains exposed to repetitive tbi. first, they found increases in ferritin and divalent metal transporter 1 (dmt1, a cytosolic iron transport protein), and a decrease in transferrin receptor in the ipsilateral cortex. this signature would typically imply iron excess; however, these findings were observed in the context of a downward trend in total iron content, suggesting perhaps that iron had previously been elevated but then restored with a regulatory response. conversely, in the contralateral cortex of mice exposed to repetitive tbi, there were no significant differences in iron regulatory proteins and rather there was an increase in iron content, which could suggest iron regulatory failure on the contralateral side. this was also coincident with increases in total zinc and copper on the contralateral side, which was not observed on the ipsilateral side. it is unclear what may account for the differences between the hemispheres, but nonetheless the authors do demonstrate chronic alterations in brain metals and related metabolism following repeated tbi. interestingly, there were no significant transcriptional or translational changes in tau or tau regulatory proteins found in either hemisphere following repeated tbi with 6and 12months of survival, respectively. given the lack of this association, and the association between the measured metals and adverse neurologic sequelae/neurodegeneration, the authors posit that many of the significantly increased neurobehavioral deficits that https://doi.org/10.17879/freeneuropathology-2024-5849 free neuropathology 5:26 (2024) priemer et al doi: https://doi.org/10.17879/freeneuropathology-2024-5849 page 10 of 15 were observed in the repeated tbi mice during the survival period were at least partially contributed to by metal excess. as such, the authors suggest that chelation therapy following repeated tbi exposure as a means to potentially prevent long-term adverse outcomes is worthy of further investigation. blast exposure damages the neurovascular unit in mice between the years 2000 and 2023, there were almost 500,000 individuals with new diagnoses of tbi among united states military personnel recorded by the defense health agency of the united states department of defense, the overwhelming majority (82.2 %) of which were classified as mild tbi.50 one aspect of the military experience that is particularly unique in comparison to the civilian population is exposure to repetitive blast overpressures in the context of both live combat and in training circumstances. of increasing recognition is the potential of so-called “low-level blast exposure” (llb), i.e. that which may be generated by firing heavy weapons, breaching exercises, or other lowintensity explosives, to produce mild tbi or subclinical damage to the brain with potential chronic implications.51-53 however, our understanding of the pathophysiology of such injury is still in development. the neurovascular unit is a functional unit of the brain that is critical in the management of cerebral blood flow and maintenance of the blood-brain barrier, being composed of the delicate and complex interplay of endothelial cells and their tight junctions, perivascular astrocytes (particularly astrocytic endfeet), pericytes, basement membranes, and finally adjacent microglia and neurons. certainly, insults to the neurovascular unit are implicated in a wide variety of neurological disease states.54 in 2023, authors li et al. tested a hypothesis that llb could induce ultrastructural abnormalities of the neurovascular unit using a mouse model.55 the authors exposed a group of anesthetized mice to a single open-field llb, during which no head or body movements were observed. among the several studies performed on the brain tissues following sacrifice, examination of ultrastructure by transmission electron microscopy (em) in mice sacrificed 7 and 30 days following llb exposure produced many of the most interesting findings, revealing a myriad of changes which suggest injury to multiple constituents of the neurovascular unit. in summary, the statistically significant findings in comparison to controls included drop-out of pericyte surface area and associated coverage of endothelial cells at 30 days survival, endothelial swelling at 7 days survival with loss and/or discontinuity of tight junctions at both 7 and 30 days survival, thickening of basement membranes at 7 and 30 days survival, swelling of astrocytic endfeet at 7 and 30 days survival, and finally detachments of astrocytic endfeet with one another as well as with basement membranes. the above changes were coincident with marked luminal constriction relative to controls at 7 days survival, and dilation at 30 days survival, potentially indicating the presence and reversal of vasospasm. many unquantified morphological changes were also observed in comparison to controls, including vacuolization of numerous neurovascular unit constituents (pericytes, endothelial cells, basement membranes, astrocytic endfeet) potentially indicative of cellular edema, detachments between endothelial cells and bulging of endothelial cytoplasm into the lumen, and bulging and fragmentation of basement membranes. with these findings, the authors demonstrate evidence of significant neurovascular unit damage and loss of structural integrity of the blood-brain barrier in the short and intermediate survival period that follows just a single llb exposure in mice. this, of course, may have profound implications with regard to military service members who may experience many multitudes of these exposures, and at short intervals, throughout the length of a career. certainly, studies with long-term survival and neurobehavioral analysis are necessary to determine the potential chronicity of these changes, or to determine how these changes may evolve over time. human autopsy studies with this research question are also paramount. nonetheless, this study serves as an indication that blast overpressure exposure in the military context, no matter how low the intensity, should be monitored and minimized where possible. https://doi.org/10.17879/freeneuropathology-2024-5849 free neuropathology 5:26 (2024) priemer et al doi: https://doi.org/10.17879/freeneuropathology-2024-5849 page 11 of 15 muhammad ali had young-onset, idiopathic parkinson disease on june 3rd, 2016 muhammad ali died of sepsis. famously, ali denied autopsy owing to his muslim faith. during approximately 34 years preceding his death, ali suffered from a progressive parkinsonian movement disorder that was and has remained among the most widely discussed anecdotes in neurodegeneration. particularly accelerated by the resurgence of dementia pugilistica as cte, speculation regarding the role of ali’s illustrious boxing career on his manifestation of parkinsonism has escalated. indeed, many lay media depictions have, at least, made implications of a strong role of tbi,56,57 with one lay book that even speculates cte.58 continuing with what has become a theme in this writing, we provide the readership one last precautionary message regarding overinterpretation and undue speculation on individual cases and bring to awareness an article published in 2023 that provides for us a succinct, yet comprehensive medical account of ali’s clinical course. this leads to an important conclusion: in the absence of an autopsy, all of the information available to us indicates that muhammad ali suffered from classic, early-onset, idiopathic parkinson disease.59 neurologists michael okun, helen mayberg, and mahlon delong, all of whom were involved in muhammad ali’s clinical care at emory university, give us an account of the two decades of clinical follow-up with ali at the institution to support their conclusion. in brief, below is a bulleted summary:  unlike what is currently understood regarding the course of cte, from which clinical manifestations tend to emerge after a latent period that most often extends into the retirement years of former athletes,60 muhammad ali’s symptomatology began while he was still active and in his mid-to-late 30’s. notably, during ali’s final fight in 1981, decreased movement on his left was demonstrable on the video footage.  following ali’s retirement, slowed movement on the left side became more apparent on televised interviews, and a rest tremor of the left hand emerged during an interview in 1991.  as the years progressed, neurologists observed chronic and progressive symptoms including the classic parkinsonian rest tremor in the left hand, slowing of motor function/bradykinesia, cogwheel rigidity, softer and dysarthric speech that eventually became hypotonic, micrographic writing (verified by signatures over time), masked facies, and characteristic sleep dysfunction. the motor symptoms were asymmetric but became more generalized over time. serial cognitive testing showed progressive frontal and memory impairment.  finally, ali developed late-stage signs and symptoms that are classic of idiopathic parkinson disease, including stooped posture, shuffling gait, postural instability, falls. though the natural history of ali’s disease is quite characteristic for idiopathic parkinson disease, several additional diagnostic findings provide further support. first, fluorodeoxyglucose positron emission tomography (pet) and fluorodopa f18 pet scans performed in 1997 and 1998 demonstrated bilateral striatal activity and low striatal uptake, respectively, both of which are consistent with and classic for parkinson disease in contrast to post-traumatic parkinsonism. polysomnography to investigate ali’s sleep dysfunction revealed rapid-eye movement sleep behavioral disorder which, though not specific, is most classically associated with synucleinopathies including parkinson disease. finally, the clinical feature that draws the most convincing contrast between parkinson disease and post-traumatic parkinsonism or cte is the fact that ali’s symptoms were “clearly” and “substantially” responsive to levodopa. in summary, muhammad ali experienced a 34-year chronic, progressive course that followed a classic natural history of parkinson disease, with compatible (if not classical) imaging, and that was responsive to levodopa. this is unlike posttraumatic parkinsonism which can be transitory, often shows kinetic tremor, and is not characteristically responsive to levodopa. though in the absence of postmortem examination it can never be stated with certainty if ali did, in fact, have cte neuropathological changes, the available (and rather complete) clinical evidence strongly suggests that at least his https://doi.org/10.17879/freeneuropathology-2024-5849 free neuropathology 5:26 (2024) priemer et al doi: https://doi.org/10.17879/freeneuropathology-2024-5849 page 12 of 15 symptomatology was driven by early-onset, idiopathic parkinson disease. in memoriam: dr. byron a. kakulas we would like to end this years’ listing to honor the legacy of dr. byron a. kakulas. in january, 2023, dr. kakulas (emeritus professor, university of western australia) died at age 90 in his native city, perth, western australia. while he was mostly known for his work on muscular dystrophy, he was also a world authority on the neuropathology of spinal cord injury. his interest in spinal cord injury began early in his career in neuropathology and over the years he accumulated and studied a remarkably large number of anatomic specimens with this lesion. for those with interest in spinal cord injury, sir ludwig guttmann is considered to be a towering figure in the history of its treatment. guttmann was an early pioneer in changing the clinical outcome for spinal cord injury patients and, among other things, introduced sports participation in their rehabilitation process. perhaps the foremost acknowledgment of one’s standing in the field of spinal cord research is to be asked to give the ludwig guttmann memorial lecture at the annual meeting of the international spinal cord society. in september, 2004, byron kakulas gave the guttmann lecture entitled: “neuropathology: the foundation for new treatments in spinal cord injury”.61 we believe he is the only neuropathologist to be so honored. his lecture has been published and remains a remarkable document, summarizing his findings and thoughts on the subject based on examining a total of 588 spinal cord injury cases! for those involved in studies of spinal cord injury who have not read this paper (and his others on the subject), it is highly recommended and shows professor kakulas’ immense talents as a neuropathologist, and his depth of knowledge and understanding of the subject. it is truly a classic paper and as he clearly demonstrates: “in the context of finding a cure for spinal cord injury, the first and foremost requirement is an in-depth knowledge of the disorder in neuropathological terms with the complexity of the spinal cord appreciated.” to read further on professor kakulas’ life and career in neuropathology, the reader is referred to his obituary published in free neuropathology (see https://doi.org/10.17879 /freeneuropathology-2023-4820).62 byron kakulas was a superb neuropathologist, a very creative scientist, a charismatic mentor to many, and a man who was devoted to his greek heritage and to his loving extended family. he was also a cherished friend of one of the authors (dpp, figure 3). he will be greatly missed. figure 3. in memory of dr. byron kakulas dr. kakulas (left) can be seen here along with his wife, valerie (middle), and dr. daniel perl (right). conflicts of interest statement: the authors do not have any conflict of interest to declare. funding statement the work of the authors is supported by the dod/usu brain tissue repository and neuropathology program, usu award #hu00012120007 (hjf award# 312159-1.00-66531). disclaimer the information/content, conclusions, and/or opinions expressed herein do not necessarily represent the official position or policy of, nor should any official endorsement be inferred on the part of, uniformed services university, the department of defense, united states uniformed services, or the united states government. https://doi.org/10.17879/freeneuropathology-2024-5849 https://doi.org/10.17879/freeneuropathology-2023-4820 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https://doi.org/10.1016/j.brainres.2012.12.038 https://doi.org/10.1093/mtomcs/mfac092 https://doi.org/10.1039/c5mt00234f https://doi.org/10.1186/s40478-023-01603-z https://doi.org/10.1093/milmed/usab394 https://doi.org/10.3390/ijerph182412925 https://doi.org/10.1161/strokeaha.108.534404 https://doi.org/10.1186/s40478-023-01636-4 https://time.com/4358063/muhammad-ali-dead-parkinsons-boxing/ https://doi.org/10.1001/jamaneurol.2022.3584 https://doi.org/10.1101/cshperspect.a024059 https://doi.org/10.1038/sj.sc.3101670 https://doi.org/10.17879/freeneuropathology-2023-4820 review introduction the neuropathology of intimate partner violence cte in young contact sports athletes evidence of vascular injury and remodeling at sulcal depths in cte assessing multiple concurrent tauopathies in the setting of cte: a way forward? the relationship between tbi, cte, and tdp43 pathology repetitive tbi elicits pathology in c9orf72 transgenic mice patterns of head and neck injury in fatal underbody blast exposures chronically altered brain metal homeostasis following repetitive impact tbi blast exposure damages the neurovascular unit in mice muhammad ali had young-onset, idiopathic parkinson disease in memoriam: dr. byron a. kakulas conflicts of interest statement: funding statement disclaimer references neuroinflammatory mechanisms may help identify candidate biomarkers in chronic traumatic encephalopathy (cte) feel free to add comments by clicking these icons on the sidebar free neuropathology 6:15 (2025) review neuroinflammatory mechanisms may help identify candidate biomarkers in chronic traumatic encephalopathy (cte) guneet s. bindra1,2, shaheryar asad1, jean shanaa1, forshing lui1, andrew e. budson2,3,4, katherine w. turk2,3,4, jonathan d. cherry2,3,4,5,6 california northstate university college of medicine, elk grove, usa va boston healthcare system, boston, usa boston university alzheimer's disease and cte centers, boston university school of medicine, boston, usa department of neurology, boston university school of medicine, boston, usa department of pathology and laboratory medicine, boston university school of medicine, boston, usa department of anatomy and neurobiology, boston university school of medicine, boston, usa corresponding author: jonathan d. cherry · va boston healthcare system · 150 south huntington avenue · ma 02130 · boston · usa · jdcherry@bu.edu submitted: 14 march 2025 accepted: 29 june 2025 copyedited by: georg haase published: 14 july 2025 https://doi.org/10.17879/freeneuropathology-2025-6832 keywords: chronic traumatic encephalopathy, repetitive head impacts, traumatic brain injury, inflammatory signature, biomarkers, neuroinflammation abstract chronic traumatic encephalopathy (cte) is a neurodegenerative disease that can only be diagnosed post-mortem via pathological autopsy. the primary risk factor for cte is a history of repetitive head impacts (rhi) received through contact sports including american football, hockey or soccer, military-related head injuries, or intimate partner violence. recent findings have demonstrated that neuroinflammation is a critical compo-nent of early cte pathogenesis and is likely part of the mechanism driving disease onset and progression. additionally, the innate specificity, or ‘signature’, of a neuroinflammatory response may function as a dis-ease-specific marker for various neurodegenerative conditions. this would suggest an enormous repository of novel cte biomarker candidates to be added to ongoing clinical trials, helping bolster diagnosis. however, few studies have truly leveraged immune mediators as candidate cte markers. in this review, we argue and provide support that inflammatory mechanisms could serve as a viable source for novel biomarkers that are specific to cte pathol-ogy. this includes an evaluation of inflammatory or damage-related markers such as ccl11 (c-c motif chem-okine ligand 11, also known as eotaxin-1), ccl21 (c-c motif chemokine ligand 21) and gfap (glial fibrillary acidic protein). we discuss the neuroinflammatory responses that give rise to these biomarkers in addition to the advantages and limitations of using each to diagnose cte with particular attention to sensitivity and specifici-ty. although further research is necessary to validate immune mediators, the latter show promise as diagnos-tic biomarkers for cte and may also eventually serve as therapeutic targets for mitigating chronic inflamma-tion in at-risk populations. abbreviations ad alzheimer’s disease, bbb blood-brain barrier, ccl2 chemokine ligand 2, ccl11 eotaxin-1, cns central nervous system, crp c-reactive protein, csf cerebrospinal fluid, cte chronic traumatic encephalopathy, damp damage-associated molecular pattern, dce-mri dynamic contrast-enhanced magnetic resonance imaging, dti diffusion tensor imaging, elisa enzyme-linked immunosorbent assay, icam1 intercellular adhesion molecule-1, fdg – fluorodeoxyglucose, ftd frontotemporal dementia, gfap glial fibrillary acidic protein, hmgb1 high mobility group box protein, nbd neurobehavioral dysregulation, nfl neurofilament light chain, opc oligodendrocyte precursor cells, p-tau phosphorylated tau, rhi repetitive head impacts, tbi traumatic brain injury, tes traumatic encephalopathy syndrome, tnf-α tumor necrosis factor-alpha, tspo translocator protein, trem2 triggering receptor expressed on myeloid cells, vcam1 vascular cell adhesion molecule-1, vegf vasoactive endothelial growth factor introduction chronic traumatic encephalopathy (cte) is a progressive neuropathological disease characterized by the perivascular accumulation of phosphorylated tau (p-tau) at the depths of cerebral sulci: (1) the largest risk factor for cte is exposure to repetitive head impacts (rhi), whether concussive or non-concussive; (2) cte pathology has been confirmed in patients with various rhi exposures including contact sports such as american football, hockey, rugby, boxing, and soccer (1,3,4); (3) although sports-related head trauma has been recognized as the primary source of cte cases in the literature, other types of exposure have been shown to be involved as well. there is emerging evidence of cte cases in military veterans. premier et al. demonstrated ten out of 225 (4.4 %) veteran brains had evidence of cte (5). although all ten of the veterans were found to also play a contact sports, other studies have observed cte in veterans without a sports background (6–9); (4) there has also been concern for cte in persons who experience long-term domestic violence. dams-o’connor et al. examined tissue from individuals exposed to intimate partner violence and did not observe cte in their cohort (10); however, other case series and autopsy studies have identified cte in individuals exposed to domestic abuse, suggesting that while less common, such exposures might still confer risk (11,12). overall, while the incidence of cte is likely much lower in exposures outside of contact sports, the risks are still not zero. however, a major limitation in our understanding of at-risk populations is that cte can only be diagnosed postmortem. although provisional in-life criteria have been suggested, coining the term traumatic encephalopathy syndrome (tes), they has not been fully validated and still remain research criteria (3). to date, there is no way to predict if rhi-exposed individuals will develop cte years after exposure, since not all individuals with rhi exposure go on to acquire cte (13,14). therefore, there is great need for biomarkers to advance ante-mortem diagnosis of cte. the current focus around cte biomarkers has primarily centered on structural protein markers, including tau proteins, neurofilament light protein (nfl) and glial fibrillary acidic protein (gfap) (15–18). despite some promising evidence, these proteins are often associated with other neurodegenerative conditions or acute injuries, hindering their specificity to cte (19,20). although certain phosphor epitopes of tau may be useful, tau markers may not generally be ideal for distinguishing between alzheimer’s disease (ad) and cte, as both are tauopathies (21). currently, no adequate biomarkers exist to differentiate cte from other forms of neurodegeneration. interestingly, recent studies have suggested that the neuroimmune response might be distinct among related tauopathies (22). these findings, coupled with the advent of high-level genomic techniques like single cell rna sequencing, have highlighted the enormous complexity and flexibility of the immune response to repetitive head trauma. it may therefore be useful to take advantage of this characteristic to identify novel sensitive and specific biomarkers that could effectively diagnose cte in life. herein we discuss neuroinflammation in cte and examine specific inflammatory processes that may involve prospective novel biomarkers. inflammation is strongly associated with cte increasing evidence supports a role for inflammation as an early modifier or initiator of cte disease progression, consistent with inflammation being a mechanistic driver of other neurodegenerative conditions. for instance, genomic studies have identified risk-stratifying immune-related loci in relation to ad, such as trem2 (triggering receptor expressed on myeloid cells 2) and cd33 (cluster of differentiation 33), (23,24), while tmem106b (triggering receptor expressed on myeloid cells 106b) and lrrk2 (leucine-rich repeat kinase 2) have been detected as immune-related variants affecting pathology in frontotemporal dementia (ftd) (25,26) and parkinson’s disease (27,28), respectively. in fact, based on post-mortem genomic analysis of cerebellum tissue, bieniek et al. reported that donors with rhi exposure and cte diagnosis at autopsy tended to have a slight increase in the mapt h1 haplotype, with less homozygous genotypes of the tmem106b rs3173615 minor allele compared to non-cte controls. furthermore, immune signaling, such as through cytokine release from activated microglia, has been reported to promote tau hyperphosphorylation and to impair amyloid-β clearance (29–31). although the absence of a validated longitudinal or animal model limits the ability to experimentally demonstrate that cte follows a similar pattern as other neurodegenerative states, post-mortem transcriptomic and computational analyses offer evidence that inflammation precedes pathology in cte. in a large-scale mrna-sequencing analysis of post-mortem cte, labadorf et al. reported upregulation of genes related to cytokine signaling, immune cell migration, and apoptosis in late stage cte, while these same categories had inverse or reduced expression in early stage cte. this suggests that early inflammatory responses could have a distinct mechanistic mechanistic function separate from the response to tauopathy. notably, activation of immune pathways in early stages of cte was positively correlated with duration of rhi exposure, suggesting inflammation as an initiating factor for cte pathogenesis (32). similarly, cherry et al. found that in post-mortem dorsolateral frontal cortex from donors with early stage cte, immune-related genes were upregulated in sulci compared to neighboring gyri compared to rhi donors without cte. these sulcal alterations in immune gene expression were also associated with rhi history and did not exclusively co-occur with pathology, further suggesting inflammation to occur upstream of tau deposition (33). additional support comes from a recent study by butler et al. who observed that in individuals under the age of 50 years with exposure to contact sports, cases that had exposure to rhi but no cte pathology still had significant neuroinflammatory changes compared to control cases. interestingly, the observed inflammatory changes were similar to those seen in cases with early stage cte, suggesting that inflammation precedes the deposition of p-tau (34). in addition, pet imaging of translocator protein (tspo), which is expressed in the mitochondria of various neural cells (i.e., microglia, endothelial cells, astrocytes), showed increased tspo in regions including right amygdala and bilateral supramarginal gyri for rhi-exposed football players (35,36). when examining tspo via post-mortem immunoassay, varlow et al. reported that despite the difference in tspo density between cte and controls not reaching statistical significance, there was a general trend of increased expression in cte (37). taken together, these findings highlight that inflammation has a critical and variable role across cte pathogenesis. future studies that more precisely target these inflammatory processes and aim to better characterize the cte immune signature may offer clearer insight into disease progression during life. this would consequently serve as a promising avenue for identifying cte-specific biomarkers. candidate neuroinflammatory markers for cte from the immune signature of cte, candidate markers may emerge allowing distinction of cte from other neurodegenerative conditions. considering the preliminary evidence that inflammation precedes and contributes to cte pathology, it could be worthwhile to evaluate proteins involved in the cte immune profile for biomarker development. here, we discuss current prospective neuroimmune biomarkers primarily derived from studies using postmortem fluids in neuropathologically confirmed cte or in fluids from living individuals with a history of playing contact sports. as cte can only be diagnosed after death, the postmortem studies are the only way to directly link a protein to confirmed cte. however, as past work has demonstrated a strong correlation between more years of contact sports play and elevated risk of cte (4), the prospective human studies offer additional insight into other targets that are likely to be involved in the cte process, pending neuropathologic follow ups. prospective markers are summarized in table 1. table 1: overview of human-based studies on prospective immune markers study immune protein(s) study design study populations and sample sizes significant findings cherry et al. 2017 (52) ccl11 elisa of postmortem dorsolateral frontal cortex (dlfc) and csf analysis 23 cte, 50 ad, and 18 non-athlete controls. all were neuropathologically confirmed. ccl11 was elevated in dlfc of cte compared to ad and controls. same results seen upon csf analysis. receiver operator characteristics (roc) curve analysis showed specificity of ccl11 to cte. ccl11 correlated with rhi exposure duration. cherry et al. 2022 (22) ccl21 ccl11 cxcl5 cxcl13 gmcsf ccl17 multiplex elisa of postmortem anterior cingulate grey matter and csf analysis 40 cte, 28 ad, 20 progressive supranuclear palsy, 20 corticobasal degeneration, 19 argyrophilic grain disease. all were neuropathologically confirmed. from 71 immune proteins, ccl21 had the strongest correlation with cte. cxcl5, cxcl13, gmcsf, and ccl17 had significant association with cte based on roc analysis. csf analysis showed ccl21 to be more significantly increased in cte samples than in ad samples. vig et al. 2023 (59) ccl11 immunoassay of postmortem vitreous humor 15 cte, 7 ad, 10 with both ad and cte, 9 controls. all were neuropathologically confirmed. ccl11 levels trended towards significance only in samples with both ad and cte, compared to controls (p = 0.09). cherry et al. 2020 (41) ccl2 immunoassay and staining of postmortem dlfc and calcarine cortex 94 rhi-exposed (20 without cte, 27 low cte, 47 high cte); 112 ad (60 low, 28 intermediate, 24 high); 18 non-rhi controls. all neuropathologically confirmed. ccl2 correlated with increased cte severity (p < 0.001) and with increased football career length (p < 0.005). ccl2 has a correlation with p-tau, which was independent of aβ42-status or age. van amerongen et al. 2024 (66) il-6 csf analysis from retired football players 104 retired athletes with nbd diagnosis, 76 retired athletes without nbd diagnosis il-6 was significantly elevated in subjects with nbd diagnosis. il-6 levels correlated with various measures of nbd. gard et al. 2023 (103) il-2 il-15 tnf-α tnf-β eotaxin tarc vegf cxcl10 csf analysis from living athletes 24 symptomatic athletes with sports related concussions (src), 12 healthy controls. levels of il-2, il-15, tnf-α, tnf-β, eotaxin, and tarc were all elevated in src-exposed athletes compared to controls. vegf levels were significantly elevated in athletes with src history. cxcl10 was increased in athletes compared to controls. asken et al. 2023 (68) il-6 ifn-γ ykl-40 plasma analysis from living subjects 33 rhi/tes [11 aβ+, 22 aβ-], 62 ad (rhi-), 59 healthy controls (rhi-) rhi/tes had significantly higher il-6 concentrations compared to controls (effect size, d = 0.67), and ad participants (d = 0.68). aβrhi/tes had significantly higher il-6 compared to aβ+ rhi (d = 1.2), ad (d = 1.1), or controls (d = 1.1). di battista, rhind, richards et al. 2016 (42) ccl2 ccl11 il-8, il-12 il-15, il-4, il-10, il-13 immunoassay of blood from living athletes 87 college-level athletes including football, field hockey, rugby, basketball, and baseball. of these, 40 participated in collision sports. ccl2, ccl11, il-8, il-12, il-15, were quantifiable in majority of participants, ranging from 77 % to 100 %. begum et al. 2020 (43) ccl2 tnfsf14 cx3cl1 il-7 plasma analysis of living professional rugby players 18 athletes with single concussion, 5 who were repetitively-concussed, 12 healthy controls reduced ccl2 was associated with severity of symptoms (p = 0.043) and increase in number of symptoms (p = 0.013). tnfsf14 levels were reduced in concussed subjects compared to repetitively-concussed subjects. il-7 levels were higher in repetitivelyconcussed subjects compared to concussed subjects. cxcl3 was increased less than one week after concussion. huibregtse et al. 2020 (54) ccl11 il-10 plasma analysis of 39 living soccer players with heading experience 22 soccer players who underwent a repetitive-heading event, and 17 soccer players who underwent a repetitive-kicking event no significant increase in ccl11 occurred after repeated-heading event. increases in ccl11 were significantly correlated with years of heading experience (p = 0.01). miner et al. 2024 (74) il-6 plasma analysis of living subjects 180 former football players, 60 asymptomatic unexposed male subjects il-6 levels did not significantly differ between rhi-exposed symptomatic subjects and unexposed asymptomatic controls. nitta et al. 2019 (67) il-6 serum analysis of football players 857 high school and collegiate football players il-6 levels increased within six hours after single concussion. levels of il-6 at six hours after concussion were associated with duration of symptoms (p = 0.031). alosco et al. 2018 (15) strem2 immunoassay of csf from former nfl players 68 former nfl players, 21 non-rhi controls levels of strem2 were significantly associated with t-tau. strem2 strengthened the relation between amount of rhi exposure and t-tau levels asken et al. 2022 (84) nfl gfap ante-mortem plasma analysis from rhi-exposed subjects, with postmortem evaluation 9 rhi-exposed subjects (5 with confirmed cte). plasma levels of gfap generally showed a longitudinal increase. baseline levels of gfap were higher for rhi-exposed subjects compared to healthy controls. bernick et al. 2023 (80) nfl gfap plasma analysis from retired athletes, active athletes, and non-rhi controls 211 active martial arts fighters, 140 active boxers, 69 retired boxers, 52 controls gfap levels correlated with cortical and sub-cortical atrophy on mri, and lower cognitive scores, for retired boxers. gfap elevation correlated with decreased corpus callosum and thalamic volumes on mri for active boxers. gfap levels were highest among retired boxers compared to active boxers, while nfl levels were highest among active boxers compared to mma fighters. shahim et al. 2022 (83) gfap plasma and csf analysis of living, symptomatic athletes 28 rhi-exposed professional athletes with persistent postconcussive symptoms, 19 age-matched unexposed controls plasma gfap moderately correlated with csf gfap (r = 0.45, p = 0.02) bazarian et al. 2024 (81) gfap plasma analysis of living football players 30 collegiate football players gfap increased from preto post-game, from 79.69 pg/ml to 91.95 pg/ml (p = 0.008), then to 99.21 pg/ml (p < 0.001) post-game gfap changes correlated with reduced functional anisotropy in right fornix (r = -0.59), and adjusted correlations with head impact metrics (r = 0.69–0.74). huibregtse et al. 2023 (82) gfap serum analysis of female water polo players 22 female collegiate water polo players gfap increased from week 1 to week 8 of preseason (p = 0.002) cte, chronic traumatic encephalopathy; tes, traumatic encephalopathy syndrome; ad, alzheimer’s disease; rhi, repetitive head impacts; dlfc, dorsolateral frontal cortex; csf, cerebrospinal fluid; nbd, neurobehavioral dysregulation; src, sports-related concussion. ccl2 ccl2 is a chemokine involved in regulating monocyte infiltration through the blood-brain barrier (bbb) in response to trauma, and it may be involved in inducing transcriptional alterations in microglia (38). ccr2 deletion or deficiency can also restrict the degree of cognitive dysfunction after tbi (39,40). despite a general association of ccl2 with tau accumulation as seen in various tauopathies, cherry et al. reported evidence of ccl2 having a vital role in mediating cte pathogenesis through microglial activation. by immunoassay, ccl2 in the dorsolateral frontal cortex was found to be elevated in both low and high-stage cte compared to controls, correlating with severity of cte pathology. notably, ccl2 levels correlated with football career length, suggesting a link between ccl2 signaling and rhi exposure duration (41). in fact, the correlation between ccl2 and p-tau, found in both cte and ad groups, was independent of aβ42 (amyloid beta 1-42) levels (41). thus, it is possible that the tauopathy in cte is more specifically driven by ccl2. this is supported by the finding that ccl2 was significantly correlated with the density of iba1(ionized calcium-binding adapter molecule 1) positive cells, which itself was found to be associated with the pathognomonic lesion of perivascular tau (41). support for ccl2 as a marker of head trauma also comes from other studies. in a blood-based biomarker analysis of university athletes from various contact sports at the beginning of competitive season, female athletes with prior exposure to multiple concussions had elevated serum levels of ccl2 compared to non-exposed controls. in fact, out of 39 markers including il-1β, il-6, ifn-γ, il-10, and tnf-α, ccl2 was the only protein that exhibited a significant relationship with multiple concussion exposure in this group (42). interestingly, begum et al. reported reduced ccl2 serum levels to be associated with an increased number and severity of post-concussive symptoms in athletes (43). however, ccl2 is also strongly associated with ad pathology (44–46), in addition to multiple sclerosis (47) and stroke (48,49). consequently, while ccl2 may not have the specificity to cte that would make it a viable biomarker, its demonstrated involvement in pathogenesis and possible sensitivity to cte suggests that ccl2 could instead have value as a marker of disease progression. this is supported by the findings of a stepwise increase across rhi exposure and cte staging continuum, although more evidence is required to validate these results. because of the role of ccl2 in microglial recruitment and subsequent tauopathy, regulation of this signaling pathway may eventually serve as a therapeutic target in order to minimize the degree of chronic inflammation. however, future investigations should still aim to better characterize the precise involvement of this signaling pathway in the cte immune signature. ccl11 ccl11, or eotaxin-1, is a chemokine that is involved in recruiting eosinophils to sites of inflammation to trigger immune responses (50). due to its ability to cross the blood-brain barrier, ccl11 can directly contribute to neuroinflammatory mechanisms. in fact, neurons, microglia, and other cns cell types are known to express receptors for ccl11 (51). in a study that used enzyme-linked immunosorbent assay (elisa) to assess csf levels of ccl11 in brain tissue from football players, cherry et al. found a significantly elevated level of ccl11 in the 23 cte-confirmed subjects compared to the 50 ad-confirmed subjects and 18 non-athlete control subjects. of note, the authors determined a significant correlation between ccl11 levels in cte subjects and football career length. length of rhi exposure and degree of tauopathy in the dorsolateral frontal cortex were more predictive of cte diagnosis compared to age (52). however, this study did not include rhi-exposed subjects without cte; therefore, it is unclear at this point whether ccl11 reflects cte or rhi exposure independent of disease status. furthermore, no notable increase in csf levels of ccl11 among the ad subjects was determined, which corroborates results from other studies (46,53). this would support ccl11 as a promising biomarker that may help differentiate cte from other pathologies (52). ccl11 has been observed in the context of sports injury in other studies as well. in the study from di battista et al., ccl11 was quantified in serum samples from college-level athletes with and without a history of multiple concussions. although this chemokine was detected, there were no significant differences in ccl11 levels between those with more than three prior concussions, and those with fewer or none, suggesting that peripheral ccl11 is not persistently elevated in young, asymptomatic athletes despite concussion exposure. additionally, correlation analyses showed no significant associations between ccl11 levels and time since last concussion, number of prior concussions, or self-reported symptoms.(42). in soccer players, huibregtse et al. (2020) investigated acute changes in ccl11 following repeated heading events, finding no significant increases in plasma levels after ten head traumas. despite no group-level acute effects from intervention, exposure duration reportedly contributed to individualized differences in ccl11 within the head trauma group. although not elevated from baseline, plasma ccl11 levels at 24 hours post-exposure were positively associated with years of heading experience, with an estimated 2.0 pg/ml increase per year of exposure. this suggests a unique sensitivity to cumulative rather than acute rhi exposure (54). however, there are several challenges with using ccl11 as a biomarker for cte. while some studies have observed more specific expression in the context of head trauma, other studies have demonstrated elevated levels in ad, huntington’s disease, and stroke (55–57). additionally, ccl11 has also been implicated in normal aging, which complicates its use in older subjects (58). furthermore, some studies have not observed increases in ccl11 in the context of cte. in a post-mortem analysis of vitreous humor, samples from cte subjects did not show significantly elevated levels of ccl11 compared to ad or healthy controls, nor were there differences in ccl11 levels across cte stages (59). there was only a significant increase of ccl11 in samples from subjects with both ad and cte pathology when compared to controls, suggesting possible synergistic effects when cte co-occurs with other neurodegenerative pathologies. although these results were found in the context of vitreous humor, it will be important to validate ccl11 levels in csf or plasma in more independent cohorts. ccl21 ccl21 or 6ckine, is a chemokine involved in mediating the migration of ccr7-expressing immune cells i.e., t-cells and dendritic cells to lymphoid organs. in the cns, ccl21 may be upregulated, facilitating lymphocyte migration through the bbb (60). using an elisa panel of 71 immune proteins to examine anterior cingulate grey matter samples from patients with various tauopathies, cherry et al. determined that ccl21 was most strongly correlated with the cte-confirmed cases, suggesting some specificity to cte pathogenesis (22). cherry et al. also examined ccl21 levels between cte-confirmed and ad-confirmed subjects via post-mortem csf analysis, showing that there was a significant elevation of ccl21 levels in ad (22). an important caveat of this study was that it only assessed the difference between different types of tauopathies and did not include non-disease control cases. however, the findings suggest ccl21 might be useful to help better segregate related neuropathologies. elevated ccl21 levels may not only assist with determining suspected cte, but can also be a point of future investigation in determining how the pathophysiology of cte might differ from mechanisms seen in other tauopathies. given its known role in trafficking immune cells across the bbb to propagate inflammation, in addition to its demonstrated specificity to cte, ccl21 may contribute directly to early immune activation and subsequent cte pathogenesis. as such, future research should investigate whether ccl21 expression is elevated during early stages of cte, as this could clarify its role in initiating or sustaining pathology. there are sparse studies examining ccl21 in the specific context of head trauma, especially in humans. in fact, the existing neurotrauma data on ccl21 primarily stems from animal model studies on spinal cord injury (sci) (61,62). of note, one human-based study on sci exists to date, reporting a negative correlation between serum levels of ccl21 and neuropsychological test scores in sci patients, suggesting ccl21 as a possible correlate for post-injury cognitive dysfunction (63). consequently, this gap in the literature highlights the need for future studies assessing bio-fluid levels of ccl21 in relation to head trauma, particularly in living subjects with rhi exposure and during post-mortem evaluation. il-6 il-6 is a multifunctional cytokine critically involved in the neuroinflammatory cascade. in the cns, il-6 is produced primarily by activated microglia and astrocytes following trauma, where it promotes leukocyte migration and cytokine signaling (64,65). a recent investigation used a csf immunoassay on retired football players for inflammatory proteins including il-1β, il-6, il-8, il-10, tnf-α, and c-reactive protein. of these markers, only il-6 was significantly elevated in subjects who had neurobehavioral dysregulation (nbd), compared to retired players without an nbd diagnosis (66). this is noteworthy since nbd, referring to behavioral changes and dysregulation of emotion, is a prominent clinical finding in the proposed criteria for tes (traumatic encephalopathy syndrome). van amerongen et al. found il-6 to be correlated with the overall nbd score, as well as with subdomains including impulsivity, emotional dysregulation, and affective lability. interestingly, il-6 appeared to have a selective relationship with certain behavioral traits, as it did not correlate with the explosiveness domain of nbd or with measures of cognitive performance. none of the tested markers in the csf, including il-6, were associated with rhi proxies i.e., football career length (66). this points to il-6 as a potential sensitive marker, particularly compared to other inflammatory cytokines, for monitoring progression of nbd symptoms in those persons at risk for cte. similarly, serum levels of il-6 were found to correlate with clinical symptom severity after a sports-related concussion, as it remained elevated at 14 days post-injury (67). additionally, il-6 levels in the plasma were significantly elevated in rhi/tes patients compared to healthy controls and ad patients without rhi exposure. furthermore, rhi/tes patients who were negative for aβ-pet negative, i.e. negative for aβ in positron-emission tomography, still had significantly higher il-6 levels than rhi/tes patients with aβ+ status and all other groups, suggesting that il-6 elevation is more reflective of chronic rhi-induced inflammation than of any potential co-morbid alzheimer’s pathology (68). while tes criteria have not been pathologically validated, these findings suggest that il-6 might play a role in neuropsychiatric symptoms observed in patients with rhi exposure, including those who may eventually develop cte. however, il-6 plasma levels have also been previously linked to disinhibition in frontotemporal dementia (ftd) (69), apathy in ad (70,71), and manic symptoms in bipolar disorder (72). thus, il-6 may non-specifically contribute to nbd symptoms in various neurologic or psychiatric conditions, including cte. future investigations could benefit from directly assessing il-6 as a possible pathological correlate for nbd in cte, such as by comparing postmortem findings with retrospective review of clinical symptoms. however, not all studies have found success with il-6 as an rhi-related biomarker. parkin et al conducted an immunoassay of blood-based immune markers up to 3 months after pediatric concussion, observing that patients with normal recovery exhibited the same gradual decrease in il-6 expression as patients with persisting symptoms showing concentration difficulty as well as cognitive and behavioral deficits (73). that said, the symptom categories in this study were broadly defined and likely included general post-concussive complaints like dizziness and headache, rather than specific components of nbd. non-specific symptom classification may have obscured potential relationships between il-6 and behavioral phenotypes when considering the findings from van amerongen et al. that suggest il-6 to have a selective association with nbd subdomains (74). additionally, il-6 plasma levels in former football athletes did not significantly differ between rhi-exposed symptomatic subjects and unexposed asymptomatic controls. furthermore, il-6 plasma levels were not significantly associated with rhi proxies including years of play and age of first exposure (74). to address uncertainty regarding il-6 as a viable marker for symptomology, future examinations should longitudinally assess il-6 in rhi-exposed subjects alongside clinical phenotypes, aiming to determine if il-6 levels reliably track symptom onset, persist independently of clinical progression, or reflect non-specific post-traumatic inflammation. gfap considering the role of astrocytes in facilitating neuroinflammation, some studies have assessed gfap in relation to rhi. gfap is a filament protein in the cytoskeleton of astrocytes and provides structural support (75). since gfap is upregulated during astrogliosis, elevated gfap levels may indicate a neuroinflammatory response, or an injury to astrocytes (76). gfap is also a potential predictor of concussion status or increased rhi severity, albeit more-so in an acute post-tbi timeframe (77,78). although postmortem studies provide evidence of altered gfap expression in cte (16,79), fluid biomarker studies in pathologically confirmed cte are rather limited. however, gfap elevations have been observed in rhi-exposed populations who may be susceptible to cte. in a longitudinal cohort study of professional boxers and mixed martial art fighters, bernick et al. (2023) measured plasma levels of gfap annually over one to four years of follow-up, examining the association of gfap levels with structural mri data and cognitive outcomes. longitudinal increases in gfap levels were significantly associated with decreased volume of certain brain regions on mri, including hippocampus and thalamus, in addition to enlarged lateral ventricles. these structural changes also correlated with declines in processing speed, memory, and reaction time, suggesting that gfap is a viable marker for long-term neurodegenerative progression in rhi-exposed populations (80). additional studies further support the relevance of gfap as a marker of astroglial activation following rhi. in a study from bazarian et al., serum gfap was measured in collegiate football players before, immediately after, and 45 minutes post-game. although no athletes sustained a diagnosed concussion, gfap levels increased significantly after the game, with magnitudes of increase correlating with both helmet-recorded head impact exposure and reduced white matter integrity measured via diffusion tensor imaging. these associations remained significant even after adjusting for physical exertion, suggesting that acute gfap elevations may reflect subclinical astrocytic injury related to repetitive, non-concussive impacts sustained during a single game (81). furthermore, in a study on collegiate women’s water polo players, serum gfap concentrations were assessed over an eight-week preseason period. the authors reported a significant linear increase in gfap over the study period, while no acute changes in gfap were observed following scrimmages, suggesting that astroglial activation may accumulate over time even in the absence of concussion diagnoses. however, this longitudinal gfap increase was not significantly associated with cumulative head impact burden calculated based on acceleration metrics from instrumented mouth guards (82). these findings support the notion that while gfap can increase longitudinally in rhi-exposed populations, the utility of gfap as a specific marker of head impact burden, particularly in subconcussive settings, may be limited. shahim et al. evaluated csf and plasma levels of gfap in a cohort of rhi-exposed professional athletes with persistent symptoms. plasma gfap levels showed a moderate correlation with csf gfap levels, but plasma levels did not significantly correlate to symptom severity or number of prior concussions. in addition, serum gfap levels did not significantly differ between rhi-exposed subjects and unexposed controls, and serum gfap levels did not relate to bbb integrity measured via the csf/serum albumin ratio. thus, gfap may have limited utility in the later stages of post-rhi neurodegeneration long after initial exposure, suggesting a temporal window where astrocytic markers could be more sensitive to injury (83). as such, future studies may aim to better characterize the long-term trajectory of serum gfap from rhi exposure to post-mortem evaluation. furthermore, asken et al. (2022) examined plasma gfap levels in a clinicopathological cohort of nine rhi-exposed individuals who were followed to autopsy. three out of five subjects with longitudinal data had a steady increase in gfap concentration over time, ranging from two to seven years, in addition to having higher levels at baseline compared to healthy controls. among the five cases with autopsy-confirmed cte, elevated gfap levels tended to co-occur with imaging evidence of medial temporal atrophy and cognitive decline. cte was the primary neuropathological diagnosis in two cases in which persistently high gfap levels were observed. while these findings suggest a possible association between gfap elevation and cte pathology, the presence of frequent co-pathologies such as ad or tdp-43 proteinopathy, underscores that gfap is not a specific marker of cte and may instead be implemented as a sensitive marker for astrocytic activation in the context of neurodegeneration (84). taken together, these findings underscore that while gfap may be a sensitive indicator of acute astroglial responses to rhi, including subconcussive impacts, it could be subject to temporal and individual variability. although promising, the utility of gfap as a long-term biomarker of neurodegenerative risk will likely require longitudinal tracking and integration with symptom trajectories and other fluid markers to improve specificity for cte. additional prospective markers of cte the above mentioned neuroinflammatory candidate markers have been examined to a limited extent for their application to cte pathology. in addition, there are several other immune mediators that may be considered for future post-mortem investigations. in general, these immune proteins have been primarily assessed through mouse models, as well as in rhi-exposed living subjects for some cases. yet, there is limited understanding of how exactly they fit into the signature for cte. future investigations could assess the specificity of these markers to cte by conducting comprehensive panels of immune proteins in post-mortem tissue. it would be particularly beneficial to directly compare csf or serum levels of these potential markers between cte and other neurodegenerative diseases that may result from head trauma i.e. ad. the prospective markers are discussed below, with pertinent findings from human-based studies also included in table 1. because of the limited literature of these markers, we propose novel proteins that could be worth examining, as the latter have not yet been applied to rhi at the human level or to post-mortem cte. strem2 based on the vital role of microgliosis in neuroinflammation, microglial markers may have some viability as diagnostic or therapeutic targets for cte, such as soluble triggering receptor expressed on myeloid cells 2 (strem2). this receptor, upregulated in activated microglia, has been implicated in various neurodegenerative diseases due to its role in regulating microglial survival, proliferation, and phagocytosis (85). it is worth noting that triggering receptor expressed on myeloid cells (trem2) refers to a transmembrane protein found in microglia which is involved in a variety of functions, such as facilitating microgliosis and regulating lipid metabolism (86). proteolysis leads to release of strem2 in csf. the protein strem2 is generally shown to be associated with tauopathy and has been primarily studied in the context of ad, but its overall function is less understood (87). by analyzing csf samples from rhi-exposed retired nfl players, alosco et al. determined that levels of strem2 significantly correlated with the amount of total tau (t-tau) in the rhi-exposed group, despite the absence of group-level differences in strem2 levels between the athletes and control subjects (15). despite an insignificant association between strem2 and cumulative rhi exposure, regression analysis showed that strem2 levels increased the strength of association between rhi exposure and t-tau levels (15). this suggests some early involvement of strem2 in facilitating cte pathology in response to rhi. a potential limitation to implementing strem2 as a marker for cte is the extensive prior evidence of strem2 having a strong association with ad pathology (88–90). consequently, future investigations may focus on assessing sensitivity and specificity of strem2 or other potential microglial markers in cte in comparison to cases of ad or other tauopathies. pro-inflammatory mediators in addition to il-6, other pro-inflammatory cytokines related to head impact exposure may serve as potential markers of cte progression. gard et al. found that among athletes with prior exposure to sports-related concussions, csf analysis revealed a significant increase in il-2, il-15, tnf-α, tnf-β, eotaxin, and tarc levels compared to those in control athletes. it is possible that these protein levels increase with post-concussive symptoms, suggesting a possible role of these cytokines in early phases of injury-related pathology (104). however, considering that the study included healthy controls without ad for comparison, it remains unclear whether these elevations represent acute or persistent changes. notably, other studies have reported more limited associations between cytokines level and rhi exposure. in one blood-sample analysis of college-level athletes, there was no significant correlation between participation in collision sports and levels of il-8, il-12, il-15, or tnf-α. in fact, only peripheral levels of tau were significantly associated with participation in collision sports, suggesting that the various cytokines may not show consistent peripheral elevation in response to trauma (42). in addition, van amerongen et al found no significant association between csf levels of tnf-α and il-1β and nbd scores in retired football players, suggesting that these markers may not be related to clinical symptom expression (66). plasma levels of ifn-γ were also not found to be significantly increased in living subjects with rhi/tes, when compared to ad and controls without rhi exposure (68). while not directly applicable to cte due to the lack of validation of tes to pathology, these findings suggest that cytokine expression following head trauma may be context-dependent, varying by injury time course, sampling method, and clinical phenotype. begum et al. conducted a serum-based comparison of inflammatory markers between athletes after single concussion and athletes who were repetitively concussed. the authors found that serum levels of tnfsf14, belonging to the tnf superfamily, and il-7 were significantly higher in repetitively concussed players than in players after single concussion, suggesting that the two proteins may reflect inflammatory responses to repeated injury (43). these findings offer preliminary support to use tnfsf14 and il-7 in order to distinguish immune profiles between single tbi and rhi. while the study defined “repetitively concussed” as two concussions within the span of three months, which may not fully represent the accumulation of minor trauma seen in rhi, the observed differences still point toward an immunological distinction between acute and cumulative injury states. as such, future studies may benefit from determining how tnfs14 and il-7 might correlate with proxies of rhi. other pro-inflammatory aspects of the cte immune signature that have not yet been explored in cte include damps (damage-associated molecular pattern) such as high mobility group box protein 1 (hmgb1). hmgb1 is a dna binding protein that can be actively secreted by leukocytes in response to cytokine activation, or it may be passively released via neuronal death (91,92). in addition to being a marker of neurodegeneration, hmgb1 can act as a damp and mediate further inflammation (93). while hmgb1 overexpression is also common to conditions like epilepsy or ad, an analysis of various hmgb1 isoforms and their potential specificity for different neurodegenerative states could help clarify the immune signature for cte (94,95). likewise, heat shock proteins such as hsp70 or hsp90 are other proteins that are released during cell death and may trigger more inflammation by functioning as damps, thus possibly serving as a measure of cellular stress in response to post-rhi neuroinflammation (96,97). although hmgb1 and heat shocks proteins have yet to be studied in either rhi or cte, future investigations analyzing their precise role in the cte immune signature, such as through immunoassay or serum analysis, may help to clarify their usefulness as markers. furthermore, ykl-40 may be a reliable measure of astrocytic-mediated inflammation in suspected cte, since it has been shown to have upregulated expression by astrocytes and microglia during trauma, including in the chronic neuroinflammatory state (98). while asken et al. 2023 did not find a significant elevation of ykl-40 in living subjects with rhi exposure, it could be worth assessing this finding in the post-mortem setting (68). notably, csf levels ykl-40 have also been shown to be elevated in autopsy-confirmed ad, including some correlation with patterns of aβ deposition (99). thus, it may be relevant to conduct a comparison between cte and ad tissue, seeking to determine whether ykl-40 can help discriminate between those two pathologies. anti-inflammatory mediators the role of anti-inflammatory proteins in post-rhi immune processes is even less understood than that of pro-inflammatory cytokines. however, since anti-inflammatory proteins tend to resolve inflammation and to promote tissue repair, related investigations may help to elucidate some of the nuances in cte progression, including any immune-related mechanisms in response to secondary injury from inflammation. while several human studies have examined anti-inflammatory factors like il-10, il-4, and il-13, few correlations could be identified (54) (42). however, there have been reports in animal studies that tgf-β could be elevated six months after repetitive head trauma (100). overall, while there is less support for anti-inflammatory proteins as novel biomarkers for repetitive head trauma, more work is needed to better clarify targets. discussion and future directions emerging evidence points to immune mediators as more specific cte biomarkers. given that inflammation contributes to cte pathology, immune mediators that could be unique to cte’s inflammatory signature, such as ccl11 and ccl21, are worth further exploration (22,52). however, a definitive answer on the reliability of immune mediators depends on a few challenges and limitations that will first need to be addressed in future studies. one of the challenges is the need to account for the prolonged latent period between rhi exposure and cte symptom onset. the progressive nature of chronic inflammation in cte complicates the identification of immune markers that are involved over the course of disease development. additionally, promising markers like ccl11 and ccl21 have been primarily assessed post-mortem, making it difficult to translate these findings to living patients. an overarching challenge with developing ante-mortem biomarkers from immune mediators is that, currently, cte can only be definitively diagnosed post-mortem, with the 2021 consensus criteria subject to future refinement (101). as such, many of the studies on cte are essentially just a snapshot in time and are limited in addressing full “causation” of the target factor versus apparent correlation. to address this, future studies should track immune markers in living rhi-exposed individuals over time, followed by cte status confirmation via autopsy. this approach is essential not only for offering clearer insights into the temporal dynamics of the cte inflammatory profile but also for revealing additional novel biomarkers that distinguish cte from other diseases. prospective studies could measure general injury markers such as nfl or gfap in rhi-exposed living individuals to find subjects in the early stages of neurodegeneration who are not yet symptomatic. subsequently, there can be an assessment of immune profiles via csfor serum-based analysis during life, followed by pathological confirmation. this would allow researchers to assess whether fluid-based detection of immune proteins corresponds to confirmed cte pathology, helping bridge fluid markers to tissue-based diagnostic criteria. the overlap of some immune mediators between cte and other neurologic conditions such as aging or ad further complicates the potential of immune mediators as biomarkers for cte. for instance, cytokines like ccl11 and il-6 can be elevated in normal aging and in psychiatric disorders, respectively, limiting specificity to cte (58,69). however, despite lack of specificity for some cytokines to cte, implementing this approach may nevertheless allow researchers to identify changes in immune mediators across the disease course, including prior to symptom onset and early and late stage cte. inflammatory markers should be viewed as central though not sole components of a comprehensive cte biomarker profile. while they may not provide absolute specificity on their own, immune mediators may enhance the disease-stage resolution of diagnostic panels when integrated with other biomarkers such as tau isoforms or general injury markers. for example, while t-tau may not distinguish cte from other tauopathies, specific tau epitopes like p-tau202 and p-tau231 may be unique to cte and warrant further study in both living patients and in post-mortem comparisons with other neurodegenerative diseases (18,102). overall, continued validation of immune markers like ccl21 and their potential role in cte is needed through fluid-based analyses in both post-mortem and ante-mortem settings. in figure 1, we provide a schematic overview of the inflammatory pathways comprising the immune signature where prospective cte biomarkers might fit. 1) repeated head trauma damages vasculature, leading to increased bbb permeability and infiltration of lymphocytes and pro-inflammatory cytokines like il-1β and ifn-γ. at this point, ccl2 and ccl21 may be upregulated by endothelial cells. 2) microglia are activated by cytokines, proliferating into phenotypes including satellite, spp1+, and hifa+ microglia. microglia then secrete factors such as cx3cr1, tnf-α, and il-6 that propagate inflammation through stimulation of other neural cell types. microgliosis may correlate with candidate markers ccl2, ccl11, ccl21, and ykl-40. 3) astrocytes proliferate and release inflammatory cytokines e.g. il-1β, il-2, il-6, and il-15) that further activate microglia. 4) reactive astrocytes contribute to an increased bbb permeability, exacerbating inflammation. ykl-40, ccl2, and ccl11 may be released by activated astrocytes. 5) the neuron-microglial crosstalk occurs, where microglia damage neurons through cytokine release, and neurons in turn secrete mediators including ros that promote more microgliosis. markers associated with this event could include ccl2, ccl11, hmgb1, hsp, and cx3cr1. 6) next, p-tau is released from damaged neurons and accumulates. 7) tau deposition is then sensed by microglia which are further activated. 8) when damaged, oligodendrocytes might cause neuronal injury through secretion of cytokines such as il-1β, il-6, tnf-α, ccl2 and also axonal degeneration. while several of these processes might also be involved in other diseases or even in single tbi, the cellular arrangement, brain region affected, regional spread, timing, and age of onset can be distinct in the context of cte. these are important processes to consider when utilizing biomarkers to help discriminate and identify pathology. for example, although ccl2 might also be implicated in ad, elevated ccl2 levels found in individuals in their 30s may rather point towards cte, given that ad changes typically start around the age of 60. figure 1: schematic overview of the immune signature for cte inflammatory pathways contributing to the immune signature for cte are highlighted, including key cellular interactions and sources of potential biomarkers. the schematic depicts the sequential activation of endothelial cells, microglia, astrocytes, neurons, and oligodendrocytes, through which chronic inflammation and neurodegeneration is maintained. created with biorender. conclusion as it is the case in many neurodegenerative diseases, neuroinflammation has a critical role in cte pathogenesis and disease trajectory. as such, neuroinflammatory changes might be useful to foster biomarker development. in this review, we have highlighted several prospective fluidic neuroimmune biomarkers that might be useful to identify cte during life. ccl21, ccl11, ccl2, il-6, and gfap have all shown some promise but have yet to be fully validated. however, several limitations surrounding specificity arise as these factors are also involved in other neuropathologies. therefore, it is not likely that a single biomarker will be sufficient for accurate cte detection. in genetics, examining multiple genes together to identify “gene signatures” has shown to be a better method to identify complex changes and disease-specific effects. in that same line of ideas, it is likely that biomarker panels consisting of several neuroimmune proteins will be able to specifically identify diseases more efficiently as previously suggested (103). in addition, it will be useful to also include other variables such as clinical symptoms, demographic details, athletic history, and imaging results to further help increase the specificity of cte detection. these types of clinical-pathologic correlation studies are currently ongoing and hoped to bridge clinical details with neuropathologically confirmed disease status and biomarker data. in conclusion, the work presented here highlights that our continued understanding of neuroinflammation offers the exiting ability to improve existing detecting techniques and to increase our future ability to identify cte during life. authors' contributions gsb: conceptualization, writing (original draft), writing (review and editing). sa: writing (original draft). js: writing (original draft). fl: conceptualization. aeb: writing (review and editing). kwt: writing (review and editing). jdc: conceptualization, writing (original draft), writing (review and editing). all authors have read and approved the final version of the manuscript. conflict of interest statement the authors declare no competing interests. funding statement this work was supported by grant funding from the national institute of aging boston university ad center (p30ag072978) and the department of veterans affairs career development awards to jc (bx004349) and kt (ik2 cx002065). references 1. mckee ac, stein td, huber br, crary jf, bieniek k, dickson d, et al. chronic traumatic encephalopathy (cte): 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https://doi.org/10.1186/s13195-015-0148-8 104. gard a, vedung f, piehl f, khademi m, wernersson mp, rorsman i, et al. cerebrospinal fluid levels of neuroinflammatory biomarkers are increased in athletes with persistent post-concussive symptoms following sports-related concussion. j neuroinflammation. 2023 aug 17;20(1):189. https://doi.org/10.1186/s12974-023-02864-0 copyright: © 2025 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. strategies to gain novel alzheimer’s disease diagnostics and therapeutics using modulators of abca transporters feel free to add comments by clicking these icons on the sidebar free neuropathology 2:33 (2021) review strategies to gain novel alzheimer’s disease diagnostics and therapeutics using modulators of abca transporters jens pahnke1,2,3, pablo bascuñana1, mirjam brackhan1,2, katja stefan1, vigneshwaran namasivayam4, radosveta koldamova5, jingyun wu1, luisa möhle1, sven marcel stefan1 1 department of pathology, section of neuropathology, translational neurodegeneration research and neuropathology lab, university of oslo and oslo university hospital, oslo, norway 2 lied, university of lübeck, lübeck, germany 3 department of pharmacology, faculty of medicine, university of latvia, rīga, latvia 4 department of pharmaceutical and cellbiological chemistry, pharmaceutical institute, university of bonn, bonn, germany 4 department of environmental and occupational health, school of public health, university of pittsburgh, pittsburgh, pa, united states of america corresponding author: sven marcel stefan · department of pathology · section of neuropathology · translational neurodegeneration research and neuropathology lab · www.pahnkelab.eu · university of oslo and oslo university hospital · sognsvannsveien 20 · 0372 oslo · norway s.m.stefan@medisin.uio.no submitted: 07 september 2021 accepted: 12 november 2021 copyedited by: biswarathan ramani published: 13 december 2021 https://doi.org/10.17879/freeneuropathology-2021-3528 keywords: abc transporter, abcb1 (p-gp), abcc1 (mrp1), abcg2 (bcrp), abca1 (abc1), abca2, abca5, abca7, multitarget inhibitor (panabc), broad-spectrum modulator, alzheimer’s disease, amyloid-beta (aβ / abeta), inhibition, activation, induction, downregulation, pet tracer (petabc), pattern analysis, polypharmacology, rational drug design and development abstract adenosine-triphosphate-(atp)-binding cassette (abc) transport proteins are ubiquitously present membrane-bound efflux pumps that distribute endoand xenobiotics across intraand intercellular barriers. discovered over 40 years ago, abc transporters have been identified as key players in various human diseases, such as multidrug-resistant cancer and atherosclerosis, but also neurodegenerative diseases, such as alzheimer’s disease (ad). most prominent and well-studied are abcb1, abcc1, and abcg2, not only due to their contribution to the multidrug resistance (mdr) phenotype in cancer, but also due to their contribution to ad. however, our understanding of other abc transporters is limited, and most of the 49 human abc transporters have been largely neglected as potential targets for novel small-molecule drugs. this is especially true for the abca subfamily, which contains several members known to play a role in ad initiation and progression. this review provides up-to-date information on the proposed functional background and pathological role of abca transporters in ad. we also provide an overview of small-molecules shown to interact with abca transporters as well as potential in silico, in vitro, and in vivo methodologies to gain novel templates for the development of innovative abc transporter-targeting diagnostics and therapeutics. introduction from mdr to neurodegeneration: abc transporters in human disease abc transporters, aβ proteins, and ad part i: status quo abca transporters: physiological function and implications for ad abca1 abca2 abca3 abca4 abca5 abca6 abca7 abca8–abca10 abca12 abca13 modulators of abca transporter function, trafficking, and regulation small-molecule interactors of abca transporters small-molecule regulators of abca transporters part ii: pipeline development to gain novel diagnostics and therapeutics in silico methodologies to predict novel lead structures structure-based drug design ligand-based drug design in vitro methodologies to assess novel lead structures host system of abca transporters functional assessment of abca transporters in vivo assessment of clinical candidates knock-out mouse models rnai models overexpression models humanized abc transporter mouse models disease models imaging techniques concluding remarks: where do we go from here? appendix abbreviations 5-fu 5-fluorouracil, aβ amyloid-β, abca atp-binding cassette transporter subfamily a, acat acyl coenzyme a cholesteryl acyl transferase, ad alzheimer’s disease, adma asymmetric dimethylarginine, adp adenosine-diphosphate, als amyotrophic lateral sclerosis, ampk camp-activated protein kinase, apoa1/e3/e4 apolipoprotein a1/e3/e4, app amyloid precursor protein, atp adenosine-triphosphate, bbb blood-brain barrier, bcsfb blood-cerebrospinal fluid barrier, bhk baby hamster kidney, big1 brefeldin 1-inhibited guanine nucleotide exchange protein, bodipy 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene, camp cyclic adenosine monophosphate, cftr cystic fibrosis transmembrane conductance regulator, cho chinese hamster ovary, cns central nervous system, cpt-camp 8-(4-chlorophenylthio)-camp, cryo-em cryogenic electron microscopy, csf cer-ebral spinal fluid, dids 4,4’-diisothiocyano-2,2’-stilbenedisulfonic acid, ec50 half-maximal effect concentration, ecd extracellular domain, ecgc epigallocatechin gallate, ed50 half-maximal effective dose, eoad early-onset ad, fpd5 fluorescigenic pyrazoline derivative 5, fxr farnesoid-x-receptor, gfp green fluorescent protein, ggpp geranylgeraniol pyrophosphate, gsh reduced glutathione, gwas genome-wide association study, hd huntington’s disease, hdac2 histone deacetylase 2, hdl high-density lipoprotein, hmg-coa-reductase 3-hydroxyl-3-methyl glutaryl-coenzyme a reductase, hts high-throughput screening, ic50 half-maximal inhibition concentration, lamp1 lysosomal-associated membrane protein 1, ldlr ldr receptor, lncrna long non-coding rna, load late-onset ad, ltc4 leukotriene c4, lxr liver-x-receptor, mdr multidrug resistance, mrna messenger rna, ms multiple sclerosis, msd membrane-spanning domain, nbd 7-nitro-2,1,3-benzooxadiazole or nucleotide binding domain, ndea n-nitrosodiethylamine, nem n-ethylmaleimide, (ox)ldl (oxidized) low density lipoprotein, pcb29-pq 2,3,5-trichloro-6-phenyl-[1,4]-benzoquinone, pd parkinson’s disease, pdb protein data bank, pg-j2 prostaglandin j2, pma phorbol 12-myristate 13-acetate, ppar peroxisome proliferator-activated receptor, prdx1 peroxiredoxin 1, rar retinoic acid receptor, rna ribonucleic acid, rxr retinoid-x-receptor, sar structure-activity relationships, shrna short-hairpin rna, sirna small interfering rna, snp single nucleotide polymorphism, sr-bi (srb1) scavenger receptor b1 (also hdl receptor), srepb sterol regulation element-binding protein, tki tyrosine kinase inhibitor, tki tyrosine kinase inhibitor, tm transmembrane helix introduction from mdr to neurodegeneration: abc transporters in human disease abc transporters are membrane-bound transport proteins that are ubiquitously present in the human body.1-4 they play a major role in determining the distribution of intrinsic and xenobiotic drugs between intraand intercellular compartments.5,6 the clinical relevance of abc transporters became pronounced when their expression was correlated to cross-resistance of cancer cells to antineoplastic agents.3,7-13 this phenomenon is called ‘multidrug resistance’ (mdr). however, despite enormous efforts and countless clinical trials to target these efflux pumps,14-17 mdr is still a major unresolved obstacle in cancer chemotherapy. to date, most abc transporters have been associated with mdr,3,7-9,11,12 but only a small minority has been studied properly and can be addressed by small-molecule modulators.18-22 amongst these are abcb1,1,18-27 abcc1,1,18,19,23,24,26,27 and abcg2.18,19,25 apart from their role in multidrug-resistant cancer, many abc transporters have been identified as key players in neurological disorders. evidence for this includes their high abundance at the blood-brain barrier (bbb) and blood-cerebrospinal fluid barrier (bcsfb) in the central nervous system (cns).28-32 additionally, their expression is altered in many pathological conditions in the brain.28-30,33-40 important players are, again, abcb1,28-30,34-36,39-44 abcc1,28-30,39,41,43,45 and abcg228,30,34,36,39-41,43in diseases like ad,28-30,41 amyotrophic lateral sclerosis (als),34,36,44 encephalopathy,45,46 epilepsy,39,40 multiple sclerosis (ms),35 and parkinson’s disease (pd).42,47 furthermore, abc transporters were also found to be associated with certain genetic neurological and psychiatric diseases such as huntington’s disease (hd),38 bipolar disorder,48,49 depression,48 or schizophrenia.48,49 table 1 summarizes the involvement of abc transporters in neurological diseases. table 1. abc transporters and related neurological and psychiatric diseases. abc transporter associated diseases abca1 ad50 hd51 abca2 ad52 abnormal sphingolipid metabolism53,54 abca4 cone-rod dystrophy55 fundus flavimaculatus56 retinitis pigmentosa57,58 stargardt disease59-62 abca5 ad28 abca7 ad63 abca13 lewy body disease64 psychiatric disorders48,65,66 stroke in mice67 abcb1 ad28 brain tumors68 hiv-associated depression and schizophrenia69,70 hiv-associated encephalopathy46 epilepsy71 ischemic stroke72 ms35 multiple systems atrophy73 pd74 progressive supranuclear palsy75 creutzfeldt-jakob disease76 abcb7 pd77 abcb9 pd78 abcc1 ad28 brain tumors79 epilepsy39 hiv-associated encephalopathy45 ischemic stroke80 abcc2 brain tumors79 epilepsy39 abcc3 brain tumors79 epilepsy39 abcc8 als81 abcc9 als81 limbic-predominant age-related tdp-43 encephalopathy (late)82 hippocampal sclerosis of aging and depression83 abcd1 cerebral adrenoleukodystrophy84 abcg1 ad85 brain metabolic disorder86 abcg2 ad87 als88 brain tumors89 epilepsy90 ms91 pd47 traumatic brain injury92 abcg4 ad93 hd51 abc transporters, aβ proteins, and ad since 2001, abc transporters have been implicated in ad pathogenesis.28-30,41,43,94,95 specifically abcb1,94 abcc1,96 and abcg297 have been suggested to directly transport amyloid-β (aβ) proteins, being involved in aβ clearance from the brain to the blood stream.94,96,97 in light of the failure of the first immunological treatment studies,98 it was already proposed that abc transporter dysfunction could explain the clearance problem of aβ.99,100 cerebral accumulation of aβ proteins interferes with neuronal metabolite homeostasis and leads to interruption of cortico-cortical circuits and hampered synaptic communication. this results in an irreversible atrophy and degeneration of specific brain regions, which further causes behavioral, cognitive, and visuospatial impairments in the progression of ad.101 the most prominent abc transporter subfamily involved in ad is the abca subfamily of cholesterol and phospholipid transporters, in which particularly abca1, abca2, abca5, and abca7 have been associated with ad.28-30,41,43,95,102 for abca1,28,41,95,103 and specifically for abca7,28,41,95,104-107 genetic variant28,41,108-111 and genome-wide association studies (gwas)28,41,106,107,112 have suggested that these transporters are risk factors in ad. these discoveries give the members of the abca subfamily a special standing within the group of ad-related abc transporters. cholesterol metabolism in the context of ad has been discussed extensively before.95,102,104,105,113-116 the contribution of cholesterol and phosphilipid transport to membrane constitution, composition, fluidity, and lipid raft formation mediated by abca transporters has already been proposed,6 presenting a putative pharmacological target.117 targeting cholesterol and lipid distribution impacts aβ production by differential activities between α-, β-, and γ-secretases, but also amyloid precursor protein (app) processing106,118-122 and aβ degradation.106,119,123-126 a contribution of abca transporters to aβ clearance from the brain was also proposed,103,106,119,124,127 but not through direct aβ transport.128,129 although abca transporters have been reviewed for the last two decades,3,130,131 little is known about their specific contribution to ad pathogenesis and their mode of action. this is mainly due to a lack of small-molecules that can be used to track, study, and impact the function of these under-studied abc transporters. the present review consists of two parts: part i provides the status quo of abca transporters in ad and small-molecule modulators – in particular intrinsic substrates, natural compounds, pharmacological drugs, and synthetic molecules – that have been reported to influence abca transporter function and expression; part ii outlines the necessary drug development pipeline for the discovery of novel lead structures as potential innovative diagnostics and therapeutics against ad. this pipeline includes cutting-edge in silico methodologies, established in vitro cell assays, and necessary in vivo models. collectively, this review contributes to a deeper understanding of small-molecule ligands that influence abca transporter function, potentially leading to the development of novel ad diagnostics and therapeutics. part i: status quo abca transporters: physiological function and implications for ad abca transporters are ubiquitously present in the human body,3,10,13 although differentially expressed.10 all of the 12 subfamily members have been associated with cholesterol and/or phospholipid transport and homeostasis,3,13,132 except for abca4, which is primarily a transporter of retinoids.133-138 in addition to the diseases listed in table 1, abca transporters have been described as key proteins in several other human disorders, including neonatal respiratory distress syndrome (abca3),139 chronic interstitial lung disease (abca3),140 cataract-microcornea syndrome (abca3),141 hypertrichosis terminalis (abca5),142 or harlequin ichtyosis (abca12).143 however, one major clinical implication for abca transporters, particularly abca1, abca2, abca5, and abca7, relates to ad.28,50,52,63 their suggested roles in this major burdensome neurodegenerative disease as well as general physiological aspects are summarized in the following sections. abca1 abca1 is the prototype of the abca subfamily,144 was first identified in 1994, and is located on human chromosome 9.145 the complete genomic sequence of human abca1 was reported in 2000. the abca1 gene spans 149 kb comprising 50 exons, and the resulting protein is 2261 amino acids long.146 abca1 is located in the plasma membrane and is also present intracellularly in the endoplasmic reticulum and golgi apparatus, where it mediates the efflux of cholesterol and phospholipids from intracellular compartments to extracellular lipid-free apolipoproteins, mainly apolipoprotein a1 (apoa1) and to a lesser extend apoa2 and apoe, to form high-density lipoprotein (hdl) particles.3,147,148 the lipidation of apoa1 is preceded by abca1 dimerization.149 abca1 thus represents the first and rate-limiting step in the reverse cholesterol transport pathway, which removes excess cholesterol from peripheral tissues via hdl and delivers it to the liver for conversion into bile acids and subsequent excretion. in contrast to peripheral tissues, the physiological role of abca1 in the brain, where it is expressed in all cell types, is not well defined.103 it has been suggested that abca1 is required for cholesterol transport from glial cells to neurons via apoe, which is secreted by glial cells and serves as the main lipid acceptor in the brain.103,125 in vitro and in vivo studies in abca1 knock-out models demonstrated that abca1 is essential for normal apoe secretion and lipidation in the cns.150,151 glial cells deficient for abca1 showed reduced lipid efflux with concurrent lipid accumulation as well as decreased apoe secretion, with apoe particles being small and poorly lipidated. in mice, abca1 knock-out resulted in dramatically decreased brain levels of apoe. moreover, examination of the hippocampi of abca1-deficient mice revealed a decrease in neurite length and number of neurite segments and branches, pointing to an importance of abca1 for neurite integrity.152 the major genetic risk factor for sporadic ad is the allelic state of the apoe genotype, with inheritance of the apoe4 allele markedly increasing disease risk.153,154 recently, rawat et al. investigated how apoe4 affected abca1 expression and function in vitro in astrocytes.155 the authors found that apoe4 decreased abca1 plasma membrane levels and increased abca1 co-localization with late endosomes via activation of adp-ribosylation factor 6, thereby reducing cholesterol efflux and lipidation of apoe particles. they corroborated their findings in blood-cerebrospinal fluid (csf) showing that csf from homozygous carriers of the apoe4 allele was less efficient in stimulating abca1-mediated cholesterol efflux compared to csf from homozygous carriers of the apoe3 allele. a recent study assessed cholesterol efflux capacity of csf by analyzing ad patients, non-ad patients, and control subjects.156 the results demonstrated that abca1-mediated csf-cholesterol efflux capacity was markedly reduced in ad but not in non-ad demented patients. however, this difference did not depend on apoe4 status. interestingly, abca1-mediated csf-cholesterol efflux capacity inversely correlated with total and phosphorylated protein tau, suggesting a link between the dysfunction of hdl-like particle in csf and neurodegeneration. apart from the indirect link via apoe, a direct link between abca1 and ad has also been subject to investigation. expression of hippocampal abca1 was elevated on both the mrna and protein levels and was positively correlated with neuro-pathological changes and dementia severity in ad patients.157 the authors of this study suggested that the observed upregulation of abca1 could be interpreted as a compensatory attempt to clear aβ from the brain. moreover, a variety of studies investigated associations between single nucleotide polymorphisms (snp) in the abca1 gene and the risk for ad,28,108-111 reporting inconclusive results.95,103 a meta-analysis of several studies identified the abca1 rs2422493 (c477t) polymorphism as a risk factor for ad while no association was found for the rs2066718 (v771m) or rs1800977 (c14t) polymorphisms.111 this risk effect for rs2422493 was confirmed in a recent genetic variant association study that, in contrast to the meta-analysis, also reported an increased ad risk for rs2066718 and a decreased ad risk for rs1800977.109 further genetic association studies and meta-analyses are necessary to search for potential associations between abca1 polymorphisms and ad risk. in a recent ad gwas, the rs1800978 polymorphism in the abca1 gene was identified as the lead snp in a new genome-wide significant locus.158 the association of genetic variants of the abca1 gene with ad risk was confirmed by exome sequencing data analysis from 32,558 individuals.158 the study identified around 120 variants that have an increased frequency in early-onset ad (eoad; 1.5%) and late-onset ad (load; 1.1%) cases, compared to 0.5% of all controls. the data demonstrated that ad-association was mainly explained by extremely rare variants, but also by a smaller number of more common variants, e.g., n1800h.159 intriguingly, loss of function and missense variants in the abca1 gene were respectively associated with a 4.7-fold (95%ci 2.2-10.3) and 2.7-fold (95%ci 1.9-3.8) increased eoad risk, and this was lower for load cases suggesting that the burden of damaging abca1 variants was concentrated in younger ad patients. additionally, some long non-coding (lnc) rnas such as lncrna loc286367 have been shown to affect abca1 expression.160 lncrna loc286367 and abca1 are located on the same chromosome but are transcribed in opposite directions. a recent study demonstrated that loc286367 reduces abca1 expression in thp-1 macrophages and increases the levels of proinflammatory cytokines.160 the role of abca1 in aβ deposition and clearance as well as in aβ deposits-related memory deficits has been extensively investigated in app-transgenic mouse models of ad. the lack of abca1 decreased brain apoe levels and either did not affect or increased aβ load.161-163 a recent study utilizing shotgun lipidomics experiments demonstrated a common apoe isoform-specific phospholipid signature between human apoe3/3 and apoe4/4 ad brains and lipoproteins isolated from astrocyte-conditioned media of apoe3 and apoe4 mice.164 interestingly, the lipoproteins derived from wild-type and abca1het mice had phospholipid content apoe3 > apoe4 > apoe3het > apoe4het suggesting that the combination of abca1 insufficiency and apoe4 genotype decreases apoe lipidation even further, thus aggravating apoe4 effect. these findings suggest that poorly lipidated apoe may promote aβ aggregation.129,161-163 in contrast, overexpression of abca1 in an app-transgenic mouse model resulted in increased lipidation, albeit reduced brain levels of apoe and decreased aβ load, implying that highly lipidated apoe may reduce aβ aggregation propensity.127 this is supported by findings of deane et al., who showed that different apoe isoforms may differentially disrupt aβ clearance from mice brains.165 a stable isotope-labelling kinetic study in an app-transgenic mouse model either lacking abca1 or overexpressing abca1 demonstrated increased apoe clearance in both abca1 knock-out and abca1-overexpressing mice, but did not reveal any effect on aβ clearance or production, suggesting that abca1 may regulate aβ deposition by a mechanism other than altering aβ metabolism.166 in contrast, a study assessing the clearance of intracerebrally injected 125i-aβ from the brain reported that abca1-deficiency decreased aβ clearance in non-app-transgenic mice.167 furthermore, knock-out of abca1 was found to augment the dissemination of intracerebrally injected, brain-derived aβ seeds in app-transgenic mice.167 haplodeficiency of abca1 led to decreased brain apoe levels and increased aβ oligomer levels but did not affect aβ deposition in app-transgenic mice.168 however, both haplodeficiency and homozygous knock-out of abca1 aggravated cognitive deficits in app-transgenic mice.152,167,168 lastly, the lack of one copy of abca1 exacerbated memory deficits, decreased aβ clearance, and increased aβ load in app-transgenic mice expressing human apoe4 but not in app-transgenic mice expressing human apoe3.169 abca2 abca2 is predominantly, but not exclusively, expressed in the brain, where it can be found in glial cells and neurons.170-173 on the subcellular level, abca2 is located in endoand lysosomal membranes, facilitating the sequestration of waste substances into intracellular vesicles.172 in addition, it is involved in myelin lipid transport, neural development, and macrophage activation.30,174,175 genetic variations of abca2 were identified as a risk factor for eoad and sporadic ad.52,176 these two studies showed a strong correlation between rs908832 and ad.52,176 however, a later study could not find a link between this snp and any form of ad.177 in addition, abca2 mrna expression was upregulated in ad patients compared to controls suggesting abca2 as a biomarker for differential diagnosis of ad.178 preclinical studies of abca2 suggested that this transporter modulates aβ production via the ldl receptor (ldlr).179,180 abca2 overexpression increased ldlr density, and ldlr deficiency has been described to enhance aβ deposition.181 chen et al. reported a co-localization of abca2 and aβ as well as aβ upregulation in cells overexpressing abca2. in addition, impairment of abca2 expression using small interfering rna (sirna) was accompanied by a decrease in aβ production.182 abca2 depletion has been shown to induce a shift from βto α-secretases and thus, a reduction of app processing by γ-secretase.182 furthermore, abca2 has been proposed to play a role in aβ production as it has been reported to upregulate sphingosine in murine cells and, therefore, to induce app transcription.183 however, another study in human cells could not confirm the modulation of aβ production or cholesterol efflux by abca2.184 thus, further research on the role of abca2 in ad pathogenesis and its potential as a therapeutic target is necessary. abca3 despite its initial report of exclusive lung expression,185 abca3 is also found in other tissues including the brain.186,187 within the brain, the highest levels of abca3 were found in oligodendrocytes.188 abca3 plays a role in producing surfactants in the lung, suggesting that the transporter may also be involved in lipid metabolism in the brain, specifically phosphatidylcholine and phosphatidylglycerol transport. interestingly, phosphatidylcholine has also been discussed in the context of ad.189 a genetic study revealed that mutations in abca3 can also cause cataract-microcornea syndrome, a rare congenital malformation of the eye.141 the actual implications of the potential connection between altered abca3 functionality and ad need to be addressed in future studies. abca4 abca4 is mainly expressed in the retina with very little presence in other tissues of the cns.190 abca4 mutation causes stargardt disease, characterized by macular dystrophy, retinal alterations, and lipofuscin accumulation.60,61,190,191 other retinal diseases, such as fundus flavimaculatus, retinitis pigmentosa, or cone-rod dystrophy, have also been associated with mutations of abca4.55,57,58,192 abca4 is expressed in brain capillary endothelial cells, as well.193 however, no link between abca4 and ad has been suggested to date. abca5 abca5 is a little-known member of the abca subfamily expressed mainly in skeletal muscle with unknown function in the brain.194 studies in peripheral tissues suggest that the function of abca5 is associated with cellular lipid metabolism.195 abca5 knock-out in mice induced signs of lysosomal storage disease in the heart and the thyroid gland.131 in the brain, abca5 is expressed in neurons and, to a lesser extent, in microglia, astrocytes, and oligodendrocytes.195 fu et al. showed that abca5 stimulated cholesterol efflux in neurons and induced a decrease in aβ production probably affecting app processing but not its expression.195 abca6 abca6 is ubiquitously expressed with high levels in liver, lung, heart, brain, and ovaries. this transporter is probably involved in macrophage lipid homeostasis as it is upregulated during macrophage differentiation and is responsive to cholesterol treatment.196 although certain missense variants of abca6 have been correlated with blood cholesterol levels,197 no link between abca6 and ad has yet been found. abca7 abca7 was first identified in the year 2000, and is located on human chromosome 19.198-200 analysis of abca7 mrna expression levels has shown that this transporter is mainly confined to the brain and the immune system.3 due to its high homology to abca1 (54%),200 abca7 was first hypothesized to play an important role in lipid trafficking, mediating cholesterol and phospholipid efflux. abca7 actively transports phosphatidylcholine, phosphatidylserine, and sphingomyelin from the cytoplasm to the exocytoplasmic leaflet of membranes.198,199,201 however, in contrast to abca1, abca7 generates only small hdl particles.202 recent research has shown that lipid trafficking by abca7 plays a secondary role. studies in abca7 knock-out models have demonstrated that abca7 is involved in the phagocytotic activity of macrophages and fibroblasts198,203-205 but not in cell cholesterol release.206-208 in 2011, hollingworth et al. identified the abca7 gene as an ad risk locus.198,209 in multiple studies, variants of abca7 have been associated with an increased risk of developing ad.198,210-212 in 2015, steinberg et al. reported that rare loss-of-function variants of abca7 confer a risk of ad in icelanders (odds ratio: 2.12; p = 2.2 ∙ 10-13), and found a similar association in study groups from europe and the united states (combined odds ratio: 2.03; p = 6.8 ∙ 10-15).213 in particular, the rare ad-related polymorphism rs200538373 was associated with an ad risk odds ratio of 1.9.210 these studies suggest that reduced levels of abca7 may increase the risk of ad. nonetheless, it is not clear how these polymorphisms affect abca7 function and contribute to ad progression. increased levels of abca7 expression were described in ad patients and were also positively correlated with cognitive decline.198,211 this finding is consistent with abca7 mrna transcription levels in j20 mice.123 the increase of abca7 may be a compensatory defense mechanism that is insufficient to stop disease progression. furthermore, the rs3764650g allele has been associated with increased neuritic plaques in human patients198,214 and a limitation of the neuroprotective effects of exercise intervention.215 these studies support a potential protective role of abca7 in ad. to date, three potential roles have been identified for abca7 contribution to ad: app processing, immune response, and lipid metabolism. chan et al. proposed an inhibitory effect of abca7 on aβ deposition after showing in vitro inhibition of aβ production independent of β-secretase activity.120 other authors proposed that abca7 is not directly linked to aβ production, but rather through lipid metabolism as abca7 mediates the transport of lipids across the bbb and abca7 loss of function may alter cholesterol transport by decreasing apoe secretion and abca1 expression. this alteration in cholesterol metabolism can also contribute to ad development.216 however, abca7 knock-out induced an increase of aβ load with no difference in clearance rate and an increase of β-secretase expression. on the other hand, abca7 overexpression led to diminished aβ production and improved cognitive function.217,218 nevertheless, abca7 is highly expressed in phagocytic cells, including macrophages and microglia, suggesting a role of the transporter in phagocytosis.188,198 phagocytosis is crucial to maintain brain homeostasis. indeed, ineffective phagocytosis may induce neuroinflammation, which is a risk factor in ad. in addition, microglial cells are involved in phagocytosis and degradation of aβ. thus, an involvement of abca7 in microglial phagocytosis of aβ may explain the contribution of this transporter to ad pathogenesis. in ad patients, increased abca7 transcription has been found in areas with plaques but not in unaltered regions such as the cerebellum.123 this increase in transcription was paralleled by microglia recruitment supporting the contribution of abca7 to microglia-mediated phagocytosis of aβ. in addition, abca7 knock-out mice showed a reduced microglia response after intracerebral aβ injection.123 kim et al. demonstrated an increased aβ load in j20/a7 knock-out mice compared to j20 mice, potentially due to an altered phagocytic function.124,198 furthermore, it has recently been shown that abca7 haplodeficiency disturbs the microglial immune response and causes enhanced aβ accumulation in microglia, probably due to alterations in endolysosomal trafficking.219 last, a new hypothesis has emerged recently, assigning abca7 a prominent role in the altered lipidostasis hypothesis in ad.104 the authors of this study proposed the existence of a neurodegenerative lipid that is naturally removed by abca7. a loss of abca7 function due to the described polymorphisms might accelerate accumulation of this lipid, inducing aβ aggregation. in fact, a link between cholesterol metabolism and abca7-mediated phagocytosis has been reported, which may also explain the protective properties of statin treatment in the development of ad.105,198,203,220 despite recent findings, the role of abca7 in ad pathogenesis remains unclear. according to in vitro and preclinical research, it may be associated with phagocytic activity by microglia, which could be linked to cell cholesterol metabolism.105,198,203 thus, further investigation is required to reveal the role of abca7 in ad pathogenesis and its potential use as a therapeutic target for this neurodegenerative disease. abca8–abca10 so far, no obvious role of abca8–10 has been elucidated for ad, neurodegenerative diseases, nor any human disease. however, several potential intrinsic substrates of abca8 have been identified.10,221,222 furthermore, a significant number of abca transporter modulators have been identified on this target.222 hence, abca8 represents a good model system for the development of potential therapeutics targeting other abca transporters taking the scarce knowledge on this transporter subclass into account. abca12 abca12 is expressed predominantly in the epidermis, and its main function is the transport of lipids.223 it is hypothesized that abca12 plays a role in skin lipid homeostasis. mutations in this gene are associated with lamellar ichthyosis type 2 and harlequin ichthyosis.143,224,225 however, a japanese study investigated common polymorphisms of abca12 and did not find an association with sporadic ad.226 abca13 abca13 is the largest abc transporter with 576 kda.227 it has been reported to be highly expressed in the brain as well as in peripheral tissues.227 a very small study found reduced neuroinflammation and altered abca13 expression in post mortem analyses of brains from patients with lewy body dementia.64 in addition, increased abca13 expression has been reported after stroke in mice.67 furthermore, two studies showed enhanced abca13 mrna expression in schizophrenic patients after different antipsychotic treatments, suggesting a role of this transporter in psychiatric disorders.48,65,66 however, no association between abca13 and ad has been found. figure 1. molecular formulas of prominent interactors of abca transporters. modulators of abca transporter function, trafficking, and regulation ‘modulation’ is a widely used term to summarize actions of small-molecules that have been reported to alter abca transporter function, trafficking, and/or regulation. modulators can be divided into ‘interactors’ and ‘regulators’. interactors summarize compounds that directly bind to abca transports, which can have either inhibiting or activating effects on the transporters. substrates are also included in this category. in terms of abca transporters, however, a direct interaction of these agents with their target(s) has in most cases not yet been comprehensively proven. therefore, compounds that are believed to directly interact with abca transporters extend the category of interactors. figure 1 represents the most prominent interactors of abca transporters and provides additional information about their mode of modulation. regulators are compounds that change abca transporter expression (transcription and/or translation) in terms of induction and/or downregulation. in addition, compounds that regulate abca transporter trafficking can be included into the category of regulators, as this effect was often observed as ‘pseudo-protein increase’ at the cell membrane. figure 2 depicts the most prominent regulators of abca transporters including proposed mode of modulations. figure 2. molecular formulas of prominent regulators of abca transporters. it must be stated that the term ‘inhibitor’ and ‘activator’ are often misused in the literature, as in most cases studies describe a downregulation or induction. in the present review, this mislabeling has been taken into account and the present review and the respective compounds have been allocated into the correct groups. as established earlier,23,24 the compounds are sorted according to their origin: (i) intrinsic substrates and substrate-like molecules, (ii) (other) natural compounds, (iii) pharmacological drugs, (iv) high-throughput screening-(hts)-derived candidates, as well as (v) compounds from synthetic/medicinal chemistry approaches. figure 3 gives a general overview of specific interactors and their postulated mode of modulation. table 2 summarizes all modulators of abca1, the most studied abca transporter, while table 3 summarizes all known modulators in terms of the other abca transporters. the stated concentration values are indicators of bioactivities of the respective compound and are strongly dependent on the testing system utilized. hence, the respective data must be interpreted with caution. figure 3. general overview of proteins participating in abca1 regulation and interaction. small-molecule interactors of abca transporters endoand xenobiotic substrates the most genuine interactors of abca transporters are intrinsic substrates of these transporters. these include cholesterol (figure 1) and other sterol derivatives,10,221,222,228 but also phospholipids (figure 1), sphingolipids228,229 and retinoids (e.g., all-trans-retinal; figure 1).133-138 in addition, certain intrinsic molecules were demonstrated to interact with abca transporters, in particular with abca1230 and abca8.10,221,222 α-tocopherol (vitamin e) was demonstrated to be transported by abca1,230 and to interfere with abca1 regulation.231 the sterol derivatives estradiol-β-glucuronide, estrone sulfate, and taurocholic acid (figure 1), but also the physiological substrate leukotriene c4 (ltc4), the natural compound ochratoxin a, as well as the chemical p-amino hippuric acid were discovered as (potential) abca8 substrates.10,221,222 specifically the abca8-mediated taurocholate export from various human pancreatic cancer cell lines was suggested as the major mechanism behind gemcitabine resistance in these cells,221 which was corroborated in hek293 cells stably expressing abca8.10 in addition, a small body of evidence suggests that abca2 and abca3 contribute to the subcellular sequestration of certain antineoplastic agents into endoand lysosomes.232-235 these agents include cytarabine (abca3),235 daunorubicin (abca3),232,233,235 etoposide (abca3),235 imatinib (abca2 and abca3; figure 1),234,236 mitoxantrone (abca3),235 and vincristine (abca3; figure 1).235 furthermore, several antineoplastic agents were described to have less effect when abca2 was overexpressed in vitro171,237,238 and in vivo.239 for example, the anticancer drug estramustine (figure 1) was effluxed from abca2-overexpressing human ovary carcinoma cells, which were less susceptible to estramustine treatment than the sensitive cell line.171,238 antisense nucleotide treatment against abca2 re-sensitized the carcinoma cells, further demonstrating a role for abca2 in mediating drug efflux.238 furthermore, abca2 knock-out mice had elevated estradiol and estrone levels when treated with estramustine.239 a similar effect in terms of susceptibility and re-sensitization was observed for abca3-mediated transport of miltefosine in leishmania,240 doxorubicin resistance in acute myeloid leukemia cells,237 and cisplatin as well as paclitaxel resistance in several lung cancer cell lines.241 table 2. currently known modulators of abca1. mode of modulation name of modulator effect concentration; concentration range; ec50; dose; ed50 (potential) substrates cholesterol phospholipids β-sitosterol sphingomyelin α-tocopherol activators ati-5261 cs-6253 1.07 µm; 30 mg/kg body weight in mice 0.73 µm; 20 mg/kg body weight in mice inhibitors blt-4 bromosulfophthaleine bumetanide cyclosporine a dids diphenylamine 2-carboxylic acid flufenamic acid furosemide glibenclamide pimecrolimus probucol sirolimus tacrolimus valspodar 150 µm 500 µm 200 µm 1–20 µm; ic50 = 5.1–7.6 µm 40–500 µm 500 µm 500 µm 200 µm 50–1000 µm 20 µm; ic50 = 7.0 µm 1.9–20 µm 20 µm; ic50 = 18.8 µm 20 µm; ic50 = 13.6 µm 5 µm; ic50 = 1.9 µm inducers a-769662 aclarubicin allicin camp butyryl-camp 8-br-camp cpt-camp atorvastatin atra az1–az9 az-1 az-2 az10606120 az876 bcd1 n-benzothiazolyl-2-benzenesulfonamides berberine bergapten bexarotene bezafibrate bms-852927 sodium-butyrate cholesterol cholic acid analog 14b celastrol chalcone derivatives chromene derivatives 2, 3, and 5 chromone analog 6 cl2-57 curcumin daidzein danthron 1,6-o,o-diacetylbritannilactone digoxin doxazosin doxorubicin efatutazone e3317 egcg homo-eriodictyol ethyl 2,4,6-trihydroxybenzoate f1 f4 fargesin fenofibrate fluvastatin fpd5 fucosterol geniposide ginsenoside (derivatives) ginsenoside compound k glycyrrhizine gq-11 gw3965 gw7845 gypenosides hesperetin-7-o-β-d-glucopyranoside hesperetin-7-o-rutinosid 20-(s)-hydroxycholesterol 4-hydroxycholesterol 22-(r)-hydroxycholesterol 22-(s)-hydroxycholesterol 24-hydroxycholesterol 24-(s)-hydroxycholesterol 25-hydroxycholesterol 27-hydroxycholesterol 3-hydroxytyrosol idarubicin kaempferol l836,978 kuwanon g l-839,867 lxr623 lycopene m2 maslinic acid metformin mevalonate mevastatin mitotane naringenin obeticholic acid ondansetron orlistat ouabain paeonol pcb29-pq pemafibrate pestalotioquinoside c phenethyl isothiocyanate tadehagi triquetrum-derived glycosides pioglitazone pitavastatin platycodin d pma ponasterone a pratensein propofol prostaglandin j2 pyrrole-imidazole-polyamide pyrromycin quercetin 9-cis-retinoic acid ro0721957/5 ro0264456 rosiglitazone rpr-5 rutaecarpine and derivatives saikosaponin a 24-(s)-saringosterol sb203580 scutellarein selenium serdemetan simvastatin spf1 spf2 soraphene a 24-(s)-stigmast-5-ene-3β,24-diol cannabis sativa-derived stilbenoids sulfoxaflor tanshindiol c taraxasterol testosterone tetradecylthioacetic acid to901317 tr1 trichostatin a troglitazone ttnpb urolithin a urolithin b urolithin b sulfate vitamin d3 vitexin way-254011 wy14643 bexarotene derivatives z10 and z36 zafirlukast 250 µm ec50 = 0.49 µm 2.5–10 µm 0.1–10 µm 300 µm 0.3–1000 µm 300–500 µm 5–10 µm; 4 mg/kg body weight in mice 0.25–10 µm ed50 = 1.49–341 µmol/kg body weight in mice 10 µm 10 µm 10 µm ed50 = 0.956 µmol/kg body weight in mice ec50 = 0.035 µm ec50 = 0.37–33.42 µm 5–20 µm 12.5–50.0 mg/kg body weight in rats 0.1–1 µm 10–200 µm ed50 = 2.10 µmol/kg body weight in mice 1000–10.000 µm; 200–400 mg/kg body weight in mice 12.9–100 µm 5–40 µm 0.1–1.0 µm; 0.5–1 mg/kg body weight in mice 5–10 µm; 20 mg/kg body weight in mice 25 µm 25 µm 10 µm; 10 mg/kg body weight in mice 5–40 µm ec50 = 3.17 µm 10–40 µm; 60 mg/kg body weight in mice 8–10 µm; 10 ml/kg body weight in mice 0.010 µm 10 µm 0.0316–1 µm; 20 mg/kg body weight in mice 40 µm 0.01–1 µm; ec50 = 0.2 µm 40 mg/kg body weight in mice 41.4–165 µm 50–100 µm ed50 = 10 µm 20 µm; 50 mg/kg body weight in mice 2.77–40 µm 1–20 µm 1 µm; 0.005–0.02 mg/kg body weight in mice 100-200 µm 515 µm; 50–100 mg/kg body weight in mice 10–30 µm 1.25 µm 60.8–243 µm 20 mg/kg body weight in mice 0.5–50 µm; ed50 = 0.969 µmol/kg body weight in mice 5 µm 5 µg/ml 107–431 µm 100 µm; 3 mg/kg body weight in mice 5–20 µm 1–20 µm 1–25 µm; ec50 = 1.0 µm 5–20 µm 20 µm 0.5–1.5 µm 2–12.4 µm 6.21 µm–10 µm 2–5 µm 0.1 µm 2.5–10 µm u.c.a 20 µm 0.1–1 µm 0.1–1 µm; ed50 = 31.5 µmol/kg body weight in mice 2.2–6.6 mg/kg body weight in ferrets 10 µm 20 µm 10 µm 5–500 µm 50 µm 20–50 µm 25–100 µm 40 mg/kg body weight in mice 1 µm 50 µm 0.010 µm 100 µm 5–10 µm 0.1–10 µm; 0.3 mg/kg body weight in mice 50 µm 30–75 mg/kg body weight in mice 10 µm 5–10 µm; ec50 = 1.28–7.474 µm; 20 mg/kg body weight in mice 0.1–10 µm 5–20 µm 0.32 µm 2–5 µm ec50 = 2.91 µm 50 µm 1–20 µm 1 µm; 1 mg/kg body weight in mice ec50 = 0.85 µm 20 µm; 12.5 mg/kg body weight in mice 0.04–10 µm; ec50 = 0.29 µm 0.050 µm 0.005 µm 0.05–10 µm; ec50 = 1.49 µm 5 µm 0.035–34.98 µm; ec50 = 0.27 µm 2–8 µm 10 µm 20 µm 50 mg/kg body weight in mice 2.5–5 µm 2–5 µm 10 µm 1 µm 1 µm 0.03–20 µm; ec50 = 0.01391 µm 10 µm 2.5–3 µm u.d.b in aphis gossypii 10 µm 3–12 µm 0.001–0.01 µm 0.75% of high-fat diet in mice 0.1–25 µm; ed50 = 4.11 µmol/kg body weight in mice 10 µm 99.2 µm; 0.5 mg/kg body weight in mice 1 µm 0.25–10 µm 20 µm 0.1–10 µm 10 µm 1 µm 50 µm ed50 = in mice 0.05–100 µm 1 µm; 40 mg/kg body weight in mice 2.5–5 µm downregulators 5cppss-50 acrolein 8-br-camp angiotensin ii asymmetric dimethylarginine atorvastatin atr-101 bisphenol a chalcone derivatives 4-{[4-(4-chlorophenyl)-2-thiazolyl]amino}phenol cholesterol dexamethasone dibutyl phthalate egcg fluvastatin ggpp gsk2033 gw6471 gw9662 desulfated holothurin a homocysteine lipopolysaccharides lovastatin ly294002 methionine mevalonate mevastatin mitotane ndea 1,2,3,4,6-penta-o-galloyl-β-d-glucose phenylalanine-proline pitavastatin pravastatin raloxifene rosuvastatin simvastatin sr9243 tamoxifene α-tocopherol γ-tocopherol toremifene troglitazone valproic acid varenicline 20 µm 5–20 µm 0.3 µm 0.0001–0.100 µm 0.5–1 µm 0.1–100 µm 10–30 µm 100 µm 10 µm 5 µm 150 µm 0.1–2.5 µm; 8 mg/kg body weight in rats 0.1 µm 100 mg/kg body weight in mice 0.1–100 µm 10 µm–200 µm 0.05–5 µm 10 µm 10 µm 2.68–4.47 µm 50–200 µm 1 mg/ml 0.1–100 µm 20 µm 17 g/kg food in mice 100 µm 0.05–50 µm 50 µm 100 mg/kg body weight in rats 25–300 mg/kg body weight in mice 1000 µm; 600 mg/kg body weight in rats 10 µm 50 µm 10 µm 5–50 µm 0.1–100 µm 1 µm 2.5–10 µm 50–100 µm 50–100 µm 10 µm 10 µm 1000 µm 10 µm; 0.5 mg/kg body weight in mice stabilizers cyclosporine a diphenoquinone erythrodiol alln leupeptin probucol spiroquinone testosterone wogonin 10 µm 0.0001–0.0005 µm 10–15 µm 50 µm 1170 µm u.c.a 0.025–0.050 µm 0.01 µm 10–40 µm destabilizers brefeldin a 2-bromopalmitate cycloheximide gö6976 monensin a serdemetan tunicamycin 17.8–36 µm 7.5–60 µm; ic50 = 15 µm 355 µm 10 µm 10 µm 2–5 µm 2.41 µm a u.c. = unspecified concentration b u.d. = unspecified dose strikingly, abca2 co-localized with the lysosomal-associated membrane protein 1 (lamp1) – an endolysosomal marker – as well as the fluorescence probe dansyl-estramustine. this co-localization indicates a direct sequestration of this antineoplastic drug into endoand/or lysosomes.171 on the other hand, the susceptibility of abca3-overexpressing ccrf-cem leukemia cells to the antineoplastic agents cytarabine, methotrexate (figure 1), vincristine, but also the anti-inflammatory drug dexamethasone, was reduced compared to their parental counterparts.242 taken together, abca2 and abca3 are contributors to mdr, and the number of potential abca2 and abca3 substrates may be even higher than currently suggested. interestingly, missense mutations of abca4 were associated with chloroquineand hydroxychloroquine-associated retinopathy,243 although contradictory studies exist.244 a direct interaction was postulated, however, not proven. nevertheless, these results suggest chloroquine and hydroxychloroquine as potential abca4 substrates. table 3. currently known modulators of abca transporters other than abca1. mode of modulation name of modulator effect concentration; concentration range; ec50; dose; ed50 abca2 (potential) substrates cytarabine dexamethasone estramustine estradiol estrone imatinib methotrexate inducers imatinib methotrexate progesterone sulfoxaflor u18666a u.c.b 1.28 µm 31.8 µm u.d.c in aphis gossypii 5 µm downregulators celecoxib 10 µm abca3 (potential) substrates cisplatin cytarabine dasatinib daunorubicin dexamethasone doxorubicin etoposide imatinib methotrexate miltefosine mitoxantrone nilotinib paclitaxel vincristine inducers dasatinib 5-fu imatinib methotrexate nilotinib vitamin c u.c.b 50 µm 0.1–12.5 µm 1.28 µm u.c.b 56.78 µm downregulators genistein indomethacin lipopolysaccharides pk11195 sirolimus 3–9 µm 2 µm 10 µg/ml; 100 µg/ml in chicken lungs u.c.b 2 µm stabilizers c13 c14 c17 genistein ivacaftor 10 µm 10 µm 10 µm 10 µm 1 µm abca4 (potential) substrates chloroquine hydroxychloroquine β-ionone 11-cis-retinal 13-cis-retinal all-trans-retinal all-trans-retinoic acid all-trans-retinol n-retinylidene-phosphatidyl-ethanolamine phosphatidyl-ethanolamine stabilizers c3 c4 c18 lumacaftor 10–20 µm 1–20 µm 10–20 µm 10–20 µm abca5 inducers atorvastatin bezafibrate cholesterol gw3965 rosiglitazone tacrolimus troglitazone 20 µm 10 µm 100–150 µm 0.5 µm 10 µm 0.04 µm 10 µm downregulators digoxin 2.5 g/kg body weight in mice abca6 inducers acitretin lovastatin mevastatin 1–10 mg/kg body weight in pigs 10 µm 10 µm downregulators lovastatin mevastatin 10 µm 10 µm abca7 inducers ponasterone a pravastatin rosuvastatin 1–5 µm 50 µm 5 µm downregulators cholesterol digoxin 25-hydroxycholesterol 2 mm 2.5 g/kg body weight in mice 2.48 µm abca8 (potential) substrates p-aminohippuric acid estradiol-β-glucuronide estrone sulfate glibenclamide leukotriene c4 ochratoxin a taurocholic acid (potential) inhibitors digoxin dofequidar glibenclamide ochratoxin a probenecid verapamil verlukast 250 µm 10 µm 250 µm 50 µm 1000 µm 1000 µm 100 µm inducers gemcitabine polyethyleneglycol-block-polyactide nanoparticles 0.05–0.8 µm 42.04 g/kg body weight in rats downregulators digoxin 2.5 g/kg body weight in mice abca9 downregulators digoxin 2.5 g/kg body weight in mice abca12 inducers ceramide n-hexanoyl-d-erythro-sphingosine ciglitazone d609 xanthate d-ddmp gi 251929x gw610742 d-mapp d-nmappd d-ppmp d-pppp 22-(r)-hydroxycholesterol to901317 troglitazone 5 µm 7.5 µm 25 µm u.c.b 10 µm 8 µm 10 µm 5 µm 5 µm 10 µm 10 µm 10 µm 7.5 µm stabilizers acitretin 1–10 mg/kg body weight in pigs a apart from cholesterol and/or phospholipids b u.c. = unspecified concentration c u.d. = unspecified dose inhibitors to date, the number of small-molecules that (are believed to) directly interact with abca transporters is very low. for example, only 14 inhibitors can be found in the literature regarding the most studied prototype of abca transporters, abca1.245-248 only four of these inhibitors are associated with half-maximal inhibition concentrations (ic50),245,249 which is the ‘golden surrogate’ to evaluate and judge inhibitory activities of small-molecules. the following section will highlight these small-molecules as well as inhibitors of other abca transporters. abca1 glibenclamide and 4,4’-diisothiocyano-2,2’-stilbenedisulfonic acid (dids) as outlined above, abca1 is the most studied and understood abca transporter, although its particular role in neurodegenerative diseases in general51,103 – and in ad in particular – is not well understood.28-30,43,95,102 however, over time, several agents were found to impact abca1 transport function. the most prominent examples are glibenclamide and dids (both figure 1), which were first shown to inhibit abca1 in 1997.247,248 these drugs blocked the abca1-mediated 125i efflux from murine peritoneal macrophages247 as well as human abca1-transfected xenopus laevis oocytes.248 glibenclamide and dids inhibited the abca1-mediated transport of cholesterol and other sterols as well as phosphoand sphingolipids. thus, these agents became the ‘standard abca1 inhibitors’ and have frequently been used in abca1 studies ever since.229,250-269 glibenclamide and dids were preferred over other discovered abca inhibitors, such as bumetanide, diphenylamine 2-carboxylic acid, flufenamic acid, furosemide, and bromosulfophthaleine.248 specifically glibenclamide was rigorously evaluated regarding its mechanism of action. it was demonstrated that glibenclamide prevented cross-linking of 125i-marked apoa1 to abca1,267,270 not interfering with abca1 location at the cell surface.267 in essence, glibenclamide and dids may play a significant role in the development of future modulators of abca transporters in general. probucol and cyclosporine a less prominent but also well characterized are the antilipidemic drug probucol246,271-278 and the immunosuppressant cyclosporine a245,249,258,279-281 (both figure 1). probucol was demonstrated to reduce the cholesterol efflux from different abca1-overexpressing murine and human macrophages,275-278 and total lipid release (cholesterol + phospholipids) from human wi-38 fibroblasts.246 vice versa, probucol increased accumulation of free cholesterol, cholesterol esters, phosphatidylcholine, and sphingomyelin in human fibroblasts.246 additionally, probucol was reported to prevent cell surface-specific binding of 125i marked apoa1 to abca1.246,278 similarly, this effect has already been demonstrated for glibenclamide before.267,270 interestingly, it was shown that total abca1 protein levels were increased after exposure to probucol due to decreased degradation.246,275 this qualifies probucol also as a stabilizer. however, as its inhibiting effect is far more pronounced, we have included it as an inhibitor here. the immunosuppressant cyclosporine a has been characterized as an abca1 inhibitor in multiple studies.245,249,258,279-281 this inhibition was shown to be direct through a radiolabeled variant of cyclosporine a and purified abca1.245 cyclosporine a not only functionally inhibited abca1-mediated cholesterol and phospholipid efflux,245,249 and caused intracellular accumulation of cholesterol,258 but also inhibited the abca1-dependent binding of alexa 546or 125i-labeled apoa1,245,249 as demonstrated for glibenclamide267,270 and probucol246,278 before. interestingly, toxicity assays demonstrated that cyclosporine a negated the positive effect of an abca1 inducer on cell viability when cells were exposed to aβ proteins.280 this was confirmed in vivo in c57bl/6 mice that had reduced hdl levels.249 interestingly, cyclosporine a was shown to decrease abca1 turnover, increasing its presence at the cell surface by a factor of two as demonstrated with a gfp-labeled abca1 variant,249 suggesting a similar mode of inhibition as for probucol.275 thus, as for probucol,246,275 cyclosporine a also appears to have a stabilizer function,275 but is included in the current section due to its pronounced inhibitory role. morevover, the cyclosporine a analog valspodar (psc833) inhibited direct binding of radiolabeled cyclosporine a to abca1, revealing that valspodar also acts as an abca1 inhibitor.245,282 furthermore, several other calmodulin antagonists inhibited abca1-mediated cholesterol efflux and binding of apoa1.245 these include pimecrolimus,245 sirolimus,245 and tacrolimus,245 suggesting these molecules as potential scaffolds for the development of future abca1 modulators. other abca1 inhibitors in terms of other small-molecules that were suggested to inhibit abca1 function, blt-4 has been demonstrated to inhibit cholesterol and phospholipid export from adipocytes and macrophages,255 and to decrease cholesterol efflux from abca1-transfected hek293 cells. blt-4 was also shown to inhibit 125i-marked apoa1-binding to abca1,270 as demonstrated for glibenclamide,267,270 probucol,246,278 and cyclosporine.245,249 other abca transporters while abca1 can be considered a less-studied abc transporter with certain knowledge about its function and interfering small-molecules,18 all other abca transporters belong to the group of under-studied abc transporters that cannot be addressed by small-molecules with very rare exceptions.18 one rare example is abca8. using the xenopus laevis oocytes model in vitro testing system,248 tsuruoka et al. reported inhibitors of this transport protein.222 while digoxin, probenecid, and verapamil (all figure 1) could be identified as very weak inhibitors of abca8-mediated estradiol-β-glucuronide transport, dofequidar (ms-209), ochratoxin a, and verlukast (mk-571; figure 1) were discovered as moderately potent inhibitors.222 in addition, glibenclamide was also suggested to (partially) inhibit abca8 function.266 activators although activators of abc transporters have been reported, as for example, for abcb123 and abcc transporters,23,283-288 these reports are somewhat scarce compared with other classified modulators of abc transporters. in terms of a subclass abc transporters, no small-molecule activators are known. however, it is well established and has been extensively demonstrated that abca1 activity depends on (co)-administration of hdl and/or apoa1.117 hdl and apoa1 are not small-molecules but peptides, and therefore fall outside of the scope of the present review. similarly, it has been shown in several reports that hdl-mimics consisting of 26 amino acids are able to increase abca1-mediated transport.289 although these molecules are also not small-molecules, the scarceness of activators of abca transporters warrants the inclusion of these middle-sized molecules here. in 2004, structural elements of apoa1 were discovered to promote abca1-mediated cholesterol efflux.290 in 2007, vedhachalam et al. discovered that the c-terminus of apoe promoted abca1-mediated efflux from murine j774.a1 macrophages.291 the latter discovery led to the development of two short-length peptides, ati-5261 and cs-6253, consisting of 26 amino acids each.289 their amino acid sequences expressed in single-letter code are evrskleewfaafrefaeeflarlks289 and evcitskleewlaalcitelaeellacit-lks (cit = citrulline),292 respectively, which is of particular interest for the development of novel lead structures. both peptides increased abca1-mediated cholesterol and phospholipid transport in murine and human macrophages.289,292 interestingly, cs-6253 decreased 125i-labed apoa1 binding to abca1,292 as demonstrated for glibenclamide,267,270 probucol,246,278 cyclosporine a,245,249 and blt-4270 before. however, cs-6253 was shown to compete with apoa1 to promote abca1-mediated transport.292 both ati-5261 and cs-6253 have a high practical relevance regarding ad and other neurodegenerative diseases, as these agents demonstrated in vivo efficacy.289,293 ati-5261 treatment of high fat diet-fed apoe knock-out mice decreased cholesterol levels in both plasma and feces and reduced atherosclerotic lesions.289 for cs-6253, a reduction of aβ42 levels and tau protein phosphorylation in transgenic humanized apoe4 mice was demonstrated, which was accompanied by improved cognitive functions.293 interestingly, an elevation of abca1 protein was also observed in treated mice.293 indeed, a stabilization and/or induction may also have contributed to the observed effects. however, the proven direct binding of these agents suggested that activation takes place as the major mode of action. nonetheless, cs-6253 has not been tested in ad mouse models so far, and being a peptide, it would not be suitable for oral application in patients. small-molecule regulators of abca transporters the herein discussed regulators interfere with abca transporter expression and/or trafficking. important representatives are depicted in figure 2 and additional information is given in terms of their mode of modulation. since many different pathways are involved in abca transporter regulation, figure 3 provides a general overview of participating proteins and protein families in terms of the most studied abca transporter, abca1. inducers abca1 lxr and rxr pathways given the findings in ad mouse models with knock-out of abca1/abca1 or overexpression of abca1, upregulating abca1 activity may be a therapeutic strategy for decreasing aβ pathology in ad. abca1 is under the transcriptional control of the nuclear receptors liver-x-receptor (lxr) and retinoid-x-receptor (rxr),294-296 which can be targeted by small-molecule agonists of lxr and rxr to induce abca1 expression (figure 3). numerous studies reported that treatment of app-transgenic mice with lxr or rxr agonists decreased aβ load126,297-301 and/or improved cognitive impairment.126,297,298,300 other studies reported cognitive improvement without significant changes in aβ load in app-transgenic mice treated with lxr agonists.302,303 lxr and rxr agonists have already been described extensively as potential therapeutics in the literature, also with respect to ad.304 the present review will focus on those agonists that were reported in clear association with abca1. oxysterols and retinoic acids 22-(r)-hydroxycholesterol (figure 2) has been established as the natural gold standard for abca1/abca1 induction through lxr activation,122,205,249,252,259,262-264,268,277,278,305-315 while 9-cis retinoic acid (figure 2) became the natural gold standard for rxr activation.122,245,249,259,262,264,277,278,309,311,313,316 the inducing effects were described both on abca1/abca1 mrna122,205,252,263,264,305,307-311,313,315-317 and abca1 protein levels.122,252,263,264,306,309-311,316,318 other oxysterols like 4-hydroxycholesterol, 20-(s)-hydroxycholesterol, 22-(s)-hydroxycholesterol, 24-hydroxycholesterol, 24-(s)-hydroxycholesterol, 25-hydroxycholesterol, 27-hydroxycholesterol, and cholesterol itself also induced abca1/abca1 mrna205,305,313,315,319-327 and abca1 protein levels.321,328 the increase in abca1 protein was functionally confirmed by an enhanced cholesterol305,306,313,315,318 and phospholipid efflux,311,318 as well as reduced total cholesterol influx.305 specifically 22-(r)-hydroxycholesterol and cholesterol induced both lxra/lxra and lxrb/lxrb.310,321 additionally, cholesterol also induced murine peroxisome proliferator-activated receptor γ (ppar-γ) mrna (pparg),321 which represents an important alternative pathway for abca1/abca1 induction. furthermore, 24-(s)-hydroxycholesterol reduced in parallel the sterol regulation element-binding protein 2 (srebp2) gene expression (srebp2).323 the sreb protein family also represents another important pathway in abca1/abca1 regulation. the 9-cis-retinoic acid derivative all-trans¬-retinoic acid (atra) significantly increased abca1/abca1 mrna and abca1 protein content in murine and human macrophages, which was paralleled by increased lxra mrna levels in human macrophages.329 this increase resulted in a subsequently enhanced cholesterol efflux from murine macrophages. atra is an agonist of the retinoic acid receptor (rar),329 which is in close relation to the rxr receptor and a potential target of retinoic acid derivatives. to901317 and gw3965 the synthetic gold standard and most studied abca1/abca1 inducer in the literature is to901317 (often referred to as ‘t0901317’; figure 2).205,245,250,252,259,260,262,264,271,272,279,280,282,308,310,317,319,322,324,326,328-345 to901317 targeted both the lxr α250,310,328,330,332,335,337-340,342 and lxr β pathways,250,310,338,342 which correlated to abca1/abca1 induction on mrna and abca1 protein levels.205,250,279,282,310,319,322,324,326,328,330-335,337-340,342,343 in addition, an induction of srebp1c/srebp1c has also been observed.336,342 functionally, to901317 increased cholesterol efflux,250,259,260,262,264,282,319,324,329,331,342 decreased intracellular aβ content, and increased aβ secretion from different murine brain cells.126,345 further, it reduced aβ25-35-mediated toxicity toward cells by induction of abca1.280 in addition, to901317 mitigated memory deficits in high-fat diet-fed app23 mice, reducing both plaque and soluble aβ protein levels.344 besides, to901317 reduced methionine-(homocysteine)-induced atherosclerotic lesions in apoe knock-out c57bl/6 mice.335 these findings were paralleled by an increase of abca1 mrna and abca1 protein content,335 suggesting a potential relevance of to901217 in ad therapy, although it must be taken into account that lxr activators, in particular to901317, were demonstrated to have severe side effects in mice, such as neutropenia, hypertriacylglycerolemia, hepatic triacylglycerol accumulation, and hepatic steatosis.271,346,347 the second most common synthetic lxr-α and lxr-β agonist is gw3965 (figure 2).255,272,317,319,321,334,348-352 gw3965 increased mrna317,319,321,348,349,351,352 and protein levels255,272,351 in different abca1-expressing cells. functionally, increased abca1 mrna and abca1 protein levels correlated with enhanced cholesterol efflux.255,351 strikingly, exposure of murine bv2 microglia to gw3965 reduced aβ42 levels due to an enhanced degradation of aβ42,126 suggesting that abca1 contributes to general aβ degradation. finally, gw3965 significantly increased abca1 transcription in c57bl/6 mice,334,351 and improved contextual memory as well as aβ pathology in tg2576 mice,126 emphasizing its high relevance in ad therapy. abca1 other lxr agonists and inducers sterane and sterane-like natural compounds several sterane derivatives were demonstrated to target lxr-α and lxr-β activation253,307,310,353 and/or lxra/lxra and lxrb/lxrb upregulation,330,332,354,355,356,357 resulting in induction of abca1/abca1. celastrol,330,332 digoxin,253 fuco-sterol,308 certain gypenosides,354 ouabain,253 platy-codin d,355 saikosaponin a,356 24-(s)-saringosterol,307 24-(s)-stigmast-5-ene-3β,24-diol,307 taxarasterol,353 testosterone,357 and tr1310 increased abca1/abca1 mrna307,308,310,330,332,353,354,356,357 and/or abca1 protein content310,253,353,354,355,357 leading to an enhanced efflux of cholesterol in vitro253,308,330,332 and decreased intracellular cholesterol and/or phospholipid levels in vitro330,332,354,356,357 and in vivo in mice.253 the effect of fucosterol was comparable to that of the standard abca1/abca1 inducer to901317.308 a correlation to srebp1(c) upregulation308,307,357 and srebp1 protein expression357 could be determined in case of fucosterol,308 24-(s)-saringosterol,307 24-(s)-stigmast-5-ene-3β,24-diol,307 and testosterone.357 in case of celastrol, the regulation of intracellular cholesterol was pinned to an activation of autophagy330,332 and lipophagy,330 which are processes that may be associated with aβ degradation. flavonoids the flavonoids naringenin,339 quercetin,358 and vitexin359 increased abca1/abca1 mrna339,359 and abca1 protein levels339,360,358 by induction of lxra/lxra mrna358,359 and lxr-α protein.339,360 the effect of naringenin and the standard abca1/abca1 inducer to901317 were additive. naringenin was shown to be dependent on the camp-activated protein kinase (ampk) regulation (ampk), as well as srebp1c regulation.339 the ampk pathway is another very important regulator of abca1 expression. functionally, cholesterol efflux from human339,360 and murine360 macrophages was increased in the presence of naringenin.339,360 in vivo, naringenin and quercetin induced abca1360 and abca1,361,362 as well as abca1-mediated cholesterol transport,360 which was reflected in reduced atherosclerotic lesions in the aorta of high-fat diet-fed c57bl/6 mice.360 in terms of quercetin, a protein increase of lxr-α and ppar-γ was observed.361 chalcones, the precursors of flavonoid biosynthesis, were also demonstrated to intervene with abca1 expression. the chalcone derivatives 1h,363 1m,363,364 and 1m-6364 were demonstrated to increase abca1 mrna and abca1 protein levels in thp-1 macrophages,363,364 which was accompanied by an increase in lxra mrna and lxr-α protein levels.363 the intracellular lipid content was decreased, while the cholesterol efflux was increased after exposure of thp1-cells to 1m-6.364 in addition, srebp1 mrna was increased by 1m-6,364 and aortic atherosclerotic plaques were reduced in ldlr knock-out c57bl/6 mice.364 polyphenols and diterpenoid natural compounds the polyphenols kuwanon g,365 paeonol,252 the celtis biondii-derived compound ethyl 2,4,6-trihydroxybenzoate,342 and the diterpenoid farnesin366 increased abca1/abca1 mrna252,342,365,366 and abca1 protein252,342,365,366 content in an lxr-α-252,366 and lxr-β-dependent342 manner, which in parallel reduced cholesterol content252 and increased abca1-mediated cholesterol efflux in various cell lines.252,342,366 in vivo, farnesin increased abca1 protein content and cholesterol efflux in apoe knock-out c57bl/6 mice in primary peritoneal macrophages and the aorta, which was reflected in reduced atherosclerotic plaques.366 other natural compounds several other natural compounds induced abca1/abca1 targeting lxr-α and lxr-β activation256,272,256,349,367 and/or lxra/lxra and lxrb/lxrb induction.331,348,350,368,369,370,371,372,373,374 the garlic ingredient allicin,350 the alkaloid berberine,256 the coumarin bergapten a,368 certain pestalotiopsis neglecta-derived chromene derivatives,348 the rheum palmatum-derived anthra-quinone danthron,369 the lacton 1,6-o,o-diacetylbritannilactone,371 epigallocatechin gallate (egcg),370 the glycoside geniposide,375 the vegetable ingredient phenethyl isothiocyanate,373 the carotenoid lycopene,372 the pestalotiopsis neglecta-derived hydroquinone pestalotioquinoside c,349 the alkaloid rutaecarpine,367 selenium,374 the macro-lactone soraphene a,272 and vitamin d3331 led to increased abca1/abca1 mrna256,272,331,348,369,256,367,370,372,373 and abca1 protein256,272,331,349,350,368,369,256,367,371,373,374 content in vitro331,349,350,369,375,374 and in vivo,368,369,370,371,372,373 enhancing cellular cholesterol efflux256,272,256,367,369 and reducing intracellular cholesterol con-tent.331,350,369,256,367,375,372,374 danthron also increased ampk protein levels,369 while egcg downregulated srebp1 mrna and srebp1 protein content.370 lycopene induced ppara mrna in tobacco carcinogenand cigarette smoke-exposed ferrets,372 while isothiocyanate induced pparg mrna as well as ppar-γ protein content in high fat diet-fed c57bl/6 mice.373 the inducing effects on abca1 expression of vitamin d3 and to901317 were additive.331 danthron, egcg, geniposide, and rutaecarpine demonstrated also reduced atherosclerotic lesions in apoe knock-out c57bl/6 mice,369,370,375,367 and isothiocyanate ameliorated the aortic injury of the high-fat diet in the same mice.373 pharmacological drugs several pharmacological drugs also demonstrated an induction of abca1/abca1 through lxr-α and/or lxr-β, including the α1-blocker doxazosin,376 the 5-ht3 receptor antagonist ondansetron,279 and the anesthetic propofol.377 consequently, increased abca1 mrna279,376 and abca1 protein279,376 levels were observed in human279,377 and murine279,376 macrophages376,377 as well as astrocytes.279 functionally, ondansetron induced apoe efflux,279 while propofol led to increased cholesterol efflux.377 in addition, propofol increased pparg mrna and ppar-γ protein content in human macrophages.377 furthermore, certain antineoplastic agents interfered with abca1 expression via lxr-α and/or lxr-β. doxorubicin demonstrated an lxr activation with subsequent induction of abca1 mrna and abca1 protein in vitro and in vivo.250 functionally, doxorubicin elevated cholesterol export in vitro. it was shown that intraand extracellular levels of cholesterol, cholesterol precursors, and several oxysterols were elevated after exposure to doxorubicin. these precursors included lathosterol, lanosterol, and desmosterol, while the oxysterols included 7-α-hydroxycholesterol, 7-β-hydroxycholesterol, 7-ketocholesterol, 24-hydroxycholesterol, and 27-hydroxycholesterol. the authors suggested that doxorubicin exposure induced cholesterol metabolism subsequently leading to an induction of abca1. besides, idarubicin augmented also abca1 mrna levels in vitro. synthetic compounds and hts hits other synthetic compounds have been shown to induce abca1/abca1 expression by lxr-α and/or lxr-β induction. the polymer pyrrole-imidazole-polyamide activated a promoter region for abca1 expression and thereby increased cholesterol and lipid efflux from raw264.7 cells.376 the authors confirmed their findings in vivo, revealing increased abca1 mrna and abca1 protein content in peripheral blood mononuclear cells and the liver in c57bl/6 mice after exposure to pyrrole-imidazole-polyamide. in addition, the lxr agonist lxr623 induced abca1 mrna and abca1 protein levels in two human renal adenocarcinoma cell lines334 as well as abca1 mrna levels in vivo in c57bl/6 mice.378 this induction was reflected in reduced intracellular cholesterol and triglyceride levels. it must be noted that several other synthetic lxr-α and lxr-β agonists induced abca1 expression in vivo: az1–az9, az876, bms-852927, f1, way254011.378 finally, an hts approach discovered two lxr-α and lxr-β agonists as novel small-molecule abca1/abca1 inducers: f4 and m2.319 synthetic approaches a few synthetic approaches have aimed toward the development of abca1/abca1 inducers.271,336,352,379-382 the cholic acid analog 14b,336 the thiophene derivative cl2-57,271 as well as derivatives of n-benzothiazolyl-2-benzene-sulfonamide,379 ginsenoside,352 and rutaecarpine,367 all induced abca1/abca1 mrna336,352,381 and abca1 protein271,336,379,381 content in vitro271,336,379 and in vivo,271 targeting the lxr-α/lxr-β pathway352 by activation271 or induction336 of lxr-α/lxra/lxra and/or lxr-β/lxrb/lxrb. in vitro, cholesterol efflux increased379,381 and intracellular cholesterol as well as lipid content were reduced,336,352 while plasma and liver triglycerides levels were reduced in vivo in high fat diet-fed c57bl/6 mice.271 interestingly, 14b induced farnesoid-x-receptor (fxr) transcription (fxr),336 and cl2-57 inhibited rxr-β, ppar-γ, and ppar-δ,271 finally, singh et al. described highly potent lxr-α and lxr-β agonists with effect at concentrations in the nanomolar range.382 the described podocarpic acid derivatives have not yet been demonstrated to induce abca1. however, these compounds were designated as potential abca1 inducers by the authors,382 and their high potency makes them interesting candidates for further evaluation. such synthetic approaches should be highlighted,271,336,352,379-382 as chemical derivatization of abca1 inducers and elucidation of their structure-activity relationships (sar) have not yet been comprehensively assessed. more reports are needed to gain innovative molecules that can be considered clinically for the treatment of various abca1-related diseases. abca1 other rxr agonists and inducers in terms of synthetic rxr agonists, the 4-chromanon derivatives spf1 and spf2 increased abcb1 mrna and abca1 protein levels and lowered aβ25–35-mediated cell toxicity in vitro.280 the same effect was observed for the rxr agonist bexarotene,280 an fda approved drug against t-cell lymphoma-related cutaneous malformations. bexarotene was used as a standard inducer of abca1/abca1 via the rxr pathway in several studies.271,272,280,319,380 induction of abca1 mrna and abca1 protein levels was maximal for bexarotene in combination with to901317.280 bexarotene is of particular practical relevance as a potential treatment against ad due to its in vivo effects. in different ad mouse models, bexarotene increased abca1 mrna and abca1 protein levels, but also reduced cerebral load of aβ and hyperphosphorylated protein tau, which is also a histological marker in ad and other dementias.297,383 this prospect led to synthetic bexarotene derivatives, specifically z10 and z36.380 both candidates induced abca1 protein expression by rxr-α activation and reduced aβ burden in the hippocampus of female app/ps1 mice. this coincided with an enhanced abca1 protein expression in bv2 cells. moreover, the pan-rar agonist ttnpb also increased abca1 protein content in murine macrophages in an rxr-α-dependent manner. however, the effect was generally smaller compared to the effect of atra.329 finally, a combination of the lxr and rxr agonists ro0721957 and ro0264456 increased abca1 mrna in thp-1 macrophages accompanied by increased cholesterol efflux.384 ro0264456 was demonstrated to increase abca1 protein content in combination with to901317.260 abca1 – protein kinase c (pkc), ampk, and p38 mitogen-activated protein kinase (mapk) an alternative approach to induce abca1 is targeting the pkc pathway (figure 3). pkc agonists were extensively used to induce abca1/abca1 mrna and abca1 protein levels.230,248,249,255,265,266,273,278,289-292,384-387 prominent pkc agonists include camp313 as well as synthetic derivatives, such as 8-bromo-camp (8-br-camp; figure 2),230,249,255,266,290,292 8-(4-chlorophenylthio)-camp (cpt-camp),273,291,384 and dibutyryl-camp.385-387 the observed effects ranged in the same order of magnitude as the combination of 22-(r)-hydroxycholesterol and 9-cis-retoic acid.313 the increase in abca1/abca1 mrna and abca1 protein levels was reflected in an enhancement of abca1-mediated cholesterol and phospholipid efflux,249,255,386 and increased apoa1 binding to murine raw264.7 macrophages.385-387 similar observations have been made for the pkc stimulant phorbol 12-myristate 13-acetate (pma), which induced abca1 protein expression and abca1-mediated cholesterol and phospholipid release.386 pma is also the standard substance used to differentiate human monocytic leukemia cells into thp-1 macrophages – a standard host system for abca transporter evaluation.231,245,249,256,268,272,275,292,308,310,312-316,321,328,335,338, ​339,341,342,360,363,364,366,377,384,388-397 regarding the ampk pathway (figure 3), the natural compound curcumin induced abca1/abca1 mrna338,388 and abca1 protein levels388,394 as well as cholesterol efflux338,388,394 in thp-1338,388,394 and raw264.7394 macrophages, which was also mediated through lxr-α activation.338 however, these lxr-α activating effects were much more pronounced in combination with the gold standard to901317.338 other ampk-targeting agents are a 769662 and metformin,398 which induced abca1/abca1,398 lxra/lxra,396,398 and lxrb/ lxrb396,398 in human398 and murine (primary) macrophages,398 leading to increased cholesterol efflux.396 concerning the mapk pathway (figure 3), the sterane glycoside ginsenoside compound k increased abca1 mrna and abca1 protein levels in murine macrophages, reducing intracellular lipid content and promoting autophagy.399 these effects were pinned to a negative impact on the mapk pathway. finally, a synthetic inhibitor of mapk, sb203580, was shown to induce abca1 protein in combination with the above mentioned geniposide in vitro in murine macrophages.375 abca1 the ppar pathway another well-known approach to induce abca1 involves the ppar pathway (figure 3).268,272,295,309,315,321,326, 327,337,343,395,400-409 certain ppar/ppar inducers and/or ppar activators have been described above, as these modulators also have effects on the lxr pathway.321,361,372,373,377 several natural compounds target the ppar pathway, such as the flavonoids homo-eriodictyol,402 hesperetin-7-o-β-d-glucopyrano-side,402 scutellarein,403 and the antimycotic trichostatin a.410 these compounds increased abca1402 and pparg402 mrna as well as abca1,402,410 ppar-α,403 and ppar-γ402,410 protein levels in vitro402,410 and in vivo.403 decreased intracellular cholesterol levels were also observed.402 trichostatin a reduced aortic atherosclerotic plaques in high-fat diet-fed apoe knock-out mice,410 and an upregulation of abca1, ppar-γ, and lxr-α/β protein levels was observed in aortic cells as well as peritoneal macrophages.410 several drugs and drug-like ppar agonists were revealed to induce abca1/abca1 mrna and/or abca1 protein content, including the ppar-α agonists fenofibrate,326,400,404 pemafibrate (k 877),405 wy14643,268,343 and rpr-5,268 as well as the ppar-γ agonists efatutazone,337 pioglitazone,272,309,326,395,407 pitavastatin,343 prostaglandin j2 (pg-j2),268,327 rosiglitazone (figure 2),268,309,315,408,409 troglitazone,268 and gw7845,315 but also the broad-spectrum ppar-α, ppar-β, and ppar-γ agonist bezafibrate268,327 and the multitarget ppar-α, ppar-γ, and ppar-δ agonist tetradecylthioacetic acid.401 this induction was observed for abca1/abca1 mrna268,315,343,401,405 as well as abca1 protein levels,268,337,343,395,405,409 and was functionally confirmed by increased cholesterol efflux.268,315 a connection between the ppar and lxr pathways has also been drawn,268,326,327,337,400 highlighting the importance of both pathways for abca1/abca1 induction. furthermore, fenofibrate had a positive impact on both the lxr-α and ampk pathways400 certain ppar agonists have been used as standard inducers of abca1, e.g., pioglitazone407 and rosiglitazone.408 synthetic ppar agonists were also reported to induce abca1.406 the benzothiazole derivative e3317 dose-dependently increased abca1/abca1 mrna and abca1 protein levels though ppar-γ activation in several cell lines.406 this was reflected in decreased cholesterol efflux and reduced intracellular cholesterol content. finally, a molecular docking approach to discover novel ppar agonists has yielded gq-11, which induced abca1 mrna in livers of c57bl/6 ldlr knock-out mice.407 abca1 the 3-hydroxyl-3-methyl glutaryl-(hmg)-coa-reductase pathway other targets for abca1/abca1 induction are the 3-hydroxyl-3-methylglutaryl-(hmg)-coa-reduc-tase and cellular cholesterol synthesis (figure 3).318,343 several hmg-coa-reductase inhibitors such as atorvastatin (figure 2),330,343,362 fluvastatin,312,411 mevastatin (compactin),318 pitavastatin,318,343 and simvastatin312,343 increased abca1/abca1 mrna312,343 and abca1 protein levels,362,411 as well as abca1-mediated cholesterol efflux.318 these data are surprising, as one might expect the loss-of-function of an enzyme in the cholesterol synthesis pathway to induce a decrease of abca1, preventing cholesterol depletion from cells.314384,412 conversely, the overproduction of cholesterol leads to the opposite effect, as demonstrated for mevalonate, which is a building block of cholesterol synthesis413 and has been demonstrated to increase abca1/abca1 mrna312,314 and to abrogate abca1 downregulation.312 pitavastatin addressed srebp-driven promotor regions upregulating abca1 mrna levels,343 and atorvastatin reduced atherosclerotic plaques in apoe knocked-out c57bl/6 mice by induction of abca1 protein content in the murine aorta.362 other abca1 inducers sterane and sterane-like natural compounds several other agents were reported to induce abca1/abca1 mrna and/or abca1 protein level(s), with some studies reporting a unique mechanism of action for these agents. such compounds include the sterane derivative ponasterone a (ecdysone; abca1 protein; abca1-mediated cholesterol and phospholipid transport),202 and the enoxolone derivative glycyrrhizine (abca1 protein).414 in addition, the sterane derivative and farnesoid-x-receptor (fxr) activator obeticholic acid induced abca1 mrna levels in vivo in the ileum of srb1-deficient c57bl/6 mice.415 in thp-1 macrophages, the sterane-like maslinic acid induced abca1 mrna levels, paralleled with an increased cholesterol efflux from these cells.390 finally, the salvia miltiorrhiza-derived tanshindiol c was demonstrated to induce peroxiredoxin 1 mrna (prdx1) and protein (prdx1) content in murine raw264.7 cells.416 prdx1 was demonstrated to regulate abca1 mrna and abca1 protein expression. a reduction of intracellular cholesterol levels in murine peritoneal macrophages could also be observed. flavonoids the flavonoids daidzein (figure 2),309 kaempferol,397 and pratensein309 induced abca1 mrna309,397 and abca1 protein levels309 as well as abca1-mediated cholesterol efflux.397in addition, hesperetin-7-o-rutinosid (hesperidin) abrogated the negative effect of varenicline on abca1 protein expression in raw264.7 macrophages.417 the authors could underpin their findings with a reduction of aortic atherosclerotic plaques in apoe knock-out c57bl/6 mice along with reduced lipid levels in peritoneal macrophages derived from these mice. polyphenols and polyphenol-like natural compounds several polyphenols and polyphenol-like compounds induced abca1 mrna408,418 and abca1 protein393,404 levels in murine393,404,408,418 and human393 macrophages, leading to an increased cholesterol efflux.404,408,418 these include certain cannabis sativa-derived stilbenoids404 as well as the tadehagi triquetrum-derived phenylpropanoid glycosides urolithin a418 and urolithin b (sulfate).393 in vivo, atherosclerotic plaques were reduced after urolothin b treatment. one phenylpropanoid glycoside was demonstrated to increase lxra, but none of the other compounds could confirm these results. given that the effect of all compounds on abca1 expression was similar, it is likely that another, yet unknown pathway was the major contributor to the observed effects. other natural compounds sodium butyrate induced abca1 mrna and abca1 protein levels in murine raw264.7 cells, accompanied by an increased efflux of cholesterol from these cells.419 this induction was reflected by increased abca1 protein content in vivo, reduced plasma cholesterol and triglyceride levels, and reduced aortic atherosclerotic lesions and hepatic steatosis in high fat diet-fed apoe knock-out c57bl/6 mice. pharmacological drugs several pharmacological drugs induced abcb1/abca1 mrna309,420,421 and abca1 protein,309,391,421 including the anti-obesity drug orlistat,391 the antibiotic sulfoxaflor,420 the leukotriene receptor antagonist zafirlukast,421 as well as the anthracyclines aclarubicin309 and pyrromycin.309 zafirlukast in particular reduced intracellular cholesterol and lipid content in oxidized ldl-(oxldl)-induced lipid-overloaded raw264.7 macrophages, and increased cholesterol efflux from these cells.421 finally, it should be highlighted that mifepristone has frequently been used in a mifepristone-inducible transfection system to stabilize and increase abca1 expression in abca1-transfected baby hamster kidney (bhk)-21 cells. this abca1 induction could be functionally confirmed by increased abca1-mediated cholesterol and phospholipid efflux.245,273,422 synthetic compounds, hts hits, and synthetic approaches the purinergic p2y7 receptor antagonists az-1, az-2, and az10606120 increased abca1 mrna and abca1 protein levels and resulted in enhanced cholesterol efflux from human ccfsttg1 astrocytoma cells.423 the polychlorinated biphenyl quinone 2,3,5-trichloro-6-phenyl-[1,4]-benzo-quinone (pcb29-pq)424 and the fluorescigenic pyrazoline derivative 5 (fpd5)425 increased abca1 mrna424 and abca1 protein425 content in raw264.7 macrophages and reduced cholesterol content in these cells.424,425 in vivo, fpd5 reduced aortic lipid and cholesterol content and atherosclerotic lesions in apoe knock-out c57bl/6 mice. inducers of other abca transporters abca2 and abca3 as detailed above, abca2 and abca3 are believed to contribute to multidrug resistance in cancer.171,232-239,241,242 in human k562 leukemia cells, it was demonstrated that the tyrosine kinase inhibitor (tki) imatinib induced increased levels of abca2 mrna and abca2 protein.236 furthermore, the tkis dasatinib, imatinib, and nilotinib increased abca3 mrna levels in various cancer cell lines as well as in tki-treated leukemia patients.426 the antimetabolite 5-fluorouracil (5-fu) induced expression of abca3 mrna in a cholangiocarcinoma cell line,427 and methotrexate increased abca2 and abca3 mrna in a leukemia cell line.242 finally, the steroid hormone progesterone,179 the antibiotic sulfoxaflor,420 and the endosomal cholesterol transport inhibitor u18666a179 induced abca2/ abca2 transcripts420 in aphis gossypii420 as well as in abca2-transfected chinese hamster ovary (cho) cells and hepg2 cells179 abca5 and abca6 as discussed earlier, cholesterol and its derivatives have been shown to induce abca1/abca1 mrna and/or abca1 protein levels.122,205,249,252,259,262-264,268,277,278,305-315,319-328 induction by cholesterol has also been demonstrated for abca5 mrna and abca5 protein levels in raw264.7 macrophages.321 this effect relied on the induction of lxra, lxrb, and pparg. consequently, several lxr and ppar agonists increased abca5 expression, including bezafibrate (ppar-α, ppar-β, and ppar-γ; abca5 mrna and abca5 protein), gw3965 (lxr; abca5 mrna), rosiglitazone (ppar-γ; abca5 mrna), and troglitazone (ppar-γ; abca5 mrna) in murine raw264.7 macrophages.321 in addition, the hmg-coa-reductase inhibitor atorvastatin increased abca5 mrna and abca5 protein levels.321 interestingly, the abca1 inhibitor tacrolimus245 showed induction of abca5 mrna in human brain microvascular endothelial cells.428 the hmg-coa-reductase inhibitors lovastatin and mevastatin resulted in an induction of abca6 mrna in the human endothelial cell line ea.hy926.429 finally, in an abca12 pig model of the rare and lethal skin disease harlequin ichthyosis, it was demonstrated that treatment with the synthetic retinoid acitretin leads to a compensatory induction of abca6 mrna.430 abca7 similarly to abca1,202 the sterane derivative ponasterone a increased both abca7 protein expression and abca7-mediated transport, mainly of phospholipids, but also of cholesterol to a small extent.202 hmg-coa-reductase inhibitors were described above to interfere with abca1/ abca1312,318,330,343,362,411 and abca5321 expression. in addition, certain compounds were also demonstrated to interfere with abca7 expression.205,431 these include pravastatin205,431 and rosuvastatin (figure 2).431 these agents increased abca7 mrna and abca7 protein levels in vitro,205,431 whilst pravastatin had the same effects in vivo in murine peritoneal macrophages.431 surprisingly, this increase of abca7 mrna and abca7 protein levels was accompanied by a downregulation of lxra and upregulation of srebp2 in vitro.431 functionally, pravastatin and rosuvastatin reduced intracellular cholesterol content431 and induced phagocytosis in vitro and in vivo.431 these effects occurred in response to an abca1 downregulation by hmg-coa-reductase inhibitors as described earlier.312,321,384,432,439,429 due to their functional similarity, the upregulation of abca7 could be a compensatory mechanism to counteract the loss of abca1.198 similarly, the observed lxra downand srebp up-regulation may be a compensatory mechanism to counteract the loss of intracellular cholesterol. finally, as described for abca1,422 exposure of abca7-transfected bhk-21 cells to mifepristone increased abca7 protein content and abca7-mediated transport of phospholipids and, to a much lesser extent, of cholesterol.422 abca8 abca8 mrna and abca8 protein content were induced by gemcitabine in panc-1 and cfpac-1 human pancreatic cancer cells.221 in rat liver, an induction of abca8 was demonstrated via microarray analysis of cdna when the rats were exposed to polyethyleneglycol-block-polylactide nanoparticles.433 abca12 several lxr and ppar agonists induced abca12/abca12 expression, such as 22-(r)-hydroxycholesterol (lxr),434 to901317 (lxr),430,434 ciglitazone (ppar-γ),434 gi 251929x (ppar-γ),434 troglitazone (ppar-γ),434 ceramide n-hexanoyl-d-erythro-sphingosine (ppar-δ),435 and gw610742 (ppar-δ).434 interestingly, inhibition of certain enzymes to prevent ceramide processing elevated intracellular ceramide content and subsequently abca12 mrna levels.435 these enzymes include, for example, the glycosyl-ceramide-transferase synthase [d threo-1-phenyl-2-hexadecanoylamino-3-morpholino-1-pro-panol (d-ppmp), d-threo-1-phenyl-2-palmitoyl-3-pyrrolidinopropanol (d-pppp / p4) and dl-threo-1-phenyl-2-de-canoylamino-3-morpholino-1-propanol (d-ddmp)], the sphingomyelin synthase [tricyclo[5.2.1.02,6]decanyl)ethanedithioic acid (d609 xanthate)], as well as the ceramidase [d-erythro-2-tetradecanoylamino-1-phenyl-1-propanol (d-mapp) and (d nmappd / b13)].435 downregulators abca1 lxr and rxr pathways – intrinsic substrates the intrinsic metabolite asymmetric dimethylarginine (adma) reduced abca1 mrna and abca1 protein levels in human and murine j744 macrophages in combination with oxldl, resulting in increased intracellular cholesterol and triglyceride levels.392 this was accompanied by decreased efflux of cholesterol from these cells. the authors suggested a negative effect on the lxr-α pathway. in this regard, the lxr-α downregulator homocysteine significantly reduced abca1/abca1 mrna and abca1 protein expression in vitro in thp-1 macrophages as well as in vivo in macrophages from apoe knock-out c57bl/6 mice.335 the cattle metabolite dipeptide phenylalanine-proline decreased abca1 mrna and abca1 protein levels in human colorectal adenocarcinoma-derived caco-2 cells.436 the observed downregulation of lxrb mrna could explain the negative impact on abca1 expression. in vivo, the jejunal abca1 mrna levels were decreased in wistar rats.436 the abca1 substrate α-tocopherol230 reduced abca1/abca1 mrna levels in vitro and in vivo.231 the same effects were observed for γ-tocopherol in vitro, most likely through the same mechanism. the authors suggested a negative impact on the lxr pathway due to deprived oxycholesterol derivatives after α-tocopherol treatment both in vitro in hep3b cells and in vivo in rat liver.231 lxr and rxr pathways sterane and sterane-like natural compounds cholesterol and its derivatives have extensively been used to induce abca1/abca1 expression122,205,249,252,259,262-264,268,277,278,305-315,319-328 however, mid-term exposure to excess cholesterol decreased abca1 expression though a negative impact on lxra, lxrb, and pparg expression.321 similar observations have been made for the sterol derivative dexamethasone, which also reduced abca1/abca1 mrna and abca1 protein expression in vitro and in vivo by downregulation of lxra/lxra mrna and lxr-α protein levels as well as upregulation of srebp2 and hmg-coa-reductase gene expression (hmgcr).437 finally, an abca1 mrna reduction was observed in murine raw264.7 macrophages for the thelenota ananas-derived saponin desulfated holothurin a.438 interestingly, hmgcr was downregulated after exposure to desulfated holothurin a, which contradicts other findings.437 lxr and rxr pathways – other natural compounds certain chalcone derivatives also caused reduced expression of abca1 protein.363 in addition, lipopolysaccharides reduced abca1 protein content in endometrial endothelial cells from c57bl/6 mice, which was accompanied by increased cholesterol levels in these cells.374 a parallel reduction in lxr-α protein was also observed. finally, the carcinogenic agent n-nitrosodiethylamine (ndea) demonstrated in vivo in wistar albino rats a downregulation of lxra and lxrb mrna as well as lxr-α and lxr-β protein levels and, subsequently, abca1 protein.368 lxr and rxr pathways – synthetic compounds and hts hits in terms of other lxr antagonists and downregulators, gsk2033 (figure 2),272,330,333 5cppss-50,357 and sr9243333 reduced abca1 mrna and abca1 protein expression.272,330,333,357 hmg-coa-reductase pathways – intrinsic substrates and pharmacological drugs the peptide hormone angiotensin ii reduced cholesterol efflux from murine peritoneal macrophages.439 this reduction could be reversed by the angiotensin ii receptor antagonist losartan. the authors concluded that abca1 was not involved in this process, as no concurrent change in abca1 expression was observed.439 however, in another report, angiotensin ii indeed demonstrated a reduction of abca1 mrna and abca1 protein levels in human podocytes.440 the authors concluded a contribution of the hmg-coa-reductase, srebp1, and srebp2.440 geranylgeraniol pyrophosphate (ggpp; figure 2), a product of the mevalonate pathway, reduced abca1 mrna expression in human macrophages, which was blocked by the prenylation inhibitors l836,978 and l-839,867.314 in addition, a reduction of abca1-mediated cholesterol export was observed, which is also true for mevalonate itself.318 ggpp was used as a standard abca1 downregulator in certain studies.279,354,366 as discussed above, atorvastatin,343 fluvastatin,312 pitavastatin,318,343 and simvasta-tin312,343 have been shown to increase abca1/abca1 mrna levels,312,343 and to enhance abca1-mediated cholesterol efflux.318 however, atorvastatin,312,321,384 fluvastatin,312 pitavastatin432 and simvastatin312,384 have also been reported to reduce abca1/abca1 transcription312,321,384,432 and abca1-mediated cholesterol efflux.384,431 these observations are in agreement with other reports on hmg-coa-reductase inhibitors that downregulated abca1.312,431 in particular, lovastatin,312 mevastatin (compactin),412 pravastatin,431 and rosuvastatin431,441 reduced abca1/abca1 mrna312,431 and abca1 protein431 levels. these findings are expected given that the loss of cholesterol by interruption of cholesterol synthesis leads to a compensatory reduction of cholesterol efflux.314384,412 the contradictory results relating to abca1 may be caused by the use of variable experimental conditions between studies, such as different cell lines, assay methodologies, or small-molecule-related aspects, such as concentration, distribution, and protein binding. finally, a similar interconnection between hmg-coa and abca1 was drawn for the antineoplastic agent mitotane, which downregulated abca1 mrna441 and increased intracellular cholesterol levels.333,441 however, mitotane in combination with lxr antagonists and lxr downregulators had an inverse effect on mrna regulation, increasing abca1 expression.327 pkc pathway intrinsic substrates interestingly, it was also demonstrated that long-term exposure to low concentrations of 8-br-camp, a standard abca1/abca1 inducer,230,249,255,266,290,292 led to decreased apoe secretion from human monocyte-derived macrophages.266 apoe secretion can be considered as a surrogate marker for abca1-mediated cholesterol transport. ppar pathway – pharmacological drugs and synthetic compounds regarding the important ppar pathway, it must be noted that troglitazone, indicated above as an abca1 inducer,268 was also reported to downregulate abca1 transcription.321 these inconsistent effects may be explained partially by the different concentrations used (1 μm vs 10 μm),268,321 but may also be related to cross-talk between the ppar, lxr, and mevalonate pathways. the ppar-γ antagonist gw9662 (figure 2) reduced abca1 protein levels.406 other abca1 downregulators – natural compounds other small-molecules have been reported to act as abca1/abca1 downregulators, acting independently of the previously mentioned lxr, rxr, ppar, and hmg-coa-reductase pathways. natural compounds such as α,β-unsaturated carbonyl derivative acrolein,442 the polyphenol bisphenol a,443 and the polyphenol 1,2,3,4,6 penta-o-galloyl-β-d-glucose444 demonstrated an abca1 mrna443,444 and abca1 protein442 downregulation in vitro443,442 and in vivo.444 the effect of acrolein could be abrogated by 3-hydroxytyrosol,442 an inducer of abca1 protein content.445 srebp2 has been demonstrated to be targeted by egcg in high fat diet-fed transgenic srebp+/+ wistar rats, resulting in abca1 mrna downregulation, while an abca1 mrna upregulation could be observed under the same conditions in srebp knock-out wistar rats.446 other abca1 downregulators – pharmacological drugs exposure of the human non-small cell lung cancer lines a549 and h358 to the antiepileptic drug valproate led to downregulation of abca1 mrna and abca1 protein levels through a histone deacetylase 2-(hdac2)-mediated mechanism. in parallel, the authors observed an increased sensitivity of these cells to cisplatin.447 the selective estrogen receptor modulators raloxifene, tamoxifen, and toremifene were reported to reduce abca1 protein content in thp-1 macrophages along with decreased cholesterol efflux and increased intracellular cholesterol levels.341 tamoxifen and raloxifene treatment decreased serum hdl-cholesterol levels in mice. in addition, tamoxifen reduced cholesterol levels in serum, liver, and feces of mice after injection with cholesterol-loaded macrophages.341 interestingly, the downregulation of abca1 protein content by these estrogen receptor modulators could not be demonstrated for murine liver, indicating a macrophage-specific effect.341 varenicline, a drug used in smoking cessation, was shown in vivo to promote aortic atherosclerotic lesions in apoe knock-out c57bl/6 mice.417,448 the authors demonstrated that intracellular lipid content in peritoneal macrophages was increased, and a decreased abca1 protein expression was confirmed in vitro in raw264.7 macrophages. finally, the antineoplastic agent gefitinib reduced abca1 protein content in various non-small cell lung cancer cell lines.400 other abca1 downregulators – synthetic compounds the plasticizer dibutyl phthalate389 and the pi3k/akt inhibitor ly294002421 reduced abca1 mrna389 and abca1 protein389,421 expression and increased cellular cholesterol and lipid levels389 in human389 and murine421 macrophages. the sphingosine kinase 1 and 2 inhibitor 4-{[4-(4-chlorophenyl)-2-thiazolyl]amino}phenol was demonstrated to downregulate abca1 protein expression in murine primary macrophages, which was dependent on the sphingosine kinase 2 as well as the sphingosine-1-phosphate receptor.306 this abca1 protein downregulation was accompanied by a reduced cholesterol efflux. the acyl coenzyme a cholesteryl acyl transferase (acat) inhibitor atr-101 reduced abca1 mrna levels and induced an increase in intracellular cholesterol content in h295r cells.251 the authors suggested that this was caused by inhibition of abca1 but provided no clear proof of direct inhibition of abca1. therefore, this compound was classified as a downregulator. other abca transporters abca2 and abca3 compared to abca1, knowledge relating to downregulators of the other abca transporters is very limited. as discussed above, human leukemia cells exposed to imatinib displayed increased abca2 mrna and abca2 protein expression.236 celecoxib abrogated this effect.236 a similar observation was reported for abca3, where the anti-inflammatory drug indomethacin and the abca1 inhibitor sirolimus245 (figure 2) downregulated abca3 mrna in various cancer cell lines.426,449,450 this treatment also resulted in a sensitization of these cell lines toward the tkis dasatinib, imatinib, and nilotinib when treated with indomethacin.426 other compounds were also reported to downregulate abca3/abca3 including the flavonoid genistein,451 lipopolysaccharides452 – already demonstrated above as abca1 protein downregulators374 – and the translocator protein ligand pk11195.453 the effect of lipopolysaccharides could be abrogated by ascorbic acid (vitamin c). abca5–abca9 interestingly, the abca8 inhibitor222 and abca1 protein inducer253 digoxin downregulated abca5 and abca7–9 in murine liver.454 the hmg-coa-reductase inhibitors lovastatin and mevastatin downregulated abca6 mrna in human umbilical vein endothelial cells.429 the cholesterol derivative 25-hydroxycholesterol, which was introduced above as an abca1 mrna inducer,327 showed the opposite effect on abca7 mrna.324 this finding is in agreement with a report stating that excess cholesterol reduced abca7 protein content in both human and murine fibroblasts.205 stabilizers of abca transporters stabilizers are compounds that promote functional activity of abc transporters through increasing their presence at the site of action (e.g., the cell membrane) either without interfering with mrna or protein levels, or in addition to these effects. the categorization is difficult, as the necessary information regarding many modulators of abca transporters is lacking and the underlying mode of modulation cannot be precisely identified. in this section, we consider only those modulators which predominantly interfere with abca1 trafficking, with relatively minor or no additional modes of action/modulation. stabilizers are of particular interest, as they may represent a novel generation of functional abc transporter activators, expanding treatment options for several diseases, particularly ad. abca1 probucol and cyclosporine a were demonstrated above to decrease abca1 turnover and increasing abca1 protein content at the cell membrane.246,275 arakawa et al. demonstrated that the probucol metabolites spiroquinone and diphenoquinone did not inhibit abca1-mediated transport like their parent compound but rather increased the fraction of functional abca1 in the cell membrane.275 this stabilization led to increased cholesterol and phospholipid efflux. both effects were observed at very low nanomolar concentrations,275 while abca1 mrna remained stable.275 strikingly, spiroquinone and diphenoquinone decreased vascular lipid deposits in vivo in cholesterol-fed rabbits,275 which may be of relevance for ad and potentially other neurodegenerative diseases. a similar mode of stabilization, albeit with less potency and no in vivo confirmation, has been observed for the flavonoid wogonin,254 the olive oil-derived compound erythrodiol,395 and certain thiol proteinase inhibitors, in particular n-acetyl-leu-leu-norleucinal and leupeptin.316,386 finally, the abca1 mrna and abca1 protein inducer testosterone was demonstrated to promote abca1 trafficking to the cell membrane.357 other abca transporters the cystic fibrosis transmembrane conductance regulator (cftr; abcc7) correctors c13,455 c14,455 c17,455 genistein,456 and ivacaftor (figure 2)456 were demonstrated to rescue abca3 mutants by increasing total abca3 mutant protein levels,455 promoting subcellular targeting of abca3 into vesicular bodies,455 and improving lipid transport function of abca3.456 furthermore, the correctors lumacaftor (vx-809; figure 2), c3, and c4, and c18 increased the presence of abca4 at the cell membrane in abca4-overexpressing hek293 cells, indicating promotion of abca4 trafficking to the plasma membrane.457,458 promotion of trafficking has already been demonstrated for other abc transporters, such as abcc123,24 and abcc7.459 hence, this mechanism represents a new potential therapeutic option for abca transporter-related ad. as proposed for abcc7,460 the authors suggested a direct binding of the correctors to the abca4 protein,457 which has not yet been proven. in an abca12 pig model of harlequin ichthyosis, acitretin (figure 2) treatment resulted in a redistribution of abca12 in the skin compared to wild-type pigs, and thus, a higher survival rate.430 destabilizers of abca transporters natural compounds in contrast to compounds that promote trafficking of functional abca1 to the plasma membrane, other compounds that have the opposite effect have been named ‘destablizers’. so far, only agents targeting abca1 are known. the lactone antibiotic brefeldin a (figure 2) interfered with abca1 cell-surface localization, recycling, and intracellular trafficking.387,461-463 these effects were at least in part dependent on the interaction with brefeldin 1-inhibited guanine nucleotide exchange protein (big1).461 this interference reduced the functional fraction of abca1 and, consequently, abca1-mediated cholesterol and phospholipid transport.255 similar observations have been made for the polyether-antibiotics monensin, which reduced abca1 turnover and trapped it inside endoand lysosomes. subsequently, monensin reduced the functional presence of abca1 at the cell surface,464 lowered cholesterol efflux,463 and increased intracellular cholesterol content.463,464 the same was demonstrated for nigericin, another polyether-antibiotic, which increased intracellular cholesterol concentration,463 and inhibited abca1-mediated cholesterol efflux from raw264.7 macrophages.385 inhibition of intracellular organelle transport as suggested for brefeldin a387,461-463 and monensin463,464 likely applies to nigericin as well.463,465 in addition, the endoplasmic reticulum stress promotor, tunicamycin, also reduced abca1 protein levels.360,466 this ‘downregulation’ is most likely mediated though stress-induced impaired abca1 trafficking and/or increased abca1 degradation.466 however, in terms of selective targeting of abca1 in particular, or abca transporters in general, these agents are less suitable as in vivo agents and serve better as in vitro controls. the palmitic acid derivative 2-bromopalmitate (figure 2) inhibited trafficking of abca1 to the plasma membrane and reduced abca1-mediated cholesterol efflux.273,467 however, the observed effect that abca1 did not translocate to the cell membrane in hek293/abca1 cells467 has not been demonstrated in bhk-21/abca1 cells.273 pharmacological drugs interestingly, the experimental anticancer drug serdemetan (jnj-26854165) was demonstrated to induce abca1 mrna levels but reduce abca1-mediated cholesterol efflux.468 the abca1 mrna induction was due to induction of lxra and lxrb. the abca1 mrna increase was also reflected at the protein level, which increased within 48 hours of exposure to serdemetan before a sudden decrease occurred. the authors also showed that abca1 turnover and degradation were increased. thus, serdemetan can be considered a destabilizer. synthetic compounds cycloheximide was frequently used to interrupt intracellular trafficking of vesicles, including abca1 containing endoand lysosomes.387,464,468 as mentioned earlier, abca1 is stabilized by n-acetyl-leu-leu-norleucinal.316,386 this stabilization could be abrogated by the protein kinase c inhibitor gö6976, which affected not only abca1 protein content, but also cholesterol and phospholipid transport.386 part ii: pipeline development to gain novel diagnostics and therapeutics in silico methodologies to predict novel lead structures rational drug design is the innovative process of identifying pharmaceutically relevant drug candidates. it is based on the information obtained in association with the drug target, e.g., abc transporters. in the following section, we will discuss computational approaches for in silico operations that help to identify novel lead molecules for potential diagnostic and therapeutic application. structure-based drug design the development of computational methodologies for structure-based drug design to understand the relationship between transporter sequence/structure and function depends on the availability of structural as well as biological information. recent advances in experimental approaches for structure determination have facilitated high-quality depictions of the structures of a growing number of abc transporters in different conformational states.469 these experimental approaches include in particular x-ray crystallography and cryo-electron microscopy (cryo-em). recently, the cryo-em structures of human abca1470 and human abca4471-473 with resolutions of 4.1 å and 3.3–3.6 å, respectively, were reported. in addition, a cryo-em structure of human abca7 has been announced474 on biorxiv (biorxiv.org), which was, however, not published to this date (pdb id: 7kqc). nevertheless, a homology model of abca7 has been recently developed.475 figure 4 shows the structures of abca1, abca4, and abca7 as determined by cryo-em as well as homology modelling. figure 4. available structures of abca transporters: the cryo-em structures of human abca1470 (very left; pdb id 5xjy) and abca4 [left (pdb id 7lkp, middle (pdb id 7e7i), and right (pdb id 7m1q)]471-473 as well as the homology model developed for human abca7 (very right).475 all three transporters are typical abca transporters with three crucial structural parts: two nucleotide-binding domains (nbds; intracellular), two membrane-spanning domains [msds (2 x 6 transmembrane helices tms); inter-membrane space], and two large extracellular domains (ecds; extracellular). considering the available structural knowledge, a ‘common’ abca transporter possesses a very long amino acid sequence (>2000 amino acids) and consists of two membrane-spanning domains (msd1 and msd2) each composed of six transmembrane helices (tm1–6 and tm7–12). these msds are followed by a cytoplasmic region comprising a nucleotide-binding domain (nbd1 and nbd2) and a small regulatory (r1 and r2) domain, which have been proposed to stabilize the interaction between nbd1 and nbd2470,473 and were found to strongly interact with each another in the absence of atp.471,472 abca transporters are ‘type ii transporters’ in which the msds indeed form a tunnel for substrate translocation from the cytosol to the lumen, however, represent separate entities without swapping/twisting of the msds, as this is the case with classical ‘type i transporters’ like abcb1.476 most tms are completely exposed to the hydrophobic environment of the membrane, which could promote the attraction and binding of fat-soluble cholesterol as well as phospholipids before guidance to and through the substrate translocation tunnel, and which hosts several cholesterol and phospholipid binding sites.470-474 a unique feature amongst abca transporters in comparison to other abc transporters is the existence of two large extracellular domains (ecd1 and ecd2). these domains together form a channel embedded in hydrophobic amino acids470-472 and are believed to facilitate intermediate storage of cholesterol470 and phospholipids. they have also been suggested as the primary binding site of apoa1,471,477 as indicated by the latest data on abca4.471 a large gap exists between the ecds and msds, pointing to strong conformational changes that are required for abca transporter function.470 another common feature amongst abca transporters are four intracellular and extracellular helices (ih1–4 and eh1–4), which are believed to provide the necessary flexibility for interaction between the msds and nbds in the substrate translocation process,478 and were suggested to enable proper folding and function of these transporters.471 of important note is that abca1 and abca4 share sequential and structural similarities with the abcg family, in particular with abcg5/abcg8,470 which is the model type ii transporter.478 this similarity suggests an evolutionary relevance amongst various abc transporter subfamilies. more importantly, conserved sequential and structural similarities also support the translation of knowledge gained on other abc transporter subfamilies to abca transporters.470,472 this is of particular interest when novel lead structures for new pharmacological targets, in this case under-studied abc transporters,18 are focused,6,18 and specific binding sites located within the msds or nbds are targeted. based on the sequence information of abc transporters within the same family, homology-modeling techniques are the preferred choice for structure determination and binding site elucidation if these subtypes do not yield x-ray or cryo-em structures. this methodology is of particular relevance for closely related homologs with high medical relevance,198 such as abca7 (similarity a1/a7: 54%; similarity a4/a7: 49%).200 the generated homology models can be refined further by molecular dynamics simulation, in which the transporter movement (‘trajectory’) is simulated to potentially unravel relevant transporter conformations. very recently, potential abca1 drug binding sites have been proposed by this methodology,479 and an abca7 homology model has been developed for molecular docking experiments.475 molecular docking is a very popular method for predicting binding orientations or poses of small-molecules within the transporter. most often, the docking programs account for full conformational flexibility of ligands within the binding site, treating the protein as a rigid body. binding site identification is an important prerequisite in the structure-based drug design implementation. in terms of abc transporters, the search for binding hot spots and cavities on the entire volume of the protein (e.g., through blind docking) is necessary due to the general lack of information on binding sites of abc transporters. recently, in search of highly effective modulators addressing abcg2-mediated mdr, derivatives of quinazolines were synthesized and biologically assessed using a hoechst 33342 accumulation assay.480 by utilizing the cryo-em structure of abcg2,481 molecular docking studies were performed using a fragment-based approach.482 this approach was used to gain insights into the molecular determinants involved in the formation of the transporter-substrate complex.480 based on the docking studies, the putative binding site of the abcg2 substrate, hoechst 33342, and its interaction with the amino acids in the binding pocket was proposed.480 the predicted binding pose was rationalized based on the mutagenesis data reported in the literature483-487 and further confirmed with kinetic studies to determine the mode of inhibition.480 this subsequent structure-based approach led to the discovery of highly potent pyrimidine-based abcg2 inhibitors,488,489 specifically by identifying a novel binding pocket of this transporter.488 in terms of abca transporters, molecular docking experiments with the newly derived abca7 homology model applying a set of diverse pan-abc transporter inhibitors revealed a putative common ‘multitarget binding site’ identified within the transmembrane domains of abca7. it must be noted that the nucleotide binding domains are the most highly conserved regions amongst all abc transporters, and hence, may also represent a(nother) multitarget binding site for certain drugs. however, the vast majority of data reported in the past hint to the transmembrane domains as the actual venue of bioactivity in terms of abc transporter modulation.472 these results as described above475,480,488,489 give this methodology a high relevance in the drug development process in terms of novel lead molecules in general, and provide the basis for rationally designed structure-guided approaches for the identification of modulators of abca transporters in particular, as recently demonstrated for abca7.475 ligand-based drug design similarity search the analysis of structure-activity relationships using ligand-based approaches is an essential component of medicinal chemistry and pharmacology of abc transporters. this becomes evident as x-ray or cryo-em structures of most abc transporter subtypes are lacking to serve as suitable templates with sufficient similarity for generating homology models. ligand-based approaches establish a correlation between the molecular structure of a small-molecule and the triggered biological response of the target. the chemical representation of the molecules is often expressed using descriptors, which are attributes that conserve the physicochemical information of the molecule. these descriptors refer to generic properties such as logp, molecular weight, polar surface area, rotatable bonds, or molar refractivity. alternatively, structural representations of the molecules can form fingerprints that portray existent molecular features of the molecule in a binary code. these fingerprints are, for example, path-like,490 or circular-based,491,492 such as maccs or ecfp4, respectively. utilizing these representations of molecules, similarity-driven virtual screenings can be applied. here, molecules are extracted from a virtual library of millions or billions of compounds compared to the bioactive template molecule(s) according to the similarity principle. the abstract representation of molecules enables clustering of compounds, which is a methodology to categorize a diverse set of molecules. moreover, these abstract representations can be used in different machine learning (artificial intelligence) approaches. pharmacophore modelling another common approach is pharmacophore modelling, which analyzes a number of ligands with a common mechanism of action. the model is the ensemble of common chemical features that are required to ensure the molecular interaction of the ligands with the target, such as hydrogen bond donors and acceptors as well as aromatic and hydrophobic centers. the pharmacophore models are generated by extracting common molecular features through flexible alignment of the active biomolecules.493,494 this can be achieved by generating all possible conformations of the ligand and aligning them to determine the essential chemical features and molecular orientation to construct the pharmacophore model. the conformational flexibility of the ligands representing the chemical features is the key factor in the pharmacophore model generation. pattern analysis in addition to these classical computational approaches, similarity search and pharmacophore modelling, a pattern analysis approach (‘c@pa’ = computer-aided pattern analysis’) has been reported recently.18,19,495 pattern analysis extracts both basic scaffolds and the statistical distribution of substructural elements amongst the template ligands. it works similarly to non-physicochemical properties-related fingerprints and conserves substructural features as they are present in the molecules. pattern analysis has specifically been derived for the development of novel potent multitarget abc transporter inhibitors. the basic operations were the categorization of bioactive molecules according to their inhibitory power against specific abc transporters and their classification according to their selectivity profile. the respective classes can statistically be analyzed for both their basic scaffolds and/or their substructural composition to extract the desired pharmacological profile and target preferences. the generated model focused multitargeting of abc transporters, and resulted in a biological hit rate of 21.7%.19 adaption of the model (‘c@pa_1.2’) through additional non-statistical and exploratory measures increased the biological hit rate to 40%,18 and an additional extension of the model enabled the discovery of the ‘outer multitarget modulator landscape’, which represented weak multitarget bioactivities (>10 μm) supporting the discovery of a larger number of multitarget agents.495 the hit rates are impressive considering that this approach takes several targets with individual ‘ligand preferences’ into account. furthermore, as several abc transporters of distinct subfamilies were considered (abcb1, abcc1, abcg2), the resultant multitarget agents open up the possibility to explore under-studied abc transporters,18 in particular abca transporters in terms of ad.6,14 combined approaches apart from the individual use of these methodologies, combined approaches may lead to improved hit rates and better prediction capabilities with respect to bioactivity of small-molecules. this has in particular been demonstrated for a combined virtual screening approach using similarity search and pharmacophore modelling for the discovery of novel abcc1 inhibitors.493 also, certain pattern analysis approaches have used a data set derived from a similarity search and pharmacophore modelling approach, and hence, can also be considered a combined computational approach.18,495 in vitro methodologies to assess novel lead structures the previous sections have already outlined the diverse testing systems that have been used to assess the modulatory effects of effectors toward abca transporters. the following section will highlight the abca transporter-expressing host systems and the related assays that can be implemented into the pipeline for the assessment of novel lead molecules as potential abca transporter diagnostics or therapeutics. host system of abca transporters the transporter host system (abca transporter carrying unit) can be categorized into (i) living-cell-based or (ii) membrane preparation-/vesicle-based (including isolated and reconstituted proteins). the vast majority of biological investigations used living cells. here, two different living cell-based transporter host systems can be differentiated: (i) native/induced/selected cells and (ii) transfected cells. native abca transporters-expressing living cells native/induced/selected cells naturally express the respective abca transporter or have been exposed to a ‘standard’ inducer, for example, the abca1 inducers 22-(r)-hydroxycholesterol,122,205,249,252,259,262-264,268,277,278,305-315 to901317,205,245,250,252,259,260,262,264,271,272,279, 280,282,308,310,317,319,322,324,326,328-345 or 8-br camp,230,249,255,266,290,292 and overexpress the respective transporter in response (e.g., abca1). most commonly, human or murine cells have been used. table 4 summarizes the cell lines used to assess the abca transporter modulators discussed in the previous sections. it must be noted that the addressed pathways regulate also the overexpression of other abc transporters. in terms of the studies of abca1, the co-expression (i.e., co-upregulation and co-downregulation) of other members, such as abcg1, has frequently been observed.160,320,335,364,366,402,410,418,421,448 table 4. non-exhaustive list of native abca transporters-expressing cell lines that have been established in the assessment of small-molecule modulators of abca transporters. cell type cell line name origin references abca1 colorectal adenocarcinoma cells caco-2 human 262,264,308,314,342,436 lung adenocarcinoma cells hcc827-gr pc9-g2 human 337 337 renal adenocarcinoma cells 786-o a498 achn hk-2 sn12c os-rc-2 human human human human human human 334 330 334,349 330 330 330 adipocytes 3t3 l-1 mouse 255 adrenocortical carcinoma cells h295r muc-1 human human 333,441 333 astrocytes human mouse rat 279 229,279 281 astrocytoma ccfsttg1 human 423 peripheral blood mononuclear cells pbmc human 411 breast cancer cells mcf-7 human 331 pancreatic β-cells ins-1 mouse 409 cardiomyocytes h9c2 hl-1 rat mouse 253 250 aortic endothelial cells haec human 263,269 endometrial endothelial cells mouse mouse umbilical vein endothelial cells huvec human 269,364,442,496 epithelial cells beas-b2 human 322 lung epithelial cells mouse 311 pigment epithelial cells human 257 mouse mammalian epithelial cells mmec mouse 350 aortic smooth muscle cells smc human 269 vascular smooth muscle cells vsmc unspecified origin 332 fibroblasts primary hip skin wi-38 (embryonic) wi38va13 (embryonic) balb/3t3 swiss 3t3 human human human mouse mouse 230,260 205,246 277 275 312 granulosa cells rat 443 hair follicles human 282 hepatoma fu5ah hep3b hepg2 mcarh7777 rat rat human rat rat 318 231 309,342,348,379 280,312,317,367,381 343 insulinoma cells ins-1 rat 405 keratinocytes human 282 embryonic kidney cells human 312 non-small cell lung cancer cells a549 h1650 h1975 h358 pc-9/gr human human human human human 322,447 400 400 447 400 liver cells l02 human 406 mantle cell lymphoma mcl human 468 macrophages primary human 268,305,339,396,398 mouse 306,312,313,320,329,341,360,366,439,448 hd11 chicken 356 j774.a1 mouse 252,254,255,259,265,271,278,289-292,384,392,393 raw264.7 mouse 249,312,313,321,336,339,342,352,360,365,367,369,375, 376,381,385,399,402,404,406,408,410,416-419,421,424, 425,438,442,448,497 thp-1 human 231,245,249,256,268,272,275,292,308,310,312-316,321, 328,335,338,339,341,342,360,363,364,366,377,384, 388-397 u937 human 307 microglia primary bv2 retinal (müller cells) rat mouse mouse 355 126,353,380 323 multiple myeloma mm human 468 neuroblastoma neuro-2a murine 359 neutrophils primary human 339 nephron cells a6 frog 258 periodontal ligament stem cells human 325 pheochromocytoma pc12 rat 280 podocytes human 440 retina cells arpe-19 human 354 oral squamous cell carcinoma cells cal27 human 371 trophoblasts bewo human 437 abca2 hepatoma hepg2 rat 179 ovary carcinoma skem human 238 abca3 cholangiocarcinoma m214-5fur human 427 lung epithelial cells mle-12 mouse 452 hepatoma hepg2 rat 451 leukemia primary (acute myeloid) bv173 k562 lama83 human human human human 234 234,236 234 235 lung cancer a549 nci-h1650 nci-h1975 human human human 241 241 241 abca5 brain microvascular endothelial cells hbmec human 428 macrophages raw264.7 thp mouse human 321 321 abca7 fibroblasts balb/3t3 wi-38 mouse human 205 205 macrophages j774.a1 mouse 431 in terms of abca1, most studies have been conducted with human thp1,231,245,249,256,268,272,275,292,308,310,312-316,321,328,335,338,339,341,342,360,363,364,366,377,384,388-397 murine j774.a1,252,254,255,259,265,271,278,289-292,384,392,393 or murine raw264.7 macrophages. 230,249,312,313,321,336,339, 342,352,360,365,367,369,375,376,381,385,399,402,404,406,408,410,416-419,421,424,425,438,442,448 in the set-up of a drug development pipeline, these cell lines are the backbone of the in vitro assessment of potential candidates. regarding other abca transporters, the situation is much more complicated due to the lack of cell lines that naturally (and almost exclusively) express the respective abca transporter. consequently, these abca transporters are much less studied and well-established. however, transfected cell lines are of great help to study one particular transporter instead of using native cell lines that may co-express several members. abca transporters-transfected living cells in terms of abca1, cell lines transfected with human abca1 have often been used, e.g., human embryonic kidney (hek) cells (hek293/abca1)171,201,202,249,260,267,270,275,329,352,386,464,467,498,499 and baby hamster kidney (bhk) cells (bhk-21/abca1).230,245,273,292,422 these transporter host systems have also been used to study other transporters, abca2,498,500 abca3,235,241,498 abca4,133-136,201,457,458,501,502 abca5,503 abca7,201,202,386,422,498 abca8,10 abca12,498 and abca13.48 transfected cells often express lower levels of the introduced transporter than native cell lines, which is a problem if the host cell lines (e.g., hek or bhk-21) naturally express other abc transporters as well. however, these transporter host systems are suitable to confirm results, and might be the only possibility to address abca transporters other than abca1. isolated abca transport proteins finally, apart from intact cells, vesicles of enriched or purified/reconstituted abca transporters have also been used to assess transporter function. compared with living-cell based assays, this kind of host system is rarely represented in the literature regarding abca transporters.133-139,201,499-502,504-506 specifically atpase assays are popular to assess functional abc transporter modulation.23,24,507-510 while transport protein purification and reconstitution in vesicles or nano discs requires advanced engineering, and is expensive and resource-consuming, membrane preparations of transporters, in particular for atpase assays, are much more feasible. however, this method has been used somewhat scarcely for abca transporter function assessment.133-135,137-139,201,499-502,504-506 functional assessment of abca transporters two groups of tracers have been established in terms of abca transporter function: (i) radiolabeled substrates,250,272,305,306,338,339,354,364,366,393,395,404,419,511,512,136,222,230,245,249,253,255,259,260,262,264,265,267-270, 273,276,278,289-292,311,313,315,318,329,341,367,377,381,384,385,464,467,499,513 and (ii) fluorescent substrates.171,201,238, 251,252,254,256,258,261,271,282,308,319,321,330,332,335,342,360,379,389,390,392,397,402,406,455,456,468,514-518  radiolabeled tracers of abca transport function in terms of radiolabeled substrates, cholesterol is by far the most frequently used genuineabca1 substrate,230,245,249,255,260,264,265,267-270,272,273,276,278,289-292,305,306,313,315,318,329,338,339,354,366,367,381,384,385, 393-395,404,408,419,464,467,499,512 followed by phospholipid(-components).249,255,267,269,273,311,464,467,514 however, other substrates have also been used. these substrates include mostly molecules with sterane scaffold, such as β-sitosterol (abca1)262 and estradiol-β-glucuronide (abca8).222 moreover, lipid-like substrates have attracted attention, like sphingosine-1-phosphate (abca1),229,496 α-tocopherol (abca1),230 and atra (abca4).136 notably, radiolabeled substrates are very effective in terms of accurate tracing of protein function, as these molecules are not changed in their molecular integrity in contrast to fluorescence probes.171,201,238,251,252,254,256,258,261,271,282,308,319,321,330,332,335,342,360,379,389,390,392,397,402,406,455,456,468, 514-518 on the downside, conducting these experiments is constrained to regulatory requirements and requires extensive staff training as well as expensive safety measures and laboratory equipment. fluorescent tracers of abca transport function regarding fluorescent derivatives of cholesterol and phospholipids, two major types can be differentiated: (i) 7-nitro-2,1,3-benzooxadiazole (nbd) derivatives201,251,252,254,256,258,261,308,335,342,360,379,389,390,392,394,397,402, 406,408,468 and (ii) 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (bodipy) derivatives.271,282,319,321,330,332,455,456, 515-517 other fluorophore-labeled dyes have been reported, too, including the sterane analog dansyl-estramustine,171,238,518 and propargyl choline, which is processed in vitro into propargylated phospholipids.514 in addition to the stated fluorescent tracers of abca transport function, several other derivatives of other substrates can be proposed. for example, n-3-oxododecanoyl-l-homoserine lactone (3oc12-hsl) was suggested as abca1 substrate, but final proof was missing.519 thus, it may be a suitable candidate for validation in a new set-up in vitro assay for abca1 (and potentially other abca transporters). other examples of potential probes are fluorescenct dyes that stand in association with cellular cholesterol and phospholipid distribution and abca1-mediated cholesterol and phospholipid transport.516 these include, for example, β bodipy fl c5-hpc, β-bodipy fl c12-hpc, bodipy tr ceramide, and red/green bodipy pc-a2, amongst many others.520-522 fluorescenct dyes are well-established tracers of abc transporter function,18,19,23,24,284,480,488,489,493,507, 523-525 and the knowledge that has accumulated regarding the well-studied abc transporters abcb1, abcc1, and abcg2 can be transferred to abca transporters as well. however, the added fluorophore changes the molecular composition of the tracing molecules. this alteration inheres the potential risk of changing affinities and even the binding site(s) of these molecules, undermining functional-kinetic analyses regarding binding site determination and elucidation of the mode of action. nevertheless, fluorescence probes are – if used and established correctly – extremely reliable, and can be used without regulatory restrictions and necessity of special equipment, except for microplate readers and/or flow cytometers. colorimetric determination of abca transport function – atpase assays as mentioned above, atpase assays have also been used to functionally analyze abca transporter function, in particular for abca1,201,499,505,506 abca2,500 abca3139,504 abca4,133-135,137,138,201,501,502 and abca7,201 although this methodology has been used somewhat rarely compared to other functional approaches. atpase assays are based on the principle that the active transport of any substrate of abc transporters consumes energy. this energy is derived from the cleavage of atp to adp and pi, and can be detected by different methodologies.23,24,507-510,526 table 5 highlights known atpase modulators of abca transporters and the associated literature reports. table 5. summary of known atpase modulators of abca transporters. transporter modulator mode of modulation references abca1 ceramide (30 mol–%) cholesterol (30 mol–%) phosphatidylcholine (30 mol–%) phosphatidylethanolamine (30 mol–%) phosphatidylinositol (30 mol–%) phosphatidylserine (30 mol–%) sphingomyelin (30 mol–%) inhibition inhibition activation inhibition inhibition activation activation 201 201,499 201 201 201 201 201 abca2 methyl-β-cyclodextrin (u.c.a) activation 500 abca4 amiodarone (20–75 µm) 2-tert-butylanthraquinone (20–50 µm) ceramide (30 mol–%) cholesterol (30 mol–%) dehydroabietylacetate (10–50 µm) digitonin (10–180 µm) n-ethylmaleimide (nem; 1000 µm) reduced glutathione (gsh; 1000 µm) β-ionone (50–100 µm) phosphatidylethanolamine (30 mol–%) phosphatidylglycerol (30 mol–%) phosphtidylinositol (30 mol–%) 11-cis-retinal (5–100 µm) 13-cis-retinal (5–100 µm) atra (5–100 µm; ec50 = 10 µm) all-trans-retinoic acid (20–100 µm) all-trans-retinol (20–100 µm) n-retinylidenephosphatidylethanolamine (40 µm) activation activation inhibition inhibition activation activation inhibition activation activation activation activation inhibition activation activation activation activation activation activation 138 138 201 201 138 138 137 137 138 201 201 201 137,138 138 133-135,137,138 138 133,138 133 abca7 ceramide (30 mol–%) cholesterol (30 mol–%) phosphatidylcholine (30 mol–%) phosphatidylethanolamine (30 mol–%) phosphatidylserine (30 mol–%) inhibition inhibition activation activation activation 201 201 201 201 201 a u.c. = unspecified concentration atpase assays have been and still are popular in terms of functional abc transporter modulation in general.23,24,507-510 strikingly, the nbds of abc transporters are – in contrast to the various binding sites identified within the transmembrane domains of abc transporters475 – highly conserved. this conservation enables targeting of abca nbds by known atpase modulators of other abc transporters. therefore, abca transporter function can be detected by methodologies that have already been established for other abc transporters.23,24,507-510,526 this transfer of knowledge will be of great use to confirm obtained results from other functional abca transporter analyses. colorimetric determination of abca transport function – other detection methodologies as a final note, it must be mentioned that other colorimetric analyses were also used to quantify the abca transporter-mediated function, specifically for transport of cholesterol or choline-containing lipids, using commercially available assay kits.202,205,246,251,268,272,275,329-332,334,336,354,365,366,369,372,374-376,386-389,392,405, 406,416,418,421,422,424,425,441,511 however, these methodologies require time-consuming extraction processes of the lipids, and hence, are less suitable to track the function of abca transporters in real-time and to determine kinetic aspects of their cholesterol and lipid transport. in rare instances, the extraction of lipid components was accomplished after incubation with a radioactive marker.246 while this is a valid methodology to accurately determine lipid components within cells, it increases workload and attracts regulatory constraints. gas-liquid chromatography has also been used in some reports.353,355 an extraction-free staining of cholesterol inside of cells was also demonstrated (filipin iii251,331,333,341,358 or oil red o staining 330,332,364,366,369,375,388,389,392,393,399,402,410,417,421,425,448). however, these systems are not suitable to track single-cell abca-mediated cholesterol or phospholipid transport. quantification of abca transporter regulation besides qpcr and western blotting, abca transporter expression was reported in several studies using fluorimetric assays. this was accomplished with either (i) green fluorescent protein-(gfp)tagged/labelled abca transporters235,241,261,275,386,422,464,504 or (ii) luciferase promotor-(luc)-transfected271,309,319,352,367,379,381,405, 406,419,436,447 abca transporter cells in luciferase reporter gene assays. in vivo assessment of clinical candidates in vivo models play a key role in drug discovery. although in vitro and cellular models are less expensive and less time consuming, in vivo models are needed to test abca modulators under physiological conditions. safety, toxicity, and efficacy of a drug candidate must be tested in an in vivo model as a last step before transferring it to clinical evaluation. however, these models also have disadvantages. animal studies are time consuming and require advanced personnel training and resources for maintaining the animals. in addition, although they are closer to humans than in vitro models, there are considerable physiological differences between species with respect to drug absorption, metabolism, and excretion, which may impede translatability. furthermore, the use of animals in research has its ethical concerns. thus, in recent years, research has been directed to reduce animal use and increase animal welfare. in vivo models have previously been used to study the role of abca transporters in physiology and disease as described above. thus, there are already available animal models for testing of abca modulators for the most prominent subtypes (table 6). as stated above, these models represent the last step before clinical evaluation of potential small-molecule therapeutics in humans. thus, after in silico identification and in vitro assessment, these in vivo models are the third column in the development of novel abca transporter diagnostics and therapeutics. in the following section, different in vivo models will be described in more detail. table 6. animal models to study the functional and pathological role of abca transporters. transporter type species phenotype references abca1 knock-out mouse reduced cholesterol and plasma phospholipid levels decreased brain apoe levels poorly lipidated apoe 161-163 https://www.jax.org/strain/003897 overexpression mouse increased lipidation of apoe 127 abca2 knock-out mouse reduced body weight, limb tremor, reduced sphingomyelin https://www.jax.org/strain/033139 54,527 abca3 knock-out mouse knocked-out pups die within 1h after birth 186,528,529 missense mutation mouse early macrophage predominant alveolitis which peaked at 8 weeks of age 530 abca4 knock-out mouse abnormal phospholipid composition, delayed dark adaptation 531,532 abca5 knock-out mouse exophthalmos and collapsed thyroid gland, early death due to cardiac insufficiency 123,131 abca7 knock-out humanized mouse mouse reduced microglia response altered phagocytosis increased β-secretase under characterization, increase aβ load 124 abca7tm1.1(abca7)pahnk mgi:6258226 abca8 knock-out mouse reduced plasma hdl 533 adenoviral overexpression mouse increased plasma hdl and cholesterol 533 abca12 not described https://www.jax.org/strain/033630 abca13 knock-out mouse monkey deficits of prepulse inhibition impaired neuronal formation, neurotransmitter alterations 48 534 knock-out mouse models a genetic knock-out mouse model is an animal model in which one or more genes of interest have been deactivated or removed by means of gene targeting. knock-out animals allow for direct investigation of the effect of a specific gene in an organism, as the loss of gene activity often causes phenotypic changes uncovering the function and biological mechanism of the targeted gene.535 knock-out mice have become one of the most useful scientific tools to analyze the human genome and its potential roles in many diseases.535 thus, knock-out animals are currently essential experimental tools for the investigation of genetic disorders and the evaluation of novel drugs.536 furthermore, the current knowledge on genome editing using the crispr/cas9 system makes generation of knock-out lines considerably faster than with the use of embryonic stem cells. to no surprise, this method has quickly become the most powerful tool for generating genetic models.537 knock-out animal models are designed with two variables in mind: (i) where and (ii) when is the gene of interest deactivated. the simplest and most common approach is a constitutive, ubiquitous knock-out, i.e., the product protein is absent permanently in all cells of an organism. to overcome limitations of this broad approach, more refined models have been developed. these conditional models use cre-lox recombination to target a gene either in specific cell populations, at specific time points, or a combination of both. here, the target gene is modified by inserting two loxp sites. the flanked gene segment can then be excised by the cre recombinase. cre activity, i.e., gene knock-out, can be limited to certain cell populations by appropriate promotor choice and/or linked to a tamoxifen-responsive element to control the exact time point at which the knock-out is induced. until now, several abca animal knock-out models have been described, which are summarized in table 6. these models are mainly mouse lines, except for abca13 (monkey).534 these animal models have contributed fundamentally to identifying the role of abca transporters in physiological conditions as well as in disease pathogenesis. in addition, these models can be used for novel drug testing, as they provide information about target specificity. if a drug is 100% specific for a transporter, knock-out of this transporter should completely abolish the drug’s effects observed in naïve animals. however, gene knock-outs often have phenotypical effects per se that need to be taken into account when evaluating drug effects. rnai models the use of rna interference (rnai) is an alternative to knock-out models. this technique is based on post-transcriptional silencing of the targeted gene using sirna molecules that are designed to bind to the target mrna.538 this process will deactivate the mrna using the cell’s own defense mechanism against pathogens. in contrast to standard knock-out models, this silencing is temporary as the sirna molecule will be degraded but the gene transcription continues.527 to avoid this temporal limitation, short-hairpin rna (shrna) has been developed. this method is based on the use of vectors that incorporate into the cell dna and encode for shrna. after transcription, these vectors are processed into sirna. these shrnas are continuously transcribed, increasing reproducibility of results.539 overexpression models similar to knock-out models, overexpression models can be used to investigate the function of a gene by evaluating the resultant phenotype. in addition, overexpression models have long been used for modeling diseases such as ad540 or pd.541 in the investigation of abca transporters, these models can resemble the effect of chronic activation of the transporters and may help to identify its physiological functions by evaluating the pathways upregulated in comparison to control animals.127 humanized abc transporter mouse models before it can be translated into clinical practice, each novel drug candidate must be tested in an in vivo model. however, the translational value of the animal model largely depends on whether the disease pathway under investigation is conserved between the two species. therefore, replacing the original (e.g., murine) gene by the respective human gene likely improves the animal model, and thus, is beneficial for evaluating a novel drug’s efficacy and specificity in clinical practice.542 with this approach, mice can be used as tools for pre-clinical screening and efficacy evaluation of new drugs, given their improved ability to predict human responses to treatments. our group has previously established a humanized abcc1 mouse model,543 and an abca7 model is under characterization. here, we generated knock-in mouse models producing a chimeric protein that is completely human except for one amino acid.543 in addition, as this gene was flanked by loxp sites, this humanized model can be knocked out in specific cell populations and at a specific age.543 models such as these represent the future of pre-clinical drug candidate evaluation. in addition, dallas et al. successfully generated a humanized abcg2 mouse model.544 however, other models, such as humanized abcb1 mice, were not successful despite multiple attempts.545 disease models in addition, all the models described above can also be used to study the role of a gene for the pathophysiology of specific diseases. for example, abca knock-out models have been crossed with transgenic mice in order to study their potential role in ad.54,123,131,161-163,527 these studies have elucidated potential disease mechanisms involving abca transporters that cannot be studied in patients. moreover, once a drug is developed and its specificity is proven, disease models enable evaluation of the role of that specific transporter in the pathophysiology of the disease. at the same time, these results may be the first step to evaluate the potential of novel transporter modulators as therapy for the respective disease. imaging techniques lastly, in vivo imaging can be used for the development of new drugs. on the one hand, labeling drug candidates with radioactive isotopes can give information about the drug distribution, drug target, and drug metabolism in vivo. in addition, it can also show whether a drug is able to cross specific natural barriers, such as the bbb. in vivo imaging can help to select candidates that appear successful or to discard drugs that seem likely to fail.546 on the other hand, drug candidates can also be used to develop new radiotracers (e.g., pet tracers) targeting abca transporters that could then be used in clinical diagnostics. radiotracers would facilitate the study of the specific gene and/or its product protein in human patients in vivo and in a longitudinal fashion, enabling a much better understanding of the role of abca transporters in human (patho)physiology.547 in this regard, knock-out animals can be used as negative controls for the development of new abca radiotracers to evaluate the specificity of the radiotracer.548 furthermore, these very same radiotracers can be used in animal disease models, enabling longitudinal studies and reducing the number of animals required.549-551 concluding remarks: where do we go from here? several in vivo studies demonstrated that modulators of abca transporters, in particular abca1, have systemic effects.231,249,250,253,271,275,289,293,297,330,335,344,350,361,362,366,368-370,376,378,383,410,415,417-419,425,431,436,448 however, the vast majority of these modulators were regulators,231,250,253,271,297,330,335,344,350,361,362,366,368-370, 376,378,383,410,415,417-419,425,431,436,448 specifically inducers,250,253,271,297,330,344,361,362,366,368-370,376,378,383,410, 415,418,419,425,431 and only very few interactors demonstrated in vivo effects.249,289,293 mostly emphasizing atherosclerosis,249,275,289,366,369,370,378,410,417-419,425,431,448 these regulators were able to demonstrate that cellular and plasma lipid content249,271,275,289,330,366,369,378,419,425,431 as well as atherosclerotic plaque formation275,289,366,369,370,410, 417-419,425,448 could be changed compared to controls (enhanced or reduced) after treatment with the respective drug. only very few in vivo approaches targeted for ad.293,297,344,383 taking the challenge of cns penetration of these drugs into account, drugs active in atherosclerosis models could generally be suggested to also have certain therapeutic relevance regarding ad. nevertheless, so far, none of these drugs has made it into clinical evaluation in humans. the underlying cause can be pinned to the fact that the principal mechanism by which abca transporters contribute to ad is still unknown. while a rationale can be found in atherosclerosis (efflux of cellular lipid to apoe and hdl resulting in lower lipid burden in the vascular system), the translation of this rationale to ad can only be achieved to a very limited extent. several questions need addressing in future evaluations: (i) what is the general function of abca transporters in the brain to ameliorate (or exacerbate) ad in patients; (ii) when does this development start; and (iii) at which stage of development can a pharmacological intervention with abca transporter modulators lead to a positive therapeutic effect? in this regard, more in vitro tests are needed with new lead structures that are rigorously assessed for their particular mechanism of action – to study vice versa the mechanism of action of abca transporters in general. one possibility to gain novel lead structures is the screening of huge analog compound libraries. however, the number of existing compounds is limited, and blind in vitro testing is resource-consuming, especially regarding time and funds. computational methodologies may help to generate novel lead structures based on the knowledge of existing modulators of abca transporters. this has led to new lead molecules in the past.18,19,493,495 particularly the knowledge on abca1 and abca8 inhibitors and substrates is of interest, because these compounds inherit the molecular-structural information that is critical for direct binding to these transporters. considering the newly developed pattern analysis methodology, c@pa,18,19,495 the scaffolds and substructural composition of this set of molecules may reveal the critical necessities for direct interaction with abca transporters. c@pa is therefore of high relevance because it was specifically developed to gain multitargeting pan-abc transporter modulators18,19,495 – molecules that particularly interact with different abc transporters of different subfamilies. assuming that a conserved multitarget binding site exists as proposed earlier,6,14,475 multitargeting may be the key to explore under-studied abc transporters in general and abca transporters in particular.6,14,18,19 several thousands of these molecules have already been predicted,18,19,493,495 and the predictions were in part biologically confirmed.18,19,493,495 additionally, selected pan-abc transporter inhibitors were analyzed in molecular docking studies, which revealed the potential existence of the multitarget binding site.475 hence, combining the existent knowledge of abca transporter modulators with (sub)structural elements of these pan-abc transporter modulators and powerful computational approaches (e.g., molecular docking or molecular dynamics simulations) could ultimately lead to the successful exploration of abca transporters in general, as well as abca1 and abca7 in particular.28,95,103-112 several drugs and drug-like compounds have already been demonstrated to be pan-abc transporter modulators interacting also with abca transporters. these drugs and drug-like compounds are, for example, cyclosporine a (9 targets of 4 subfamilies: abca1,245 abcb1,20 abcb4,552 abcb11,553 abcc1–2,24,554 abcc10,26 and abcg1–2555,556), glibenclamide (8 targets of 4 subfamilies: abca1,270 abcb11,553 abcc1,24 abcc5,557 abcc7–9,558-560 and abcg2554), imatinib (6 targets of 4 subfamilies: abca3,426 abcb1,561 abcb11,553 abcc1,561 abcc10,561 and abcg2561), probenecid (8 targets of 2 subfamilies: abca8,222 abcc1–6,24,26,562-564 abcc10565), verapamil (9 targets of 4 subfamilies: abca8,222 abcb1,20 abcb4–5,552,566 abcb11,567 abcc1,24 abcc4,568 abcc10,565 and abcg2554), and verlukast (11 targets of 4 subfamilies: abca8,222 abcb4,552 abcb11,553 abcc1–5,24,554,557,564,569 abcc10–11,26,570 abcg2554). in silico analyses with verapamil and verlukast supported the notion of addressing the multitarget binding site in abca7.475 taking their structural peculiarities in a pattern-based rational drug design approach into account may yield novel lead structures for functional in vitro studies of abca transporters. this may ultimately result in the development of innovative ad diagnostics and therapeutics. appendix author information corresponding author sven marcel stefan, department of pathology, section of neuropathology, translational neurodegeneration research and neuropathology lab, university of oslo and oslo university hospital, sognsvannsveien 20, 0372 oslo, norway; orcid: 0000-0002-2048-8598 email: s.m.stefan@medisin.uio.no authors jens pahnke, department of pathology, section of neuropathology, translational neurodegeneration research and neuropathology lab, university of oslo and oslo university hospital, sognsvannsveien 20, 0372 oslo, norway; lied, university of lübeck, ratzeburger allee 160, 23538 lübeck, germany; department of pharmacology, faculty of medicine, university of latvia, jelgavas iela 1, 1004 rīga, latvia; orcid: 0000-0001-7355-4213 pablo bascuñana, department of pathology, section of neuropathology, translational neurodegeneration research and neuropathology lab, university of oslo and oslo university hospital, sognsvannsveien 20, 0372 oslo, norway; orcid: 0000-0003-2186-8899 mirjam brackhan, department of pathology, section of neuropathology, translational neurodeg-eneration research and neuropathology lab, university of oslo and oslo university hospital, sognsvannsveien 20, 0372 oslo, norway; lied, university of lübeck, ratzenburger allee 160, 23538 lübeck, germany; orcid: 0000-0002-0753-6292 katja stefan, department of pathology, section of neuropathology, translational neurodegeneration research and neuropathology lab, university of oslo and oslo university hospital, sognsvannsveien 20, 0372 oslo, norway; orcid: 0000-0003-3544-2477 vigneshwaran namasivayam, department of pharmaceutical and cellbiological chemistry, pharmaceutical institute, university of bonn, an der immenburg 4, 53121 bonn, germany; orcid: 0000-0003-3031-3377 radosveta koldamova, department of environmental and occupational health, school of public health, university of pittsburgh, 130 de soto street, pittsburgh, pa 15261, united states of america; orcid: 0000-0002-6761-0984 jingyun wu, department of pathology, section of neuropathology, translational neurodegeneration research and neuropathology lab, university of oslo and oslo university hospital, sognsvannsveien 20, 0372 oslo, norway; orcid: 0000-0002-5137-4614 luisa möhle, department of pathology, section of neuropathology, translational neurodege-neration research and neuropathology lab, university of oslo and oslo university hospital, sognsvannsveien 20, 0372 oslo, norway; orcid: 0000-0002-4535-9952 conflict of interest the authors declare that they have no conflict of interest. funding jp received funding from deutsche forschungsgemeinschaft (dfg, german research foundation; germany; 263024513); latvian council of science (latvia; lzp-2018/1-0275); helsesø (norway; 2019054, 2019055); barnekreftforeningen (norway; 19008); eea grant/norway grants kappa programme (iceland, liechtenstein, norway; tačr tarimad to100078); norges forskningsråd [norway; 260786 (prop-ad), 295910 (napi), and 327571 (petabc)]; european commission (european union; 643417). prop-ad and petabc are eu joint programme neurodegenerative disease research (jpnd) projects. prop-ad is supported through the following funding organizations under the aegis of jpnd – www.jpnd.eu: aka #301228 – finland, bmbf #01ed1605 – germany; cso-moh #30000-12631 – israel; nfr #260786 – norway; src #2015-06795 – sweden). petabc is supported through the following funding organizations under the aegis of jpnd – www.jpnd.eu: nfr #327571 – norway; ffg #882717 – austria; bmbf #01ed2106 – germany; msmt #8f21002 – czech republic; viaa #es rtd/2020/26 – latvia; anr #20-jpw2-0002-04 – france, src #2020-02905 – sweden. the projects receive funding from the european union’s horizon 2020 research and innovation programme under grant agreement #643417 (jpco-fund). ks receives a walter benjamin fellowship of the dfg (germany; 466106904). rk is funded by the national institute of health (nih; united states; ag056371, ag057565; ag066198). lm is supported by the norwegian health association (nasjonalforeningen for folkehelsen; norway; #16154). sms receives a walter benjamin fellowship of the dfg (germany; 446812474). acknowledgement the authors would like to cordially thank joseph mark robertson 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kaewkitichai, s. tenofovir disoproxil fumarate is a new substrate of atp-binding cassette subfamily c member 11. antimicrob agents chemother 2017, 61, e01725-16. copyright: © 2021 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. fluid preservation in brain banking: a review feel free to add comments by clicking these icons on the sidebar free neuropathology 5:10 (2024) review fluid preservation in brain banking: a review andrew t. mckenzie1, oge nnadi2, kat d. slagell3,4,5, emma l. thorn3,4,5, kurt farrell3,4,5, john f. crary3,4,5 apex neuroscience, salem, oregon, usa brain preservation foundation, ashburn, virginia, usa department of neuroscience, icahn school of medicine at mount sinai, new york, new york, usa friedman brain institute, departments of pathology, neuroscience, and artificial intelligence & human health, icahn school of medicine at mount sinai, new york, new york, usa neuropathology brain bank & research core and ronald m. loeb center for alzheimer's disease, icahn school of medicine at mount sinai, new york, new york, usa corresponding author: andrew t. mckenzie · apex neuroscience · 3265 marietta st se · salem · or 97317 · usa amckenzie@apexneuro.org additional resources and electronic supplementary material: supplementary material submitted: 12 february 2024 accepted: 29 march 2024 copyedited by: s. radhika published: 23 april 2024 https://doi.org/10.17879/freeneuropathology-2024-5373 keywords: biobanking, postmortem brain, fluid preservation, formaldehyde, overfixation, storage artifact, tissue clearing, glycerol abstract fluid preservation is nearly universally used in brain banking to store fixed tissue specimens for future research applications. however, the effects of long-term immersion on neural circuitry and biomolecules are not well characterized. as a result, there is a need to synthesize studies investigating fluid preservation of brain tissue. we searched pubmed and other databases to identify studies measuring the effects of fluid preservation in nervous system tissue. we categorized studies based on the fluid preservative used: formaldehyde solutions, buffer solutions, alcohol solutions, storage after tissue clearing, and cryoprotectant solutions. we identified 91 studies containing 197 independent observations of the effects of long-term storage on cellular morphology. most studies did not report any significant alterations due to long-term storage. when present, the most frequent alteration was decreased antigenicity, commonly attributed to progressive crosslinking by aldehydes that renders biomolecules increasingly inaccessible over time. to build a mechanistic understanding, we discuss biochemical aspects of long-term fluid preservation. a subset of lipids appears to be chemical altered or extracted over time due to incomplete retention in the crosslinked gel. alternative storage fluids mitigate the problem of antigen masking but have not been extensively characterized and may have other downsides. we also compare fluid preservation to cryopreservation, paraffin embedding, and resin embedding. overall, existing evidence suggests that fluid preservation provides maintenance of neural architecture for decades, including precise structural details. however, to avoid the well-established problem of overfixation caused by storage in high concentration formaldehyde solutions, fluid preservation procedures can use an initial fixation step followed by an alternative long-term storage fluid. further research is warranted on optimizing protocols and characterizing the generalizability of the storage artifacts that have been identified. table of contents introduction methods and literature search biochemistry of long-term fluid preservation polymer properties of formaldehyde formic acid ph buffering initial aldehyde crosslinking of biomolecules chemical gel formation chemical gel-based preservation of biomolecules over time aldehyde storage empirical studies of formaldehyde storage effects on proteins effects on lipids effects on nucleic acids effects on small molecules summary of storage effects on biomolecules effects of formaldehyde storage on tissue morphology formaldehyde formulation storage in other aldehydes summary buffer storage degree of residual fixative preventing microbial growth effects on cellular morphology effects on brain volumes summary alcohol storage mechanisms effects on biomolecules effects on morphology summary storage after tissue clearing empirical effects of brain clearing comparison to buffer storage summary cryoprotectant storage glycerol other cryoprotectants kinetics of cryoprotectant brain penetration summary other storage conditions temperature storage container fluid refreshing summary comparison to alternatives cryopreservation paraffin embedding resin embedding summary relationship with the post-mortem interval implications for brain banking areas for further research comparison to other reviews strengths and limitations of this review conclusions abbreviations author contributions acknowledgements funding conflict of interest data availability supplementary material references   introduction fluid preservation, or storage in liquid, is a common method of preserving biological specimens intended for research and education. in global natural history collections, a substantial percentage of the billions of specimens are maintained in a fluid state, often referred to as “wet collections” (marte et al., 2003; hilton et al., 2021). in brain banking, fluid preservation stands alongside paraffin embedding and cryopreservation as one of the major preservation methods. fluid preservation is commonly used because it is simple to perform, inexpensive, and suitable for many investigations. worldwide, there are many thousands of brains preserved in fluid. for example, the university of geneva collection contains over 10,000 human brains preserved in formalin (kövari et al., 2011). despite the ubiquity of fluid preservation, no one method for preserving brains in the fluid state has yet established itself as obviously the best choice. instead, many different methods are used, each with advantages and disadvantages. the long-term storage outcomes of these fluid preservation methods in comparison with each other and with non-fluid state storage methods remain unclear. therefore, there is a critical need to comprehensively investigate the effect of fluid preservation methods in the long-term storage of brain tissue, including their impact on preserving cellular and molecular structures, subsequent data analysis, and the accumulation of any storage artifacts over time. in this review, our focus is the fluid preservation of fixed brains, drawing upon a distinction between initial fixation methods and long-term storage or preservation methods outlined in the biobanking literature (hartman, 2019). “fixation” is used in the specific sense of covalent crosslinking of biomolecules with a fixative such as formaldehyde (stoddart, 1989). this initial fixation process, which can involve immersion or perfusion, is largely independent of the long-term storage method (mcfadden et al., 2019; mckenzie et al., 2022). a practical working definition dividing initial fixation from long-term preservation is the period after which the tissue can be removed from the fixative but remain intact. however, there is often a grey area between the initial fixation and long-term storage. for example, brains are sometimes left in the same fixative medium for long-term storage. furthermore, there may be no clear boundary when fixation is “complete”, as fixation strength is on a spectrum without clear thresholds. to understand a complex field such as fluid preservation, it is helpful to review the evolution of the methods (carlos et al., 2019). frederik ruysch, a dutch anatomist and pioneer in biospecimen preservation, was among the first to use a fluid containing alcohol for preservation during the 17th century (luyendijk-elshout, 1970). alcohol was the preferred preservative until the 1890s, when the fixative effects of the newly commercially available formaldehyde were discovered by ferdinand blum (baird, 1859; fox et al., 1985; herbin et al., 2021). formaldehyde was preferred because it was less expensive, non-flammable, and led to better preservation of morphology, so its use rapidly expanded (fish, 1895). formaldehyde also began to be used for long-term fluid preservation (herbin et al., 2021). the choice of ideal preservation fluid for long-term brain banking has not received as much attention in recent years, but this is worthy of re-evaluation given the increased emphasis on reproducibility in modern research. the ideal method involves striking a balance between various goals: safeguarding the specimen's biomolecular structure, maintaining chemical stability, and ensuring safe handling via low flammability, toxicity, and volatility. it should also be cost-effective, user-friendly, and compatible with downstream applications, which may not always be pre-defined in prospective biobanking projects. novelty is also considered a downside for preservation methods, as long-term preservation outcomes are best evaluated with time. several candidate chemicals or chemical combinations have shown promise in meeting these criteria, each with their own advantages and drawbacks. in this review, we aim to dissect the current body of knowledge surrounding fluid preservation techniques for brain specimens, with an emphasis on the maintenance of morphomolecular characteristics for brain mapping studies. in recent years, several next-generation brain mapping technologies have emerged, allowing for the 3d visualization of the cellular and molecular organization of the brain (shapson-coe et al., 2021; patel et al., 2022). although existing reviews have focused on the long-term fluid preservation of biomolecules such as dna, it is less clear how fluid preservation in brain banking will affect the neural structures visualized with these cutting-edge brain mapping methods (lou et al., 2014; gustafsson et al., 2015). accurate and reliable mapping requires specimens that have been preserved in a manner that maintains their original cellular and molecular structures as closely as possible. this review may be useful to professionals engaged in the development and management of brain collections, as it offers guidance in selecting preservation techniques. moreover, researchers studying tissue from existing brain banks, particularly those with an interest in unraveling neural circuitry from archival brain samples, may find this review helpful in guiding experimental design. this review is aimed to help ensure the reliability of data generated from these invaluable resources, ultimately supporting the development of more effective therapies for neurobiological disorders.   methods and literature search we conducted a realist synthesis review with the goal of developing a theoretical understanding of fluid preservation, an approach that combines aspects of a systematic review with a focus on theory and applicability (wong et al., 2013). this style was selected given the broad and variably defined nature of fluid preservation. we followed the rameses reporting standards (see supplementary file 1) (wong et al., 2013). before formalizing our search methodology, we first scoped the literature by searching pubmed, google scholar, biorxiv, and medrxiv, and by holding discussions among the authors. the review protocol was preregistered here: https://osf.io/jdyvs. additional review methods are available in supplementary file 2. through our formal search process, we screened 1080 abstracts, reviewed 136 full texts, and included 50 studies (figure 1). we also identified 41 studies through citation analysis or ad hoc searches, after which we eventually included 91 total studies (supplementary files 3-6). we categorized the included studies into five types of chemicals used for storage: formaldehyde-containing solutions (n=68), buffer (n=5), alcohol-containing solutions (n=3), storage after tissue clearing (n=5), and cryoprotectant-containing solutions (n=10). collectively, these studies contain 197 distinct observations about the effects of fluid preservation on cellular structure. prior to discussing outcomes for each of these storage options, we first review some relevant biochemistry. figure 1. study selection flow diagram. studies were screened and selected using the web-based software sysrev. the search results are available online for the abstract screening stage (https://www.sysrev.com/p/123382) and the full-text review stage (https://www.sysrev.com/p/123457).   biochemistry of long-term fluid preservation   polymer properties of formaldehyde formaldehyde is by far the most common initial preservative used in brain banking. molecular formaldehyde is a gas that dissolves rapidly in water. in water, it is rapidly hydrated to form methanediol, also known as methylene glycol or formaldehyde monohydrate (figure 2). in aqueous solutions, methanediol is abundant, while unhydrated monomeric formaldehyde is almost completely absent (boyer et al., 2013; walker, 1944). in turn, methanediol oligomerizes or polymerizes very rapidly, making it difficult to isolate in the pure state (schmitz et al., 2015). specifically, methanediol reacts to form 1,3,5-trioxane, a stable cyclic trimer, or polyoxymethylene glycol, which is a polymer form with a variable number of subunits. the equilibrium of monomeric and polymeric formaldehyde hydrates in a solution is governed by factors such as the temperature and concentration of formaldehyde. at higher temperature and more dilute concentrations, methanediol is favored, thus leading to the depolymerization of polyoxymethylene glycol. additionally, in an alkaline environment, hydroxyl end-groups of the polymer form are more rapidly degraded, leading to the progressive cleavage of formaldehyde units from the extremities of the linear molecular chains (french and edsall, 1945). figure 2. schematic diagram of chemical alterations of formaldehyde in aqueous solutions. when dissolved in water, formaldehyde is rapidly hydrated to methanediol, in an equilibrium that heavily favors methanediol under standard conditions. methanediol can polymerize in several ways, including into 1,3,5-trioxane or into linear chains, called polyoxymethylene. polyoxymethylene chains tend to be less water soluble and are increasingly insoluble if the degree is 4 or greater (walker, 1944). paraformaldehyde is a mix of polyoxymethylene chains with typical degree of 8-100. methanol decreases the rate of polymerization via reactions with methanediol or the ends of polyoxymethylene chains, forming hemiacetals or acetals such as dimethoxymethane. formic acid can be formed by the oxidation of formaldehyde or by the cannizzaro reaction (latter not shown). this figure was made using chemdraw. paraformaldehyde is a commercial storage form of polyoxymethylene, with a mixture of polymerization products with degrees of 8-100 formaldehyde units (walker, 1944). it is thought to be the monomeric form that is reactive and crosslinks biomolecules. as a result, the polyoxymethylene polymers in paraformaldehyde must be depolymerized prior to use as a fixative. this usually involves heating and the addition of sodium hydroxide to increase the ph of the solution and stimulate cleavage of formaldehyde units. when longer polyoxymethylene polymers form in aqueous solutions of formaldehyde, which is more likely to occur during storage at low temperatures, they can become insoluble and precipitate out of the solution. methanol is often added to formaldehyde as a stabilizer. for example, the commercial product formalin often contains 37 % formaldehyde by weight and approximately 10 % of methanol. methanol is believed to prevent polymer precipitation in formaldehyde solutions through the reaction of the alcohol group in methanol with the aldehyde group in formaldehyde, leading to the formation of hemiacetals or acetals such as dimethoxymethane (norris et al., 2010; walker, 1944). these hemiacetals interfere with the polymerization process of methanediol, thereby stabilizing them in solution and preventing the formation and precipitation of solid polymers. notably, other chemicals that also contain an alcohol group, such as ethanol, glycols, and glycerol, can also stabilize formaldehyde and limit the extent of its polymerization. the reason that methanol is most commonly used as a stabilizer appears to be partially a historical consequence of the fact that formaldehyde was manufactured from methanol (walker, 1944). a complicating factor is that methanol itself acts a fixative and may also contribute to tissue decomposition over time, especially of lipids. some investigators use depolymerized paraformaldehyde that lacks any methanol. the practical effect of the methanol stabilizer in fixation and long-term storage of biospecimens is not well established.   formic acid in addition to polymerization, formaldehyde solutions stored at room temperature can undergo several chemical other reactions, leading to the formation of methanol, hydroxyaldehydes, sugars, and formic acid (walker, 1944). for our purposes, the most important chemical reaction is thought to be the formation of formic acid. this can occur through two pathways. first, formaldehyde can be oxidized via oxygen in the solution to formic acid. second, even if substantial oxygen is not present, the cannizzaro reaction can occur, which is a disproportionation reaction wherein one molecule of formaldehyde is oxidized to formic acid, and another is reduced to methanol. formic acid formation is a problem for brains stored in formaldehyde solutions over the long-term for several reasons. first, formic acid has the potential to solubilize proteins, especially hydrophobic ones, such as those found in myelin (zheng and doucette, 2016). second, formic acid can also induce chemical modifications to proteins, which limits the use of formic acid in proteomics (zheng and doucette, 2016). as an example of this, formic acid has been found to destroy prion proteins in a way that formaldehyde itself does not (taylor et al., 1997). although formic acid may not adversely affect histology after short exposure times of one hour, its long-term effects on brain tissue preservation are still a concern (brown et al., 1990). one source notes that the long-term storage of biospecimens in formaldehyde is corrosive because it breaks down into formic acid (eichhorn et al., 2018). one study used a novel formulation of formaldehyde that removed formic acid with an ion-exchange basic resin, which they described as acid-deprived formaldehyde (berrino et al., 2022). in this study, after six months of storage, they reported significantly less dna fragmentation in the samples stored in formic acid-deprived formaldehyde compared to standard formaldehyde solutions. notably, rna was stable over the six-month period tested when stored in either solution.   ph buffering in addition to directly damaging tissue, the formation of formic acid over time will also lower the ph of a solution containing formaldehyde. in turn, lower ph can damage tissue by causing or accelerating protein denaturation, protein aggregation, and the hydrolysis of biomolecules. as a result, it is commonly thought that more acidic conditions are associated with worse tissue degradation (van duijn et al., 2011). to prevent acidification of formaldehyde solutions, a neutral buffer is often used, which is often in the form of a phosphate buffer. a ph buffer system involves the use of weak acids and their corresponding conjugate bases, or vice versa. in the case of phosphate buffer, monohydrogen phosphate, [po3(oh)]2-, acts as the conjugate acid and dihydrogen phosphate, [h2po4]-, acts as the conjugate base. these ions form a buffer system that can slow changes in ph. formalin with a phosphate buffer added is called neutral buffered formalin (nbf). unbuffered formalin has a ph of approximately 3.7, while that of buffered formalin can be tuned, usually to a ph of approximately 7.0 (nuovo and richart, 1989). historically, nbf was not widely used, but it has been more commonly used in the past several decades. nbf is expected to lead to less tissue damage during storage by preventing a drop in ph as the formaldehyde-preserved solution forms acids. however, the capacity of the buffer system will eventually be reached, and with additional acid formation, the ph will decrease regardless of the initial buffer. multiple empirical studies have examined the acidification of formalin solutions, both buffered and unbuffered, in the preservation of human brains. these studies have found that the ph of brain tissue fixed with buffered formalin tends to decrease over time. for example, one study found that the ph of buffered formalin solutions was 6.4 after 1 month, 5.7 at 8 years, and 4.5 after 10 years (pikkarainen et al., 2010). another study, profiling brains with fixation times of 7 months to more than 50 years, found that the ph of the formalin solutions had a ph range of 5 in the more recently preserved brains to 4 in the older cases (sheaffer et al., 1999). other studies have reported similar ph ranges from 4-6 that decrease over time (ploeger et al., 1993; rosoklija et al., 2003). factors contributing to the differences in the rate of ph decline across collections likely include the volume and concentration of fixative, the type of brain tissue, the container used, and the amount of oxygen exposure. notably, one source notes that this drop of ph in stored formalin solutions only occurs in the presence of tissue (weil, 1929). this finding suggests that there is an interaction between formaldehyde solutions and tissue biomolecules that promotes acid formation. taken together, the use of phosphate buffer can slow, but not prevent, the lowering of ph in formalin solutions when they are used to preserve brains for the long-term.   initial aldehyde crosslinking of biomolecules the initial fixation step in brain banking is typically performed using formaldehyde, which is favored over alternative aldehyde fixatives due to its high diffusion rate. formaldehyde covalently crosslinks intracellular and extracellular biomolecules, forming both intramolecular and intermolecular bonds (walker, 1944). the exact chemical mechanisms remain somewhat uncertain and context-dependent, but some principles can be identified (gustafsson et al., 2015; tayri-wilk et al., 2020). formaldehyde predominantly crosslinks arginine and lysine side chains of proteins, followed by tyrosine and aspartic acid (tayri-wilk et al., 2020). although methylene bridges between two amino acids (r1-ch2-r2) were previously believed to be the primary crosslinks, recent studies suggest a higher prevalence of formaldehyde reacting to form imine bonds (r-n=ch2) on distinct amino acids, which then crosslink through an unknown mechanism (tayri-wilk et al., 2020). the ability of formaldehyde to react with the amino and imino groups of dna also enables it to crosslink dna with proteins, making it integral to chromatin studies (hoffman et al., 2015). for example, there is minimal free dna (<10 %) detected after just minutes of formaldehyde fixation (as cited in hoffman et al., 2015). prolonged or concentrated exposure to formaldehyde leads to the formation of insoluble higher-order crosslinked chromatin complexes (hoffman et al., 2015; nilsen, 2014). the effects of initial fixation on different lipid species in brain tissue is variable. phosphatidylethanolamines, and to a lesser extent phosphatidylserines, which contain primary amines, readily undergo crosslinking with proteins and other biomolecules through their amine groups (carter et al., 2016; vos et al., 2019; bien et al., 2021; kotnala et al., 2021). these lipids likely form an interconnected mesh with other cross-linked proteins, making them challenging to detect in their free form after fixation using approaches like mass spectrometry that require dissociated molecules, in the absence of antigen retrieval steps (carter et al., 2016; denti et al., 2020). in contrast, most other lipid types lacking amines, such as phosphatidylcholine, sphingomyelin, and cholesterol, have minimal direct crosslinking upon initial fixation, as suggested by the minimal changes in their abundance on mass spectrometry or magnetic resonance after fixation (purea and webb, 2006; bien et al., 2021; kotnala et al., 2021). however, some of these lipids may be indirectly retained in the tissue through the protein-amine phospholipid-crosslink mesh, though this depends on the harshness of subsequent processing steps (maneta-peyret et al., 1999). in addition, initial fixation does not cause significant changes in the spatial localization of lipids, though species that have more membrane fluidity, such as cholesterol, can migrate during subsequent processing steps (carter et al., 2011; vos et al., 2019; bien et al., 2021). chemical alterations like hydrolysis, peroxidation, and methylation of amine head groups also likely occur for some lipids during initial fixation (carter et al., 2016; kotnala et al., 2021; dannhorn et al., 2022).   chemical gel formation because fixatives induce gel formation in tissues, the properties of gels are essential to understand in fluid preservation. a gel is a material primarily composed of liquid – often accounting for over 99 % of its total mass – constrained by a three-dimensional immobilizing matrix (adams, 2022). the matrix bestows upon gels its key solid-like characteristic of elasticity, which enables the gel to resume its original form after deformation (clark, 1991). gel robustness or strength can be evaluated based on the extent of its structural deformation in response to mechanical stressors, such as inversion (jia et al., 2011). gels can be classified into chemical and physical types. chemical gels originate from covalent crosslinks between chains, while physical gels originate from non-covalent interactions, such as electrostatic or hydrophobic forces (gulrez et al., 2011; richtering and saunders, 2014). the crosslinks in chemical gels disintegrate at a significantly slower pace than in their physical counterparts, owing to the tough nature of covalent bonds. as a result, the covalent bonds in chemical gels are sometimes considered "essentially permanent" (adams, 2022). on a physical level, gel formation triggers a steep increase in viscosity and effectively halts molecular motion among the crosslinked molecules, mirroring other liquid-solid phase transitions like colloidal aggregation or the glass transition (trappe et al., 2001). even prior to fixation, cells and extracellular matrix in biological tissues tend to have gel-like properties, which is not surprising because gels are the quintessential form of soft matter (douglas, 2018). for example, the cytoskeleton of dendritic spines has been found to behave as a gel (eberhardt et al., 2022). following crosslinking fixation, these native gel-like networks are dramatically strengthened and stabilized (wang and minassian, 1987). as an example of this, crosslinking of chitosan with glutaraldehyde induces the formation of a chemical gel that strengthens with increased concentration of glutaraldehyde and time (argüelles-monal et al., 1998). an unfixed and sufficiently decomposed brain does not, in practice, act as a chemical gel. however, following the covalent crosslinking of proteins in the brain with formaldehyde, a fixed brain could be considered to have formed a chemical gel network. indeed, once the brain has been fixed with formaldehyde and delipidated, it has been found to act macroscopically as a hydrogel, insofar as it can reversibly swell and shrink in water (susaki et al., 2020). for a chemical gel in general, the number of covalent crosslinks, the distance between them, and its resulting strength, can be controlled by the chemistry used to create the gel matrix (adams, 2022). therefore, the crosslinking properties and the strength of the gel created by fixation depends on the aldehyde fixative(s) used and the duration of fixation. there are many trade-offs involved in determining the optimal duration, and thus the strength, of fixation. longer fixation times are associated with more retention of antigens in the tissue prior to long-term storage or subsequent tissue processing steps (romijn et al., 1999). on the other hand, longer fixation times are also associated with substantially decreased antibody penetration and reduced antigenicity for immunohistochemistry, which is a phenomenon called “overfixation” (romijn et al., 1999; mcfadden et al., 2019). in practice, 1-2 weeks of formaldehyde immersion is often considered necessary for “complete fixation” of the human brain at room temperature (romijn et al., 1999). one group tested various fixation times ranging from 3 days to several months, finding that 6-14 days of fixation was the best duration of fixation for their goals (romijn et al., 1999). taken together, the duration of initial fixation is an important variable to consider in evaluating the literature on long-term fluid preservation.   chemical gel-based preservation of biomolecules over time we can consider two features of biomolecules that can be preserved: their chemical and location properties. chemical refers to whether the atomic composition or conformation of a molecule has been modified during the fluid preservation process. this is relevant in fluid preservation, because in the liquid state, chemical reactions will still be occurring. location refers to the relative position of a biomolecule in the tissue. changes in biomolecular location during storage of fixed brains are dependent on the extent to which the biomolecule is incorporated into the crosslinked mesh. a pure chemical gel typically contains two components: an immobile network of crosslinked molecules and a mobile solvent. in contrast, we can categorize biomolecules in the fixed brain into three groups: (a) an immobile network of directly crosslinked biomolecules, which primarily consists of proteins, (b) a mobile solvent, which is usually water, but this can be replaced, and (c) entangled biomolecules that are not directly crosslinked but are indirectly confined in the protein mesh. this entangled category includes many lipids. if a protein is directly crosslinked, then the main question for maintenance of its location is the stability of its covalent bonds. in aqueous environments, the primary mechanism for the degradation of peptide bonds and crosslinking bonds is expected to be hydrolysis (shiurba et al., 1998). uncatalyzed peptide bond cleavage occurs at a slow rate; at neutral ph, hydrolysis of peptide bonds has demonstrated half-lives on the order of hundreds of years (mahesh et al., 2018; radzicka and wolfenden, 1996). enzymatic catalysis, which theoretically could accelerate the process, is anticipated to be substantially impeded by the crosslinking procedure. as a result, there is a strong theoretical rationale to expect that proteins exhibit considerable stability during fluid preservation. hydrolysis of crosslinking bonds can certainly occur, especially at higher temperatures (barker et al., 1980; hoffman et al., 2015). however, in many contexts, a subset of aldehyde crosslinks are also expected to last for a substantial period (shiurba et al., 1998). this is difficult to measure when the brain remains in fixative, because in that case, there will be a long-term, dynamic process of covalent crosslinking and loss of crosslinking bonds. as a result, stability is better assessed in specimens that have been removed from fixative. it was shown early in the era of formaldehyde that gelatin fixed with excess formaldehyde forms an insoluble gel, which does not change structure even after being exposed to water for 10 months (hardy, 1899). one study using radiolabeled formaldehyde found that a portion of formaldehyde-collagen bonds cannot be removed even after up to 19 weeks of washing (barker et al., 1980). in this study, the percentage of non-removable formaldehyde reached a plateau at 12-20 %, suggesting these formaldehyde molecules had formed stable formaldehyde-collagen bonds. as another example, glutaraldehyde crosslinked heart valves can last for 12-15 years in vivo prior to structural degeneration, which occurs either due to calcification or tearing at suture points (tam et al., 2017). as a result, there is reason to think that aldehyde-based crosslinking bonds can be stable for long periods of time. however, this is a complex question that undoubtedly depends on the context. for example, the stability of crosslinks depends on the concentration of aldehyde used prior to the removal from fixative (barker et al., 1980; lyon et al., 1991). additionally, the stability of the crosslink depends on the type of chemical bonds formed between formaldehyde and the crosslinked biomolecules (gavrilov et al., 2015; kamps et al., 2019). other factors include the fixative used, the tissue type, and the storage medium. indirectly entangled biomolecules likely include lipids such as phosphatidylcholines and sphingomyelin that are not directly crosslinked by fixatives but appear to be retained as a part of the membrane protein-lipid complex (denti et al., 2020). if a biomolecule is indirectly entangled in the chemical gel, then there are a few possible ways that its location could change during long-term storage. namely, the biomolecule could (a) become chemically modified and covalently bound to the mesh by residual aldehyde, (b) remain confined long-term in the absence of a covalent bond, (c) diffuse to a significantly different location within the mesh, or (d) leak out of the mesh and dissolve in the solvent. we can think of an entangled biomolecule as a guest molecule embedded in a chemical gel matrix (arends et al., 2015; kowalczuk et al., 2016; chen and muthukumar, 2021). in certain scenarios, the biomolecule may become effectively immobilized due to barriers that hinder its diffusion (chen and muthukumar, 2021). the propensity of a guest molecule to disperse is an empirical question dictated by multiple factors, including its radius of gyration relative to the local mesh size, the gel's biochemical composition, non-covalent interactions such as electrostatic bonds, crosslink density, and the temperature. for instance, during aldehyde fixation, triglycerides, cholesterol, and glycogen may remain physically trapped without direct crosslinking (lyon et al., 1991).   aldehyde storage after the initial step of fixation in the brain preservation procedure, the most straightforward and common method for long-term fluid preservation is to simply leave the brain in the same fixative solution (feirabend and ploeger, 1991; vonsattel et al., 2008; kövari et al., 2011). some previous authors have suggested that storage can be effective for the long-term in formaldehyde solutions. for example, two sources note that brains can be stored in formalin for an indefinite period (fish, 1895; voogd and feirabend, 1981). however, even though brains can be stored for long periods in solutions containing formaldehyde, the key question is the quality of the preservation over time, such as the degree to which specific features can still be distinguished on microscopy as expected with a given visualization technique (koehler et al., 2024). therefore, understanding the empirical effects of this storage method is essential.   empirical studies of formaldehyde storage we built a database of studies measuring the effects of formaldehyde storage on cellular morphology in nervous system tissue, employing a wide range of methodologies (supplementary file 7; example results in figure 3). observations extracted from these studies were independently graded by two raters for storage artifact severity on a subjective 0-2 scale, with “0” indicating no or minimal artifact, “1” partial, and “2” severe or total. inter-rater reliability for these grades was excellent, as indicated by an intraclass correlation statistic of 0.958 (f-test p-value = 8.9 * 10-70) (koo and li, 2016). among the 155 observations, 60.8 % reported no or minimal storage artifact, 22.2 % a partial storage artifact, and 17.1 % a severe or total storage artifact (figure 4). we identified one type of biomolecular artifact – loss of antigenicity (n=47 observations) – and five types of morphological artifacts: decreased silver staining (6.9 %), decreased structural preservation (n=2 observations from (lai et al., 2018)), areas of empty neuropil (n=2 observations from (van duijn et al., 2011)), myelin-like whorls (n=2 observations from (robards and wilson, 1993)), and nuclear degeneration (n=2 observations from (cook et al., 2014)). we first discuss the biomolecular alterations and then the morphological alterations observed during storage in formaldehyde solutions. figure 3. example images showing morphology preservation from tissue stored long-term in solutions containing formaldehyde. a-c: images from (bouvier et al., 2016). a: staining for axonal neurofilaments with the antibody smi312 in cortical sections stored in fixative for 25 years. b: staining for vglut1-positive presynaptic boutons (magenta) and psd95-positive postsynaptic structures (green) and high-resolution imaging allows synapse visualization in cortical tissue stored in fixative for 15 years. c: staining for calbindin in layers i/ii of the cortical tissue stored in fixative for 19 years and 3d reconstruction shows the morphology of calbindin-expressing interneurons. d-e: images from (lai et al., 2018). d: staining for zo-1 (green) and dylight 649-labelled lectin (red) allows visualization of blood vessels in cortical tissue stored in fixative for 45 years. e: color depth-coded, z-stack image of cleared cortical tissue stained with gfap following 50 years of storage in fixative. f: image from (phillips et al., 2016) shows neurofilament h staining in cerebellar tissue from two separate brains (upper and lower) stored in fixative for 3 years each. g: image from (larsen et al., 2022) shows a 3d reconstruction of pyramidal cells from tissue stored at least 17 years in formalin, with yellow lines indicating orientation and solid circles the cell centroids. scale bars = 50 μm (a), 200 μm (b), 5 μm (c), 200 μm (d), 50 μm (e), 100 μm (f), 20 μm (g). all images reproduced under a creative commons attribution 4.0 international license, available here: https://creativecommons.org/licenses/by/4.0/. figure 4. severity grades for effects of formaldehyde storage on cellular morphology in brain tissue. this scatterplot shows the severity of storage artifacts in brain tissue stored in solutions containing formaldehyde. each data point is an observation, which there could be multiple of from a single study. storage artifacts were graded on a subjective 0-2 scale, with “0” indicating no or minimal artifact, “1” indicating a partial artifact, and “2” indicating a severe or total artifact. data points are colored by the histologic visualization method used: biomolecular staining (pink) or morphologic staining (green). the x-axis shows the storage duration on a logarithmic scale.   effects on proteins the effect of fixative storage on proteins depends strongly on the protein and the techniques used for detection (thacker et al., 2021). in immunohistochemistry, the duration of fixation needs to be timed precisely for optimal staining. underfixed tissues have altered tissue morphology and poor antigen retention, while overfixed tissues have poor antigen staining, largely because excessive crosslinks hinder antibody penetration and binding to antigens (beckstead, 1994). numerous studies found that there is a loss of staining for certain antigens following storage in formaldehyde for months, years, or decades (beach et al., 1987; dwork et al., 1998; sheaffer et al., 1999; pikkarainen et al., 2010; lundström et al., 2019; wu et al., 2022; lin et al., 2023). for example, one study found that fixation times need to be precisely controlled when staining formaldehyde-sensitive antigens such as dcx, psa-ncam, and neun (flor-garcía et al., 2020). in this study, fixation times of more than 12 hours were shown to have strong effects on staining properties, and staining could be completely abolished with more than 6 months of fixation time. antigen retrieval methods can often remove excessive crosslinks and allow for binding of antibodies to proteins despite long durations of fixation (evers and uylings, 1997; liu et al., 2010). for example, one study found that tissue clearing using clarity led to substantially increased immunostaining for camkiia in a brain that had been fixed in formalin for 18 years (woelfle et al., 2022). the authors speculated that this was because the clearing led to mild disruption of highly crosslinked formaldehyde-protein networks, thus making the epitopes more accessible. however, there are also limits to current antigen retrieval methods. one study of brains stored in formalin for up to 14 years found that while antigen retrieval helped to improve the visualization of some antigens, other antigens still had diminished or absent staining as a result of storage (pikkarainen et al., 2010). other sources have used unbiased profiling methods to analyze numerous proteins at once. one study used proteomics following antigen retrieval techniques, finding that mouse brain tissue fixed for 3 years did not yield more proteins than human brain tissue fixed for 7 years (rahimi et al., 2006). indeed, they found that human tissue fixed for 7 years detected a similar number of proteins as was detected in fresh frozen human brain tissue (rahimi et al., 2006). another study developed a method of mass spectrometry on fixed tissue following brain clearing (bhatia et al., 2022). they compared tissue that had been formalin fixed for more than 5 years to pfa fixed control samples, with more than 5000 proteins identified, and pearson correlations between the proteomes from these two conditions ranging from 0.91 to 0.96. this result suggests that overall protein content is largely maintained during storage in formaldehyde, for at least 5 years. notably, other studies have reported biochemical changes in proteins as a result of formaldehyde fixation; for example, hydrolysis of certain proteins is thought to occur (matsuda et al., 1998; hackett et al., 2011). however, the extent of these chemical changes remains unknown. taken together, extant data shows that most proteins remain present in fixative-stored brain tissue over a timescale of years, albeit potentially difficult to access with antibodies and with the potential for chemical alterations.   effects on lipids one early study investigated the effect of formalin storage on the ability to extract and visualize lipids with chromatography from several types of animal tissues, including brain and spinal cord (heslinga and deierkauf, 1961). they found that phosphatidylethanolamine was no longer visualized after 93 hours of fixation. in samples fixed for more than one year, there were further changes, including a reduction of lecithin. on the other hand, the levels of other lipids, such as cholesterol and sphingomyelin, were not substantially affected. the authors proposed that the observed changes might result from modifications in the chromatographic and staining characteristics of the lipids, or possibly due to changes in lipid extractability after they had more time to interact with tissue proteins. a more recent study using mass spectrometry found that there was a loss of phosphatidylethanolamine and phosphatidylserine detection in fixed brain tissue, while sphingolipids remained intact after years of storage in formalin (gaudin et al., 2014). on this basis, they suggested that phospholipids are largely degraded by hydrolysis, oxidation, and covalent modifications. however, a later study argued that their result was not due to the chemical degradation of the phospholipidome, but rather that the aminophosholipids are rendered inaccessible to their method of mass spectrometry, which is a rapid effect that occurs after only overnight fixation (carter et al., 2016). consistent with the notion that many lipids are not chemically degraded during fluid preservation, a recent study used mass spectrometry imaging on fresh frozen and formalin-fixed brain samples to detect gangliosides, which are a type of sphingolipid often found on the surface of neurons (harris et al., 2020). they found that although immersion in formalin initially decreases lipid signal compared to fresh frozen tissue, there was a similar signal enhancement in rat brains fixed for 15 minutes and human brains fixed for up to 15 years, suggesting that long-term storage in formalin does not significantly affect ganglioside content. notably, following long-term storage in fixative for many decades, brain tissue has been reported to be “virtually non-clearable” (lai et al., 2016). this suggests that long-term chemical modifications may cause a subset of lipids that are at first indirectly confined to become more strongly bound to the crosslinked biomolecular mesh over time. in addition, it has been speculated that the degree of chemical alterations to lipid species, such as hydrolysis and oxidation, likely correlates with the storage duration of specimens in formalin (dannhorn et al., 2022). overall, there may be significant chemical alteration of tissue lipids when brain tissue is stored in fixative for the long term, but it seems that a substantial subset of lipids is retained.   effects on nucleic acids formaldehyde is well-known to cause chemical changes in nucleic acids, such as cytosine deamination and depurination, which become more common over time (raxworthy and smith, 2021). the ability to sequence nucleic acids is rapidly lost during the initial days of the fixation process, because sequencing relies on dissociating the molecules, and crosslinking prevents this (guo et al., 2023; vitošević et al., 2023). however, nucleic acid profiling is still possible after many decades or even more than a century. for example, one study sequenced 1918 pandemic influenza rna from samples fixed in formalin for close to a century, after using heat treatment to partially reverse the formaldehyde crosslinks (patrono et al., 2022). in addition, one study found that the distribution of dna sizes extracted from brains stored in formalin was not altered when comparing storage times of 3 years up to 46 years, suggesting that long-term storage does not lead to a linear decrease in molecular preservation quality (savioz et al., 1997). as with proteins and lipids, extraction methods play a critical role in determining the success of profiling nucleic acids in long-term formalin-fixed tissue (herbin et al., 2021). for example, one study found that there was a decreased in situ hybridization (ish) signal for dna following fixation in 7 % neutral buffered formalin for 79 weeks (mostegl et al., 2011). however, through an increase in the concentration they used of proteinase k – which digests crosslinks and renders the dna more accessible – prior to performing the ish assay, the signal returned to the same level as day one of fixation time. another study found that mirnas can be profiled in brain tissue that has been stored in formaldehyde for more than 20 years (herai et al., 2014). mechanistically, acidic conditions in the tissue, attributed to formaldehyde degradation into formic acid and the degradation of fats into fatty acids, are thought to accelerate dna hydrolysis, leading to the loss of nucleobases from the dna molecule (kösel and graeber, 1994; herbin et al., 2021). dna sequencing in archival tissue is a highly active area of research. emerging methods have had success in extracting longer dna molecules from long-term fixed tissue, suggesting that much of the dna content remains present in cells and tissues even after long-term storage in fixative, and that this can be accessed with the proper techniques (savioz et al., 1997; fang et al., 2002; hykin et al., 2015; hassani and khan, 2015; hahn et al., 2022).   effects on small molecules regarding small molecules, they are liable to leech out of the fluid preserved brain tissue over time. for example, levels of the cocaine metabolite benzoylecgonine were found at high levels in the formalin solution after 30 d of storage of several tissues in formalin (hilal et al., 2009). in this study, the leaching was the smallest in brain tissue compared to the other tissues tested, but it is likely still present. bioelements also can shift substantially during fluid preservation. tissues fixed with buffered formalin and profiled with mass spectrometry imaging show a shift towards sodium adducts compared to potassium adducts in fresh tissues, likely due to the sodium content of buffered formalin solutions (carter et al., 2011). one study investigated the levels of 19 elements in brains stored in formalin for approximately 20 years (gellein et al., 2008). they found that there was a substantial leaching out of some elements, such as as, cd, and mg. however, the concentration of most of the bioelements was still much higher in the brain tissue than in the formalin solution they were stored in. bioelements that are known to be strongly bound to the sulfhydryl groups found in proteins, such as ag, hg, and ni, leached out from the tissue less than others.   summary of storage effects on biomolecules in summary, some chemical species that are not directly attached to the formaldehyde-induced crosslinking meshwork, small molecules and bioelements especially, appear to slowly migrate out of the tissue over time (figure 5). but the preponderance of data suggest that most biomolecules become increasingly trapped in the formaldehyde meshwork over time. this inhibits our ability to visualize the molecules, leading to the phenomenon of overfixation. there are limits in the extent of clearing and antigen retrieval that may be possible with contemporary technology, but this may improve in the future, further “unlocking” these biomolecules within archival tissue stored in formaldehyde solutions (thacker et al., 2021; hahn et al., 2022). additionally, the biomolecules may undergo chemical reactions such as oxidation and hydrolysis that can alter their chemical composition. figure 5. conceptual diagram of the dynamic changes in biomolecules during long-term storage in fixative. during the initial fixation process, three primary states are identified: biomolecules covalently bonded into the biomolecule-aldehyde crosslinking mesh (termed “crosslinked”, black); biomolecules confined by the steric properties of this mesh without being covalently bonded (termed “confined”, grey); and aldehyde molecules, which can be either part of intramolecular or intermolecular crosslinks, bonded to a biomolecule as an adduct without forming a crosslink, or free in solution (purple). here, the term “biomolecules” primarily refers to proteins, lipids, nucleic acids, and other macromolecules. bioelements, although not biomolecules per se, are also included in this category. regarding long-term storage alterations, there is: a general increase in the number of crosslinks; a conversion of many confined biomolecules to crosslinked states; hydrolysis of some existing crosslinks (red); fragmentation of certain biomolecules (blue); migration or leaching, which primarily happens to confined biomolecules (yellow); and chemical alterations of biomolecules, which could be due to composition or conformation changes (green). note that the relative proportion of each alteration is not meant to be precise but solely for illustration purposes.   effects of formaldehyde storage on tissue morphology the most severe storage artifact we identified was in van duijn et al. 2011, who reported numerous areas of white discoloration on gross examination and corresponding hypointensities on mri (van duijn et al., 2011). no such areas were identified in brains stored in formalin for up to 1 year, but all brains stored over 6 years showed them, with increasing frequency with longer storage. these areas are on the order of hundreds of micrometers in size. on light microscopy, they contain granular, basophilic neuropil changes with some tissue rarefaction, and decreased kluver's staining, thereby indicating a lower density of myelin. the authors noted that cells and vessels are unaltered in these areas. on electron microscopy, these areas contain spaces with absent or minimal neuropil, and varying amounts of lamellar structures, interpreted as membrane remnants and/or degenerated myelin sheaths. notably, the brains in this study were stored in sealed plastic bags with a small excess of 10 % formalin, as opposed to the more common method of storage immersed in fluid in a glass or plastic container. the storage artifacts identified in van duijn et al. 2011 were found throughout the cortex, and should be detectable by many other studies, including as white discolorations on gross examination, hypointensities on mri, areas with basophilic neuropil on light microscopy, and localized empty spaces on electron microscopy. however, other studies did not report these artifacts, including other electron microscopy studies (dykstra, 2010; liu and schumann, 2014; tsutsumi, 2018), and studies that could account for sampling bias by using neuroimaging (herbin et al., 2021; wiggermann et al., 2023). as an additional test, we grossly examined several brain samples stored via fluid preservation in the neuropathology brain bank at mount sinai for between 3-5 years and we did not identify any white discolorations (figure 6). taken together, we suggest that the neuropil decomposition this study found during storage may be associated with the plastic bag storage method that likely leads to more air exposure, while also pointing out that this is a critical area for further research. figure 6. gross examination of postmortem human brain tissue reveals an absence of macroscopic discolorations in the cortex. the coronal slabs of four left hemibrains, each paired with a higher magnification view on the right, are at the level of the anterior genu of the corpus callosum. images b, d, f, and h highlight the superior frontal gyrus (ba 8/9) of these slabs. these samples were initially stored in 10 % nbf for 3.2 years (a-b), 3.1 years (c-d), 2.6 years (e-f), and 2.7 years (g-h), prior to transfer to pbs with 0.1 % sodium azide, where they were stored for an additional 2 years (a-b), 2 years (c-d), 2 years (e-f), and 1.7 years (g-h), respectively, prior to imaging. photo brightness has been adjusted to ensure visual consistency across samples. upon assessment, no white discolorations, potentially indicative of storage artifacts, were observed. rulers to the right of brain tissue are centimeter scale. one study found that storing rat brain tissue in 2 % paraformaldehyde and 2.5 % glutaraldehyde buffered by sodium cacodylate for 1 month led to more frequent intracellular and extracellular “myelin-like” whorls on electron microscopy than were seen after 3 hours of fixation (robards and wilson, 1993). by 12 months, the whorls appeared less membranous and were associated with areas of lucency, suggestive of lipid leaching. the authors suggest that this artifact may be due to incomplete fixation of phospholipids, causing them to dissolve, coalesce, and then take on this myelin-like appearance after reacting with osmium tetroxide. they note that the preservation of cellular organelles was stable with long-term storage and that the length of fixation is thought to be less critical for morphology than other variables like the fixative used, the buffer, the ph, and the osmolarity. another study found that similar myelin-like whorls were more common inside and outside of cells in skin tissue on electron microscopy after storage for 4 weeks in 2.5 % glutaraldehyde (lindberg, 1984). myelin-like figures have also been associated with mitochondrial degeneration (le beux et al., 1969). it has been speculated that they result from the reaction of extracted phospholipids with osmium tetroxide (ericsson et al., 1965). unless they become severe, the myelin-like whorl artifacts seem to be small enough that they would be unlikely to substantially affect the mapping of neural circuitry. in addition to these morphological artifacts, there are several others to consider. one study notes that nuclei degrade in brain tissue stored in formalin and that this progressively worsens from 15 to 80 years of storage (cook et al., 2014). the authors suggest that for the samples fixed for a longer period, different formulations of formalin may have been used, such as a non-buffered version. in contrast, other studies did not describe a loss of nuclei even after many decades of storage in formaldehyde (sheaffer et al., 1999; herbin et al., 2021). several studies note that silver staining can be altered following extended formalin fixation (wilcock et al., 1990; heinsen et al., 2000; rosoklija et al., 2003). however, this appears to be dependent on the method of silver staining. for example, one study notes that the rapid golgi method gave inadequate results, while the golgi–kopsch method yielded excellent quality staining comparable to tissue fixed for a few years, even on tissue stored in formalin for up to 55 years (rosoklija et al., 2003). one study that used cresyl violet staining to visualize neuronal morphology reported that “structural preservation” was not as good in tissue fixed in formalin for many years, without providing more detail on what aspect of the structure was altered (lai et al., 2018). as in immunostaining studies, overfixation might help to account for decreased morphologic staining intensity after prolonged fixation. because morphological stains non-specifically bind to many types of biomolecules, if there is a global decrease in biomolecular binding capacity due to excessive crosslinks, then this will lead to a global decrease in the signal to noise ratio of morphological staining. some biomolecules may also be extracted from cells over time, making the cytoplasm of cells appear more pale (lindberg, 1984; robards and wilson, 1993). additionally, because long-term fixation can render tissues more acidic, the staining quality of anionic dyes may be especially decreased (puchtler and meloan, 1985). in addition to histologic effects, long-term storage in fixative can also have macroscopic and biomechanical effects. upon initial fixation, fixatives often cause cell shrinkage, which is accentuated for stronger fixatives such as glutaraldehyde (nei, 1981). one source finds that after the initial fixation, the volume of canine brain structures was not altered due to additional storage in formalin for up to 12 months (del signore et al., 2022). on the other hand, other reports have suggested that specimens do have secondary shrinkage if they are stored in fixative fluids for long periods (bahr et al., 1957; heinsen et al., 2000). this discrepancy may be due to the type of brain tissue, the fluid properties, and the duration of storage. macroscopic shrinkage may correspond to widespread microscopic strengthening of chemical gels in the tissue over time. strengthening of microscopic interactions, however, is associated with changes in the material properties of the brain, insofar as it becomes more stiff, brittle, and difficult to section (dannhorn et al., 2022). prolonged storage in fixative can also promote the accumulation of formalin pigment (sacchini et al., 2022).   formaldehyde formulation the concentration and formulation of formaldehyde may play an important role in long-term storage. several studies have used a lower concentration of formaldehyde for storage, such as 0.1 % paraformaldehyde (lyck et al., 2008), 1 % formalin (papageorgopoulou et al., 2010; wiggermann et al., 2023), 2 % paraformaldehyde (wu et al., 2002), or 2 % formalin (monteiro, 2008). using a lower concentration of formaldehyde during storage is appealing because it may help to prevent the negative effects of formaldehyde while still preventing microbial growth and any theoretical loss of morphologic cohesion. in terms of additives, there is a concern that storage in commercial formalin solutions, which contain a small amount of methanol in addition to formaldehyde, may lead to lipid damage, because methanol is a solvent for lipids (öztürk and koç, 2022). however, one study found that even after 18 years of storage in methanol-stabilized formaldehyde, staining with a lipophilic dye was still possible, and that there was no difference in the distance of nerve tracing possible as a result of storage (öztürk and koç, 2022).   storage in other aldehydes in addition to formaldehyde, other aldehyde fixatives can also be used for long-term storage. although this is less well studied, these storage solutions are expected to have many of the same effects as formaldehyde. tissue stored in other aldehyde fixatives will undergo overfixation. other aldehyde chemicals also have the potential to convert to acid forms. for example, concentrated glutaraldehyde oxidizes to form an organic acid during storage, which has been found to decrease the ph of the solution over time (boucher, 1978; ranly, 1984). when glutaraldehyde is used in the fixation of nerves, studies have reported switching the solution to formaldehyde or phosphate buffer for long-term storage (magoon and robb, 1981; morgello et al., 2001). one source notes that nervous system tissue “deteriorates rapidly” in glutaraldehyde; however, they do not specify the outcome measure they used to make this evaluation (voogd and feirabend, 1981). because glutaraldehyde is a stronger fixative than formaldehyde, problems of overfixation and inaccessibility of biomolecules are expected to occur more quickly during storage in glutaraldehyde. however, one source reports that storage in glutaraldehyde is compatible with good long-term structural preservation, with no evidence of ultrastructural alterations to kidney tissue stored in 4 % formaldehyde and 1 % glutaraldehyde at 4 °c for up to 23 years (dykstra, 2010). one publication reported using glyoxal for long-term storage, but did not report the outcomes of tissue stored in this manner (thomas et al., 2022).   summary the most common storage artifact identified in the studies reviewed is a loss of antigenicity for certain proteins over time, likely due to the accumulated crosslinking making antigens inaccessible. while some antigens become undetectable after days, months, or years of storage, other proteins can still be visualized even after decades in fixative. other biomolecules like nucleic acids and a subset of lipids also frequently become more heavily crosslinked and confined over time, making them more difficult to profile, but usually not impossible to profile with specialized techniques. besides effects on biomolecules, morphological artifacts were less frequently reported. one study found localized empty spaces in neuropil and after more than six years of storage, but this finding was not reported by other studies with similar or longer storage times, raising the question of whether the severity of this artifact was associated with their storage method in plastic bags (van duijn et al., 2011). one study noted the accumulation of “myelin-like” whorls during storage that was associated with incomplete fixation of lipids and lipid leaching (robards and wilson, 1993). another study reported nuclear degradation after decades of storage, but others did not note this effect (cook et al., 2014). more commonly, nonspecific decreased staining intensity was noted in some studies, potentially also related to chemical alterations of biomolecules. overall, while there have been some storage artifacts reported following-long term storage, the extent to which aspects of the underlying neural circuitry information is irreversibly lost, or simply more difficult to measure, as well as how this varies between different fixative storage methods, are open questions worthy of further investigation.   buffer storage buffer solutions such as sodium phosphate buffers, phosphate-buffered saline (pbs), and cacodylate buffer are commonly used for maintaining a stable ph during the preservation of fixed brains (supplementary file 8). pbs is designed to be an isotonic, non-toxic solution that mimics the extracellular fluid, and is among the most common buffers used in the field of brain banking. cacodylate buffer is a heavy metal buffer commonly used in electron microscopy that avoids the risk of calcium phosphate precipitates and microorganism contamination that can occur with the use of phosphate buffers (ivanchenko et al., 2021). a common method for long-term fluid preservation of fixed brain tissue is to transfer brains to a buffer solution, without any fixative. as discussed above, the ph buffering capacity of these solutions can diminish over time, rendering long-term storage in buffers essentially equivalent to storage in water. two other factors that are important to consider is the degree of residual aldehyde fixative remaining in the solution and the prevention of microbial contamination.   degree of residual fixative one option to decrease the amount of formaldehyde in the solution is to attempt to wash it out. however, even washing formaldehyde fixed tissue for weeks has been found to be insufficient to remove all formaldehyde crosslinking bonds (barker et al., 1980). washing tissue is an even a greater problem in the study of intact whole brains because of their size. for example, washing human brains with buffer for 48 hours has been found to be clearly insufficient for buffer penetration to interior brain regions, leading to a contrast boundary artifact (miller et al., 2011). as a result, despite the imaging artifacts associated with formaldehyde, washing fixed whole human brains is often not performed prior to neuroimaging because removing formaldehyde would take a prohibitive amount of time (tendler et al., 2021). one source suggests that washing tissues in water for an extended number of years does eventually dislodge more formaldehyde (puchtler and meloan, 1985). because washing fixed tissue for extensive periods of time can still yield formaldehyde in the effluent, it is tricky to distinguish the bound formaldehyde from that which can be removed (french and edsall, 1945). however, there appears to be thresholds beyond which additional washing does not yield a substantial amount of additional fixative (french and edsall, 1945). effectively, storage in buffer constitutes storage in a much lower concentration of fixative solution, with the concentration depending on the amount of time that the tissue was washed prior to long-term storage.   preventing microbial growth one of the major potential downsides of removing the brain from fixative storage is the potential for microbial growth. one source reports that this is a common occurrence in buffer solutions, as proxied by bacterial or fungal growth in the absence of a host immune reaction (dykstra, 2010). as a result, it is commonly thought that storage in buffer requires adding an additional chemical agent to prevent microbial growth and resulting tissue damage. the most common agent for this is sodium azide (nan3), an antimicrobial preservative that acts to inhibit mitochondrial respiration, largely by binding to cytochrome c oxidase (ishii et al., 2014). for example, one source reports that if fixed brain tissue is stored in pbs at 4 °c for longer than a week, 0.02 % of sodium azide should be added to the pbs solution to prevent bacterial growth (de prisco et al., 2022). another study reports that the addition of sodium azide can help with maintenance of histochemical staining in brain sections (morán and gómez-ramos, 1992). thymol is another antimicrobial agent that can be used, which acts to disturb the integrity of bacterial and fungal membranes (hammer et al., 2012). the question of how long sodium azide retains its antimicrobial activity during storage is not well established in the literature. notably, sodium azide is used at a range of concentrations in preservation solutions; for example, it has been used at 0.1 % (beach et al., 2015) or 0.01 % (micheva et al., 2023). however, concentrations lower than 0.01 % may not allow for effective antimicrobial activity (snyder and lichstein, 1940). at relatively higher concentrations, there is more leeway against the potential for sodium azide to break down chemically over time and no longer protect against microbial growth.   effects on cellular morphology one of the major advantages of buffer storage is in mitigating or preventing overfixation, which damages antigenicity and makes sections difficult to process. one brain bank reports that they store fixed brain blocks in 0.1 mol/l phosphate buffer with 0.1 % sodium azide at room temperature, with excellent results for immunohistochemistry studies after years of storage (beach et al., 2015). one study stored brain tissue for around 1.5 to 15 months in 0.1 m cacodylate buffer ph 7.4 or 0.1 m phosphate buffer with sodium azide at 4 c prior to embedding for electron microscopy (sele, 2020). they reported that there was no difference in the ultrastructural preservation quality when comparing tissue that was stored for this length of time or not, suggesting that there were no adverse effects of the storage on tissue quality. storage in phosphate or cacodylate buffer also has been recommended for storage of nerve tissue fixed with glutaraldehyde (morgello et al., 2001). finally, phosphate buffer has been used as a control storage medium when studying brain clearing methods (furuta et al., 2022). overall, because it is not as common of a method, not as much long-term preservation data is available, but the available data suggests that buffer storage is not associated with damage to neural structures over the time scale of years.   effects on brain volumes a decreased effect on brain volumes is thought to be an advantage of fluid preservation in phosphate buffer compared to storage in formaldehyde, which has been associated in some studies with long-term shrinkage due to cross-link formation and the partial leaching of soluble solids (hackett et al., 2011; de guzman et al., 2016). this might be useful for studies attempting to measure the volume of a particular brain region. however, the effect of buffers on brain volume sizes depends on the osmotic concentration. one study found that five months of storage of fixed brains in a standard concentration of pbs led to a slight decrease in volume in several brain regions (de guzman et al., 2016). on the other hand, three weeks of storage in distilled water led to increases in volumes across the brain, while three weeks of storage in 10x pbs led to a dramatic shrinkage in volumes across the brain (de guzman et al., 2016).   summary buffer solutions are commonly used for long-term storage of fixed brain tissue. a major advantage is preventing overfixation and associated loss of antigenicity. however, washing fixative from large brains is challenging, so residual aldehyde may remain. microbial growth also needs to be prevented, which is often performed using sodium azide. there is theoretical reason to expect that buffer storage would prevent overfixation and morphological alterations associated with formaldehyde storage, but long-term data is limited. the long-term breakdown of the stabilizing chemical gel without additional crosslinking agents is also a theoretical concern. taken together, buffer storage helps address some limitations of aldehyde storage, but open questions remain about very long-term preservation quality.   alcohol storage alcohols, especially ethanol, have long been recognized as a useful medium for fluid preservation (supplementary file 9). ethanol is still commonly used today in museum collections, where a low concentration of formaldehyde is often used for initial fixation, followed by long-term preservation in ethanol (straube et al., 2021). the use of other alcohols like methanol and isopropanol has also been documented, but ethanol tends to be the most used due to its relatively lower toxicity. a notable concern is the flammability of alcohol, which requires careful storage procedures to mitigate safety risks.   mechanisms the main mechanism by which alcohols act as preservatives is to dehydrate tissues, eliminating water from cells and consequently leading to protein denaturation and prevention of enzymatic degradation. in this context, it is sometimes called a coagulative fixative. it also possesses antimicrobial properties, which helps to prevent contamination of stored specimens. the elimination of water can lead to severe shrinkage of the tissue, affecting morphology. many investigators use an ethanol concentration of around 70-80 % for the long-term preservation of fixed brains, balancing preservation efficacy with less damage to tissue morphology. however, the optimal concentration of ethanol depends heavily on the context and desired use, with more morphological damage but less biomolecular damage occurring at higher concentrations (marquina et al., 2021).   effects on biomolecules in contemporary use, alcohols are sometimes favored over aldehydes as a preservative due to their less damaging effects on certain classes of biomolecules, mainly dna and proteins. for example, the yield of dna from brain specimens was altered after one month of storage in formaldehyde, but not after the same duration of storage in 75 % ethanol (zamenhof et al., 1972). more recent studies have confirmed that spirit-preserved specimens have higher quality dna when stored in ethanol as compared with formaldehyde, although contemporary retrieval techniques following formaldehyde storage have improved substantially (straube et al., 2021; hahn et al., 2022). regarding proteins, one study of preserved fish found that specimens initially fixed in formaldehyde and then transferred to isopropyl alcohol or ethanol for storage for many years (up to 70) had high-quality histology (kwan et al., 2022). they found that specimens preserved in formaldehyde alone for long periods of time had higher autofluorescence and challenges with achieving high histologic quality. the likely reason that alcohols are less damaging to dna and proteins is likely that they avoid the overfixation that occurs during storage in aldehydes (zamenhof et al., 1972). ethanol has also been used as a storage medium after 24 hours of formaldehyde fixation to prevent overfixation during transport (stumm et al., 2012). although alcohol preservation avoids overfixation, it comes with the disadvantage of extracting certain classes of biomolecules from the tissue, especially lipids. ethanol can dissolve lipids out of the specimen, which can cause them to accumulate as fatty acids at the top of the storage container (hartman, 2019). this can make the preservative solution cloudy (stoddart, 1989). the increased acidity due to the accumulation of fatty acids is also thought to contribute to specimen damage (straube et al., 2021). due to its denaturing properties, ethanol storage can also damage the conformation and fluorescence of proteins, such as green fluorescent protein (becker et al., 2012).   effects on morphology evaluating the literature on fluid preservation with alcohols, particularly ethanol, is challenging due to its historical use as an initial fixative rather than solely as a long-term preservative following formaldehyde fixation. as fixatives, alcohols are generally less effective than crosslinking aldehydes at initial fixation, often resulting in lower quality morphological preservation. for example, one study that perfused 70 % ethanol to preserve a human brain found that it did not harden and displayed a red coloration even two months after fixation, suggestive of inadequate preservation (grinberg et al., 2008). another study found that brains preserved in formaldehyde for decades have much better histology than brains preserved in ethanol (herbin et al., 2021). in this study, tissue preserved in ethanol alone was found to have numerous holes when visualized by light microscopy. investigations using neuroimaging provide additional context. for instance, spiders stored in ethanol for 70-90 years showed no macroscopic changes in neural architecture, with the exception of two specimens that appeared to have suffered from desiccation (rivera-quiroz and miller, 2021). this is consistent with a study on a human brain preserved in a german collection for 150 years, which also showed satisfactory preservation at the macroscopic level (schweizer et al., 2014). however, a more in-depth mri study of this collection identified the presence of sub-millimeter tubular cavities in the white matter of human brains preserved in ethanol for long periods of time (helms et al., 2014). these tubular cavities were not seen in a bovine brain preserved in ethanol for four months, suggesting that they are an artifact of long-term storage (helms et al., 2014). these findings led the authors to suggest that ethanol preservation is associated with rapid water and cholesterol loss, followed by longer-term macroscopic deterioration that could be due to a slow loss of lipids. however, ethanol might serve much more effectively as a long-term preservative following fixation. as discussed above, studies in non-brain tissue have found that immunohistochemistry is well-preserved following storage in ethanol as a secondary agent (stumm et al., 2012; kwan et al., 2022). one early source suggested that ethanol could serve as an appropriate storage medium for museum specimens following formalin fixation (fish, 1895). even prior to the use of formaldehyde in pathology, one source noted that if brain tissue was not adequately hardened in muller's fluid (a dichromate solution acting as a fixative), then long-term preservation in strong alcohol of greater than 80 % could lead to significant damage (van gieson, 1889). specifically, the alcohol would dissolve out the fixative salts and alter the myelin over a period of 1-2 years. this process would result in the formation of cholesterol crystals in the alcohol, and microscopic cavities or vacuoles in the specimens, which did not stain well. van gieson noted that even if the tissue were adequately hardened, there would still be problems with histology after storage in alcohol, such as neuroglia not being visualized well, compared to tissue stored in water that had a low concentration of muller's fluid. another source notes that storage of fixed tissues in alcohol over just one weekend in can lead to extensive vacuolization of white matter, although this likely depends on the concentration used (garman, 2011). a different source reports that tumor tissue can be stored in 70 % ethanol for up to 4 weeks with no significant changes of histology and biomolecular staining patterns (stumm et al., 2012). finally, one source notes that fixed sperm cells tend to have membrane damage when they are stored in ethanol without secondary fixation in osmium, consistent with the known role of ethanol in lipid extraction (fahey and healy, 2003).   summary alcohol storage is a cost-effective, practical, and time-tested method for preserving fixed brains. storage in alcohol has several advantages, including antimicrobial effects and avoiding overfixation. however, it can cause several problems, including tissue shrinkage leading to altered morphology, biomolecular extraction, cavity formation, and vacuole formation. there is sometimes thought to be a trade-off, wherein storage in ethanol has worse morphological preservation, but better biomolecular preservation, compared to storage in formaldehyde. however, that trade-off is likely a practical one based on our currently available biomolecular profiling technology, rather than fundamentally driven by the underlying biochemistry. overall, following the development of buffer solutions and antimicrobial agents such as sodium azide that can prevent overfixation without causing biomolecular extraction, alcohols seem relatively less useful than they were in the past. they are not commonly used in brain banking and would not be ideal for use in brain mapping studies. however, the extant literature on this topic is not extensive, making it difficult to draw firm conclusions.   storage after tissue clearing brain tissue clearing is primarily used for imaging intact brain structures in detail by removing parts of the tissue that interfere with 3d visualization, such as lipids and calcium, thus rendering it more transparent (ueda et al., 2020). tissue clearing processing procedures are often quite harsh, involving powerful solvents and often high temperatures. for example, the switch protocol uses temperatures of 80 °c for up to 4 days in tissue fixed with glutaraldehyde, equivalent to slightly more than 2 years of storage at 4 °c, assuming a q10 value of 2 used in shelf life studies (murray et al., 2015). although not primarily developed for long-term storage, several types of brain clearing techniques have also been reported to provide high-qualitylong-term storage of brain tissue, so they merit consideration as fluid preservation methods (supplementary file 10).   empirical effects of brain clearing there are three classes of brain clearing approaches, each with different effects on preservation quality: hydrophobic, hydrophilic, and hydrogel methods (ueda et al., 2020). hydrophobic clearing methods use organic solvents to clear the tissue, which hardens them and allows for long-term preservation, re-analysis via immunolabeling, and stabilization of endogenous fluorescent signals. notably, hydrophobic clearing methods sometimes use ethanol as a dehydrating agent, but also use additional steps for delipidation and refractive index matching, thus differentiating these techniques from fluid preservation using ethanol on its own. one example of a hydrophobic brain clearing approach is shanel, which has been reported to allow for long-term storage and future histologic studies of the same tissue (zhao et al., 2020). as another example, sdisco has been found to retain structural details and protein fluorescence after storage for 22 months (hahn et al., 2019). methyl salicylate is another hydrophobic reagent that is the active ingredient of wintergreen oil and was a component of the first clearing method used by spalteholz in 1911 (liu et al., 2016). one study found that storage of fixed human choroid plexus in methyl salicylate for up to 25 years allowed for high-quality immunohistochemical staining of several antigens (sufieva et al., 2023). another study found that methyl salicylate combined with permanent mounting allowed storage of insect brains for well over a year at room temperature with intact immunohistochemistry (bekkouche et al., 2020). in hydrophilic clearing methods, reagents can be used for decolorization, delipidation, and refractive index matching, but water is not removed from the specimen (ueda et al., 2020). these techniques usually have reagents that form hydrogen bonds with proteins, which can aid in the preservation of protein conformation and associated fluorescence (ueda et al., 2020). in one study of a modified version of the hydrophilic brain clearing method cubic, the authors reported that the samples were washed and stored in pbs with 0.01 % sodium azide in case re-imaging was needed in the future (pinheiro et al., 2021). finally, in hydrogel brain clearing methods, chemicals are introduced to strengthen tissue gels, either via introducing molecules to create a synthetic gel, or via epoxy crosslinkers to fortify existing tissue gels (ueda et al., 2020). for example, in the clarity method, acrylamide is used to create a hydrogel-tissue hybrid that aids in retaining proteins and maintaining structural integrity during subsequent processing steps such as delipidation (malkovskiy et al., 2022). the extent to which the extra stabilization provided by hydrogel methods might improve structural maintenance in long-term fluid preservation of brains is an open question, warranting additional research.   comparison to buffer storage as discussed above, protocols for brain clearing sometimes recommend transferring the specimen back to pbs for long-term storage after clearing (ke and imai, 2014). it is interesting to compare this to the buffer storage only approach. clearing followed by storage in pbs allows for the removal of potentially damaging biomolecules, without the clearing reagents themselves potentially damaging brain tissue over time. for example, lipids are known to be a major source of oxidants during storage of tissue, so removing them could decrease the accumulation of oxidative species during fluid preservation (huang and ahn, 2019). removing lipids also prevents their long-term breakdown into fatty acids and associated decrease in specimen ph. one source reports that the storage of cleared brain tissue in pbs with 0.1 % triton-x and 0.01 % sodium azide can be performed “indefinitely” (liu et al., 2016). from the perspective of brain mapping, obvious downsides of delipidation prior to storage in pbs are that the lipids cannot be profiled and the ultrastructure will be dramatically altered (malkovskiy et al., 2022).   summary brain tissue clearing techniques like clarity and sdisco have shown promise for long-term storage while enabling intact imaging. clearing removes light-scattering elements like lipids, which could otherwise damage tissue over time. this is often followed by storage in buffer, avoiding unnecessary further extraction by clearing reagents. compared to many storage methods relying on embedding in polymers, clearing approaches are more practical, while still achieving many of the same benefits (sufieva et al., 2023). though delipidation obviously causes information loss, clearing may better maintain protein structure when compared with other techniques. clearing techniques also differ in the extent to which they can be practically used in large volumes such as an intact human brain (zhao et al., 2020). overall, clearing techniques are an emerging approach that may enable high-quality preservation in addition to allowing intact imaging, but with limited long-term data.   cryoprotectant storage cryoprotectants, such as glycerol, ethylene glycol, and sucrose, can improve the preservation of biospecimens even above freezing temperatures (supplementary file 11). while cryoprotectants are best known for protecting against ice damage during subzero temperature storage, we focus here on their biostabilization properties above 0 °c. indeed, cryoprotectants often act as osmolytes as well, which are small organic molecules that tend to accumulate in cells to help them survive under various stress conditions, such as temperature changes, pressure, and osmotic stress (rumjanek, 2018). by substituting for the water molecules that typically bind to biomolecules, cryoprotectants tend to increase intracellular viscosity, reduce molecular motion, and slow down reactions, preserving biomolecular integrity without causing significant extraction. they also help preserve hydrophobic interactions and the native structural properties of biomolecules, like protein fluorescence (nürnberg et al., 2020). reactions that depend on water, such as hydrolysis, will not occur in pure cryoprotectant solutions, because water is not present. however, chemical reactions not relying on water will still occur, potentially contributing to tissue degradation over time. for example, one study found that when dissolved in a 99.8 % glycerol solution, the enzyme lysozyme still retained one-seventh of its catalytic activity compared to water (rariy and klibanov, 1997). finally, certain cryoprotectants such as sugar alcohols have been found to play a role in stabilizing gel structures for the long-term, consistent with their role in protein stabilization (baydin et al., 2022).   glycerol glycerol is a nontoxic cryoprotectant that was used as an early preservative for biological specimens (van dam, 2020). its antiseptic and humectant properties can help slow decomposition. glycerol began to be used in the late 1800s as an alternative or complement to ethanol for storage in museum collections. carlo giancomini used glycerol for storing zinc chloride-fixed and alcohol-dehydrated brains, with good reported efficacy in both dry and fluid preservation (thomson, 1880). glycerol also gained use for stabilizing formaldehyde-fixed tissues, with some human organs reportedly stored in this manner with excellent macroscopic condition after more than 100 years (macleod et al., 2018). the effects of glycerol on tissue microstructure and morphology are quite variable between studies. it depends on multiple factors like the tissue type, glycerol concentration, how rapidly the concentration is increased, and the degree of hydration (armitage, 1986; nowacka et al., 2012). however, many studies have found that gradual increases of glycerol do not cause major structural changes like shrinkage. for example, cell membranes are thought to be the structures most vulnerable to damage during dehydration (westh, 2003). glycerol helps preserve cell membranes during dehydration through a mechanism that appears to involve interacting with polar lipid headgroups (nowacka et al., 2012; pocivavsek et al., 2011). consistent with the idea that glycerol is able to maintain cellular structure, one study found that glutaraldehyde and osmium fixed sural nerve biopsies had intact ultrastructure and collagen content after storage in 100 % glycerol at 4 °c for up to 9 months (myers et al., 1977). the authors stated that tissues could be "stored indefinitely" in this manner. kaiserling iii is a glycerol-containing fluid preservation solution introduced in the late 18th century, with the primary goal of maintaining the color of formaldehyde fixed specimens over time (kaiserling, 1900; ajileye and adeyemi, 2020). although kaiserling iii is not a pure glycerol solution, as it also contains potassium acetate and chloral hydrate, the state of tissues preserved in it can act as a proxy for evaluating glycerol as a preservative agent. although kaiserling originally recommended 25 % glycerol, today, 40 % glycerol has been reported as a better concentration for fluid preservation (ajileye and adeyemi, 2020). one of kaiserling's contributions was identifying the need to minimize air exposure and acid formation within the preservation medium to the greatest extent feasible (potaliya and ghatak, 2016). one study evaluated the histologic quality of fixed brain tissue that had been preserved in kaiserling iii for up to 55 years (monteiro, 2008). when an archival specimen was pre-treated in 10 % formalin prior to histologic processing, it had similar histology as control brain tissue, including intact cellular structure, except for having less distinct nucleoli. immunostaining was also possible, with neurofilament staining allowing the identification of individual neuronal processes and gfap staining allowing for the identification of astrocytes. glycerol has even been used to preserve biospecimens for extended periods without fixatives. for example, skin tissue is often preserved in skin banks using high concentrations of glycerol, up to 98 %. this leads a loss of cellular viability but retention of ultrastructure for years (de backere, 1994; richters et al., 1996). dura mater has been preserved for up to 7 weeks in 98 % glycerol, allowing for the retention of normal ultrastructure, with the exception of more finely divided collagen fibers (mcgarvey et al., 1984). nerve tissue has been preserved in 98 % glycerol at 4 °c for months prior to transplant (wolff et al., 1993). regarding brain tissue, one study found that samples of brain tissue can be stored in phosphate-buffered 50 % glycerol at temperatures of 4 °c or at –20 °c, for at least 15 months (lembo et al., 2006). this storage procedure allowed the tissue to be successfully immunostained for rabies antigen against a neuronal background. rather than suggesting that glycerol be used as a solitary preservative in brain banking, these studies show the power of glycerol preservation and why combining it with the technique of fixation can be helpful. one downside of glycerol preservation is that, at intermediate concentrations of glycerol of less than 65 % and relative humidities of greater than 75 % in the room used for storage, specimens can be susceptible to mold growth (van dam, 2020). a small concentration of fixative, such as 0.5 % formaldehyde, or microbial inhibitors such as camphor or thymol can be added to prevent mold growth (ajileye and adeyemi, 2020). glycerol is also expected to eventually break down into reactive impurities during the storage process, such as via oxidation to methylglyoxal (sugiura et al., 2020). finally, storage in glycerol may promote chemical reactions, such as the maillard reaction, which occurs between amino acids and reducing sugars (smarrito-menozzi et al., 2013).   other cryoprotectants ethylene glycol has also been used as a fluid preservative, most commonly for marine species (williamson and russell, 1965). compared to glycerol, ethylene glycol has a lower risk of causing osmotic damage, allowing for stronger solutions to be used (williamson and russell, 1965). however, one source reports that it can cause precipitates over time when used as a fluid preservative (luisa, 1982, p. 33). disaccharides are also potential alternatives to glycerol for storage in the liquid state. trehalose has been found to stabilize biomolecules such as proteins when dissolved in aqueous solutions, potentially due to its effect of raising the surface tension of water (kaushik and bhat, 2003). another source compares heart valve storage in 50-80 % sucrose to pure glycerol at 4 °c, finding that the sucrose solution was able to maintain structural preservation for 52 weeks while glycerol was able to maintain preservation for 12 weeks (as cited in vásquez-rivera et al., 2018). when stored in sucrose, some sources recommend to also add sodium azide (strnad et al., 2022).   kinetics of cryoprotectant brain penetration many of these studies are performed on small biopsy samples or small organisms. however, it is often desired for the human brain to be stored intact, for example for the purpose of ex vivo neuroimaging, or so that long-range neural connections can be mapped. in this case, there is a need to implement a protocol allowing for the cryoprotectant to penetrate throughout the brain, without taking too long or causing damage due to rapid changes in osmotic concentration. one study performed an immersion of 10 % glycerol and 2 % dmso into one hemisphere of a fixed rhesus monkey brain for 1 day, followed by immersion in 20 % glycerol and 2 % dmso for 3 days (sloane et al., 2000). however, the rhesus brain volume is about 10-15 times less than the human brain volume, meaning that this process may be time consuming in a human brain banking setting. notably, evidence suggests that osmotic damage is not as significant in tissue that has been adequately fixed (paljärvi et al., 1979). one alternative possibility would be to perfuse the preservative chemicals, but that is challenging to perform consistently in a postmortem setting, especially because it would need to be done evenly to avoid inducing osmotic damage (mcfadden et al., 2019).   summary cryoprotectants such as glycerol, ethylene glycol, and sucrose have shown promise for room temperature storage of fixed brain tissue while minimizing biomolecular extraction. glycerol has been used since the late 1800s, with studies generally reporting intact morphology after years or even decades of storage, except for a loss of nucleolar or collagen fiber detail. however, there are not many studies on this topic, and optimal protocols for penetrating large intact human brains have not been established. overall, cryoprotectants are a promising fluid storage approach in combination with an initial fixation step, warranting further investigation.   other storage conditions   temperature temperature is a critical factor influencing the rate of chemical reactions during fluid preservation. lower temperatures slow reaction kinetics, enabling longer preservation durations. as a rule of thumb, the 10-degree rule estimates that chemical and biological reaction rates decrease 2 fold per 10 °c temperature drop (hukins et al., 2008; orlowski et al., 2014). based on this estimate, a brain stored at a standard refrigerator of 4 °c would be expected to have a compared to have a 4-fold slower decomposition rate than one stored at a room temperature of approximately 24 °c. in brain banking, it is common but certainly not ubiquitous to store fixed tissue at refrigerator temperature of around 4 °c to slow degradation (insausti et al., 1995; wiggermann et al., 2023). one study reported that storage at 4 °c, alongside a substantially lower concentration of fixative, led to a lesser degree of antigen degradation over time for several antigens compared to storage at higher temperatures and a higher concentration of fixative (lyck et al., 2008). for alcohol preserved specimens, storing them below the flash point of absolute ethanol of 12 °c is often recommended for safety reasons. however, low temperatures can introduce new problems. storage below 0 °c risks ice crystal formation and associated tissue damage, unless cryoprotectants are used. even above 0 °c, lower temperature storage favors the precipitation of polyoxymethylene from formaldehyde (howe et al., 1995). the main downside of lower temperature storage is that it can substantially increase the costs due to the need for storage infrastructure and labor for maintenance. as a result, despite the expectation that it will slow degradation, storage at low temperature needs to be balanced by cost-benefit considerations.   storage container the container used for fluid preservation plays an important role in preventing evaporation and oxidation, which can lead to severe tissue damage over time. glass and plastic are the most common materials used. glass is impermeable to oxygen and moisture but is vulnerable to cracking over time, especially if stressed (stoddart, 1989; hiebert et al., 2021). plastic containers vary in their oxygen and moisture permeability. in long-term fluid preservation, one of the major risks is evaporation leading to tissue desiccation and potential irreversible damage (eichhorn et al., 2018). any compromise in the container, such as fissures in the glass, can result in evaporation and specimen desiccation (williamson and russell, 1965). for both glass and plastic containers, the integrity of the seal is also critical in minimizing oxygen permeability. preventing oxygen exposure is essential because oxygen causes oxidative damage to brain tissue and is associated with decreases in ph. oxidation affects unsaturated linkages in lipids, certain amino acids in proteins, and pigments (stoddart, 1989). to minimize oxygen exposure, the container can be filled with fluid or otherwise degassed (stoddart, 1989). if a specimen is stored in an oxygen-sealed container, then every time it is opened, the fluid preservative and specimen will be re-exposed to oxygen (barnes et al., 2000). as a space-saving measure, some sources recommend storing tissue in heat-sealed plastic bags, but this risks desiccation and oxidative damage over time (høyer et al., 1991). adding antioxidants to the preservative solution is another approach to mitigate oxidative damage (silvestre et al., 2021; sugiura et al., 2020). another parameter that containers affect is the amount of light exposure. minimizing exposure to light is often recommended in biospecimen storage (maki et al., 2021). uv radiation can cause photodamage to biomolecules via covalent bond formation, such as thymidine dimer formation in dna (kansagara et al., 2008). exposure to light can also cause pigment fading (monteiro, 2008). storing specimens in uv-protective containers or avoiding light exposure is recommended. overall, the container plays a crucial role in fluid preservation by regulating evaporation, oxidation, and light exposure.   fluid refreshing brain banking protocols often include steps to periodically refresh the preservative fluid. when using formaldehyde solutions, this has be used in an attempt to prevent a decline in ph (rahimi et al., 2006). for example, multiple sources report refreshing formaldehyde solutions every 2 years (rahimi et al., 2006; qiu et al., 2019). however, over time, the rate of biomolecular extraction decreases as the fluid preservative reaches equilibrium with the biospecimen (simmons, 2019). as a result, a key downside of fluid refreshing is that it will disrupt this equilibrium. additionally, fluid refreshing will lead to the loss of any small molecules that have leached out into the solution. this is why one source recommends that cocaine levels should be tested in the formalin solution, not just in the tissue (hilal et al., 2009). because different tissue types, containers, and fluid compositions will all lead to degradation at different rates, any fluid change schedule should be adapted to the needs and circumstances of the brain bank. more durable containers and fluids will need less frequent refreshing. the optimal schedule of fluid refreshing, if it is netbeneficial at all for expected future research applications, is unknown.   summary temperature significantly influences the rate of chemical reactions in fluid preservation, with lower temperatures slowing down these reactions. the type of storage container is crucial for preventing tissue damage due to evaporation and oxidation. glass and plastic are common materials, but their integrity and sealing are vital. periodic fluid refreshing is sometimes performed in brain banking, often aiming to minimize the accumulation of acidity. however, it can disrupt the brain tissue’s chemical equilibrium and may result in the loss of certain molecules from the solvent.   comparison to alternatives   cryopreservation cryopreservation uses subzero temperatures to decrease the rate of molecular motion and thereby improve long-term preservation outcomes. cryopreservation can be performed either without fixation or following fixation, thereby either avoiding entirely or substantially mitigating the problem of overfixation occurring in fluid preservation. consistent with this, data suggests that the imaging signal for proteins and nucleic acids is improved when comparing cryopreservation to long-term fixation protocols (kingsbury et al., 1999; hrabovszky et al., 2007). when used without fixation, cryopreservation also preserves more enzyme activity and allows for the use of many biomolecular profiling techniques that are more difficult to perform on fixed tissue (verhaert et al., 1990). however, cryopreservation introduces the issue of ice artifact, which can severely damage brain cell morphology (itoyama et al., 1980; vonsattel et al., 2008). ice artifacts can be mitigated or prevented by cryopreserving small tissue segments at a fast rate or by using cryoprotectants. indeed, fixed and cryoprotected tissue allows for high-quality morphologic preservation (sloane et al., 2000; van herp et al., 2005; estrada et al., 2017). after long-term storage at moderate subzero temperatures of approximately -20 °c, fixed and sufficiently cryoprotected tissue is expected to be in the fluid state, yet these protocols have shown good preservation of morphology and antigenicity for years (watson et al., 1986; otubo et al., 2021). however, cryoprotection in the absence of fixation is challenging, because cryoprotectant immersion requires a slow ramp-up to prevent osmotic damage, and perfusion quality is often limited in the postmortem setting (mcfadden et al., 2019). one source reports that even when the cryoprotectant immersion procedure was performed carefully, some freezing damage still occurred; however, the cryoprotectant solutions they used were likely not at the concentration required to vitrify (romijn et al., 1999). cryopreservation of large tissue blocks or the entire brain also poses the risk of thermal stress fracturing and physical aging (chang and baust, 1991; gangwar et al., 2022). perhaps most importantly, cryopreservation is substantially more expensive and less reliable than storage via fluid preservation (dwork et al., 1998; leboeuf et al., 2008).   paraffin embedding embedding specimens in paraffin wax was first introduced in the 1860s (van der lem et al., 2021). following the introduction of formaldehyde fixation in the 1890s, formaldehyde fixed paraffin embedded (ffpe) tissue blocks have grown to become one of the most common methods for preserving biospecimens. paraffin embedding involves multiple steps that can cause damage, including heating and dehydration. paraffin is made up of a mixture of straight chain alkanes. when the tissue cools, the paraffin crystallizes, allowing the tissue to be stored in the solid state (rhodes et al., 1927; zocher and machado, 1959). antigenicity preservation can be improved in ffpe by removal of residual water and storing tissue in low humidity environments (xie et al., 2011). ffpe tissue is very commonly used in archival settings, with demonstrated preservation of biological structures for decades (bradl and lassmann, 2012; rauch et al., 2018; guerra et al., 2020). numerous studies have shown that ffpe demonstrates better antigenicity than tissue stored long-term in formaldehyde solutions, consistent with its ability to prevent overfixation (sillevis smitt et al., 1993; dwork et al., 1998; lin et al., 2023). also, the localized areas of empty neuropil seen in the one study of brains stored in formalin that identified this artifact were not seen in matched ffpe samples from the same cohort (van duijn et al., 2011). however, there are drawbacks to paraffin embedding. first, the tissue processing required, especially dehydration, can lead to the loss of numerous biomolecules that are not sufficiently crosslinked, including structural lipids and amino acids (vos et al., 2019; dannhorn et al., 2022). while some lipid species are solvent-resistant, possibly due to direct formaldehyde cross-linking, the overall biochemical landscape is irreversibly altered by dehydration (denti et al., 2022). additionally, it is not yet possible to perform paraffin embedding on whole human brain samples en bloc. to our knowledge, the largest specimens that have been shown to be embedded in paraffin are less than 60 cm3, such as a macaque brain hemisphere (zhanmu et al., 2020). this means that cutting damage will occur during to dissection and there will be constraints on the size, type, and orientation of the tissue preserved (dwork et al., 1998). finally, paraffin embedding is more complex and expensive than simply allowing the brain to remain in fluid preservative for the long term.   resin embedding embedding resins such as epoxies and acrylates into brain tissue is commonly used as sample preparation for microscopy studies. as with paraffin embedding, this allows for storage in a solid state, which minimizes overfixation and chemical reactivity over time. however, also as with paraffin embedding, the extensive dehydration required for resin embedding often leads to loss of lipids and other biomolecules that are not directly crosslinked following fixation. the extent of dehydration required varies based on the resin, with some acrylates able to dissolve in up to 10-12 % of water by weight (newman and hobot, 1999). the extent of lipid extraction also depends on the resin. for example, the acrylate resin lowicryl hm20 extracts fewer lipids, because it is nonpolar (glauert and lewis, 1998). as they are used for electron microscopy, resin embedding clearly offers excellent morphological preservation. the stability of resin embedded specimens over time is not well established in the literature. there are some theoretical reasons to expect that preservation durations may not be unlimited in the very long term. for example, polymerized epoxy resins are found to act in some circumstances as viscous fluids, allowing for displacement of tissue elements over time (mollenhauer, 1993). however, most sources suggest that resin embedded tissue has good long-term storage longevity, especially when stored in a desiccator (glauert and lewis, 1998, p. 276; celis, 2006, p. 294; becker et al., 2014). resin embedding is often only practical on small tissue samples and the chemicals required can be relatively expensive, proprietary, and challenging to use properly, limiting their widespread use in brain banking.   summary compared to fluid preservation of brain tissue, cryopreservation offers better antigen preservation and biomolecular profiling capabilities, but faces the downsides of ice artifact and high costs. paraffin embedding is reliable for long-term storage and antigenicity but leads to the loss of a large subset of biomolecules. resin embedding is best for morphological preservation but is limited by sample volume and expense. most brain banks utilize a multifaceted strategy, storing some tissue samples with each of fluid preservation, cryopreservation, and paraffin embedding. this allows brain banks to capitalize on the unique advantages of each method and provide a versatile resource for a range of future experimental needs.   relationship with the post-mortem interval in addition to storage time, another key technical variable in brain banking is the postmortem interval, i.e. the amount of time between when death occurs and when the preservation procedure begins. there are several histologic artifacts known to manifest during the postmortem interval, such as pericellular rarefaction, vacuolization, and cell death due to oncotic necrosis (krassner et al., 2023). in general, these are distinct from the storage artifacts that we identified as potentially associated with fluid preservation. an important exception is decreased antigenicity on immunohistochemistry, which is a multifactorial process that can be caused by both decomposition associated with the postmortem interval and overfixation due to long periods of preservation in fixative-containing solutions. rapid fixation approaches such as perfusion fixation or ventricular injection may be able to help ameliorate damage associated with the postmortem interval, by minimizing any additional degradation that occurs prior to the fixative reaching that area of the tissue (mcfadden et al., 2019; mckenzie et al., 2022). however, we are not aware of any substantial evidence suggesting that different long-term fluid preservation options may interact with the postmortem interval in a similar way. notably, for many applications, the age of the fluid-preserved tissue, even after many years, is generally considered to be less impactful on research outcomes than the postmortem interval (wu et al., 2002; herbin et al., 2021).   implications for brain banking investigators operating brain banks and smaller brain collections face many constraints, including limited time and resources. the notion of the “shelf life” of brain tissue is important in brain banking (erslev, 2018). our review suggests that fluid-preserved brain tissue can be used in research applications for at least several decades, depending on the research goals. for antigen preservation, long-term immersion in formaldehyde solutions clearly results in overfixation and diminished antigenicity for immunohistochemical studies (liu et al., 2010). although contemporary antigen retrieval techniques can mitigate some of the effects of overfixation, they cannot always fully restore the original antigenic properties (ramos-vara and miller, 2014). therefore, recording and communicating the fixation time is crucial for researchers who will use the tissue. for morphological preservation, the existing literature presents limitations in drawing conclusions. as a result, it is still unclear whether brains stored for long periods of time in fluid preservative will be appropriate for many types of brain mapping study, and this will require further experimental research. most of the extant histology data is available for long-term storage in formaldehyde solutions. there is relatively less of a database to recommend the use of alternatives such as buffer storage or storage in cryoprotectants. there are also many unknowns in the field, such as the longevity of antimicrobial activity conferred by sodium azide in solution. as a result, there is currently no universally accepted “gold standard” method or performance metrics for fluid preservation in brain banking, with trade-offs among existing preservation methods (table 1). regardless of the preservation fluid used, maintaining a low temperature likely improves preservation outcomes. for example, morphological preservation has been reported to be intact after 92 years of storage in formalin at room temperature (herbin et al., 2021). based on an assumed q10 value of 2, this implies a refrigerator temperature storage length of approximately 4 times longer, or 368 years. if refrigeration is not feasible, a cool room with stable temperatures is recommended. finally, a sturdy container that reduces the risks of evaporation, desiccation, oxidative damage, and light exposure can further enhance tissue quality over the long-term. method upsides downsides evidence level aldehyde-based fluid preservation • simple and inexpensive • generally good reported morphologic preservation, although potential for artifacts • proteins and most other molecules are expected to be retained • overfixation causes antigen masking and chemical changes • acidity can damage tissue • extraction of a subset of lipids, small molecules, and other molecules that are not directly crosslinked moderate buffer-based fluid preservation • simple and inexpensive • mitigates overfixation • likely less acid formation over time • much less data for morphological preservation over decades of storage • microbial contamination without biocides low alcohol-based fluid preservation • widely used long-term fluid preservative agent • mitigates overfixation • antimicrobial effects • tissue and cell shrinkage • biomolecular extraction • white matter cavity formation • highly flammable and hazardous low fluid preservation after tissue clearing • allows imaging of intact specimens • minimizes lipid-associated oxidative damage • delipidation causes biomolecular information loss • novel methods without long-term track record minimal cryoprotectant-based fluid preservation • mitigates overfixation • limits biomolecule extraction • easy conversion to cryopreservation • chemical changes still occur in biomolecules • tissue penetration is slow • risk of osmotic damage low cryopreservation (sub-zero temperature) • best antigenicity preservation • allows for compatibility with molecular biology techniques used on fresh tissue • potential for function preservation • ice artifacts can occur in absence of complex vitrification protocols • thermal stress damage for large specimens • highest cost moderate paraffin embedding • avoids overfixation, excellent antigen preservation • excellent morphology preservation • minimal chemical reactions occur during storage • no protocols yet established for intact human brains • paraffin processing requires high temperature that can damage tissue • biomolecular extraction moderate resin embedding • allows for highest quality ultrastructural imaging possible • can retain more lipids if osmium post-fixation is used • minimal chemistry during storage • usually performed on tiny samples, not close to human brain scale • chemicals used tend to be expensive • biomolecular extraction low table 1. trade-offs among the methods discussed for brain banking. this table summarizes the key advantages, disadvantages, evidence level for each of the different methods discussed in this review that can be used for long-term brain banking and neuroanatomical preservation. in the "evidence level" column, "moderate", "low", and "minimal" describe the relative abundance of supporting scientific literature for each preservation method.   areas for further research one high-priority concern is better characterization of storage artifacts that could impact both short-term and long-term research objectives. critical among these are the areas of empty spaces in neuropil, whose prevalence and impact on neural circuit mapping are yet to be thoroughly examined (van duijn et al., 2011). it remains an open question whether the type of container used is a factor in accelerating or mediating this artifact. given that these areas of empty space in neuropil are associated with white discolorations observable at the macroscopic scale, initial investigations into this artifact could be conducted in different cohorts in a relatively straightforward manner. advanced imaging techniques like mri, along with correlative histological analyses, could offer another way to identify these artifacts and any related ones. another important area is to research why nuclei, nucleoli, and dna have been found to be vulnerable to alterations during storage in some studies but not others (cook et al., 2014; monteiro, 2008; herbin et al., 2021; berrino et al., 2022). the stability of other biomolecules, especially lipids, also requires further study. further research could identify which lipid species are extracted, modified, or cross-linked during storage, and what implications these changes have for research using banked brains. finally, research comparing the extent of storage artifacts when using different fluid preservation methods would be a valuable contribution to the field.   comparison to other reviews as far as we know, no previous review has solely focused on fluid preservation in brain banking, although several sources have discussed it as a part of a broader focus. one source performs a general review of storage of biospecimens in fluid preservatives (simmons, 2019). they have a thorough discussion of container choice and note that the storage environment is critical, including temperature control, humidity control, minimizing light and uv radiation exposure, minimizing vibrations, preventing evaporation, and preventing physical damage to the specimen. one review focuses on room temperature storage methods for biospecimens, emphasizing the space, cost, and environmental downsides of low temperature storage (lou et al., 2014). one review notes that for developmental neurotoxicity testing, nervous system specimens are recommended to be embedded in paraffin or resin for long-term storage, in order to avoid shrinkage artifacts potentially associated with prolonged storage in fixative (garman et al., 2016). one study searched pubmed and found six studies evaluating the effect of prolonged fixation on neural and glial cell marker antigenicity, covering a partially overlapping literature when compared with our review (wu et al., 2022). they found that there were clear timeand antigen-dependent effects of prolonged fixation.   strengths and limitations of this review one strength of this review is that we have included a large set of studies, allowing for findings from many years ago to be contextualized with current paradigms. also, we explored results using both biomolecular profiling and histology, highlighting the connections between these two linked fields. this review also has several limitations. first, we did not review in-depth the data from non-brain organs, even though the results for other organs are likely to be relevant to those from the brain. second, the included studies usually have qualitative assessments of morphologic preservation, not quantitative ones, which means that subtle shifts in neural circuitry during long-term storage might not be detected. on a related note, studies may be susceptible to sampling bias in the choice of brain tissue they profiled, which is more challenging to address when analyzing qualitative metrics of preservation. third, we did not perform quality ratings of individual studies, so it is more difficult to appropriately weight the findings of individual studies based on the quality of their evidence. however, with such a heterogenous, small field, it is not clear how to rate the quality of studies. finally, our search strategy was not exhaustive and likely did not identify all the relevant studies on this topic. however, we believe the studies we identified are likely representative of the broader literature.   conclusions archival brain tissue is a valuable resource, often providing well-characterized specimens that may be more useful for certain studies than freshly acquired samples (bradl and lassmann, 2012). formaldehyde is the most comprehensively studied preservative and has been found capable of maintaining the intricacies of neural circuitry, such as dendritic protrusions, even over extended storage durations. however, long-term storage in formaldehyde causes overfixation, making tissue difficult to process and hindering antigenicity. additionally, there appears to be extraction of some biomolecules that are not directly crosslinked, including a subset of lipids and small molecules. other artifacts have also been suggested to occur in formaldehyde solutions that may be specific to the method of storage or imaging employed. in contrast, alternative fluid preservation solutions, such as cryoprotectants and buffers with sodium azide, could avoid the pitfalls associated with storage in formaldehyde. simply using a lower concentration of formaldehyde for storage may also help to mitigate these problems. a lack of solid data on these alternative methods, however, means that they cannot yet be recommended as obviously superior. in short, the drawbacks of long-term formaldehyde storage are largely known but not yet fully quantified, and whether alternatives carry their own hidden issues remains an open question in need of further research.   abbreviations ba brodmann area, camkiia ca2+/calmodulin-dependent protein kinase ii a isoform, clarity clear lipid-exchanged acrylamide-hybridized rigid imaging/immunostaining compatible tissue hydrogel, cubic clear unobstructed brain/body imaging cocktails and computational analysis, dcx doublecortin, d day, dmso dimethyl sulfoxide, ffpe formaldehyde fixed paraffin embedded, gfap glial fibrillary acidic protein, ish in situ hybridization, mri magnetic resonance imaging, mirna micro rna, n sample size, nbf neutral buffered formalin, neun neuronal nuclear protein, psa-ncam polysialylated-neural cell adhesion molecule, psd95 postsynaptic density protein 95, q10 temperature coefficient, rameses realist and meta-narrative evidence syntheses evolving standards, sdisco stabilized 3d imaging of solvent cleared organs, shanel small-micelle-mediated human organ efficient clearing and labeling, smi312 a commercial antibody staining for neurofilaments from sternberger monoclonals incorporated, switch system-wide control of interaction time and kinetics of chemicals, vglut1 vesicular glutamate transporter 1, zo-1 zonula occludens-1.   author contributions a.m., o.n., k.f., and j.c. conceptualized the study. a.m. performed abstract and article screening. a.m. performed data extraction from the included studies and o.n. reviewed the data extracted from a subset of the studies. a.m. and o.n. performed grading of the storage artifacts. k.s. and e.t. performed imaging of the gross brain specimens and associated data analysis. a.m. wrote the initial draft of the manuscript. all authors reviewed the manuscript and approved the final manuscript.   acknowledgements we would like to thank louise van der weerd, andrew dwork, thomas beach, patrick hof, zhuhao wu, john smart, kenneth hayworth, martin baldan, trevor christensen, aschwin de wolf, aurelia song, anna lavergne, and borys wrobel for helpful personal communications regarding this topic. the icahn school of medicine at mount sinai provided access to library resources.   funding the study was supported by nih grants p30ag066514, rf1mh128969, r01ag054008, rf1ns095252, rf1ag060961, r01ns086736, r01ag062348, k01ag070326, and the rainwater charitable foundation. the funders had no role in the design of the study or in the collection or interpretation of the data.   conflict of interest andrew mckenzie is employed by oregon brain preservation, a non-profit brain preservation organization. the management of oregon brain preservation had no role in the design of the study or in the collection or interpretation of the data.   data availability the generated database of studies and extracted data is available in table format at https://github.com/andymckenzie/fluid_preservation this repository also contains the code required to reproduce the figure created in r.   supplementary material supplementary material 1 (download) rameses checklist (docx) supplementary material 2 (download) additional review methods (docx) supplementary material 3 (download) list of pubmed ids from initial literature search (txt) supplementary material 4 (download) articles selected after abstract screening (xml/zip) supplementary material 5 (download) articles selected after full text review (xml/zip) supplementary material 6 (download) information about the included studies 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which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. neuropathology in 1984: a deadly shot into the heart of europe feel free to add comments by clicking these icons on the sidebar free neuropathology 5:33 (2024) flashback neuropathology in 1984: a deadly shot into the heart of europe herbert budka division of neuropathology and neurochemistry, department of neurology, medical university vienna, vienna, austria corresponding author: herbert budka · division of neuropathology and neurochemistry · department of neurology · medical university vienna · akh wien 4j · währinger gürtel 18–20 · 1097 vienna · austria herbert.budka@meduniwien.ac.at submitted: 19 october 2024 accepted: 25 november 2024 copyedited by: georg haase published: 10 december 2024 https://doi.org/10.17879/freeneuropathology-2024-5976 keywords: border incident, fugitive victim, refugee killing, iron curtain, spinal cord injury, spinspinal cord transverse laesion, gunshot injury abstract just 40 years ago, europe was divided into the eastern communist bloc, which included the czechoslovak socialist republic (čssr) and was dominated by the now historical soviet union, and the western bloc comprising democracies such as austria. the iron curtain, a heavily guarded and deadly border zone, separated the two blocs and constrained, in prison style, the populations of the eastern bloc. the present neuropathological article relates the sad fate of františek faktor, a 33 years-old czech who was shot by čssr border guards when attempting to flee to austria at the border between česke velenice and gmünd. františek faktor was found dead on november 5th, 1984, on austrian soil some 500 meters from the border. čssr authorities claimed that he was shot when still within their territory, then ran some 900 meters to the other side of the border and died there. neuropathology demonstrated a gunshot injury of the spinal canal, with transverse lesioning of the spinal cord predominating at th10 that must have resulted in immediate paraplegia. this finding proved that čssr border guards had shot on austrian territory, resulting in a major diplomatic éclat between both countries. after the implosion of the communist governments of the eastern bloc in 1989, relations between czechia and austria started to normalise. by now, the gmünd/česke velenice region has developed an exemplary good local neighbourhood and the former border has become virtually irrelevant. attempts to bring justice have started in czechia as well as other countries behind the past iron curtain, and some former czechoslovak officials held responsible for border killings were legally prosecuted. the present article demonstrates how a small medico-scientific discipline such as neuropathology can contribute to assess critical political events in our world. "who controls the past controls the future: who controls the present controls the past." – george orwell (1903-1950), nineteen eighty-four (1984) [1] background imagine. imagine the world of 1984, yes, nineteen eighty-four. imagine not exactly the orwellian scenario of big brother, but a rather similar one; a world of two huge blocs bristling with weapons, which could pulverise our planet to an uninhabitable desert, a possibility continuing until present. indeed, after world war ii (ww ii), the communist east separated from the democratic west by construction of a heavily guarded and deadly border zone, the iron curtain, running through the very heart of europe from near the north cape to the black sea [2]. originally claimed by the east to be a military defence installation, the iron curtain soon served in prison style to contain its own population. this situation, 40 years ago, a blink of an eye in the history of our planet, may at present seem rather unimaginable to most younger europeans. science needs stories, and here is one that offers even more. imagine a person like orwell’s main character winston who wrestles with oppression in his dictatorial part of the world where he was unlucky to be born in. imagine no orwellian type of happy ending, where winston finally gets to love big brother and to transform into conformity. imagine our person here as a hero who voted with his feet against the ruthless totalitarian system in which he had to live, and fell as a victim of inhumanity in the midst of europe. imagine an early afternoon of late october 1984 in an idyllic landscape of forests with interspersed meadows and fields after the harvest in the northern part of lower austria [3]. this place is near to gmünd, an originally medieval town founded by the kuenringer knights, famed to have imprisoned richard the lionheart in dürnstein in the wachau danube river valley. the town of gmünd perfectly illustrates the historical developments in central europe, reflecting the turmoil europe suffered in the 19th and 20th centuries. by 1900, gmünd had become an industrial centre in the austrian part of the multi-ethnic habsburg austro-hungarian double monarchy, with some 10.000 inhabitants [4]. gmünd was located at the important emperor-francis-joseph-railway line in the midst between vienna and prague. this location favoured the construction of a huge refugee camp during ww i aimed to take care of masses of expelled and displaced persons from the war-ravaged parts of the habsburg monarchy, mostly from galicia (now western ukraine) and istria (now croatia). over the years, the camp housed a total of 200.000 persons, of which 30.000 were killed by measles, typhoid fever or typhus and buried at the still existing camp cemetery [5]. ww i left gmünd as a border town with a new frontier in its backyard, man-drawn by the peace treaty of st. germain, without the important railway station nor its surrounding backyard area nor its infrastructure that now belonged as české velenice across the river to the newly founded czechoslovak republic. franz kafka (1883-1924) documented the new border situation in his “letters to milena” (jesenská) whom he met in gmünd in 1920 [6]. after the annexation (“anschluss”) of austria as “ostmark” to the nazi-governed german reich in 1938, the local frontier was abolished and the backyard area added again to the gmünd municipality. about 400 inhabitants of gmünd then relocated to the former czechoslovak part [6]. in 1944, 1700 jews were imprisoned in a camp in gmünd, and one third of them did not survive. as railroad centre, gmünd suffered a devastating bombardment by air raid in march 1945, with 336 persons killed [7]. after ww ii, history repeated itself, the frontier returned to its post-ww i location, and gmünd became again the receptacle for the expelled and displaced [4]. thus gmünd and its transfluvial czech twin town české velenice were again separated, now by the iron curtain between the republic of austria and the czechoslovak socialist republic (československá socialistická republika, čssr), a member of the communist eastern bloc dominated by the former soviet union. imagine the inhabitants of české velenice during the communist era that is now called “totality” in czech. the inhabitants later on participated in a sociological study on the impact of the border situation including the topic of border guarding. the study concludes that “everybody adopted to the rule at one level or another, which implied, according to the circumstances, both compliance and resistance at the individual level.”; thus, české velenice encapsulates the basic features of communist rule in a particularly condensed way, with regard to both its ‘publicly available’ and its ‘hidden transcripts’ [8]. imagine wielands (velenice in czech), a small farming austrian village located at a few kilometres to the southwest of gmünd. as an exception, wielands' border to its north was not heavily fortified although permanently patrolled by čssr border guards. a fugitive from čssr to austria would have had to cross about 3 km of forest with some fences, to avoid touch signalling devices designed to alert the border guards, then to finally arrive at an open field already in austrian territory. living nearby, you would have known perfectly. yet, you would probably not have known nor wanted to know how close tragedy can be. the victim figure 1. františek faktor’s picture from his railways id card, available in articles by several austrian newspapers at the time, as well as on various internet sites, e.g. https://www.facebook.com/photo/?fbid=918904573600872&set=a.478515610973106, last accessed on nov. 14, 2024, https://www.pametnaroda.cz/sites/default/files/styles/930x520/public/1.%20frantisek%20faktor.jpeg?itok=qanf8mw5, last accessed on oct. 19, 2024 (with comment “zdroj: archiv m. petráčka”); https://www.extra.cz/zlociny-komunistu-frantiska-32-zastrelili-jako-posledniho-cloveka-na-hranicich-umiral-v-lese-dlouhe-hodiny-f59d0/galerie/5 (with comment “paměť národa, m. petráček, publikováno se souhlasem”), last accessed on oct. 19, 2024; as well as in the exhibition of the house of contemporary history in gmünd. imagine a 33-year-old mechanic from the city of české budějovice or budweis, world famous for budweiser budvar beer. františek faktor’s name and picture are in the public domain, showing a young man with a somewhat self-effacing facial expression (fig. 1). at that time, he lived in vyšné, a hamlet a mere one kilometre north of the border with austria, and worked at the economically important czech railways workshops in české velenice. not much more is known about his personal life, except that he allegedly boasted about being able to flee to austria, according to reports by people close to him. on nov. 5, 1984, františek faktor was found dead by a farmer from wielands on the edge of a field to a small forest. františek faktor was half-leaning against a big tree (fig. 2), and 16 empty bullet cartridge casings were found in the field nearby. importantly, this occurred on austrian soil at a distance of some 500m from the border, as documented by austrian authorities [9], and recently also by photographic imaging of the site (figs. 2–4). the detailed location on a map is given in fig. 5. františek faktor had suffered a bullet wound in the back that hit his vertebral column and was apparently already dead for a few days. he had an id card from czech railways, a piece of soap and some money in his pockets. another farmer from wielands and his son later reported to austrian police that in the early afternoon of oct. 30 they had heard several rounds of gunshots, something that was not unheard for people living there. they further reported that they had seen čssr border guards running on the free field from austria back towards their country. figure 2. a. edge of a grove where františek faktor’s body was found in half-sitting position, leaning against the trunk of the large spruce at right. photograph by h. budka (2024). b. a memorial plate that had been fixed to the trunk was recently removed by unidentified persons. its picture is still available on the web https://www.extra.cz/zlociny-komunistu-frantiska-32-zastrelili-jako-posledniho-cloveka-na-hranicich-umiral-v-lese-dlouhe-hodiny-f59d0/galerie/4. immediately, the issue emerged whether františek faktor was shot on čssr territory but still able to run or drag himself to austria, or whether he was killed on austrian soil. austrian newspapers and tabloids were full of discrepant data and speculations. of course, shooting him in austria would have constituted a severe violation of austrian sovereignty, with potential bilateral and international legal consequences, as recognised by international newspapers [10]. austria had traditionally been a neutral country between the two blocs but imagine the consequences of the shooting violating the border of a european nato member or the us. figure 3. view from the trees depicted in fig. 2 towards the čssr, showing a field of some 500 m distance to another forest where the border is situated. a tiny white spot represents a border warning sign. photograph by h. budka (2024). figure 4. view from a few metres from the čssr border in direction to austria, the same sight that františek faktor perceived when crossing the border. the field in the foreground is already austrian territory. the arrow indicates the spruce where his body was found, depicted in fig. 2. photograph by h. budka (2024). figure 5. map of the region with a mark showing where františek faktor’s body was found north-west of wielands (48°45'47.9"n 14°54'42.3"e, shown by apple maps in satellite view). the red line represents the frontier. not surprisingly, czechoslovak authorities immediately argued for an incident on their own soil, but emerging data soon showed the contrary. at this moment, neuropathology joined the tragic theatre. a complete legal autopsy was ordered by the gmünd district court and performed at the department of legal medicine of the university of vienna by the very experienced forensic pathologist georg bauer, then a docent/lecturer and later on a professor. georg bauer documented a completely penetrating gunshot wound of the lower thoracic vertebral column and sent, on nov. 6, 1984, a fixed specimen in toto to me for neuropathologic assessment of the spinal cord. neuropathology the fixed specimen comprised thoracic vertebral bodies 8 to 11 with proximal parts of ribs and surrounding soft tissues in toto. the vertebral arcs had been removed at the general autopsy, hence the spinal canal was open from the posterior. i removed the spinal cord from the spinal canal. the spinal cord had a length of 12,5 cm, ranging from 1 cm cranially of the dural exit of root th 7 to 1 cm caudally of root exit th 11. the essential finding was an elliptic dural defect of 6 to 8 mm in diameter on the left side, with slightly ragged margins and a small subdural haemorrhage located between dural exits of dorsal roots 9 and 10, i.e. at the level of vertebral body th 10 (fig. 6). the adjacent surface of the cord lacked the pia mater and was irregularly humped and softened in a diameter of 1 cm. at this level, most of the dorsal root th 10 was missing, while more proximal parts were present but appeared retracted and flexuous. the anterior root and spinal ganglion of th 10 were preserved. transverse sectioning of the spinal cord revealed hemorrhagic and softened lesions between the levels of dural root exits th 8 and 11, with the maximum of haemorrhagic lesions at the level of the round dural defect (fig. 7). figure 6. anterior (at left) and posterior (at right) view of spinal contents dissected from the vertebral column specimen. the large arrow points to the dural hole by the bullet. the arrowhead at left points towards the intact anterior root th 10. at right, the small arrow indicates the intact anterior root th 10. the cranial and caudal parts of the posterior root th 10, shot into two pieces, are indicated by arrowheads. figure 7. transverse sections of the spinal cord at six levels as indicated between dural root exits th8/9 and th11. there are multiple and extended haemorrhages and softenings. the largest haemorrhage is present at th10, at the level of the dural defect (upper right). photograph by h. budka (1984). histology confirmed extensive spinal cord haemorrhages, oedema and necroses, comprising at maximum both the gray matter and large parts of the white matter (fig. 8). there was focal early leukodiapedesis with some perivascular granulocytes and a few round cells. the proximal and distal stumps of the dorsal root th 10 showed some axonal swelling and early myelin destruction, as well as tiny fragments of textile fibres and bone. given the political importance of, and public interest in, the case, i hastened to complete my investigation and sent my complete neuropathology report to the investigating court of the city of krems-on-danube 10 days after františek faktor’s body had been found. figure 8. histological transverse sections of the spinal cord at the level of dural exit th 9 (upper row), at the level of the dural defect (middle row), and caudally adjacent to the preceding level (lower row). there are multiple and extended haemorrhages, oedema and softenings. left side haematoxylin & eosin, right side luxol fast blue & nuclear fast red. original magnification x 1,6. interpretation the neuropathological results fully concorded with a gunshot lesion of the spinal canal: a dural hole by the bullet, grazing of the left and dorsal cord surface and extensive transverse damage of the spinal cord with haemorrhages at the dural root exit levels th 9 and 10. such a spinal cord lesion appeared compatible with secondary effects of the bullet due to temporary cavity and possibly also due to damage from the initial shock wave [11]. type and extent of the transverse spinal cord lesion must have resulted immediately in complete yet non-fatal paraplegia. the early tissue reactions demonstrated limited survival after the shooting, estimated between 12 to 48 hours. follow-up of this case led to the inhumane and hard-to-swallow conclusion that the involved čssr border guards did not take care of the critically wounded františek faktor after the shooting and abandoned him to suffer a terrible death after some day-long most painful agony. political sequels after retrieval of františek faktor’s body and the nearby bullet cartridge casings on austrian territory, the austrian side urgently asked for an official meeting of the ‘austrian-czechoslovakian commission for investigation of incidents on the shared national border’. the meeting was held on 7th november 1984 in gmünd and its protocol is available in czech language on the internet [9]. the čssr delegation had eight members, the austrian seven, involving on both sides mostly administrators from the ministries of interior, external affairs, and defence, with georg bauer as austrian forensic medicine expert. for the first time in these commission meetings, no consensus was achieved on what had happened. the čssr side maintained that františek faktor was already shot on čssr territory, some 400 m from the border despite the fact that no bullet cartridges were found on that site. the austrian side countered that františek faktor, critically wounded by gunshot, must have been unable to run 400 m to the austrian border and farther on across the field, in total some 900 m. the austrian side further countered that the bullet cartridges found in the field, the bullet stuck in a tree trunk and the position of the body were all by far within austria, arguing for the austrian scenery of the shooting. the neuropathological report with the final proof for the austrian version was provided only after the border commission meeting. between ww ii and the implosion of the soviet bloc in 1989, františek faktor’s shooting was not the first or unique incident, neither on the čssr border nor on other borders of the iron curtain. what singled out this incident as special was the proven fact that františek faktor was not killed on the territory of his own east bloc country but on the territory of a foreign state after a successful flight across the border. imagine a neutral country like austria where political implications were considerable, causing a sort of diplomatic mini-ice crisis with the čssr. the čssr ambassador to vienna was called in to receive a harsh protest. čssr authorities claimed an "anti-czechoslovak hate campaign" raging in austria after the shooting and recalled their ambassador back to prague [10]. the austrian minister of external affairs gave a formal statement to parliament about this “most severe violation of the austrian-čssr border since years”. a general debate about murder by čssr border guards and violation of the helsinki treaty of 1975 ensued. finally, the members of the austrian parliament unanimously accepted a declaration that condemned the violation of austrian sovereignty and asked for full clarification and punishment of the persons involved [12]. there is no evidence that čssr authorities complied with any of these demands. imagine the autopsy with neuropathology examination would not have been done. there would have been rumours instead of proof, and questions whether čssr border guards would appease their merciless behaviour. in retrospect however, the behaviour of the čssr border guards seemed to have changed since františek faktor was the last person killed at the čssr-austrian border [13]. final justice and relief imagine a post-1984 world that would be able to persecute all types of crime, including those claimed as “necessary” for governmental or state interests, irrespective of political influence, ideology, religion, ethnicity or nationality. indeed, the international court of justice at the hague was founded by the united nations to take care of such a transnational ideal, but it rules only between states. by contrast, the international criminal court (icc), also seated at the hague, has the mandate to prosecute individuals for international crimes of genocide, crimes against humanity, war crimes and crimes of aggression. surprisingly however, the icc has been and continues to be under heavy fire from many sides, including political movements, civil groups, states and governments. systemic border killings on foreign territory as in františek faktor’s case would align with the mandate of the icc on crime of aggression by “invasion or attack by armed forces against territory”. yet, only crimes committed after 2002 can be persecuted by the icc [14]. despite this, some national governments that succeeded communist regimes and some national civil societies of central european countries brought up the historical issue of justice for the border shootings at their iron curtain. a culture of remembrance has grown, historians have documented the evidence, and court proceedings have started in 2023. of note, criminal prosecutions on the border killing of františek faktor and other persons were conducted against vratislav vajnar, the former minister of the interior of the čssr between 1983 and 1988 [15,16], and against jan muzikář, the former colonel and chief-of-staff of the čssr border guards [17]. the minister, aged 92 years, did not attend the court proceedings and died one day after their opening [17]. the colonel, aged 90 years, claimed to be not guilty on the first day and announced not to attend further court proceedings [18]. this illustrates that the age and health state of these and other potential defendants make the outcome of such criminal prosecutions a race against time. it is therefore essential for democracies to do their best to rapidly clean up the evil past. in this respect, remarkable recent news is the first conviction in berlin of a former stasi officer of east germany (german democratic republic, "ddr"), martin manfred naumann, now 80 years old, who shot a polish fugitive at the former intra-german border 50 years ago [19]. imagine finally human beings who have learned from history – in contrast to a well-known negative phrase. learning from history is evident in the gmünd-české velenice region since 1989, particularly since both nations joined the european union (eu). the former border has become virtually irrelevant and in practice non-existing. austrians use to shop in czechia, and czechs walk across the bridge to visit and enjoy local austrian wine and company. in gmünd, an exemplary house of contemporary history tries to remind visitors of their shared tumultuous past – never to forget [20]. another example is the iron curtain trail, the european bicycle route of the year 2021 between gmünd and bratislava, allowing to savour new life and nature in the once deadly border zone [14]. for the present and future, there is another exemplary enterprise, the first binational health centre in the eu at the very border called healthacross med gmünd which admits indiscriminately both czech and austrian patients in a best practice region recognized by who [21]. indeed, all this has become a very long shot from the one in 1984. imagine – would it have been imaginable 40 years ago? and at the end, please "...imagine there's no countries it isn't hard to do nothing to kill or die for and no religion, too...."  – john lennon (1940-1980, imagine [22]) acknowledgements prof. georg bauer, centre for forensic medicine, medical university vienna, performed the general autopsy of deceased františek faktor and made the forensic medicine report in 1984. yet, he chose not to contribute to this article. the author is greatly indebted to mr. harald winkler of the city of gmünd direction and principal of the house of contemporary history of gmünd, for generously helping with abundant local information including the exact location on the map, and to two local contemporary witnesses in wielands, mr. rupert kitzler, the owner of the field and grove where františek faktor died, and mr. franz zeller, for local guiding during a fact-finding visit on oct. 2, 2024 (fig. 9). mr. kitzler and mr. zeller committed themselves to arrange for re-installing a memorial plate at the site of františek faktor’s death. ms. carmen haider and susanne schmid deserve credit for helping with archival work, and dr. sigrid klotz with microphotographs, all from of the division of neuropathology and neurochemistry, department of neurology, medical university vienna. special thanks are due to my wife ivana for translating text passages from documents in czech, and enduring my absorption with history-related matters. funding statement no funding was received for the present work. conflict of interest statement no conflict of interests is to be stated. the author is associate editor of this journal. figure 9. photograph of contributors to a recent fact-finding visit on oct. 2, 2024 to the area where františek faktor died. from left: herbert budka, rupert kitzler, franz zeller and harald winkler. photograph by ivana budka (2024). references 1. orwell g (1949) nineteen eighty-four, part i, chapter iii (“party slogan”). london: martin secker & warburg ltd. 2. iron curtain. in: wikipedia, the free encyclopedia. https://en.wikipedia.org/wiki/iron_curtain, last accessed on nov. 16, 2024 3. the upper waldviertel. https://www.waldviertel.at/en/upper-waldviertel, last accessed on nov. 16, 2024 4. geschichte, gmünd (history of gmünd) https://www.gmuend.at/de/events/kultur/freizeit/geschichte, last accessed on nov. 16, 2024 5. gmünd-neustadt, ehemaliges flüchtlingslager (former refugee camp). https://www.gmuend.at/de/gmuend-neustadt_ehemaliges_fluechtlingslager_, last accessed on nov. 16, 2024 6. gmünd (niederösterreich). wikipedia, the free encyclopedia, 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banking: methods of assessment and relationship to the postmortem interval feel free to add comments by clicking these icons on the sidebar free neuropathology 6:20 (2025) original paper mechanical perfusion in brain banking: methods of assessment and relationship to the postmortem interval macy garrood1*, alicia keberle1*, gabriel a. taylor1, emma l. thorn2,3, claudia de sanctis2,3, kurt farrell2,3, john f. crary2,3, jordan s. sparks1, andrew t. mckenzie1 apex neuroscience, salem, oregon, usa friedman brain institute, departments of pathology, neuroscience, and artificial intelligence & human health, icahn school of medicine at mount sinai, new york, new york, usa neuropathology brain bank & research core and ronald m. loeb center for alzheimer's disease, icahn school of medicine at mount sinai, new york, new york, usa * these authors contributed equally to this manuscript corresponding author: andrew t. mckenzie · apex neuroscience · 3265 marietta st se · salem · or 97317 · usa amckenzie@apexneuro.org submitted: 07 august 2025 accepted: 04 october 2025 copyedited by: georg haase published: 21 october 2025 https://doi.org/10.17879/freeneuropathology-2025-8880 keywords: brain banking, mechanical perfusion, perfusion fixation, preservation quality, no-reflow phenomenon abstract the mechanical perfusion of solutions through the cerebrovascular system is critical for several types of postmortem research. however, achieving consistently high-quality perfusion in this setting is challenging. several previous studies have reported that longer postmortem intervals are associated with decreased perfusion quality, but the mechanisms and temporal progression of perfusion impairment are poorly understood. in this study, we describe our experience in developing a protocol for in situ perfusion of the postmortem brain in human whole-body donors (n = 77). through the evaluation of different approaches, we found that cannulation of the internal carotid arteries combined with clamping of the vertebral arteries allows targeted perfusion of the brain. we evaluated perfusion quality through three complementary methods: gross anatomical appearance, ct imaging, and histology. these quality assessment measures were partially correlated across donors, indicating that they offer complementary perspectives on perfusion quality. correlational analysis of our cohort of banked brains confirms that perfusion quality decreases as the postmortem interval increases, with a heterogenous pattern across brain regions. our findings provide data for optimizing brain banking protocols and suggest future directions for investigating the mechanisms of postmortem perfusion impairment. abbreviations aca anterior cerebral artery, bbb blood-brain barrier, cb cerebellum, csf cerebrospinal fluid, ct computed tomography, fctx frontal cortex, h&e hematoxylin and eosin, icc intra-class correlation, ijv internal jugular vein, mca middle cerebral artery, nbf neutral buffered formalin, octx occipital cortex, pca posterior cerebral artery, pmi postmortem interval, tctx temporal cortex, thal thalamus, twm temporal white matter, wsi whole slide image. introduction in postmortem brain research, mechanical perfusion i.e. the delivery of solutions through blood vessels using external pressure from pumps, gravity systems, or syringes rather than the natural heartbeat has proven valuable for several specialized research applications. these include the delivery of fixatives for tissue preservation (brown, 1939; mcfadden et al., 2019), latex or other compounds for vascular mapping (duvernoy et al., 1981; alvernia et al., 2010), radio-opaque substances for angiography (turkoglu et al., 2014), and detergents or other chemicals for brain clearing (zhao et al., 2020). mechanical perfusion can be performed with the brain both in situ (inside of the skull) and ex situ (removed from the skull). it can be an effective way to deliver chemicals throughout large organs such as the brain because it utilizes the existing vascular network to distribute substances widely. however, even in ideal, controlled laboratory animal settings, mechanical perfusion remains a challenging problem, often leading to heterogenous perfusion across the brain (bodian, 1937; cahill et al., 2012; schwarzmaier et al., 2022). it is even more challenging to achieve consistently high-quality perfusion in the postmortem human brain, because of the diverse physiological changes that occur in the agonal and postmortem periods. we define perfusion impairment as the observed inability to mechanically perfuse tissue as typically occurred during life. perfusion impairment shares many similarities with the clinical phenomenon of "no-reflow", wherein there is a failure of microvascular perfusion after a period of ischemia, despite restoration of arterial flow (kaul et al., 2022). while no-reflow has been extensively studied in the context of reperfusion after stroke and myocardial infarction, the phenomenon of perfusion impairment in the postmortem brain is not yet well understood. numerous studies have reported that the ability to drive solutions through the brain’s blood vessels decreases with longer intervals between the cessation of blood flow and the initiation of perfusion (mcfadden et al., 2019; mignucci-jiménez et al., 2024). that perfusion of the postmortem body in general is impaired by a longer period of ischemia has been known for centuries. for example, in the 18th century, william hunter recommended initiating perfusion with preservative solutions for anatomical studies within eight hours after death in the summer and within 24 hours after death in the winter (bryant, 2003, p. 537). studies on the brain in particular have reported that perfusion quality begins to deteriorate just 5 minutes after death (bodian, 1936; karlsson and schultz, 1966). however, the precise rates of this decline are unclear, especially for human brain specimens (mcfadden et al., 2019). the progression of perfusion impairment can also be heterogeneous across brain regions. for example, one study found that after 10–15 minutes of ischemia, perfusion defects in the rabbit brain developed in a patchy distribution, with some areas showing complete perfusion failure while adjacent regions remained perfusable (ames et al., 1968). the extent to which this heterogeneity of perfusion impairment is haphazard or regionally patterned is unclear, with some previous research finding a tendency for perfusion defects in certain regions (ames et al., 1968). the regional heterogeneity could depend on multiple factors, including the relative degree of blood flow in those regions during life, the patterns of cerebrovascular pathology that developed prior to death, the route of mechanical perfusion, differences in postmortem edema, postmortem lividity, or gravitational effects (du et al., 2022; frigon et al., 2024). assessing the degree to which the perfused fluid actually traverses through the blood vessels throughout the brain – i.e., the quality of perfusion – also presents challenges. investigators have approached the quality assessment problem in several ways. at the macroscopic level, perfusion quality can be evaluated on the basis of tissue color, firmness, swelling or shrinkage, consistency during cutting, and the clearance of blood from surface vessels (jenkins et al., 1979; mcfadden et al., 2019; monroy-gómez et al., 2020). however, reliance on surface appearance alone can be misleading, as it may not reflect successful perfusion of deeper brain regions. it has been reported that major vessels may be perfused but while the perfusate still fails to penetrate into the small vessels or most of the tissue itself (palay et al., 1962). assessment methods during the perfusion procedure include monitoring flow rates, venous return characteristics, changes in perfusion pressure, and ocular tension (donckaster and politoff, 1963; schwarzmaier et al., 2022). histological evaluation is generally considered the gold standard, but is resource-intensive when applied to large tissue areas. specific histological metrics include the presence of red blood cells in vessels, vessel dilation, and perivascular space morphology (scharrer, 1938; palay et al., 1962; wohlsein et al., 2013). since slower preservation leads to more postmortem changes, the extent of autolysis can serve as an indirect proxy for perfusion quality (frigon et al., 2022). finally, some studies have added chemicals to the perfusate to aid in visualizing perfusion quality. for example, garcia et al., 1977 used colloidal carbon as an electron-dense tracer, to directly visualize capillary perfusion via electron microscopy (garcia et al., 1977). neuroimaging approaches have also enabled non-destructive, three-dimensional assessment of perfusate distribution (böhm, 1983). despite this wide array of quality assessment methods, direct comparisons between them remain limited – particularly in the context of human brain banking, where imperfect perfusion is expected. although the mechanisms of postmortem perfusion impairment are not yet fully understood, insights can be drawn from adjacent fields. mechanistic factors that contribute to perfusion impairment after temporary cerebrovascular ischemia include blood coagulation, vessel constriction, pericyte contraction, and cellular edema (kaul et al., 2022). an important early source of parenchymal fluid accumulation after ischemia appears to be backflow from the cerebrospinal fluid (csf). csf begins to enter brain tissue within two to three minutes after circulatory arrest, with peak influx around 7 minutes (du et al., 2022). this influx may be associated with pressure gradients created by the loss of blood pressure and coincides with the onset of anoxic spreading depolarization (weed, 1914; du et al., 2022). the early postmortem accumulation of fluid tends to occur in astrocyte processes, which become swollen, including around blood vessels and certain neurons (krassner et al., 2023). these swollen astrocyte processes can externally compress capillaries, contributing to perfusion impairment and possibly helping to explain why perfusion failure develops more rapidly in the brain than in other tissues (majno et al., 1967). finally, blood-brain barrier (bbb) alterations during ischemia also contribute to perfusion impairment. one study found that bbb disruption, as indicated by extravasation, occurs rapidly during reperfusion following 30 minutes of global cerebral ischemia in mice (ju et al., 2018). notably, in this study, venules and capillaries were found to have the most leakage, with the leaky vessels were predominantly venules (48 %) and capillaries (25 %). together, these mechanisms appear to act synergistically to progressively impair perfusion in the postmortem brain. while the postmortem interval (pmi) is obviously a key factor affecting perfusion quality, several other donor characteristics can also significantly influence perfusion success as well. the agonal state – i.e. the physiological condition preceding death – appears to be particularly important. prolonged agonal states can cause blood vessel inflammation, thrombus formation, and bbb disruption (perry et al., 1982; hardy et al., 1985; hansma et al., 2015). as a result, donors with extended periods of hypoperfusion, hypoxia, or multi-organ failure may have more severe perfusion impairment for a given pmi compared to those who die suddenly. advanced age and various disease processes can also affect perfusion quality through mechanisms such as atherosclerosis and altered vascular compliance, independently of the pmi. donor use of anticoagulant medications such as heparin or apixaban prior to death is likely to decrease the extent of thrombus formation during the agonal phase and postmortem period. environmental factors such as ambient temperature and humidity during the pmi likely also play a large moderating effect on the degree of perfusion impairment. the complex interplay between these factors – agonal state, age, disease history, medications, and environmental circumstances during the pmi – likely contributes to substantial variability in postmortem brain perfusion beyond the pmi alone. in this study, we describe our methods for performing mechanical perfusion fixation of the postmortem brain and for assessing perfusion quality. after testing multiple methods for perfusion, we adopted an in situ approach on the isolated cephalon, involving cannulation of the internal carotid artery and clamping of the vertebral arteries. we evaluated perfusion quality using multiple modalities, including gross examination, neuroimaging, and histology. in our experience, perfusion fixation is of variable efficacy, with heterogeneity observed both across donor brains and across areas of the same brain. because of our limited sample size and lack of systematic comparisons, we do not claim that our method for postmortem perfusion is superior to others. instead, we provide data and practical insights that will help other investigators in optimizing their own methods of mechanical perfusion, whether for fixation or other purposes. methods whole body donation procedures anatomical whole-body donations were performed by a partner whole body donation organization operating under oregon health authority regulations. all donations were processed after obtaining informed consent. the apex neuroscience brain and tissue bank operates under an exemption determination issued by the pearl institutional review board (irb) after submission of our protocols for review (pearl irb id #2023-0260). the cohort was a convenience sample of whole-body donors with no specific exclusion criteria, other than confirmed or suspected transmissible disease such as hiv, hepatitis b/c, or prion disease. all cases where perfusion was performed were included in this data set, provided that data from at least one modality (gross examination, ct scan, or histology) were available to assess the quality of perfusion. perfusion procedure in our initial experiments, we used an anterior cervical approach with cannulation of the common carotid arteries or internal carotid arteries. subsequently, the method of perfusion was adapted for use on isolated cephalon specimens, following neck dissection at the level of c4–c5. the cephalon was secured in a container filled with ice, aiming to cool the brain during the perfusion. the internal carotids were identified superior to the carotid bifurcation as the more posterior of the two major arteries (the more anterior vessel being the external carotid). after a brief dissection with scalpel and forceps to isolate approximately 2 cm of the vessel from surrounding tissue, 10g or 12g dispensing needles, used as cannulae, were inserted into the internal carotid arteries and secured via zip ties (table 1). once both carotid cannulae were in place, the perfusion was initiated. occasionally one of the zip ties was not tied securely tightly enough, in which case the perfusion was momentarily stopped, and the cannulation of that artery was redone prior to restarting perfusion. table 1: supplies and reagents for perfusion equipment / chemical supplier product identifier masterflex l/s economy drive cole-parmer 7518-10 masterflex easy-load pump head cole-parmer 7518-10 silicone tubing, 1/4" inner diameter novosci sdb08 straight barbed connector, 1/4" inner diameter vwr mflx40614-43 pump roller tubing, c-flex vwr mflx06424-24 y-connector, 1/4" inner diameter cole-parmer mflx30726-05 male luer lock barb for 1/4" id tubing qosina 11567 pendotech pressuremat sensor monitor biopharm world pmat-s pressure monitor pendotech pmat4a polycarbonate carboy nalgene 2322-0020pk neutral buffered formalin azer scientific nbf55g mannitol lab alley manpu-1lb omnipaque™ 300 mgi/ml (iohexol) med-plus 0407-1413-63 green colored dye kroger 0001111002073 dispensing needle, 10g or 12g labaider b0b5d28cbm dispensing needle, 14g bbtcty b07dzc225b zip tie have me td b08tvlyb3q zip tie gun rv rhodes b0938pvfm9 plastic tubing clamp bel-art f18228-0000 plastic syringe, 140 ml cardinal health 8881114055 plastic syringe, 200 ml a akraf b0cjjdqhph once the perfusion through the carotid arteries had begun, the bilateral vertebral arteries were usually clamped with hemostats to prevent outflow through them. however, in cases where no or only minimal flow was visualized returning through the vertebral arteries, the latter were not clamped, as this was not deemed necessary. occasionally the external carotid arteries were also found to have flow of perfusate through them, most likely due to collateral circulation pathways. in that case, the leaking external carotid arteries were clamped with hemostats. in a small minority of cases (n = 5), the vertebral arteries were also cannulated and perfused through. in these cases, the vertebral arteries were cannulated with 14g dispensing needles functioning as catheters. the same perfusate solution and a separate perfusion circuit with the same design were used for the vertebral artery perfusion. perfusion pressure was applied using a peristaltic pump system or by manual syringe injection. for pump-driven perfusion, we used an open circuit setup (figure 1). for the peristaltic pump, we primarily used the masterflex l/s economy drive (cole-parmer, model #7554-80) with the masterflex easy-load pump head (cole-parmer, 7518-10). for syringe-based perfusion, we manually injected fixative into the internal carotid arteries in a controlled, slow manner. we used multiple syringes, which were sized 140 ml, 200 ml, or both, and which were pre-filled with perfusate prior to the procedure. figure 1. diagram of one version of the perfusion circuit used. batches of perfusate were made in polycarbonate carboys (thermo scientific, 2322-0020pk). the base solution was 10 % neutral buffered formalin (nbf; azer scientific, nbf55g). in some cases, we added mannitol (lab alley, manpu-1lb), primarily at a concentration of 10 % (v/v), although in a minority of cases 20 % (v/v) mannitol was used. for contrast-enhanced ct imaging studies, we used iohexol (omnipaque™) at a concentration of 3 mgi/ml, although in a small minority of cases a concentration of 6 mgi/ml was used. we also added colored dye, typically green, to be able to better visualize the color changes of brain tissue on gross examination. although in initial experiments the carboys were sometimes stored at room temperature prior to use, we later switched to storing the carboys at 4 °c until just before use, so that the solution was cold during perfusion. this was done to aid in more rapidly cooling the brain, and because we expected that cold perfusate might help to decrease tissue edema during perfusion. for the cases with peristaltic pump-driven perfusion, which provides a constant flow, pressure was monitored using an inline pressure sensor previously connected to the perfusion circuit. prior to cannulation, the circuit was primed by rapidly distributing fluid through all of the tubing to completely remove of visible air bubbles. we typically began with flow rates of approximately 16 ml/min (corresponding to a speed of 0.5 on the pump, with negligible differences when using a cannula size of 10g or 12g), and gradually increased the flow rate to 38 ml/min (speed of 1), 94 ml/min (speed of 2), or 147 ml/min (speed of 3), provided that the pressure was not too high and no spinal cord swelling or herniation were observed. it should be noted that these flow rates are relative to operating conditions such as the state of the tubing, air bubbles, or temperature. in initial experiments, the target perfusion pressure was between 50 to 100 mm hg (0.97 to 1.93 psi), although this was a loose guide. the primary factors used for adjusting the speed and deciding whether to stop perfusion were (a) the absence of rising pressure over time, which would indicate progressive resistance to perfusion and potential edema, (b) the absence of visible spinal cord bulging or herniation, and (c) complete clearance of blood from the draining effluent. occasionally, especially in earlier cases, the pressure sensor malfunctioned most likely due to water contamination introduced during cleaning – leading to erroneously high-pressure readings. when such malfunction was suspected, the recorded pressure data were excluded from the analysis. venous return during perfusion was monitored through the internal jugular veins (ijvs) and within the spinal canal. in most cases, a gradual transition in the color of return fluid from dark red to clear was observed. however, we noticed that this rapid clearing of the return fluid could also occur when perfusion was limited to a small area of the brain, rather than being widespread. therefore, from a venous return perspective, the best sign for widespread perfusion across the brain was a slow, progressive transition of the return fluid from blood-colored to clear over the course of the procedure. in addition to the variations in the approach described above, we also tried (a) different cannula sizes for the internal carotid arteries (10g or 12g) and (b) alternating perfusate delivery through a single carotid at a time by temporarily clamping the contralateral line. qualitatively, neither of variation produced substantial differences in perfusion outcome, and hence were not considered in subsequent analyses. ct images we aimed to perform neuroimaging both before and after perfusion, with the brain remaining inside of the skull. the ct scanner we used was the omnitom® elite (neurologica, danvers, ma), a 16-slice scanner. images were viewed with the osimis web viewer. occasionally, one or both of these ct scans was not performed, for example if the ct scanner was not working at the time. for each of eight major vascular territories – corresponding to the territory supplied by the left and right anterior cerebral artery (aca), middle cerebral artery (mca), and posterior cerebral artery (pca), as well as the cerebellum, we graded the extent of contrast visualized on the post-perfusion ct scans in a given territory on a 0–3 scale, with 0 indicating < 5 % extent of perfusion, 1 indicating 5–50 %, 2 indicating 50–95 %, and 3 indicating > 95 %. in some cases, the grades could not be completed for some of the areas in the brain because the available ct scan contained only part of the brain, not the whole brain. the extent of air bubbles in the ct scan across the brain was also graded on a 0–3 scale, with 0 indicating no apparent air bubbles, 1 minimal, 2 moderate, and 3 substantial. this grading was performed on all of the post-perfusion ct scans available via the collaboration of two graders. for a brain-wide assessment measure, we generated a separate score based on the sum of the grades across all evaluated areas, excluding cases with missing data from any vascular territory. brain extraction procedure following perfusion, we performed brain extraction following standard autopsy procedures (adams and murray, 1982), with slight modifications aimed at minimizing tissue damage during removal. we used an oscillating saw to create a circumferential skull cut extending below the occipital protuberance to have better access to the posterior cranial fossa (felle et al., 1995; hlavac et al., 2018). in addition, we performed a median sagittal cut to aid in decreasing trauma during removal (robertson et al., 2024). after carefully cracking the inner table using an osteotome and mallet, we removed the calvaria, incised the dura along the saw lines, and transected the falx cerebri and tentorium. the brain was then gently extracted by sequentially transecting the connecting cranial nerves and arteries. when the brain was well perfused, it became firmer, which appeared to aid in mitigating damage during removal. gross examination following extraction, digital photographs of the brain were taken from multiple angles prior to immersion fixation, to avoid fixation-induced alterations in tissue color. after the photographs were taken, the brains were completely immersed in a plastic container with 10 % neutral buffered formalin (nbf), and stored at 4 °c (mckee, 1999). the gross examination images were graded analogously to the ct images, evaluating the territories supplied each the left and right aca, mca, and pca, as well as the cerebellum. the features used for estimating the extent of perfusion on the images were (a) clearance of blood from surface blood vessels, (b) apparent stiffness of tissue, and (c) color change of tissue, from pink in the case of no perfusion to pale or pale/colored dye-tinged in the case of well-perfused tissue. in some cases, specific brain areas could not be graded due to incomplete visualization in the available images. as with the ct scans, two independent raters evaluated the same subset of data. for a brain-wide assessment measure, a separate score was created based on the sum of the grades across all of the graded areas, removing cases with missing data from any region. histology in a subset of 11 cases, we sectioned the brains and dissected approximately 1x1x1 cm samples for histology. specifically, we collected grey matter samples from the left and right cerebellum, frontal cortex, occipital cortex, temporal cortex, thalamus, and white matter samples from the left and right temporal cortex, yielding a total of 12 distinct brain regions. as a control, one additional brain (from an 83-year-old donor with a pmi of 27 hours) that was immersion-fixed rather than perfusion-fixed, was processed to obtain samples from the same 12 brain regions. brain tissue sampled for light microscopy was placed into cassettes for processing and embedded in paraffin. paraffin-embedded brain sections 6 μm thick were baked, deparaffinized, and stained for hematoxylin and eosin (h&e). digital images of the stained sections were captured at 40x as whole slide images (wsis) using the aperio gt450 high-resolution scanner (leica biosystems). to analyze the data, we graded the extent of vessel clearance across each of the wsis on a 0–3 scale, with 0 indicating < 5 % clearance of blood vessels, 1 indicating 5–50 %, 2 indicating 50–95 %, and 3 indicating > 95 %. vessel clearance refers to the absence of intravascular material from both small and large vessels, as in some cases there was clearance from the large vessels but not the small vessels. as an additional brain banking cohort for a comparison to immersion fixation, we also graded the vessel clearance in the frontal cortex from brain hemisphere specimens that were immersion fixed with 10 % neutral buffered formalin, stained for h&e, and captured as wsis (garrood et al., 2025). we graded these wsis using the same 0–3 vessel clearance scale as above. the grades from the perfusion fixed samples and the immersion fixed samples in this separate cohort from the frontal cortex (the only region available for comparison in both cohorts) were then compared with a t-test. interrater reliability scores for both the gross examination images and ct scans, on a subset of the data, perfusion quality grades were performed independently by two raters. for the ct scans, this subset included the air bubble assessment as well. for all of the histology wsis, perfusion quality grades were performed independently by the two raters. the interrater reliability for each of these types of grades was then calculated using the intraclass correlation coefficient (icc), applying a model with agreement estimation, single unit of analysis, and two-way random-effects. the icc values were interpreted using previously established guidelines (koo and li, 2016). if instances of discrepancies between the raters, the final grade was determined via a consensus review between the two raters. results approach to brain perfusion in preliminary experiments, we cannulated and perfused the common carotid arteries using an anterior cervical approach that has previously been demonstrated to provide high-quality perfusion of the brain (insausti et al., 2023). however, in our experience, especially in donors with pmis longer than 24 hours, this approach sometimes resulted in substantial perfusion of facial tissues, causing facial edema but minimal or no perfusion of the brain. additionally, when using the anterior cervical approach for carotid cannulation, backflow was observed from the caudal severed ends of the carotid arteries. this was attributed to perfusion through the circle of willis, and subsequent backflow into the systemic circulation through the vertebral arteries, and out through the low-pressure system created by the open carotid arteries. therefore, instead of the fixative perfusing the brain parenchyma and returning through the intended venous drainage pathways, the vertebral arteries created a shunt that diverted perfusate away from this route to the systemic circulation. in order to more quickly access the internal carotid arteries and prevent the problem of vertebral artery shunting, we switched to performing perfusion on the isolated cephalon, a method previously used in neuroanatomical studies and neurosurgical training (sanan et al., 1999; alvernia et al., 2010; benet et al., 2014; turkoglu et al., 2014; mignucci-jiménez et al., 2024). this approach facilitates rapid visualization and clamping of the vertebral arteries, effectively preventing perfusate from shunting through this pathway. even with this more focused approach, we sometimes observed perfusion to the superior facial tissues, particularly in the periorbital region. this phenomenon likely reflects collateral circulation via the ophthalmic artery, as previously described (kalimo et al., 1974). the extent of periorbital perfusion appeared to be inversely related to the degree of cerebral parenchymal perfusion, although in most cases, periorbital distribution of perfusate remained minimal. with cannulation of the internal carotid arteries and clamping of the vertebral arteries, the majority of the perfused fluid appeared to flow through the brain vasculature. using this approach, total perfusion volumes ranged from 0.1 to 2.8 liters per case (mean: 1.16 liters), delivered over 4 to 45 minutes (mean: 19.9 minutes). spinal canal fluid return during perfusion with the isolated cephalon approach, we observed substantial fluid outflow through the open spinal canal in most cases. this occurred rapidly, often within a few seconds after the initiation of mechanical perfusion, and sometimes before fluid returned from any other site. moreover, outflow through the spinal canal often represented substantial source of perfusate return, often exceeding the volume returning through the ijvs. consistent with perfusate entering the csf compartments, some degree of contrast was frequently visible at least in the ventricles on post-perfusion ct scans. importantly, superficial cerebral veins were often cleared even in cases where nearly all fluid outflow occurred through the spinal canal. additionally, when the cephalon was repositioned during the procedure, outflow from the ijvs occasionally ceased completely, possibly due to constriction. when the ijv flow stopped, spinal canal outflow appeared to increase proportionally, without any apparent change in the measured perfusion pressure or the color of the returning fluid. this shift in relative flow through the spinal canal was reversible upon further repositioning of the cephalon. these observations suggest functional communication between the venous and csf compartments within the intracranial cavity. one possible explanation for this phenomenon is retrograde flow through arachnoid villi, which has been suggested to allow communication between venous and csf compartments under certain conditions, including postmortem (potts et al., 1972; alvernia et al., 2010; proulx, 2021). another possible route involves the perivascular spaces surrounding cerebral veins, which have been found to communicate with the csf compartment postmortem (ma et al., 2019). regardless of the exact mechanism, which remains unclear, these findings indicate that spinal canal outflow can effectively substitute for venous outflow, and is therefore not necessarily indicative of perfusion failure. in some cases, we also observed sudden displacement of the spinal cord or adjacent nervous tissue during perfusion. this finding is indicative of brain herniation, which in turn reflects edema in at least some regions of the brain (alvernia et al., 2010). herniation occurred in 14 out of the 66 cases (21.2 %) using the isolated cephalon approach for which the presence or absence of this outcome was recorded. qualitatively, it appeared to be more likely in cases with longer pmis, but this was not a statistically significant effect in our sample (average pmi of cases with herniation = 53.4 hours, average pmi of cases without herniation = 32.5 hours, t-test p-value = 0.11). herniation often occurred shortly after an increase in perfusion flow rate. the mechanism by which postmortem perfusion may cause edema in some cases is not clear. we propose that it most likely involves increased bbb permeability, which is known to increase progressively due to ischemia during the postmortem interval (krassner et al., 2023). when bbb integrity is compromised, perfusate may extravasate into the parenchyma more readily than it circulates through the vascular system. this process could theoretically affect different brain regions heterogeneously, depending on variations in baseline bbb integrity and the rate of bbb degradation. some protocols designed for vascular visualization have suggested occluding the spinal canal with bone wax during isolated cephalon perfusion to prevent fluid loss through this route (sanan et al., 1999). however, we intentionally maintained an open spinal canal as we expected that maintaining an outlet for fluid to leave the cranial cavity may help to decrease edema. additionally, this configuration allowed the observation of spinal cord bulging as a proxy for increased intracranial pressure, which we consider a useful marker for when perfusion should be slowed or terminated to prevent further parenchymal damage from edema and associated herniation (alvernia et al., 2010). quality of perfusion in gross images we developed a semi-quantitative rating scale to measure the extent of tissue perfusion in areas corresponding to each of six major cerebral arteries (i.e. the left and right anterior cerebral artery, middle cerebral artery, and posterior cerebral artery), as well as both sides of the cerebellum. perfusion quality was assessed based on degree of pallor, clearance of surface blood vessels, visible tissue stiffness, and the presence of colored dye when used at a concentration sufficient for visualization (figure 2). for a subset of data, perfusion quality based on these images was graded separately by two independent raters. these grades had an icc of 0.691 (95 % ci 0.542–0.799), indicating good interrater reliability. qualitatively, we found that in nearly all cases, perfusion appeared patchy achieved across the surface of the brain – both between and within vascular territories, for reasons that remain unclear. figure 2. representative gross examination images showing perfusion rated to be each of the four grades (0, 1, 2, and 3) with in the vascular distributions of the anterior cerebral artery (aca), middle cerebral artery (mca), and posterior cerebral artery (pca). for grade 0, we did not have a single brain with a grade of 0 in all three regions, so different brain samples were used for each representative image. donor ids: 169 (a), 183 (b, c), 205 (d, e, f), 197 (g, h, i), 126 (j, k, l). the mean perfusion quality grades across both hemispheres were 1.94 ± 0.09 (standard error of the mean) for the aca distribution, 1.75 ± 0.08 for the mca, 1.75 ± 0.08 for the pca, and 1.29 ± 0.08 for the cerebellum (figure 3). there was no significant difference between the grades in the aca and mca distributions (t-test, p-value = 0.11). the grades in the aca and mca were each significantly higher than those in the pca (t-test, p-values = 1.2e-5 and 0.003, respectively) and the cerebellum (t-test, p-values = 1.1e-7 and 7.7e-5, respectively). this indicates that perfusion quality was relatively higher in areas supplied by the anterior circulation (aca and mca) compared to those supplied by the posterior circulation (pca and cerebellum). on the other hand, no consistent difference was found in perfusion quality between the left and right hemispheres, although some brains exhibited a slightly greater perfusion on one side than the other. figure 3. histogram of the perfusion quality grades based on gross examination. quality is graded on a 0–3 scale, where 0 indicates minimal perfusion and 3 indicates maximal perfusion in each region. red bars represent right hemisphere; blue bars represent the left hemisphere. aca: anterior cerebral artery; mca: middle cerebral artery; pca: posterior cerebral artery; cb: cerebellum. to further analyze differences in perfusion between the anterior and posterior circulation, we focused on the cases for which perfusion of the isolated cephalon was performed solely through the internal carotids, which was the majority of total cases (62/77). in such cases, the primary way that perfusate can reach the posterior circulation is via the circle of willis, which may however be absent, stenotic, or have perfusion impairment due to agonal or postmortem factors. after the initiation of mechanical perfusion through the carotid arteries, the degree of flow through the circle of willis can be ascertained based on the amount of fluid that returns through the vertebral arteries, which we initially left patent. although we recorded the presence or absence of flow in a subset of cases, we more commonly recorded whether we clamped the vertebral arteries, which we only did when a sufficient amount of flow was going through them. as a result, clamping of the vertebral arteries can serve as a proxy for the presence of flow through the circle of willis in a given case. we created a measure of the divergence between the perfusion quality in the anterior and posterior circulations, which was the difference of the sums of the grades in the areas supplied by the aca and mca minus the sums of the grades in the area supplied by the pca and the cerebellum. we measured the average difference in the anterior-posterior divergence in perfusion quality between the cases in which at least one of the vertebral arteries were clamped (46/59) and cases in which none were clamped (13/59), and found no significant difference (t-test, p-value = 0.32). these results suggest that, when fluid is perfused through the internal carotids, the observed flow through the vertebral arteries is not a significant predictor of divergence in the perfusion quality between the anterior and posterior circulations of the brain, at least in this sample. quality of perfusion in ct images perfusion quality based on ct scans was graded on a semi-quantitative scale, reflecting the estimated percentage of contrast agent present in different brain regions (figure 4). we also developed a semi-quantitative grading scale to assess the extent of air bubbles observed in some ct scans (figure 5). for a subset of cases, the images were graded separately by two independent raters, yielding an icc of 0.536 (95 % ci: 0.293–0.705), indicating fair interrater reliability. consistent with the observations from gross examination images, the ct scans revealed patchy distribution of contrast in nearly all cases, both across and within vascular territories. figure 4. representative images of ct scans that demonstrate the grading schema. all images show the grading for both the left and right sides of the respective region, except for image(l), in which only the left side represents the correct grading. images follow the standard radiological convention, with the right side of the image corresponding to the donor’s left side and vice versa. donor ids: 136 (a), 71 (b), 185 (c), 179 (d), 197 (e), 201 (f), 195 (g), 5 (h), 84 (i), 203 (j), 206 (k), 142 (l). figure 5. representative images of ct scans showing the grading of air bubble extent across the brain. donor ids: 202 (a, b, c), 207 (d, e, f), 184 (g, h, i), 160 (j, k, l). the mean perfusion quality grades based on ct scans across both hemispheres were 1.94 ± 0.09 for the aca distribution, 1.80 ± 0.09 for the mca, 1.33 ± 0.09 for the pca, and 1.32 ± 0.07 for the cerebellum (figure 6). there was no significant difference between the grades in the aca and mca distribution (t-test p-value = 0.27). the grades in the aca and mca were each significantly higher than those in the pca (t-test, p-values = 2.6e-6 and 0.0003, respectively) and the cerebellum (t-test, p-values = 2.3e-7 and 6.8e-5, respectively). therefore, as with the gross examination data, average perfusion quality graded on ct scans was found to be higher in the regions supplied by the anterior circulation than in those supplied by the posterior circulation. figure 6. histogram of perfusion quality grades based on ct images. perfusion quality is graded on a 0–3 scale, where 0 indicates minimal perfusion and 3 indicates maximal perfusion in each region. red bars represent the right hemisphere; blue bars represent the left hemisphere. aca: anterior cerebral artery; mca: middle cerebral artery; pca: posterior cerebral artery; cb: cerebellum. in four of the cases in which herniation of tissue through the spinal canal was observed, we had ct scans of the brain following perfusion available, which we analyzed further. in one of these cases, there was evidence of a midline shift; however, this donor had a history of an arteriovenous malformation in the frontal lobe, and no pre-perfusion ct scan was available for comparison, so the arteriovenous malformation may have been present before the perfusion. in another case, the donor died of an aneurysm, which is visible on the ct scan, but there was no midline shift and the ventricles were of normal size. in another case, the brain appeared to have smaller-than-normal ventricles, but there was no midline shift, and there was no pre-perfusion ct scan available for comparison. finally, in one of these cases, the donor died from a hemorrhagic stroke, and there was a large clot visible on the pre-perfusion ct scan, but after perfusion, there was no change in the ventricles or evidence of midline shift (figure 7). taken together, these results are difficult to interpret, because many of the cases in which herniation occurred had a history of brain pathology, which may be a predisposing risk factor for herniation. however, our data suggests that the herniation event does not necessarily lead to significant changes in the anatomical condition of the brain as visualized on ct. figure 7. ct scans of a brain donor with observed herniation of tissue through the spinal canal during perfusion, without no clear evidence of significant anatomical sequelae. a: pre-perfusion axial ct image of a donor with a known hemorrhagic stroke, showing a large intraventricular clot (red arrow). b: post-perfusion coronal ct image showing partially successful perfusion, as evidenced by iohexol distribution, with persistent intraventricular clot (red arrow). during perfusion of this brain, a suspected herniation event occurred, as tissue abruptly emerged from the spinal canal, prompting immediate termination of the perfusion. however, there was no clear evidence of anatomical damage due to the herniation on the ct scan, as the ventricles remained of the same size and no midline shift was observed. donor id 179, pmi of 70.5 hrs. quality of perfusion based on histology the quality of perfusion on histology was graded based on the clearance of intravascular material from blood vessels, with each vessel as not cleared, partially cleared, or fully cleared (figure 8). the extent of clearance across all blood vessels in the wsi was then graded on a 0–3 scale to estimate the perfusion quality of that tissue sample (figure 9). these grades were found to have an icc of 0.804 (95 % ci 0.675–0.885), indicating excellent interrater reliability. as a control, we also performed histology on the brain of one donor (#166) that was exclusively preserved via immersion fixation, which yielded grades of 0 in six brain regions and grades of 1 in the remaining six brain regions. for additional comparison, we graded the vessel clearance in a larger set (n = 36) of exclusively immersion-fixed brain frontal cortex from a previously described cohort (garrood et al., 2025). we found that vessel clearance was significantly higher in perfusion-fixed samples compared with immersion-fixed samples (mean perfusion-fixed sample grade = 1.59, mean immersion-fixed sample grade = 0.42, t-test, p-value = 1.56e-7). these results demonstrate that vessel clearance – and therefore our grading metric – is not entirely specific to perfusion, and that the partial clearance of intravascular material from blood vessels can also result from other mechanisms. however, vessel clearance is significantly greater in perfusion-fixed samples, consistent with the expected effect of perfusion in removing intravascular material. figure 8. representative histology images showing the degree of blood vessel clearance. this degree of clearance was determined across the entire wsi to determine the perfusion quality of each tissue sample. donor ids 147 (a, b), 78 (c, d), and 142 (e, f). scale bars: 700 μm (a, c, e) and 200 μm (b, d, f). scale bars: a, c, e: 700 μm; b, d, f: 200 μm. figure 9. representative histologic perfusion quality grades in wsis. grades were assigned on a 0–3 scale, where 0 indicates < 5 % clearance of intravascular material from vessels, 1 indicates 5–50 %, 2 indicates 50–95 %, and 3 indicates > 95 %. grade 0: donor #166, left frontal cortex (a, b, c). grade 1: donor #147, right occipital cortex (d, e, f). grade 2: donor #107, right occipital cortex (g, h, i). grade 3: donor #142, right occipital cortex (j, k, l). scale bars: 200 μm (a, c, d, f, g, i, j, l), 8 mm (b), 7 mm (e, h), and 6 mm (k). the mean perfusion quality grades based on histology, averaged across both hemispheres, were 1.91 ± 0.15 for the temporal white matter, 1.59 ± 0.16 for the frontal cortex, 1.41 ± 0.16 for the temporal cortex, 1.32 ± 0.15 for the thalamus, 1.27 ± 0.15 for the occipital cortex, and 0.45 ± 0.11 for the cerebellum (figure 10). the cerebellum exhibited significantly lower perfusion quality than the other regions, including the thalamus and occipital cortex (t-test, p-values = 6.4e-5 and 0.0002, respectively). the occipital cortex, in turn, had significantly lower average grades than the temporal white matter, but not the frontal cortex (t-test, p-values = 0.0059 and 0.167, respectively). these data suggest that average perfusion quality with our approach is relatively lower in the cerebellum, a region supplied by the posterior circulation. figure 10. histogram of histology perfusion quality grades. higher grades correspond to a greater extent of vessel clearance across the whole slide image. red bars represent the right hemisphere; blue bars represent the left hemisphere. cb: cerebellum; fctx: frontal cortex; octx: occipital cortex; tctx: temporal cortex; thal: thalamus; twm: temporal white matter. in the histology data, we found that, frequently but not always, larger vessels were cleared of intravascular material while adjacent capillary networks predominantly remained largely filled with blood cells and other aggregated blood elements. this suggests that, even when large surface vessels appear cleared on gross examination and contrast is visible on ct imaging, portions of the capillary bed may remain largely unperfused. notably, in such cases, it is unclear whether the subset of capillaries showing evidence of perfusate flow is sufficient for tissue perfusion, or whether arteriovenous shunts may allow significant portions of the perfusate to bypass the capillary beds entirely (duvernoy et al., 1981; grabherr et al., 2008). correspondence between the quality metrics qualitatively, we found that in many cases, there was a spatial correlation between the perfusion profiles visualized via gross examination and those detected on ct scans (figure 11). we also provide a representative example of perfusion correspondence across all three modalities: a brain from a 54-year-old donor with a pmi of 36.5 hours, which exhibited similar perfusion quality grades throughout the brain as measured by gross examination, ct imaging, and histology (figure 12). figure 11. representative matched gross examination and ct images illustrating spatial correlations in perfusion quality across modalities. on ct images (b, d, f), following standard radiologic convention, the right side of the image corresponds to the left side of the brain. in one of these perfused brains, a clear anterior-to-posterior gradient of perfusion is observed, with relatively better perfusion in the anterior regions and relatively worse perfusion in the posterior regions; this gradient is visible in both gross examination images (c) and ct scans (d). donor ids: 205 (a, b), 207 (c, d), 185 (e, f). figure 12. representative gross examination, ct scan, and histology findings in a single brain. this brain was assessed a perfusion quality grade of 2 in all regions across all three modalities. gross examination images: right lateral view (a), inferior view (b), left lateral view (c), and coronal ct images (d–g). h&e histology from the right frontal cortex (h, zoom in l), right temporal cortex (i, zoom in m), left temporal cortex (j, zoom in n), and left frontal cortex (k, zoom in o). donor id 107, pmi of 36.5 hours. scale bars: 6 mm (h, i, j, k) and 200 μm (l, m, n, o). to more comprehensively compare the perfusion quality scores across different methods, we next measured the correlations between quality scores and other relevant variables (figure 13). we also created metrics of whole-brain perfusion quality for gross examination and ct scans by summing the grades across all assessed regions (insufficient cases with histology were available to perform similar quantitative correlations). we found no significant correlation between the sum of gross image grades and the sum of ct scan grades (correlation coefficient r = 0.12, p = 0.57, degree of freedom df = 23), which may be due in part to the limited number of samples available for comparison. however, the grades for gross examination and ct scores did have a significant positive correlation in several individual regions, such as the left aca area (r = 0.48, p = 0.0096, df = 26), although notably these p-values were not adjusted for multiple comparisons. regarding pmi, there was a significant negative correlation between the pmi and the sum of the gross examination grades across areas (r = –0.45, p = 0.0004, df = 57), while the correlation between the pmi and the sum of the ct scan grades across areas was negative without reaching statistical significance (r = –0.29, p = 0.11, df = 29). figure 13 correlation matrix of perfusion quality metrics across brain donors. pearson correlation coefficients between perfusion quality assessments (gross examination and ct imaging), technical parameters (lowest and highest perfusion pressures recorded, total perfusate volume, and donor characteristics (age, postmortem interval (pmi)). perfusion quality was assessed in multiple areas: the vascular territories supplied by the anterior cerebral artery (aca), middle cerebral artery (mca), and posterior cerebral artery (pca), as well as the cerebellum (cbm), with left (l) and right (r) hemispheres evaluated separately. sum scores correspond to the aggregate perfusion quality grades across all evaluated regions for both gross examination (gross.sum) and ct imaging (ct.sum). air.bubbles.ct indicates the grade for the extent of air bubbles visible on post-perfusion ct scans. circle size and color intensity indicate correlation strength, with blue representing positive correlations and red representing negative correlations. asterisks (*) denote statistically significant correlations (p < 0.05, unadjusted). regarding other technical variables, several correlations stand out. first, we found that the pmi was significantly correlated with the lowest recorded perfusion pressure (r = 0.50, p = 0.002, df = 32). we usually initiated flow at a consistent, relatively low rate, and the lowest recorded perfusion pressure is generally the perfusion pressure at this initial flow rate. therefore, this result likely indicates lower vascular resistance in brains with a shorter pmi, which may reflect less time for the accumulation of factors that impair perfusion. the lowest recorded perfusion pressure also had a significant negative correlation with the sum of ct perfusion grades (r = –0.61, p = 0.002, df = 32), indicating that, under the same procedural protocol, the eventual quality of perfusion may be predicted to some extent based on the initial pressure reading at the start of the procedure. notably, the grade for the extent of air bubbles observed on ct images was also significantly negatively correlated with pmi (r = –0.43, p = 0.015, df = 30). a shorter pmi may be associated with more air bubbles because the same factors that prevent adequate perfusate flow may also the introduction of air bubbles into the brain. comparison of syringe and pump-based perfusion we compared two commonly used methods for driving perfusate flow: (a) a peristaltic pump and (b) manual syringe injection. a number of previous studies have performed postmortem perfusion via syringe injection in the isolated cephalon (mignucci-jiménez et al., 2024; pérez-cruz et al., 2024) or whole brain (smirnov et al., 2023). the syringe method has some advantages, for example being less resource-intensive and allowing manual assessment of pressure based on resistance felt during injection. in contrast, the pump method provides more continuous flow and the possibility to use higher pressures. we used peristaltic pump-driven perfusion in 61 cases and manual syringe injection in 16 cases. we found no significant difference between the two methods in the sum of perfusion quality grades on the gross examination images (13.2 for pump and 13.3 for syringe, t-test p-value = 0.97) or ct images (12.7 for pump and 13.0 for syringe, t-test p-value = 0.84). similarly, there was no significant difference in the extent of air bubbles on ct images (1.45 for pump and 1.50 for syringe, t-test, p-value = 0.92). among cases using the cephalon isolation approach, there was also no significant difference in the proportion of cases with herniation (24 % or 12/50 cases for pump, 14.2 % or 2/14 cases for syringe, two proportions z-test p-value = 0.68). these data suggest that the simpler syringe-based method may be a useful option when laboratories do not have access to a pump, although further research is needed to corroborate this finding. discussion in this study, we assessed perfusion quality across a sample of donated brains that were perfusion-fixed prior to immersion fixation. our results highlight significant variability in perfusion quality both within and across brain specimens. the assessment methods we employed – gross examination, ct imaging, and histology – were partially correlated, but not perfectly, supporting their use as complementary approaches to evaluate perfusion outcomes. consistent with the existing literature, we also found that the perfusion quality as assessed by gross examination images decreases with longer pmis. naïvely, one might expect that fluid perfused into the arteries should follow a physiological path through the capillaries, with balanced hydrostatic and oncotic pressure gradients controlling limited exchange of fluid across vessel walls, before the majority effluxes through the ijvs. however, we found that the physiology of postmortem perfusion clearly differs from in vivo circulation, indicating the need to study it as a distinct phenomenon. one of the main differences between postmortem mechanical perfusion and in vivo circulation is the regional heterogeneity of perfusion quality, which we observed across all perfused specimens. the underlying reasons are unclear and likely multifactorial. in vivo, active compensatory and autoregulatory mechanisms help to maintain uniform perfusion. in contrast, the postmortem brain lacks these homeostatic mechanisms, leading to heterogeneous perfusion impairment across the brain. one contributing factor may be the uneven accumulation of clots that obstruct fluid flow through different vascular territories, which could be self-reinforcing. specifically, areas that receive more initial perfusate may become progressively cleared of intravascular debris, while vascular territories with no initial perfusion remain obstructed and continue to accumulate factors that impair flow over time. another aspect of postmortem perfusion is the risk of edema, which we also noticed in previous cases using ex situ perfusion fixation (mckenzie et al., 2022). to mitigate the risk of edema and associated herniation, we began adding mannitol to the perfusate, which was included in the fixative perfusate for the majority of our cases. this approach was based on evidence that the osmotic concentration of the buffer in fixation solutions primarily determines its effective osmotic pressure (pallotto et al., 2015). although some members of our team subjectively thought that mannitol might slightly mitigate the risk of edema, we did not have enough observations about brain volume changes or other parameters with and without mannitol to assess this quantitatively. nevertheless, mannitol clearly did not completely prevent edema and herniation. one potential explanation is that mannitol is not a large enough molecule to increase effective osmotic concentration when the bbb is damaged. previous data indicates that in regions where the bbb is severely damaged, mannitol can leak through tight junctions into the brain parenchyma rather than creating the intended osmotic gradient in the blood vessels, negating its beneficial effects (videen et al., 2001; liu et al., 2022). air bubbles are another important variable to consider in postmortem perfusion. in liver transplantation, air bubbles can be introduced during organ procurement, reducing the extent to which the organ is perfused, and can be mitigated by avoiding the exposure of arteries to air (liu et al., 2010; izamis et al., 2014). we previously demonstrated via neuroimaging that ex situ perfusion-fixation of the human brain can introduce air bubbles (mckenzie et al., 2022). one potential concern with the isolated cephalon approach is that it might introduce air bubbles that hinder perfusion (bolliger et al., 2018). the dose of air bubbles is likely important, as very small amounts of air seem both difficult to avoid in mechanical perfusion and unlikely to cause significant problems. one study on the perfusion of the isolated cephalon does not describe air bubbles being introduced or causing problems following a 10-minute-long period before carotid artery cannulation and perfusion, despite extensive imaging (vrselja et al., 2019). numerous other studies on the isolated cephalon or brain have also demonstrated that the adequate perfusion of such specimens is in principle possible (lee et al., 1968; gilboe, 1982). in this study, we found that air bubbles were frequently visible on ct scans following perfusion, to varying degrees, but that the burden of air bubbles was not correlated with perfusion quality. some of the variability in air bubbles may be attributable to differences in the amount of residual blood in vessels following cephalon isolation, which could affect how easily air enters the brain. procedural differences may also contribute, as we did not consistently implement measures to mitigate the introduction of air emboli. such measures to could include inverting the cephalon, clamping or irrigation of vessels prior to their cannulation, and performing initial retrograde perfusion to displace air already trapped in the proximal arteries. one potential confound is that air bubbles could also be introduced after perfusion, for instance when cannulae are removed. in future research, this technical confound could be minimized by maintaining perfused fluid within the blood vessels using plastic clamps that do not interfere with ct imaging. one potential avenue for future research is the use of a washout solution prior to fixative perfusion. although we perfused a washout solution in our first two perfusion cases, in subsequent cases we immediately perfused fixative solution. our findings suggest that a washout solution may not be necessary, as we were able to effectively remove blood from the vessels with fixative alone. the duration required for formaldehyde to act as a chemical fixative is much longer than the time during which the fluid physically pushes out intravascular material, suggesting that theoretical concerns about red blood cells or clots being fixed in place are unlikely to affect perfusion outcomes, consistent with previous literature (pease, 1964). one potential advantage of a washout solution is that it could incorporate chemicals, such as detergents, specifically designed to break down intravascular material that impedes perfusion, thereby potentially improving the quality of subsequent fixative perfusion (tobin, 1970; bradbury and hoshino, 1978; grabherr et al., 2008; frigon et al., 2024). however, if such chemicals are to be used in a brain banking context, it would be essential to test whether they alter cellular structure. for example, detergents can damage lipid membranes, making them a potential double-edged sword: while they may help perfusion quality by facilitating the removal of blood clots, they also could compromise the visualization of cellular structures. we acknowledge that perfusion fixation will almost certainly remain a specialized technique, rather than a routine practice in most brain banks. instead, immersion fixation is likely to remain the standard method, as it has several advantages, including lower cost, higher reproducibility, and the possibility to flash freeze the other hemisphere unfixed. additionally, immersion fixation may provide adequate preservation quality on its own, depending on the research application (garrood et al., 2025). however, in the right research context, perfusion fixation may prove particularly useful. for example, a brain mapping study that requires high-fidelity preservation of cellular architecture across regions, such as light-sheet-microscopy-based reconstruction of neural circuits, may benefit from an intact brain with more uniform fixation throughout. perfusion may also be useful for applications other than accelerating the process of fixation, such as mapping the vasculature with angiography, brain clearing, or distributing cryoprotective agents (mckenzie et al., 2024). the benefits of perfusion are particularly likely to be realized if the right brain donors are chosen, such as those with low pmi and a minimal agonal state. furthermore, if methods can be developed to improve the quality of postmortem brain perfusion, then the technique could become more reliable and therefore more useful in applications requiring uniform and reproducible perfusion across the brain. there are several limitations to our study. first, our analysis was qualitative or semi-quantitative rather than fully quantitative, which limits our ability to draw precise statistical conclusions about the relationships between perfusion variables and outcomes. future studies incorporating more quantitative image analysis, as well as expanding the sample size, would strengthen these findings. second, our perfusion methods were refined iteratively over the course of the study, introducing variability that may have confounded some of our comparisons. while this iterative approach was necessary for the development and optimization of the protocol, it limits our ability to isolate the effects of specific procedural variables. third, we did not grade the severity of the agonal state of the donors prior to their death, as we often lacked sufficient information to do so. this is a limitation because the agonal state and cause of death likely have a substantial impact on perfusion quality. finally, another limitation concerns the potential variability in the concentration of iohexol contrast agent across specimens. this variation may have affected the perceived intensity of regional perfusion in ct images, potentially confounding our assessment of perfusion quality. although iohexol does not cross vessels with intact bbb in most circumstances, it can traverse a compromised bbb in the context of ischemia (mariajoseph et al., 2024). however, the bbb clearly does not fully break down immediately after cardiac arrest (du et al., 2022). because postmortem bbb integrity is expected to deteriorate progressively with increasing pmi, the iohexol distribution likely represents a combination of contrast agent confined within blood vessels and contrast agent that has penetrated into the parenchyma through a compromised bbb. the rate and extent of this penetration likely varied among cases with different pmis, adding another layer of complexity to the interpretation of contrast distribution. while this is not a major confound for our primary analysis of the presence versus absence of perfusion in different parts of the brain, it does limit our ability to make quantitative comparisons of perfusion intensity across brains. conclusions the quality of human brain specimens is crucial for their adequate study, which in turn is essential to better understand the mechanisms of neurobiological disorders and improve our treatments for them. existing methods for tissue preparation are often inadequate, and mechanical perfusion is one proposed way to improve the quality (beach et al., 198; mcfadden et al., 2019). in this study, we investigated the quality of perfusion across the brain using a single perfusion-fixation protocol. we found that in some cases, especially those with relatively shorter pmis, mechanical perfusion successfully reached at least a subset of blood vessels throughout the brain. however, in other cases, perfusion quality was relatively poor, with the perfusate failing to reach most regions of the brain. in a significant minority of cases, perfusion also caused brain edema, leading to herniation and requiring interruption of the perfusion to prevent further tissue damage. we also found that the pmi is correlated with our perfusion quality assessment methods, but far from perfectly so, suggesting that other factors related to the condition of the brain at the time of perfusion also play a major role in determining perfusion quality. further optimization of perfusion protocols may facilitate more rapid and uniform fixation of the brain and also performing other types of tissue preparation in the postmortem brain. additional research in this area may also clarify the mechanisms of perfusion impairment after ischemia and enable the testing of methods to mitigate such impairment in human brains. author contributions j.s.s., k.f., j.f.c., and a.t.m. conceptualized the study. e.t. and c.d.s. performed light microscopy experiments. m.g., a.k., and a.t.m. performed data analysis. a.t.m. wrote the initial draft of the manuscript. all authors reviewed the manuscript and approved the final manuscript. acknowledgements we would like to acknowledge the neuropathology brain bank & research core at the icahn school of medicine at mount sinai for their histology and tissue processing services. the icahn school of medicine at mount sinai provided access to library resources. funding statement this work was supported by the rainwater charitable foundation as well as nih grants p30 ag066514, k01 ag070326, rf1 ag062348, p30 ag066514, rf1 ns095252, u54 ns115266, and rf1 mh128969. the funders had no role in the design of the study or in the collection or interpretation of the data. conflict of interest statement macy garrood, alicia keberle, gabriel taylor, jordan sparks, and andrew mckenzie are employees of sparks brain preservation, a non-profit brain preservation organization. data availability whole slide image data can be accessed in a public repository on zenodo, available here: https://zenodo.org/communities/mechanicalperfusioninbrainbanking. code and data used for data analysis are available at 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https://doi.org/10.17879/freeneuropathology-2024-5848 page 1 of 12 copyright: © 2024 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. neurodegeneration: 2024 update john f. crary department of pathology, nash family department of neuroscience, department of artificial intelligence & human health, neuropathology brain bank & research core, ronald m. loeb center for alzheimer's disease, friedman brain institute, icahn school of medicine at mount sinai, new york, ny, usa corresponding author: john f. crary · icahn school of medicine at mount sinai · 1 gustave l. levy place box 1194 · new york, ny 10029 · usa john.crary@mountsinai.org submitted: 09 september 2024 · accepted: 18 november 2024 · copyedited by: jerry lou · published: 09 december 2024 abstract this review highlights a collection of both diverse and highly impactful studies published in the previous year selected by the author from the neurodegenerative neuropathology literature. as with previous reviews in this series, the focus is, to the best of my ability, to highlight human tissue-based experimentation most relevant to experimental and clinical neuropathologists. a concerted effort was made to balance the selected studies across neurodegenerative disease categories, approaches, and methodologies to capture the breadth of the research landscape. these studies employ a range of classical and state-of-the-art methodologies ranging from clinical pathoanatomical correlative studies to single-cell rna sequencing, artificial intelligence, and patient-derived human induced pluripotent stem cell models. key studies include demonstration of the earliest pathological changes in young patients with repetitive head impacts (rhi), elucidation of the longitudinal trajectory of extra pyramidal symptoms in lewy body disease subtypes, mapping of cell-type specific polygenic risk in alzheimer’s disease to neuropathology, a novel measure of histological brain age acceleration using artificial intelligence, trends in cerebrovascular pathologies over 25 years, associations between rhi and tdp-43 / hippocampal sclerosis, microglia / t-cell interaction in neurodegeneration, the impact of viral exposures on neurodegenerative disease risk, and polyglutamine repeat expansion disorders. this sampling of the literature collectively displays the breadth of the progress being made in the neuropathology of neurodegenerative diseases. keywords: neurodegeneration, neuropathology, aging, alzheimer’s disease, tauopathy, α-synucleinopathy, tdp-43 proteinopathy, traumatic brain injury review https://doi.org/10.17879/freeneuropathology-2024https://creativecommons.org/licenses/by/4.0/ https://creativecommons.org/publicdomain/zero/1.0/ mailto:john.crary@mountsinai.org free neuropathology 5:31 (2024) john f. crary doi: https://doi.org/10.17879/freeneuropathology-2024-5848 page 2 of 12 1. chronic traumatic encephalopathy in young athletes’ post-repetitive head impacts concussions are extremely prevalent, especially among individuals exposed to repetitive head impacts (rhis) through contact sports, military service, intimate partner violence, and other means. while these impacts may be symptomatic or asymptomatic in the short term, long-term exposure can lead to severe neurological outcomes, including chronic traumatic encephalopathy (cte), a degenerative brain disease with varied clinical and neuropathological features. most research on rhi has focused on older populations, leaving a critical gap in our understanding of how rhis affect younger individuals. investigating changes in young athletes is crucial, as it may uncover underlying mechanisms for neuropsychiatric symptoms and identify early triggers for tau accumulation and associated toxicity, potentially informing preventive strategies and interventions. a study by mckee et al. published in jama neurology investigates the neuropathologic and clinical consequences of repetitive head impacts (rhis) in young contact sport athletes (mckee et al., 2023). analyzing data from 152 brain donors younger than 30 years from the unite brain bank, the study characterized the presence and extent of chronic traumatic encephalopathy (cte) and other neuropathologic abnormalities. the authors found that 41.4 % of the athletes showed neuropathological changes diagnostic for cte, predominantly mild (stages i or ii). notably, athletes with cte were more likely to be older and have longer exposure to rhi, particularly american football. cte positive subjects showed key neuropathologic features, including ventricular enlargement, cavum septum pellucidum, thalamic notching, and the presence of perivascular pigmentladen macrophages in the frontal white matter (figure 1). further, the study also examined the clinical symptoms reported by informants using standardized scales, showing that cognitive, behavioral, and mood disturbances were prevalent among the brain donors, regardless of cte status. common symptoms included executive dysfunction, impulse control issues, depression, and apathy. this study provides critical insights into the early manifestations of cte and other brain pathologies in young athletes, emphasizing the need for prospective studies to better understand the specific impacts of rhis. the study's findings are potentially pivotal for developing strategies to mitigate the long-term effects of rhis in contact sports. figure 1. neuropathological changes in young contact sports athletes. a. immunohistochemistry for hyperphosphorylated tau showing the pathognomonic perivascular lesion. b. lh&e showing robust perivascular hemosiderin deposition in the white matter. c. lh&e demonstrating white matter rarefaction. d. activated microglial clusters are prominent. reproduced with modifications under the terms of the cc-by license (http://creativecommons.org/licenses/by/4.0/). https://doi.org/10.17879/freeneuropathology-2024http://creativecommons.org/licenses/by/4.0/ free neuropathology 5:31 (2024) john f. crary doi: https://doi.org/10.17879/freeneuropathology-2024-5848 page 3 of 12 2. hippocampal sclerosis with tdp-43 inclusions following repetitive head impacts hippocampal sclerosis of aging, characterized by neuronal loss and gliosis primarily in the ca1 region and occurring independently of hypoxic ischemic injury, is prevalent in the aging population. most cases also exhibit inclusions positive for transactive response dna-binding protein with 43 kda (tdp-43), a protein that, since its identification as the primary inclusion in amyotrophic lateral sclerosis (als) and frontotemporal lobar degeneration (ftld), has been linked to other disorders including alzheimer's disease (ad), limbic age-related tdp-43 encephalopathy neuropathologic change (late-nc), and chronic traumatic encephalopathy (cte). the association with cte is particularly notable and understudied, raising questions about whether these conditions represent co-morbidities or if they have synergistic effects. a study by nicks et al. published in acta neuropathologica directly addresses the neuropatholo gical association between repetitive head impacts (rhi), chronic traumatic encephalopathy (cte), and the presence of tdp-43 inclusions and hippocampal sclerosis (hs) (nicks et al., 2023). the researchers analyzed brain samples from 401 participants with a history of rhi and neuropathologically diagnosed cte and compared them with 33 individuals diagnosed with hs without cte. the study found that hs was present in 23.4 % of the cte cases and that tdp-43 inclusions were present in 43.3 % of the cte cases. notably, hs in cte occurred at a younger mean age (77 years) and was significantly associated with a longer history of contact sports exposure compared to cte without hs. tdp-43 inclusions were frequently observed in the frontal cortex and often co-occurred with limbic tdp-43 pathology. structural equation modeling demonstrated a significant association between years of rhi exposure and the presence of hippocampal tdp-43 inclusions through increased cte stage. the study also showed that tdp-43 inclusions in cte were predominantly limbic but also involved the frontal cortex, distinguishing it from typical age-related tdp-43 patho logies such as late-nc. the presence of tdp-43 inclusions in the hippocampus was significantly associated with hs in cte, suggesting a pathogenic link between repetitive head impacts, cte patho logy, and the development of tdp-43 proteinopathy and hs. these findings highlight the importance of considering rhi history in the assessment of hippocampal sclerosis and suggest potential therapeutic targets for mitigating the effects of rhi on neurodegeneration. 3. longitudinal motor decline in diffuse lewy body disease, parkinson’s disease with dementia, and alzheimer’s disease lewy bodies, a common neuropathological finding, are the hallmark of parkinson's disease (pd) when localized to the substantia nigra, but they can progress to a diffuse pattern leading to parkinson's disease dementia (pdd). when lewy bodies are found in the neocortex and associated with dementia as the presenting symptom, this condition is termed diffuse lewy body disease (dlbd). in patients with dlbd, extrapyramidal parkinsonian features may or may not develop. lewy bodies are also seen in alzheimer's disease (ad). however, the extent to which the rates of extrapyramidal motor decline progress in these different contexts is unclear due to the limited number of longitudinal studies with neuropathological endpoints, which are crucial for prognostication, clinical trial design, and further research. a study by choudhury et al. published in alzheimer’s & dementia investigates the progression of motor deficits in dementia with lewy bodies (dlb), parkinson’s disease dementia (pdd), and alzheimer’s disease dementia (ad) using data from the arizona study of aging and neurodegenerative disorders (choudhury et al., 2023). the study included 193 participants with autopsy-confirmed diagnoses: 98 with pdd, 48 with dlb, and 47 with ad. within the dlb cohort, participants were further categorized into those with parkinsonism (dlb+) and without parkinsonism (dlb-). the researchers utilized the unified parkinson’s disease rating scale (updrs) parts ii and iii to assess motor function over an eight-year period, employing non-linear mixed effects models to analyze the data. the results revealed that motor deficits progressed most rapidly https://doi.org/10.17879/freeneuropathology-2024free neuropathology 5:31 (2024) john f. crary doi: https://doi.org/10.17879/freeneuropathology-2024-5848 page 4 of 12 in the dlb+ group, with significant worsening in gait and limb bradykinesia compared to pdd and other groups (figure 2). the study found that baseline updrs-ii and iii scores were highest in the pdd group, followed by dlb+, ad, and dlb-. over time, the dlb+ group showed a faster progression in updrs-iii scores compared to pdd, driven by significant declines in gait and limb bradykinesia. the study also highlighted that the presence of parkinsonism in dlb (dlb+) was associated with more rapid motor decline than in dlbor ad, emphasizing the importance of recognizing and monitoring these symptoms for better clinical management and prognostication. the findings underscore the need for targeted interventions to address motor deficits and suggest that patients may benefit from specific therapeutic strategies aimed at mitigating motor decline. the research provides valuable insights into the differential progression of motor impairments in neurodegenerative dementias, with implications for clinical trial design and patient care. figure 2. updrs-iii total scores during off stage examination for each participant and their trajectories for groups: pdd (a), dlb+ (b), dlb− (c), and ad (d). each point represents one movement exam visit and corresponding score. blue lines depict each participant. red line represents a mixed model fit line. the shaded area (funnel) surrounding red fit line represents 95 % confidence interval for the mixed model fitted curves. ad, alzheimer’s disease; dlb+, dlb with parkinsonism; dlb−, dlb without parkinsonism; dlb, dementia with lewy bodies; pdd, parkinson’s disease dementia. reproduced with modifications under the terms of the cc-by license (http://creativecommons.org/licenses/by/4.0/). https://doi.org/10.17879/freeneuropathology-2024http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ free neuropathology 5:31 (2024) john f. crary doi: https://doi.org/10.17879/freeneuropathology-2024-5848 page 5 of 12 4. cell-type-specific alzheimer’s polygenic risk scores large-scale genetic studies have uncovered numerous genetic loci associated with alzheimer's disease (ad), but understanding how the corresponding causal genes contribute to the cellular and molecular mechanisms driving ad remains a major challenge. since neurodegeneration in ad is a complex, non-cell autonomous process involving interactions between multiple cell types, deciphering how these genes act across different cells is essential for identifying therapeutic targets. genes expressed by microglia and astrocytes are notably over-represented in genome-wide association studies (gwas) of ad. however, how these cell-specific genetic changes connect to the neuropatholo gical hallmarks of ad, such as neurofibrillary tangles and amyloid plaques, and ultimately to cognitive decline, remains unclear—a critical gap in our knowledge. a study by yang et al. published in nature communications investigates how cell-type-specific polygenic risk scores (adprs) for ad are associated with distinct pathological processes (yang et al., 2023). by deriving adprs from large-scale genomewide association studies (gwas) and leveraging single nucleus rna sequencing (snrna-seq) data, the researchers assessed the impact of genetic risk localized to different brain cell types, focusing on microglia and astrocytes, on various ad endophenotypes. using data from the religious orders study and the rush memory and aging project (rosmap), as well as the anti-amyloid treatment in asymptomatic alzheimer’s (a4) study, the study found that astrocytic adprs were primarily associated with amyloid-β (aβ) pathology, while microglial adprs were linked to both aβ and tau pathologies, as well as cognitive decline. the findings suggest that genetic risks associated with astrocytes contribute to early aβ accumulation, whereas microglial genetic risks drive later-stage pathological changes and cognitive impairment. yang et al. utilized causal modeling to map the contributions of cell-type-specific adprs to the sequence of ad pathophysiology. the study suggests that astrocytic genetic risks affect ad primarily through aβ accumulation in diffuse and neuritic plaques, whereas microglial genetic risks have broader effects on neuritic plaques, tau neurofibrillary tangles, and cognitive decline. this was supported by data from the a4 study, which showed that microglial adprs were significantly associated with in vivo tau pet measures, indicating an early role in tau pathology. the research underscores the importance of considering cell-type-specific genetic risks in understanding the progression of ad and highlights potential targets for therapeutic interventions. these insights pave the way for future studies to explore genetically guided approaches to ad treatment, focusing on modulating specific glial cell functions to mitigate disease progression. 5. histological brain age acceleration using digital neuropathology and artificial intelligence understanding mechanisms of human brain aging is increasingly important as the elderly population grows, given the complexity of structural changes that arise from a spectrum of normal and pathological processes affecting functional impairment. one highly effective approach to studying aging is through biological clocks, initially developed using dna methylation analysis. dna methylation clocks estimate biological age by measuring methylation patterns at specific cpg sites in the genome, reflecting cellular aging processes. this method can be used to reveal discrepancies between biological and chronological age, providing insights into age acceleration or deceleration that can be linked to environmental and genetic factors. in a study by marx et al. published in acta neuropathologica, and led by the author of this review, the authors leverage digital pathology to deploy multiple instance learning (mil) to estimate brain age from histopathological whole slide images (marx et al., 2023). employing an attention-based deep mil model, the team analyzed digitized postmortem hippocampal sections to develop an advanced brain age estimation tool, achieving a mean absolute error of 5.45 years (figure 4). the integration of spatially resolved graph convolutional networks (gcns) allowed the model to consider neuro https://doi.org/10.17879/freeneuropathology-2024free neuropathology 5:31 (2024) john f. crary doi: https://doi.org/10.17879/freeneuropathology-2024-5848 page 6 of 12 figure 3. association of cell-type-specific ad polygenic risk scores. rosmap, religious orders study/memory and aging project; ad, alzheimer disease; prs, polygenic risk score; ex, exitatory neurons; in, inhibatory neurons; ast, astrocytes; mic, microglia; oli, oligodendrocytes; opc, oligodendrocyte precursors. reproduced in under a creative commons attribution 4.0 international license (http://creativecommons.org/licenses/by/4.0/) anatomical context, resulting in a significant improvement over traditional methods. marx et al. identified robust associations between histopathologic brain age acceleration and clinical and pathological outcomes, which were not evident with gold standard epigenetic measures. attention heatmaps generated by the model highlighted aging-vulnerable brain regions, such as specific white matter areas and hippocampal subfields, as critical for accurate age estimation. this study emphasizes the potential of histopathological markers to provide deeper insights into brain aging mechanisms. additionally, the combination of mil with gcns presents a novel methodology for predicting brain age, offering promising implications for understanding and diagnosing neurodegenerative diseases and their progression. this innovative approach paves the way for more precise and informative assessments of age-related neuropathological changes. in this context, histoage represents a cutting-edge advancement—an ai-driven digital neuropathology tool designed to study brain aging more precisely, leveraging advanced computational techniques to enhance our understanding of age-related changes in the brain. 6. decline in cerebrovascular pathologies over 25 years recently, several studies have suggested that the incidence rate of dementia may be declining, offering hope that improved healthcare and lifestyle factors are influencing this global challenge. however, dementia remains a highly complex disease with multiple underlying causes that are difficult to diagnose in the clinical setting. both genetic and environmental risk factors play significant roles in the development of dementia, and understanding their interplay over time is critical. across birth epochs, an enormous number of factors have changed, any of which could potentially influence dementia risk. these include environmental exposures (e.g., air pollution, pesticides), infectious diseases (e.g., herpes, hiv), socioeconomic shifts (e.g., malnutrition), medical advances, dietary and lifestyle trends, technological changes, genetic variations, access to healthcare, longevity, and even epigenetic modifications. despite recent findings pointing toward declining dementia rates, the precise neuropathological underpinnings of these trends remain unclear. this gap in knowledge presents a major barrier to https://doi.org/10.17879/freeneuropathology-2024http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ free neuropathology 5:31 (2024) john f. crary doi: https://doi.org/10.17879/freeneuropathology-2024-5848 page 7 of 12 figure 4. the “histoage” pipeline. a. digital whole slide images were broken up into smaller tiles, then run through a neural network that extracts features. these features were linked to adjoining tiles using graph structure. b. the features were then used to train a new neural network that predicted age, which was compared to chronological age to estimate age acceleration. reproduced under the creative commons attribution 4.0 international license (http://creativecommons.org/licenses/by/4.0/). developing targeted interventions and public health strategies, particularly as the population continues to age and the global burden of dementia grows. a study by grodstein et al. published in jama neurology examines trends in postmortem neurodegenerative and cerebrovascular neuropathologies over 25 years using data from the religious orders study and the rush memory and aging project (grodstein et al., 2023). this comprehensive investigation includes 1,554 deceased participants with complete brain autopsies and neuropathologic evaluations. the study analyzes four birth cohorts spanning from 1905 to 1930 to assess changes in neuropathological outcomes such as alzheimer's disease (ad), amyloid load, tau tangles, neocortical lewy bodies, limbic-predominant age-related tdp-43 encephalopathy, atherosclerosis, arteriolosclerosis, and brain infarcts. strikingly, they found no significant differences in the prevalence of pathologic ad diagnoses across birth cohorts, with age-standardized prevalence ranging between 62 % and 68 %. similarly, there were no marked changes in global ad pathology or other neurodegenerative pathologies over time. however, an increase in tau tangle density was noted in more recent birth cohorts, suggesting a potential rise in this specific pathology. in contrast, the authors found a dramatic decrease in cerebrovascular pathologies, particularly atherosclerosis and arteriolosclerosis, over the observed period. age-standardized prevalence of moderate to severe atherosclerosis decreased from 54 % in the earliest cohort (1905–1914) to 22 % in the latest cohort (1925–1930). arteriolosclerosis showed a similar decline. these findings highlight the impact of improved vascular health on brain aging and suggest that reductions in clinical dementia observed in other studies may be associated with enhanced resilience to neuropathology rather than a decrease in the underlying neurodegenerative pathologies. https://doi.org/10.17879/freeneuropathology-2024http://creativecommons.org/licenses/by/4.0/ free neuropathology 5:31 (2024) john f. crary doi: https://doi.org/10.17879/freeneuropathology-2024-5848 page 8 of 12 additionally, the study emphasizes the importance of considering both neurodegenerative and cerebrovascular factors in understanding dementia trends and developing public health strategies to address the growing aging population. the results underscore the need for continued efforts to improve vascular health as a means to mitigate the impact of dementia. 7. microglia-t cell interaction in alzheimer’s disease and tauopathy the immune system's involvement in neurodegeneration continues to gain increasing attention, with compelling evidence pointing to its critical role in disease progression. the innate immune system, particularly through the activation of microglia, has been at the forefront of research efforts, with these cells being implicated in the inflammatory processes that contribute to neuronal damage. however, a significant gap remains in understanding the role of the adaptive immune system, which, despite being central in autoimmune and infectious diseases, has been comparatively understudied in neurodegenerative disorders. t cells, key players in the adaptive immune response, have received far less focus in neurodegenerative diseases like alzheimer's. this oversight represents a critical barrier to fully understanding the immune-mediated mechanisms of neurodegeneration. although not as abundant as in conditions such as multiple sclerosis, t cells may still play an important role in these diseases, offering a potential but underexplored therapeutic target. a study by chen et al. at the washington university school of medicine published in nature investigated the relationship between microglia, t cells, and tau-mediated neurodegeneration (chen et al., 2023). using mouse models of amyloid deposition and tau aggregation, they systematically compared the immune responses in the brains of transgenic mice and humans, revealing that tauopathy, but not amyloid deposition, triggers a unique adaptive immune response. depletion of either microglia or t cells significantly blocked tau-mediated neurodegeneration, suggesting a critical role these immune cells play in the disease process. the study identified a marked increase in t cells, especially cytotoxic t cells, in regions with tau pathology in both mice and human alzheimer's disease brains, correlating with neuronal loss and dynamic transformations from activated to exhausted states. the researchers utilized single-cell rna sequencing (scrna-seq) and single-cell tcr sequencing (sctcrseq) to map the cellular and molecular signatures of immune cells in the brain. they found that in tauopathy, t cells and microglia formed an immune hub characterized by increased expression of interferon-γ and pdcd1 signaling pathways. notably, inhibiting these pathways significantly reduced brain atrophy. the study also highlighted the role of microglia in presenting antigens to t cells, promoting their infiltration and activation. this interaction was found to be critical in driving neurodegeneration, as evidenced by the reduction in tau pathology and brain atrophy upon depletion of t cells. these findings underscore the potential of targeting microglia and t cell interactions as a therapeutic strategy for tauopathies and alzheimer's disease, offering new avenues for treatment by modulating the immune response. 8. viral exposures linked to neurodegeneration growing evidence has increasingly spotlighted the potential causal role of viruses in neurodegenerative diseases (ndds). historical examples, such as post-encephalitic parkinsonism, first hinted at this link, and subsequent research has suggested associations with viruses like herpes simplex virus (hsv). the covid-19 pandemic further intensified speculation regarding viral contributions to neurodegeneration. most notably, recent studies have strongly linked epstein-barr virus (ebv) to multiple sclerosis (ms). this connection is significant because viral infections can be mitigated through vaccination or treated with antivirals, presenting vast new possibilities for preventing or slowing neurodegenerative processes. however, the specific mechanisms by which viral exposures contribute to ndds remain unclear, representing a critical gap in our understanding and a barrier to developing targeted interventions. a study published in neuron by levine et al. examines the association between viral exposures https://doi.org/10.17879/freeneuropathology-2024free neuropathology 5:31 (2024) john f. crary doi: https://doi.org/10.17879/freeneuropathology-2024-5848 page 9 of 12 and the risk of developing neurodegenerative diseases (ndds) using large-scale biobank data from finngen and the uk biobank (levine et al., 2023). the researchers identified 45 significant viral-ndd associations in the discovery phase with finngen, and successfully replicated 22 of these associations in the replication phase using the uk biobank. notably, the strongest association was observed between viral encephalitis (not elsewhere classified/unspecified) and alzheimer’s disease (ad), with a hazard ratio of 30.72 in finngen and an odds ratio of 22.06 in the uk biobank. other significant associations included influenza with pneumonia, which was linked to an increased risk of five of the six studied ndds, including ad, amyotrophic lateral sclerosis (als), dementia, parkinson's disease (pd), and vascular dementia (vas). the analysis highlighted the long-term impact of viral exposures, showing that some associations persisted up to 15 years prior to ndd diagnosis. the study also replicated the association between epstein-barr virus (ebv) and multiple sclerosis (ms). importantly, the findings suggest that vaccination against certain viruses, such as influenza and varicella-zoster, might reduce the risk of developing ndds. it is important to note that the reverse causality has not been eliminated. the possibility that patients in the presymptomatic phases of neurodegenerative disease might be at risk for viral infection needs to be addressed more closely. the study emphasizes the need for more research into the role of viral infections in the pathogenesis of neurodegenerative diseases and suggests that leveraging vaccination and antiviral strategies might offer new avenues for prevention and treatment. these findings underscore the importance of considering viral exposure history in ndd risk assessments and highlight the potential for public health interventions to mitigate these risks. 9. polyglutamine (polyq) diseases: ribosomal proteins and tauopathy polyglutamine (polyq) diseases, such as huntington's disease (hd) and various spinocerebellar ataxias, represent an important yet understudied group of neurodegenerative disorders. these diseases are caused by cag-repeat expansions in different genes and are characterized by neuronal intranuclear inclusions (niis), which play a key role in disease pathology. niis are not only central to polyq disorders but are also observed in normal aging, as evidenced by marinesco bodies in the substantia nigra and locus coeruleus. however, the formation of niis and their precise role in neurodegeneration remain poorly understood, representing a critical gap in knowledge. addressing this gap through focused research on polyq disorders and related inclusions is essential for advancing our understanding of these complex diseases and their broader implications for neurodegeneration. few neuropathological studies focusing specifically on polyq disorders are published each year, but two notable studies were recently conducted. one study by yagita et al. published in neuropathology investigated the ribosomal protein sa (rpsa) in neuronal intranuclear inclusions (niis) associated with polyglutamine (polyq) diseases and marinesco bodies (mbs) in normal aging brains (yagita et al., 2024). utilizing immunohistochemical and biochemical analyses, the researchers examined brain samples from patients with huntington disease (hd), spinocerebellar ataxia type 3 (sca3), and normal elderly controls (ncs). the study revealed that rpsa is a common component of niis in polyq diseases and mbs in normal aging. in polyq diseases, rpsa co-localizes with polyq aggregations and other proteins such as p62, ubiquitin, and huntingtin, forming a mosaic-like distribution within the niis. the nuclear fraction of hd patients' brain samples contained higher levels of rpsa compared to ncs, suggesting an upregulation of rpsa in the diseased state. in contrast, cytoplasmic rpsa expression was reduced in neurons with niis, which could imply sequestration of rpsa into the inclusions, leading to its depletion in the cytoplasm. the study also identified that mbs in the substantia nigra of ncs share similar protein components with niis, including rpsa and p62, indicating common mechanisms may drive the formation of these intranuclear inclusions. these findings highlight the role of ribosomal dysfunction in both neurodegenerative diseases and normal aging, providing new insights into the pathogenesis of polyq diseases and the potential impact of ribosomal proteins in neuronal health and disease. https://doi.org/10.17879/freeneuropathology-2024free neuropathology 5:31 (2024) john f. crary doi: https://doi.org/10.17879/freeneuropathology-2024-5848 page 10 of 12 figure 5. tauopathy in sca8. (a) in the dentate nucleus of the cerebellum, grumose degeneration was observed. (b–h) immunohisto chemistry for p-tau demonstrateed numerous globose-type nfts, threads, and tufted astrocytes in the dentate nucleus (b), internal globus pallidus (c), subthalamic nucleus (d), precentral gyrus (e, f), and cerebellum (g, h). in the precentral gyrus, betz cells were diffusely granular positive for p-tau (f). purkinje cells were also positive for p-tau (h). he staining (a); immunohistochemistry for p-tau (b–h). scale bars: 100 μm in (a–d), 500 μm in (e, g), 25 μm in (f, h). reproduced under the creative commons attribution 4.0 international license (http://creativecommons.org/licenses/by/4.0/). https://doi.org/10.17879/freeneuropathology-2024http://creativecommons.org/licenses/by/4.0/ free neuropathology 5:31 (2024) john f. crary doi: https://doi.org/10.17879/freeneuropathology-2024-5848 page 11 of 12 a second study also published in neuropathology by yonenobu et al. examined tauopathy in spinocerebellar ataxia type 8 (sca8), suggesting that this variant might display unique tauopathic features (yonenobu et al., 2023). the researchers analyzed post-mortem brain samples from patients diagnosed with sca8. sca8 is characterized by neuronal loss and gliosis predominantly in the cerebellum and brainstem, which aligns with the clinical manifestations of the disease. additionally, purkinje cell loss is a consistent finding, accompanied by bergmann gliosis and dentate nucleus degeneration. intriguingly, the investigators uncovered a unique tauopathy. unlike other spinocerebellar ataxias, sca8 exhibited tau-positive neuronal and glial inclusions, particularly in the brainstem and cerebellum. this finding not only expands the neuropathological spectrum of sca8 but also provides new insights into its underlying mechanisms, paving the way for future research on targeted therapeutic strategies. intriguingly, sca8 is an atypical polyglutamine disorder, with the polyq tract encoded from the anti-sense strand, raising a possible explanation for why the tauopathy described for sca8 might be unique. the study emphasizes the importance of recognizing these unique pathological features to improve the diagnosis and understanding of sca8. funding statement this work was supported by funding from the nih (r01ag054008, r01ns095252, r01ag062348, r01ns086736, u54ns115266, u54ns115322) as well as the rainwater charitable trust / tau consortium, the alexander saint amand scholar award, the karen strauss cook research scholar award, and a generous gift from stuart katz and dr. jane martin. conflict of interest the author reports no conflicts of interest. disclosure statement generative artificial intelligence tools, specifically openai's gpt-4, were used in the preparation of this manuscript. these tools assisted in drafting and refining portions of the text. the final content has been reviewed and edited by the author to ensure its accuracy, originality, and compliance with the journal's authorship and integrity guidelines. the author takes full responsibility for the content of the manuscript. references chen, x., firulyova, m., manis, m., herz, j., smirnov, i., aladyeva, e., wang, c., bao, x., finn, m. b., hu, h., shchukina, i., kim, m. w., yuede, c. m., kipnis, j., artyomov, m. n., ulrich, j. d., & holtzman, d. m. 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(2023). cell-type-specific alzheimer’s disease polygenic risk scores are associated with distinct disease processes in alzheimer’s disease. nature communications, 14(1), 7659. https://doi.org/10.1038/s41467-023-43132-2 yonenobu, y., beck, g., kido, k., maeda, n., yamashita, r., inoue, k., saito, y., hasegawa, m., ito, h., hasegawa, k., morii, e., iwaki, t., murayama, s., & mochizuki, h. (2023). neuropathology of spinocerebellar ataxia type 8: common features and unique tauopathy. neuropathology: official journal of the japanese society of neuropathology, 43(5), 351–361. https://doi.org/10.1111/neup.12894 https://doi.org/10.17879/freeneuropathology-2024https://doi.org/10.1186/s40478-023-01649-z https://doi.org/10.1111/neup.12927 https://doi.org/10.1038/s41467-023-43132-2 https://doi.org/10.1111/neup.12894 rewiew 1. chronic traumatic encephalopathy in young athletes’ post-repetitive head impacts 2. hippocampal sclerosis with tdp-43 inclusions following repetitive head impacts 3. longitudinal motor decline in diffuse lewy body disease, parkinson’s disease with dementia, and alzheimer’s disease 4. cell-type-specific alzheimer’s polygenic risk scores 5. histological brain age acceleration using digital neuropathology and artificial intelligence 6. decline in cerebrovascular pathologies over 25 years 7. microglia-t cell interaction in alzheimer’s disease and tauopathy 8. viral exposures linked to neurodegeneration 9. polyglutamine (polyq) diseases: ribosomal proteins and tauopathy funding statement conflict of interest disclosure statement references neurooncology: 2025 update free neuropathology 6:10 (2025) michel mittelbronn doi: https://doi.org/10.17879/freeneuropathology-2025-6316 page 1 of 19 copyright: © 2025 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. neurooncology: 2025 update michel mittelbronn1,2 1 department of life sciences and medicine (dlsm); university of luxembourg; esch sur alzette, luxembourg 2 faculty of science, technology and medicine (fstm), university of luxembourg, esch-sur-alzette, luxembourg corresponding author: michel mittelbronn · department of life sciences and medicine (dlsm) · university of luxembourg · 6, avenue du swing · l-4367 belvaux · luxembourg · michel.mittelbronn@uni.lu submitted: 23 february 2025 · accepted: 17 march 2025 · copyedited by: joão gama · published: 26 march 2025 abstract this collection of studies highlights groundbreaking advancements in brain tumor research, particularly primary cns tumors and brain metastasis. one major focus is the tumor microenvironment, where alterations in cerebral microcirculation and hypoxic-ischemic conditions have been shown to influence metastatic progression. in glioblastoma, recurrent tumors exhibit distinct dna methylation profiles, and global dna methylation has emerged as an independent diagnostic marker for idh-wildtype glioblastoma. a whole-tumor perspective further emphasizes the extensive intratumoral heterogeneity driving glioblastoma evolution. the immune landscape of glioblastoma is another key area of research. cranioencephalic functional lymphoid units have been implicated in tumor progression, while time-dependent single-cell phenotyping offers novel insights into immune cell dynamics within glioblastoma. additionally, histone serotonylation has been identified as a critical epigenetic regulator in ependymoma tumorigenesis. diagnostic and prognostic innovations are paving the way for improved patient care. histomorphological features provide enhanced prognosis prediction for glioblastoma patients. confocal laser microscopy enables real-time intraoperative histopathological diagnostics, and sequencing of cerebrospinal fluid-derived cell-free dna presents a promising non-invasive diagnostic approach. together, these top studies of 2024 underscore the complexity of brain tumor biology and the integration of epigenetics, immune interactions, and advanced diagnostics into clinical practice. these insights mark significant progress toward personalized treatment strategies and improved outcomes in neurooncology. keywords: neurooncology, neuropathology, brain tumors, glioblastoma, brain metastasis introduction artificial intelligence is currently a hot topic and is intended to make work easier for humans. however, there are still numerous challenges in academic literature research, particularly due to hallucinations that lead to the generation of inconsistent or completely incorrect references [albuck et al., 2024]. nevertheless, the author of the current article wanted to give it a try to see whether his work could be simplified through the use of artificial intelligence, or more specifically, large language models review https://doi.org/10.17879/freeneuropathology-2025-6316 https://creativecommons.org/licenses/by/4.0/ https://creativecommons.org/publicdomain/zero/1.0/ mailto:michel.mittelbronn@uni.lu free neuropathology 6:10 (2025) michel mittelbronn doi: https://doi.org/10.17879/freeneuropathology-2025-6316 page 2 of 19 (llms), or at least gain some additional inspiration about interesting primary research articles in the field of neurooncology published in 2024. therefore, the following very generic question was posed to three llms (https://chatgpt.com/; https://gemini.google.com/app and https://www.perplexity.ai): ‘could you please provide me with the 10 most influential primary research papers published in neurooncology with a link to neuropathology in the year 2024?’. chatgpt proposed several non-existent articles, including a review article supposedly authored by the writer of the present review. however, it altered the author list as follows: 'neurooncology: 2023 update'; authors: wisoff jh, sanford ra, heier la, et al. published in: free neuropathology, 2023 [mittelbronn, 2023]. in addition, 5 of the 10 proposed 'primary research papers' were actually podcasts. by rephrasing and adjust ing the prompt, some improvements and correct ions were achieved; however, the results improved only partially and gradually. perplexity suggested exactly the 10 papers from the top 10 series in neurooncology published in free neuropathology last year but re-dated all articles from 2023 to 2024 [mittelbronn, 2024]. after being re-prompted, perplexity explained that it would be inappropriate to speculate or provide inaccurate information about influential papers from 2024. only gemini – both initially and after several attempts or re-prompting – refused to provide some suggestions of real scientific papers, instead recommending journals, search engines such as pubmed (https://pubmed.ncbi.nlm. nih.gov/) or google scholar (https://scholar.google. com/). as a result, unfortunately, not a single of the different llms was useful for the compilation of influential primary scientific papers in the field of neurooncology of this year’s edition. therefore, the author returned to a more traditional approach, screening thousands of titles and abstracts related to key search terms in the field (e.g., brain tumor, glioma, meningioma, brain metastasis, neurooncology, etc.). in contrast to last year, however, fewer novel treatment approaches in neurooncology were identified. of note, with 29 retractions of papers related to the keyword 'glioblastoma' in 2024 (compared to 22 in 2023 and 9 in 2022, and similarly high number of publications), along with multiple corrections, a worrisome increase in potentially sloppy scientific behavior in this field was observed. nevertheless, numerous excellent primary research articles were identified and subjective selection was a challenge. as usual, the goal was to provide a collection of articles ranging from basic research findings to novel diagnostic or prognostic criteria, technical and methodological innovations, and therapeutic approaches. with this, the “top ten” series in neurooncology for 2024 reads as follows: 1. impact of cerebral microcirculation alteration and hypoxic-ischemic microenvironment on brain metastasis development [roesler et al., 2024]. 2. global dna methylation as independent diagnostic marker in idh-wildtype glioblastoma [eckhardt et al., 2024]. 3. change in dna methylation profile in recurrent glioblastoma [drexler et al., 2024]. 4. glioblastoma evolution and heterogeneity from a whole tumor perspective [mathur et al., 2024]. 5. cranioencephalic functional lymphoid units in glioblastoma [dobersalske et al., 2024]. 6. prognosis prediction via histomorphological features in glioblastoma [kirishima et al., 2024]. 7. confocal laser microscopy for intraoperative histopathological diagnostics of intracranial tumors [wagner et al., 2024]. 8. brain tumor diagnostics from cell-free dna from cerebrospinal fluid [afflerbach et al., 2024 & hickman et al., 2024]. 9. time-dependent single-cell phenotyping of immune cells in glioblastoma [kirschenbaum et al., 2024]. 10. histone serotonylation regulates ependymoma tumorigenesis [chen et al., 2024]. https://doi.org/10.17879/freeneuropathology-2025-6316 https://eur01.safelinks.protection.outlook.com/?url=https%3a%2f%2fchatgpt.com%2f&data=05%7c02%7cmichel.mittelbronn%40ext.uni.lu%7cba0ec810e19c4ccdec7d08dd64484652%7c445a9c950f9d49539db1bc4a45dd1220%7c0%7c0%7c638776983780334547%7cunknown%7ctwfpbgzsb3d8eyjfbxb0eu1hcgkionrydwusilyioiiwljaumdawmcisilaioijxaw4zmiisikfoijoitwfpbcisilduijoyfq%3d%3d%7c0%7c%7c%7c&sdata=rshrttsmi9sswrqdpt0ikvgyhoxt8gcgfkgzfnwsrfu%3d&reserved=0 https://gemini.google.com/app https://gemini.google.com/app https://www.perplexity.ai/ free neuropathology 6:10 (2025) michel mittelbronn doi: https://doi.org/10.17879/freeneuropathology-2025-6316 page 3 of 19 1. impact of disturbed cerebral microcirculation and hypoxic-ischemic microenvironment on brain metastasis development [roesler et al., 2024] although the basic steps of brain metastasis formation, including – amongst others – detachment of cancer cells from the primary tumor and intravasation into the blood stream, followed by adhesion to brain endothelial cells and finally extravasation into as well as colonization of the brain is reiterated, the exact mechanisms and time-dependent sequences are not fully understood. in a previous groundbreaking work of kienast et al., the metastatic ways of individual cancer cells could be traced over several months in a murine model with real-time multiphoton laser scanning microscopy [kienast et al., 2010]. with this, the authors could identify the initial arrest of cancer cells at cerebral blood vessel branches followed by angiogenic processes or vessel cooption after early extravasation. using a similar methodological approach, roesler et al. were now able to show a similar intravascular tumor cell arrest in brain microvessels before extravasation and formation of micrometastases [roesler et al., 2024]. of note, as a most novel finding, already 24h after intracardiac injection of tumor cells, prominent hypoxic-ischemic tissue alterations were observed in the brain that were partly well corresponding with areas, in which metastasis formation was detected at a later timepoint. in the hypoxic-ischemic areas, an upregulation of ang-2, mmp9 and vegf was observed in brain endothelial cells leading to the hypothesis that those hypoxiaassociated molecules might be at least partly responsible for the seeding of metastatic cells in the brain. to corroborate this hypothesis, the authors also used a transgenic, endothelial-specific ang-2 gain-of-function approach that revealed an increase of numbers and volumes of brain metastases compared to wild-type animals thereby indicating that extravasation of cancer cells into the brain parenchyma is ang-2 dependent. the findings of the study were in line with previous findings showing that ang-2 expression correlated with bbb disruption, impaired tight junctions and increased blood vessel permeability in a murine triple-negative breast cancer model [avraham et al., 2014]. the findings of roesler et al. indicate that ang-2 and vegf are crucial for the formation of brain metastases via shaping a tumor-supporting pre-metastatic niche, therefore being potential treatment targets, especially in an early phase. with a novel, early pre-metastatic inhibition of ang-2 (via the amg 386 peptibody) and/or vegf (via aflibercept, a fusion protein with vegfr1 and vegfr2 domains serving as a so-called “vegf trap”), the metastatic cerebral tumor cell load could be considerably reduced (see figure 1). similar findings were obtained in other previous murine brain metastasis models in which a combined inhibition of ang-2 and vegf reduced the number and volume of brain metastases while extracranial metastases did not show a positive treatment response [bohn et al., 2017; kovalchuk et al., 2020]. in summary, roesler et al. present a novel mechanism for brain metastatic processes linked to an increase of ang-2 and vegf in the cerebral pre-metastatic niche which are induced by focal hypoxic-ischemic events most probably caused by occlusion of brain capillaries by tumor cells. showing a significantly reduced cerebral tumor cell load by early dual inhibition of ang-2 and vegf, this treatment approach could be highly promising as a prevention strategy in patients suffering from highly malignant tumors with a high risk to develop cerebral metastases. 2. global dna methylation as independent diagnostic marker in idhwildtype glioblastoma [eckhardt et al., 2024] large population-based studies still report median survival times of slightly more than 6 months for glioblastoma patients that could be considerably increased to a median of 14.8 months in cases with maximum treatment including neurosurgical resection followed by radiochemotherapy [brodbelt et al., 2015]. of note, approximately 30 % of all glioblastoma patients survive 2 years or longer after initial diagnosis. apart from age, mgmt promoter methylation, extent of resection and followup treatment, there is not much known about glioblastoma-intrinsic tumor biological factors that might well predict patient prognosis. nowadays, neuropathological diagnostics in the field of neuro https://doi.org/10.17879/freeneuropathology-2025-6316 free neuropathology 6:10 (2025) michel mittelbronn doi: https://doi.org/10.17879/freeneuropathology-2025-6316 page 4 of 19 figure 1: concept of brain metastasis formation and therapeutic prevention oncology is most precisely performed by dna methylation profiling [capper et al., 2018]. within the class of idh wildtype glioblastoma, mainly 3 subclasses could be defined: receptor tyrosine kinase i (rtk i), rtk ii and mesenchymal subclass, however without showing any relevant survival differences [drexler et al., 2023b]. in general, only poor data is available about the potential prognostic impact of methylation differences within distinct tumor entities. to address this question, eckhardt et al. assessed 492 primary and 31 relapse idh wildtype glioblastomas that all received similar standard treatment regimen. although no unequivocally clear methylation signature associated with patient survival could be detected, the median methylation rate of 0.49 significantly split the cohort in long (median methylation above 0.49; median overall survival of 18 months) and short (median methylation level equal or below 0.49; median survival of 13 months) time survivors. the optimal cut-off level was determined as a beta value of 0.458 that allowed for identifying a smaller subgroup of 73 patients with a median overall survival of only 9.2 months. of note, the mean methylation level was an independent prognostic marker in a multivariate analysis together patient age, mgmt promoter methylation status and extent of resection, all factors that are known as prognosticators for a long time. the authors further showed that rtk i subclass glioblastoma had the worst median survival reaching 14.2 months while displaying significantly lower mean methylation levels compared to its counterparts that reached 15 months (rtk ii) and 18.8 months (mesenchymal). however, the mean methylation levels were still significantly associated in rtk i and ii subclasses. combined risk classes including mean methylation level, age, extent of resection and mgmt promoter methylation could further increase the prognostic prediction by splitting the cohort into a risk factor-free subgroup reaching 39 months of median survival while the worst subgroup only reached 8.3 months (figure 2). while immune https://doi.org/10.17879/freeneuropathology-2025-6316 free neuropathology 6:10 (2025) michel mittelbronn doi: https://doi.org/10.17879/freeneuropathology-2025-6316 page 5 of 19 figure 2: risk stratification for idh wildtype glioblastoma patients. patient age over 65 years, extent of less than 90 % of the contrastenhancing tumor area, an unmethylated mgmt promoter and a low mean global methylation beta value of less than 0.49 are considered as negative prognostic factors. median overall survival times are as follows: risk group 1: 39 months; risk group 2: 19 months; risk group 3: 8.3 months. cell infiltration could be bioinformatically ruled out as underlying explanatory factor, higher mean methylation levels were associated with better radiosensitivity, therefore at least providing a hypothesis why the patient subgroup with high beta values survived longer. these findings were also corroborated in a glioma cell line panel exhibiting better radiosensitivity in cells with a higher beta value. in summary, the authors provide a novel, highly important risk stratification for idh wildtype glioblastoma patients that might also pave the way for new treatment stratifications in the future. however, a comparison with other tumor entities could not confirm high methylation levels as being associated with tumor aggressiveness in general. 3. change in dna methylation profile in recurrent glioblastoma [drexler et al., 2024] dna methylation profiling has become a highly reliable and unbiased reproducible diagnostic tool in neuropathological routine diagnostics [capper et al., 2018]. however, only poor data exist about a temporal change of dna methylation upon treatment in malignant brain tumors that might have an impact on treatment resistance. to address this question, drexler et al. assessed paired primary and recurrent glioblastomas of 47 patients by dna methylation from tumor bulk and plasma, mass https://doi.org/10.17879/freeneuropathology-2025-6316 free neuropathology 6:10 (2025) michel mittelbronn doi: https://doi.org/10.17879/freeneuropathology-2025-6316 page 6 of 19 spectrometry and multiplex bead-based immunoassay [drexler et al., 2024]. a potential change even in glioblastoma subclasses might be of importance as previous analyses revealed that mesenchymal, rtk i and rtk ii subclasses differed in patient survival with 15.5 months, 16 months and 27 months, respectively [dejaegher et al., 2021]. in the primary glioblastoma cohort of drexler et al., dna methylation profiling revealed the following subclass distribution: 23.4 % rtk i, 40.4 % rtk ii and 36.2 % mesenchymal while this composition changed to 12.8 % rtk i, 29.8 % rtk ii, 40.4 % mesenchymal and 17 % with no match with a known class (figure 3). in total, 40 % of all recurrent gbm did not match with the subclass obtained in the primary tumors (including 17 % of cases with no match). the highest percentage of transitions was towards the mesenchymal subclass (72.7 %). thirteen percent of all patients showed a switch of the mgmt promoter methylation status, mostly from a methylated to a nonmethylated status, a finding that is in line with previous analyses performed in primary and recurrent glioblastoma [choi et al., 2021]. the authors also analyzed multiple clinical -pathological para meters but mostly found incomplete resection of contrast-enhancing tumor parts as a significant factor for subclass transition in the recurrent situation. while the cellular composition did not considerably vary between the groups with and without subclass change in general, the cohort undergoing a switch towards a mesenchymal phenotype showed for example a decreased number of immune cells in the primary tumor tissue, however did not reach the level of significance when the analyses were repeated for immune cell subtypes. in contrast, higher methylation signatures were observed for monocytes, b cells and neutrophils in blood of patients in the primary situation later showing a subclass switch towards a mesenchymal phenotype compared to the cohort with stable glioblastoma subclass in the figure 3: temporal molecular change between primary and recurrent glioblastoma https://doi.org/10.17879/freeneuropathology-2025-6316 free neuropathology 6:10 (2025) michel mittelbronn doi: https://doi.org/10.17879/freeneuropathology-2025-6316 page 7 of 19 recurrent situation. proteomic analysis revealed a stronger metabolic and catabolic processes in the primary tumor tissue of patients later undergoing a switch to mesenchymal subclass. of note, no association of subclass changes with patient survival was observed. this study is of high importance for the understanding of temporal molecular changes in glioblastoma and underscores the necessity to re-perform full molecular assessment in the recurrent situation, especially as also broadly accepted molecular biomarkers such as mgmt promoter methylation status may change over time. to which degree radiochemotherapy induces the temporal switch towards a mesenchymal subclass phenotype has to be further elucidated. 4. glioblastoma evolution and heterogeneity from a whole tumor perspective [mathur et al., 2024] glioblastoma is a highly heterogenous neoplasm composed of both tumor and non-neoplastic cells of the microenvironment. in addition, little is known about the overall molecular heterogeneity including genetic, epigenetic and / or metabolic features as diagnostics is mostly performed on smaller bulk tissue parts. in a previous large-scale approach, a glioblastoma atlas linking classical histomorphological features with transcriptional profiles, the latter assessed by in-situ hybridization, revealed a strong regional molecular heterogeneity partly related to microscopic characteristics [puchalski et al., 2018]. however, as these previous findings were also derived from preselected tissue blocks not representing the entire regional spectrum of glioblastoma, mathur et al. sampled multiple tissues (n = 103) from the 10 patients using 3d surgical neuronavigation to obtain representative areas of the tumor core, periphery and contrast-enhancing regions [mathur et al., 2024]. furthermore, the biopsy positions were correlated with preoperative mri scans to generate a representative 3d model of the entire glioblastoma. the small biopsy samples were assessed by histopathological analyses, highthroughput chromosome conformation capture (hi-c), whole exome sequencing (wes), rna sequ encing (rna-seq) as well as assay for transposaseaccessible chromatin with high-throughput sequencing (atac-seq, for tissue and single nucleus) (for summary, see figure 4). the authors detected a vast genetic and transcriptional heterogeneity within the patients. only one out of the ten patients showed a consistent signature in all biopsies regarding the previously published, gene expression-based molecular classification into proneural, neural, classic or mesenchymal subtypes while all other patients displayed mixed intraindividual signatures [verhaak et al., 2010]. of note, tumor biopsies from the tumor cores more frequently showed a mesenchymal or classic signature while more peripheral biopsies revealed rather neural or proneural signatures. the authors also discovered distinct neurodevelopmental transcriptomic signatures such as for example glioblastoma with a gain of chromosome 7 (with the egfr, ptprz1 and ptn genes) being associated with glioblastoma cells in an intermediate progenitor cell (ipc) transit-amplifying state or – in contrast glioblastoma with a primitive neuronal component, however without a gain of chromosome 7, showing transcriptional signatures typically observed in cells differentiating from ipcs to neurons. additional signatures in the tumor center comprised an immune-hot microenvironment with interferon signaling and t cell infiltration versus immune-cold areas with a mesenchymal transcriptomic signature. while most mutations were highly heterogenous, chromothripsis seems to be a very early event in gliomagenesis with virtually no intraindividual heterogeneity. most importantly, the authors presented several novel potential diagnostic biomarkers and treatment targets, such as the activator protein 1 (ap-1) pathway or several gene fusions that were missed by clinical ngs analyses as breakpoints were outside exons. with this work, mathur et al. considerably contributed to the understanding of glioblastoma evolution and heterogeneity and provide promising therapeutic targets for further assessment. for more in-depth exploration of the findings, a publicly accessible interactive online platform is available: https://3d-gbms.shin yapps.io/search/. https://doi.org/10.17879/freeneuropathology-2025-6316 free neuropathology 6:10 (2025) michel mittelbronn doi: https://doi.org/10.17879/freeneuropathology-2025-6316 page 8 of 19 figure 4: 3d glioblastoma evolution and heterogeneity: methodological approach and key findings. abbreviations: org: outer radial glia; ipc: intermediate progenitor cell; opc: oligodendrocyte precursor cell; neurod1: neuronal differentiation 1 (transcription factor); ap-1: activator protein 1 (transcription factor). 5. cranioencephalic functional lymphoid units in glioblastoma [dobersalske et al., 2024] bridging veins allow for an immunological communication between cns and skull bone marrow crossing the arachnoid barrier by connecting cns blood vessels with the meningeal blood circulation and finally the sinusoidal vasculature of the bone marrow [smyth et al., 2024]. as hematopoietic stem and progenitor cells are located in the skull bone marrow, however not well studied in the context of human diseases, dobersalske et al. intended to assess the immune cell properties in those areas in regional proximity to human glioblastoma, a condition known for its highly immunosuppressive nature [dobersalske et al., 2024]. using a cxcl12 analog radioligand tracer for cxcr4, a marker for hematopoietic and immune cells, the authors found surprisingly high activity in the ipsilateral skull bone marrow and meninges related to the glioblastoma localization in usually older glioblastoma patients. in a single-cell rna sequencing approach from corresponding (a) glioblastoma samples, (b) ipsilateral bone marrow of the skull, (c) peripheral blood mononuclear cells (pbmcs) and (d) distal hip bone marrow, considerable differences in immune cell compositions were found. regarding the myeloid cell compartment, mostly naïve monocytes were found the ipsilateral skull bone marrow contrasting anti-inflammatory monocytes and macrophages, predominantly found within glioblastoma. of note, a strong accumulation of effector-type, glioma-reactive cd8+ t cell activity was detected in the skull of ipsilateral bone marrow whereas a lower activity was found in https://doi.org/10.17879/freeneuropathology-2025-6316 free neuropathology 6:10 (2025) michel mittelbronn doi: https://doi.org/10.17879/freeneuropathology-2025-6316 page 9 of 19 figure 5: cranioencephalic functional lymphoid units in glioblastoma. left side: methodological set-up. right side: major findings according to compartment. intratumor cd8+ t cells. both cd8+ t cells from pbmcs and ipsilateral bone marrow were able to experimentally reproduce memory and effector t cells, while intratumor t cells failed. of note, the antitumoral activity of skull bone marrow derived cd8+ t cells was higher compared to counterparts derived from pbmcs, distal hip bone marrow or the glioblastoma microenvironment. similarly high frequencies of tumor-reactive t cells were found in skull bone marrow compared to the intratumor microenvironment, indicating an immune cell trafficking between both compartments. using the aforementioned radioligand tracer, a significantly longer survival benefit for glioblastoma patients with stronger intratumor and skull bone marrow immune cell activity was observed already in a relatively small patient cohort (n = 52). taking the clinical impact into account, these findings imply that the skull bone marrow immune cell niches should be absolutely protected during neurosurgical and subsequent radiotherapeutic intervention. in summary, dobersalske et al. shed a new light of the local antitumoral immune cell activities in glioblastoma patients that needs to be considered for future immunotherapies (for summary, see figure 5). 6. prognosis prediction via histomorphological features in glioblastoma [kirishima et al., 2024] neuropathological tumor diagnostics is more and more shifting away from the previous gold standard of microscopic analysis to more unbiased molecular methods mostly including genetic and epigenetic assessment for both diagnostic precision as well as prediction for treatment response and patient survival. as molecular, especially epigenetic features seem to be more stable than the highly variable morphological features in glioblastoma, it is extremely important to always sample enough tissue from all different tumor areas (e.g. core, periphery, contrast-enhancing areas, necroses etc.) [mikkelsen https://doi.org/10.17879/freeneuropathology-2025-6316 free neuropathology 6:10 (2025) michel mittelbronn doi: https://doi.org/10.17879/freeneuropathology-2025-6316 page 10 of 19 et al., 2021]. it is very interesting for neuropathologists if complex histomorphological assessments can still compete with modern molecular diagnostics. kirishima et al. undertake the endeavor to explore if distinct morphological features may still be of prognostic value in glioblastoma. as for today, a few studies showed worst survival for some glioblastoma subtypes such as epithelioid or rhabdoid variants, no unequivocally clear association of any specific morphological phenotype with clinical prognosis could be detected [mallick et al., 2022]. furthermore, most classical microscopic characteristics necessary for the diagnosis of glioblastoma, such as pseudopalisading necrosis or microvascular proliferations, are no longer sufficient for this diagnosis as they might also be found in idhmutant astrocytoma [who classification of brain tumors, 2021]. therefore, the authors of the present study analyze 227 primary glioblastoma according to the who criteria for the classification of tumours of the central nervous system from 2021 and additionally assessed the following microscopic features in 10 % increments: pleomorphic, astrocytic, gemistocytic, rhabdoid, oligodendroglioma (odg)-like, small, giant, spindle, epithelioid, primitive neuronal differentiation(pnd)-like and lipidized [kirishima et al., 2024; who classification of brain tumors, 2021]. for histomorphological assessment to construct a survival prediction model, the largest, most representative areas were analyzed by two board-certified pathologists. of note, also ngs panel analysis for genomic profiling, including assessment of gene mutations, copy number variation and 1p/19q co-deletion as well as methylation-specific pcr to determine mgmt promoter methylation status were performed leading to an integrated final diagnosis together with histological and immunohistochemical (e.g. for idh1-r132h) features. as major morphological variants, pleomorphic (33 % of total tumor area) and astrocytic (29.1 %) differentiation were reported. most interestingly, if glioblastoma showed epithelioid tumor cells in more than 30 % of total tumor cells, patients displayed an unfavorable prognosis independent of clinical parameters, treatment or molecular alterations. the most favorable prognostic values for glioblastoma patients could be achieved by calculating a combined score taking into account both gemistocytic and epithelioid areas as follows: % area of gemistocytic cells minus 4 x % area of epithelioid cells. a value of less than 20 % of this index was an independent significant factor for worse survival in a multivariate analyses together with age, medullary dissemination, chemotherapy and mgmt promoter methylation status (for summary see figure 6). the prediction for patient survival could be further increased by combining this index with the mgmt promoter methylation status, showing worst patient survival for the combination of a low index together with an unmethylated mgmt promoter. as only 6 out of 227 displayed an epithelioid phenotype, the results certainly have to be considered with caution, nevertheless, the prognostic impact of such a differentiation seemed to be strongly associated with negative patient survival even in a multivariate analysis. although a detailed definition of cell morphology is provided by the authors, the reproducibility of such an analysis with still suffer from the subjective interpretation of individual neuropathologist. if this approach should enter the diagnostic routine, future automated digital image analysis would be preferable to undoubtfully recognize and grade the morphological parameters 7. confocal laser microscopy for intraoperative histopathological diagnostics of intracranial tumors [wagner et al., 2024] intraoperative diagnostics performed on frozen sections is time-consuming, often lasting around 20 minutes from tissue sampling out of the surgical cavity to the final transmission of the diagnosis [novis and zarbo, 1997]. in addition, only single, mostly very small tissue fragments can be assessed at a time. even in highly specialized tertiary medical centers, a diagnostic shift of 18 % between intraoperative and post-operative diagnoses was reported, actually leading to a second subsequent neurosurgical intervention in 3 % of all brain tumor patients [harms et al., 2023]. therefore, there is an urgent need for faster methods that can also repeatedly analyze larger tissue areas, ideally in real-time and/or directly in situ. to address this issue, wagner et al. conducted a phase ii clinical study to test the https://doi.org/10.17879/freeneuropathology-2025-6316 free neuropathology 6:10 (2025) michel mittelbronn doi: https://doi.org/10.17879/freeneuropathology-2025-6316 page 11 of 19 figure 6: prognostic prediction of histological features in glioblastoma noninferiority of fluorescein-stained intraoperative confocal laser endomicroscopy (cle) compared to the classic assessment of frozen sections by neuropathologists in 210 adult brain tumor patients. cle (using the zeiss convivo system) is a non-invasive/non-traumatic technique that enables histopathological assessment in vivo almost in real time. in addition, fluorescein sodium acts as a contrast-enhancing intraoperative dye for an easier identification of the tumor-suspicious areas. of note, no serious adverse events were reported, neither in relation to cle nor to fluorescein. compared to the final neuropathological diagnoses, a correct result was obtained in 87 % of cle-assessed samples, while classic frozen sections achieved 91 %, demonstrating the noninferiority of the cle method (figure 7). the concordance rate between cle and classic frozen sections was 76 %. most importantly, the median time until cle diagnosis was communicated was 3 min compared to 27 min in classic frozen sections, resulting in a nearly 10-fold reduction of time required for intraoperative neuropathological assessment. in summary, in vivo cle assessment for intraoperative neuropathological diagnostics is a highly reliable and significantly faster approach that could at least partially replace frozen section diagnostics in the future. in addition, a considerably higher amount of tissue images could be processed thereby further increasing its diagnostic validity. 8. brain tumor diagnostics from cellfree dna from cerebrospinal fluid [afflerbach et al., 2024 and hickman et al., 2024] precise brain tumor diagnostics still relies on highly invasive procedures to obtain tissue samples for microscopic or molecular diagnostics. as high rates of intraoperative adverse events have been reported for neurosurgical interventions reaching up to 40 %, there is a strong need for nonor less invasive diagnostic procedures in neurooncology https://doi.org/10.17879/freeneuropathology-2025-6316 free neuropathology 6:10 (2025) michel mittelbronn doi: https://doi.org/10.17879/freeneuropathology-2025-6316 page 12 of 19 figure 7: diagnostic set-up and major results for clinical phase ii non-inferiority study for intraoperative neuropathological assessment using fluorescein-stained confocal laser endomicroscopy. [drexler et al., 2023a]. ideally, a non-operative tumor classification could even prevent patients from invasive follow-up procedures as in the case of cns lymphoma. a second important reason for why non-invasive diagnostics would considerably improve patient care would be related to the detection of early recurrences or minimal residual tumor load. some studies have shown the suitability of sequencing techniques to detect tumor-specific mutations from cell-free dna of the csf in some tumor entities such as gliomas [miller et al., 2019]. for other brain tumor entities, such as medulloblastoma, non-invasive molecular diagnostics from cell-free dna from cerebrospinal fluid (csf) has already been proven to be superior to cytological diagnostics with regard to both the detection of residual tumor and the prediction of patient prognosis [liu et al., 2021]. to evaluate the suitability of molecular diagnostics on csfderived cell-free dna across broader brain tumor cohorts, different research teams employed various approaches. consequently, two exceptional papers were selected under a single topic. afflerbach et al. analyzed a larger cohort of 110 samples from 99 patients with suspicion of primary brain tumor using nanopore sequencing (at least 5ng dna per sample was needed), a technique which, in addition, also considerably reduces the time needed for sample preparation and sequencing [afflerbach et al., 2024]. in 45 % of the samples, circulating tumor dna (ctdna) could be detected in the csf. among the samples in which ctdna was successfully detected, only the copy number variation (cnv) profile could be defined in 56 % (n = 28), only dna methylation profiling in 12 % (n = 6), and both types of information in 32 % (n = 16). the cnv profile revealed several diagnostically relevant amplifications as well as larger chromosomal gains and losses. of note, corresponding cytological analyses detected tumor cells in only 9 cases. with their groundbreaking study, afflerbach et al. provided evidence that nanopore sequencing is a fast and reliable diagnostic technique allowing for determining both cnv and https://doi.org/10.17879/freeneuropathology-2025-6316 free neuropathology 6:10 (2025) michel mittelbronn doi: https://doi.org/10.17879/freeneuropathology-2025-6316 page 13 of 19 dna methylation profiles from ctdna from csf in brain tumor patients. in another approach, hickman et al. performed mutational analyses of csf circulating ctdna from 711 brain tumor patients using an fda-authorized targeting next-generation sequencing platform [hickman et al., 2024]. similarly to the findings of afflerbach et al., also hickman and colleagues detected tumor-specific mutations in 53 % of all patient samples with clinically documented cns tumors, whereas none of the control samples showed a false positive result, therefore constituting a highly specific diagnostic approach. considering the technical limitations of various approaches, nanopore-based sequencing is particularly constrained by low genomic coverage, making it unsuitable for the reliable detection of point mutations [emiliani et al., 2025]. moreover, since short-read next-generation sequencing techniques have successfully identified tumor-specific mutations with as little as 0.1 ng of cell-free dna from csf, a methodological comparison of different sequencing techniques is essential to determine the most suitable approach [miller et al., 2022]. these approaches will considerably change our initial diagnostic approach and treatment survey in neurooncology in the future. 9. time-dependent single-cell phenotyping of immune cells in glioblastoma [kirschenbaum et al., 2024] single cell rna sequencing techniques revolutionized the understanding of individual cell states about which one could only speculate from data derived from bulk sequencing [ramsköld et al., 2012; tang et al., 2009]. however, it is still challenging to decipher how cell states change over time under distinct conditions, such as for example the effect of the tumor microenvironment on infiltrating immune cells. to address this question, kirschenbaum et al. developed a new method called zman sequencing (“zman” is the phonetic spelling of the hebrew word for “time”) which combines the use of fluorophore pulse labels in the blood as temporal stamps with single cell rna sequencing [kirschenbaum et al., 2024]. the authors intravenously applied four times an identical antibody directed against cd45 that, however, was labeled each time with a different fluorophore (n = 4 in total) in 12h intervals (between 24-60h or 12-48h before mice were sacrificed). after having confirmed that the fluorophore-coupled antibodies (a) almost exclusively stain immune cells in the blood stream, (b) can be reliably detected on immune cells over 96h, (c) do not penetrate the brain and (d) are virtually completely degraded in the blood within 60-90 min, transcriptional molecular trajectories of natural killer (nk) and myeloid cells in the syngeneic gl261 murine glioblastoma model were assessed. one can assume that cd45-positive immune cells labeled with one of the four distinct fluorophores encountering in the glioma tumor microenvironment mostly infiltrated the brain via the blood stream within a time frame of +/12h before the application of the next, subsequent fluorophore-labeled antibody. with this approach, a temporal transcriptional kinetic profile can be calculated for each immune cell type within the glioma microenvironment. of note, primary microglia are mostly not stained by this approach as the intravenously injected fluorophore-coupled antibodies did not penetrate the brain. with this approach, the authors showed that nk cells changed from a cytotoxic to a dysfunctional, and myeloid cells from classic monocytes to im munosuppressive tumor-associated macrophages (tam) via activated tgf-1 signaling within 24h and 36-48h, respectively. as tgf-1 signaling was associated with trem2 activation in immune cells of the myeloid lineage, an immunotherapeutic approach with an anti-trem2 antibody was performed in the gl261 glioma model. this treatment prevented the differentiation to immunosuppressive tams leading to a pro-inflammatory cell state that is supposed to possess a much stronger anti-tumoral properties by recruiting t and nk cells [allen et al., 2017]. with their study, kirschenbaum et al. provide a revolutionary technique allowing for deciphering the transcriptional trajectories of immune cells from the blood stream. they nicely apply this method to assess the impact of the glioma microenvironment on infiltrating immune cells showing that the tumor mostly escapes from an immune attack by inhibiting nk and myeloid cells via tgf-1-activated pathways. finally, they provide a smart initial therapeutic approach by preventing myeloid cells to acquire a pro-tumorigenic cell state applying an anti-trem2 antibody (for summary see figure 8). https://doi.org/10.17879/freeneuropathology-2025-6316 free neuropathology 6:10 (2025) michel mittelbronn doi: https://doi.org/10.17879/freeneuropathology-2025-6316 page 14 of 19 figure 8: time-dependent immune cells transcriptomics in glioblastoma. left side: methodological set-up. right side: major findings of nk and myeloid cell/tam transcriptional trajectories. 10. histone serotonylation regulates ependymoma tumorigenesis [chen et al., 2024] ependymomas constitute a heterogeneous group of cns tumors that are currently classified into at least nine distinct molecular subtypes and are partly considered an epigenetic disorder [stuckert et al., 2020]. as recent studies have revealed a direct epigenetic modification of histone h3 by serotonin, a process termed 'serotonylation,' a potential link between serotonin-induced histone alterations and the development and progression of ependymoma has been hypothesized [farrelly et al., 2019]. to address this hypothesis, chen et al. generated ependymomas by performing in utero electroporation to insert the zfta-rela fusion gene and knock out trp53, a tumor suppressor gene. additionally, a designer receptor exclusively activated by designer drugs (dreadd) approach was employed to modulate neuronal activity in cortical neurons and dorsal raphe nucleus neurons. this was achieved by injecting non-naturally occurring receptors into distinct brain areas via viral vectors, which were subsequently activated by a synthetic ligand. the authors showed that the activation of excitatory cortical neurons promoted the development of zfta-rela fusion-driven ependymomas and identified serotonergic activity in the dorsal raphe nucleus – an area remote from the ependymoma – as a suppressive factor for ependymoma progression. however, established, both murine and human ependymoma cells also displayed a considerable higher amount of serotonylated h3 compared to normal cns tissue and histone serotonylation was associated with tumor progression, providing evidence that serotonin signaling may have a rather disparate role in ependymoma tumor biology. in the experimental setting, https://doi.org/10.17879/freeneuropathology-2025-6316 free neuropathology 6:10 (2025) michel mittelbronn doi: https://doi.org/10.17879/freeneuropathology-2025-6316 page 15 of 19 figure 9: disparate effects of serotonin in ependymoma tumorigenesis. on the one hand, serotonin signaling from the dorsal raphe nucleus inhibits remote ependymoma growth, most likely by reducing neuronal activity in the tumor microenvironment. on the other hand, tumor cell-intrinsic serotonylation promotes ependymoma growth through epigenetic modification and subsequent etv5 overexpression, ultimately leading to neuropeptide y (npy) inhibition. as a result, the inhibitory effect of npy on brain activity and its tumor-suppressive function are abolished. ets transcription variant factor 5 (etv5), a transcription factor with binding sites for both serotonylated h3 and the zfta-rela fusion, was identified as a tumor-promoting factor. the authors further demonstrated that etv5 was associated with a repressive h3k27 trimethylation signature and decreased neuropeptide y (npy) expression in ependymoma. moreover, etv5 overexpression abrogated npy-mediated synaptic inhibition in the tumor microenvironment, which was linked to reduced tumor growth (figure 9). discussion the body of research published in 2024 reveals significant advancements in the understanding of glioblastoma and brain metastasis, with a particular emphasis on tumor heterogeneity, diagnostic advancements, and therapeutic implications. the studies covered in this overview contribute to refining our approaches to both the molecular and clinical management of brain tumors, highlighting critical challenges and innovative solutions. a major theme emerging from this year's literature is the growing recognition of the microenvironment's role in tumor progression. roesler et al. demonstrate the pivotal impact of altered cerebral microcirculation and a hypoxic-ischemic microenvironment in the development of brain metastases [roesler et al., 2024]. their findings underscore the importance of understanding how changes in the vascular network and oxygen availability influence tumor spread, highhttps://doi.org/10.17879/freeneuropathology-2025-6316 free neuropathology 6:10 (2025) michel mittelbronn doi: https://doi.org/10.17879/freeneuropathology-2025-6316 page 16 of 19 lighting potential targets for therapeutic intervention, especially using prevention strategies inhibiting brain metastasis seeding by combined inhibition of vegf and ang-2. this approach will change our therapeutic view as oncological patients are usually treated when symptoms or lesion occur. on the primary malignant brain tumor side, mathur et al. explored glioblastoma's evolution and heterogeneity from a whole tumor perspective [mathur et al., 2024]. the dynamic nature of glioblastoma, with its diverse cellular populations and the continuous evolution of its genetic and epigenetic landscape, presents a substantial challenge for both diagnosis and treatment, however there is still a considerable debate about how hierarchical or rather reversible such an evolution is, the latter more speaking in favor of highly dynamic, tumor-intrinsic plasticity [dirske et al., 2019]. dna methylation continues to emerge as a crucial mechanism in brain tumor biology. drexler et al. and eckhardt et al. both highlight the significance of dna methylation profiles in glioblastoma [drexler et al., 2024; eckhardt et al., 2024]. drexler et al. demonstrate the alterations in dna methylation in recurrent glioblastoma, providing valuable insights into the epigenetic shifts that occur during disease progression, especially that methylation classifier subclasses have to be considered with caution as they may considerably change over time. meanwhile, eckhardt et al. present global dna methylation as an independent diagnostic marker for idh-wildtype glioblastoma, offering a potential non-invasive tool for early diagnosis and prognosis. of note, they provide an easily applicable risk score that can be integrated into routine diagnostic procedures, offering significant prognostic value by distinguishing between a high-risk group with a median survival of only 8.3 months and a lowrisk group with a median survival of 39 months. these findings underscore the growing interest in epigenetic biomarkers for glioblastoma, which may ultimately improve diagnostic accuracy and allow for more personalized therapeutic strategies. in addition to dna methylation, histopathological and molecular features are increasingly used to predict prognosis. kirishima et al. demonstrate how histomorphological features can provide valuable prognostic information in glioblastoma, supporting the role of tissue-based diagnostics in clinical settings [kirishima et al., 2024]. a considerable limitation of this study is that morphological criteria – despite being carefully and precisely defined – are still subject to interpretation bias. this will need to be considered in the future when using artificial intelligence algorithms applied to digital images, which are often annotated by human (neuro)pathologists during the training phase. the utilization of cuttingedge technologies such as confocal laser microscopy in intraoperative diagnostics, as outlined by wagner et al., further enhances the speed and hopefully also the precision in the future of tumor identification and delineation, offering real-time guidance during surgery and improving patient outcomes [wagner et al., 2024]. this technological advancement, alongside the development of non-invasive diagnostic techniques like those reported by afflerbach et al. and hickman et al., who used sequencing techniques of cell-free dna from cerebrospinal fluid, marks a significant step forward in the non-invasive diagnosis and monitoring of brain tumors [afflerbach et al., 2024; hickman et al., 2024]. their work presents exciting prospects for liquid biopsy as a means to detect and track tumor progression in real-time. immune cell dynamics are also a focal point of recent studies. kirschenbaum et al. explore the time-dependent phenotyping of immune cells in glioblastoma, providing valuable insights into the temporal changes in immune cell populations within the tumor microenvironment. these findings could have significant implications for immunotherapy, as the ability to track immune cell profiles over time may inform the development of more targeted immune-based therapies [kirschenbaum et al., 2024]. however, at least as important as the scientific findings of this study is the technical development of tracking immune cells through time-dependent transcriptomics using the novel zman sequencing technique. at present, this approach is primarily suited for cells infiltrating other tissue areas, previously labeled via the bloodstream. an important next step in the development of time-dependent transcriptomics would be expanding its applicability to cells that remain within a distinct cellular collective, such as brain tumor cells. this ties in with the work of dobersalske et al., who investigated the role of cranioencephalic functional lymphoid units in glioblastoma, expanding our understanding of how immune surveillance and local immune responses may influence tumor behavior and response to https://doi.org/10.17879/freeneuropathology-2025-6316 free neuropathology 6:10 (2025) michel mittelbronn doi: https://doi.org/10.17879/freeneuropathology-2025-6316 page 17 of 19 treatment [dobersalske et al., 2024]. to further decipher the temporal dynamics of 'glymphatic' drainage, zman sequencing could also be of interest in expanding upon the findings of the study by dobersalske et al. finally, the role of post-translational modifications in tumorigenesis is underscored by the study of chen et al., which examines histone serotonylation in ependymoma tumorigenesis [chen et al., 2024]. this work highlights how serotonin-mediated alterations in histone modifications can regulate tumor growth and suggests that targeting epigenetic regulators may offer a novel therapeutic avenue for ependymomas and other brain tumors. it remains to be determined if serotonylation will be a reproducible, important factor in brain tumor progression in the future, however there is an increasing body of evidence that a crosstalk between neurons and brain tumor might be implicated [venkataramani et al., 2025]. together, these studies reflect a shift toward more comprehensive, multifactorial approaches to understanding and treating brain tumors. in general, there currently seems to be a disproportion between scientific studies focusing on the 'bad guys' – the brain tumor cells that proliferate, migrate, and infiltrate – and the large number of studies examining the tumor microenvironment. while the microenvironment may contribute to tumor progression, it lacks the malignant oncogenic driver mutations. it may be easier to study tumor microenvironmental factors, possibly due to superior methodological tools and simpler tracking methods, but it is crucial not to lose sight of tumor cells as the main players in future research. the integration of molecular and immune profiling, advanced imaging technologies, and epigenetic markers has the potential to revolutionize both the diagnosis and treatment of primary and secondary brain tumors. as our understanding of the complex interplay between tumor biology, the microenvironment, and the immune system deepens, we can anticipate more targeted, individualized therapies that may significantly improve patient outcomes in the future. acknowledgements the figures were prepared using https://biorender.com figure creation tool. single images using for the figures were freely available tagged with creative common license. funding statement mm would like to thank the luxembourg national research fund (fnr) for the support (fnr pearl 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https://doi.org/10.1093/neuonc/noab106 https://doi.org/10.17879/freeneuropathology-2025https://doi.org/10.4103/0028-3886.359222 https://doi.org/10.1016/j.cell.2023.12.013 https://doi.org/10.1007/s00701-020-04608-y https://doi.org/10.1038/s41586-019-0882-3 https://doi.org/10.1093/neuonc/noac035 https://doi.org/10.17879/freeneuropathology-2023-4692 https://doi.org/10.17879/freeneuropathology-2024-5809 https://pubmed.ncbi.nlm.nih.gov/9199619/ https://doi.org/10.1126/science.aaf2666 https://doi.org/10.1038/nbt.2282 https://doi.org/10.1093/neuonc/noae094 https://doi.org/10.1038/s41586-023-06993-7 https://doi.org/10.1007/s11060-020-03562-0 https://doi.org/10.1038/nmeth.1315 https://doi.org/10.1158/2159-8290.cd-24-0194 https://doi.org/10.1016/j.ccr.2009.12.020 https://doi.org/10.1093/neuonc/noae006 https://doi.org/10.1093/neuonc/noab106 review introduction 1. impact of disturbed cerebral microcirculation and hypoxic-ischemic microenvironment on brain metastasis development [roesler et al., 2024] 2. global dna methylation as independent diagnostic marker in idh-wildtype glioblastoma [eckhardt et al., 2024] 3. change in dna methylation profile in recurrent glioblastoma [drexler et al., 2024] 4. glioblastoma evolution and heterogeneity from a whole tumor perspective [mathur et al., 2024] 5. cranioencephalic functional lymphoid units in glioblastoma [dober-salske et al., 2024] 7. confocal laser microscopy for intraoperative histopathological diagnostics of intracranial tumors [wagner et al., 2024] 8. brain tumor diagnostics from cell-free dna from cerebrospinal fluid [afflerbach et al., 2024 and hickman et al., 2024] 9. time-dependent single-cell phenotyping of immune cells in glioblastoma [kirschenbaum et al., 2024] 10. histone serotonylation regulates ependymoma tumorigenesis [chen et al., 2024] discussion acknowledgements funding statement conflict of interest references free neuropathology: a bibliometric impact analysis feel free to add comments by clicking these icons on the sidebar free neuropathology 6:22 (2025) editorial free neuropathology: a bibliometric impact analysis georg haase1, marta margeta2, ralf mersmann3, werner paulus3 inserm and aix-marseille university, 13005 marseille, france department of pathology, university of california san francisco, san francisco, ca 94143, usa institute of neuropathology, university hospital münster, 48149 münster, germany corresponding authors: georg haase · inserm and aix-marseille university · 13005 marseille · france georg.haase@univ-amu.fr ralf mersmann · institute of neuropathology · university hospital münster · 48149 münster · germany ralf.mersmann@ukmuenster.de https://doi.org/10.17879/freeneuropathology-2025-9124 keywords: bibliometry, impact factor, scopus, google scholar, citation index introduction free neuropathology (fnp) is a scientific journal that covers all aspects of human and experimental neuropathology, including morphological or molecular analyses of tissues, cells, biofluids, and other biospecimens. fnp has several unique features: it is free for both authors and readers, free from publisher demands, and free from excessive formal requirements; it encourages free opinion by publishing opinion, reflection and flashback papers in addition to more traditional categories of papers; it publishes the annual proceedings of several neuropathological societies; and it has a very rapid turnaround time from paper submission to publication. since its launch in 2020, fnp has published a total of 154 papers at a rate of 24 to 35 papers per year. to evaluate the scientific impact of fnp, we here performed a detailed bibliometric analysis using the fnp, pubmedcentral (pmc), scopus and google scholar databases. impact, not impact factor the most frequently used parameter to measure a journal's impact is the impact factor (if). the impact factor, first proposed by eugene garfield in 1955 [1], is defined as the yearly number of citations of papers that were published in the previous two years, divided by the number of papers (or citable items) published by the journal in the same two-year period [2]. the impact factor is published yearly by clarivate, a commercial non-academic organization formerly known as the institute for scientific information. the if is based on the clarivate’s proprietary database web of science, which covers more than 21 000 journals and more than 90 million indexed articles [3]. free neuropathology has not yet received an impact factor since fnp papers are not yet indexed in the web of science. as an alternative to the impact factor, we used the bibliometric index citescore [4]. the citescore is based on the same principle as the if but is calculated differently, i.e. as the sum of citations received by papers over a four year-period divided by the sum of papers published in the journal over the same period [5]. the citescore is published yearly by elsevier for more than 23 000 journals indexed in the scopus database [6] and is updated monthly by citescore tracker [4]. the citescore of fnp was 2.8 in 2023 and 3.8 in 2024 (fig. 1a), indicating an upward trajectory of fnp’s scientific impact over time. however, many fnp papers 38 out of the 154 were not indexed in the scopus database (fig. 1b). these hidden fnp papers comprised meeting abstracts and editorials, as expected, but also citable items such as original papers and reviews, probably due to incomplete indexing in scopus (fig. 1b) as reported elsewhere [4, 5]. to overcome this issue, we turned to the google scholar database [7, 8], which was found to contain all 154 published fnp papers (fig. 1b). fig. 1. citation of fnp papers. a. citescore values for fnp from 2020 to 2024. the citescore is defined as the number of a journal’s citations during a four-year period divided by the number of citable items published during the same period. b. number of fnp papers (in black) indexed in the scopus database (in pink) or the google scholar database (in green). c. cumulative plot showing the number of citations received by fnp papers. fnp papers published in 2020 were cited 232 times by 2024. fnp papers published in 2021 or before were cited 502 times by 2024. taken together, published fnp papers received a total of 669 citations by 2024. d. top 15 cited fnp papers according to google scholar and the number of their citations. using google scholar, we determined that fnp papers received a total of 669 citations until 2024, with a progressive increase in citations over time (fig. 1c). the hitherto most cited fnp paper with 149 citations is entitled ‘neuropathology of covid-19 (neuro-covid): clinicopathological update’ by lou et al. (2021) [9], reflecting the high impact of the covid-19 pandemic on the field of neuropathology (fig. 1d). the other most cited fnp papers concern neurodegenerative diseases with an emphasis on alzheimer disease, brain tumors, and neuroinflammatory diseases including multiple sclerosis (fig. 1d). taken together, the 15 top cited fnp papers received 466 citations, meaning that about 10 % of the published fnp papers account for 70 % of the journal’s citations. this is comparable to the journal nature, where the 25 % most cited papers contributed 89 % citations to its impact factor of 32.1 in 2005 [10, 11]. electronic access of fnp papers in addition to the citation analysis, we evaluated the extent to which fnp papers were electronically accessed. we defined access to an fnp paper as any electronic consultation of its html full text or download of its pdf. this operational definition does not discriminate between partial and complete reads or between single and multiple accesses by the same user (i.e., ip address), which should however not significantly change the outcome of the analysis. we analyzed access to all 154 fnp papers published from 1st january 2020 to 31st december 2024 through either the fnp website or the pubmed/pmc website. we found that fnp papers were accessed 407 000 times in total, reflecting a mean number of 2 647 accesses to each individual paper. among the 15 most accessed papers (fig. 2a), there was one entitled "aβ plaques" by walker (2020) [12] with 23 573 accesses and another one entitled "multiple sclerosis: 2023 update" by kuhlmann et al. (2023) [13] with 12 250 accesses. we wondered whether these papers were primarily accessed through the journal's website or through pubmed/pmc which has been indexing fnp papers since may 2023. we found that the monthly number of accesses has been significantly increased by pubmed/pmc (fig. 2b–c), rewarding the long-term efforts to get fnp papers indexed in the pmc database. however, electronic access via the fnp website remained considerable, suggesting that the fnp website will continue to be a major gateway to fnp papers in the future. fig. 2. electronic consultation of fnp papers. a. the 15 most accessed fnp papers with the corresponding number of accesses. b and c. access over time to the two most accessed fnp papers from 2020 (b) and 2023 (c) via the fnp website (in dark red) or the pmc website (in light red). access to fnp papers was analyzed through an excel file merging five separate datasets: (1) an fnp dataset comprising author name, title, publication date, document identifier (doi) and pmc id, (2) an fnp dataset comprising global bibliographic metrics for each paper, (3) an fnp dataset comprising monthly bibliographic metrics, (4) a pmc dataset comprising monthly bibliographic metrics, and (5) a google scholar dataset comprising bibliographic metrics. fnp research topics fnp aims to publish research papers across the entire spectrum of neuropathology. we therefore determined how different neuropathology research topics were covered by fnp through original papers or reviews (fig. 3a–b). we found that the number of papers (not accesses) concerning neurooncology and neurodegeneration corresponded to approximately 29 % and 23 % of all papers, respectively. fifteen percent of fnp papers focused on neuroinflammation, primarily on the neuropathology of covid-19. papers concerning neuromuscular, neurodevelopmental, neurovascular, or neurotrauma topics represented between 6 % and 9 % of papers, while 2 % of papers concerned epilepsy (fig. 3a). all research topics were equally well accessed, achieving a mean of about 3 000 to 4 500 accesses per topic (fig. 3b). thus, fnp papers efficiently cover the entire spectrum of neuropathology. fig. 3. fnp papers per topic. a. percentage of fnp papers concerning different neuropathology topics. abstracts from neuropathological societies and papers that cover personal recollections, flashbacks, or neuropathological techniques are not included. b. mean number of accesses to fnp papers per neuropathological topic. categories of fnp papers fnp papers fall into traditional categories (reviews, original papers, letters, and case reports), as well as less traditional categories such as opinion papers, reflection papers, flashback papers and meeting proceedings. we found that original papers were well consulted with a mean of about 2 500 accesses per paper (fig. 4a). fnp reviews were highly accessed, with clinicopathological updates, which achieved a mean of 7 500 accesses per paper, having a particularly large impact (fig. 4a). remarkably, opinion, reflection, and flashback papers also received a wide attention (fig. 4a). for instance, the opinion paper by cevik et al. on covid-19 neuropathology [14], the reflection paper by budka on neuropathology through the ages [15], and the flashback paper by kasper on taylor's focal cortical dysplasia [16] were each accessed more than 3 500 times (not shown). furthermore, we found that the annual meeting proceedings (abstracts) of the german, french, canadian, indian, and australian/new zealand neuropathological societies were accessed a mean of 900 times (fig. 4a–b). fig. 4. consultation of fnp papers belonging to different categories. a. mean number of accesses to fnp papers in each publication category. b. accesses to abstracts of the annual meetings of the german, french, canadian, indian/asian and australian/new zealand neuropathological societies, as a fraction of the total. c. the number of accesses (x-axis) and the number of citations (y-axis) are significantly correlated (r = 0.696). two papers with a very large number of citations (see d) are excluded from the graph for clarity. d. number of accesses (x-axis) and citations (y-axis) for fnp papers representing clinicopathological updates (turquoise), top ten papers (orange), or other categories (black). the ratio between the number of citations and the number of accesses differs significantly among the three categories of papers (p < 0.01 by mann-whitney test). the analysis included only the papers published prior to 2023 to ensure at least two years of data. finally, we analyzed the extent to which papers of the different categories were both accessed and cited. overall, the access and citation numbers were strongly correlated (r = 0.696), (fig. 2c). however, significant differences were observed between categories. indeed, clinicopathological updates received a mean of 6 citations per 1 000 accesses, whereas annual research updates (top ten papers) received tenfold fewer citations (0.6 citations per 1 000 accesses). papers in all other categories had a mean of 1.6 citations per 1 000 accesses (p < 0.01, mann-whitney test), (fig. 4d). we conclude that clinicopathological updates, which generally focus on a specific disease or scientific question, are both highly accessed and highly cited, indicating their continuing relevance for researchers working in a particular field. by contrast, annual research updates (top ten papers), although widely consulted, are not frequently cited, perhaps due to their annual focus and primarily educational nature. conclusions we demonstrate that free neuropathology (fnp) is now an established, widely read, and broadly accepted journal that covers all aspects of neuropathology. we hope that the detailed bibliometric data presented here will be useful to readers, reviewers, potential contributors, and neuropathological societies, thereby sparking additional interest in this unique scientific journal. 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https://doi.org/10.17879/freeneuropathology-2021-3324. pmc10240934. copyright: © 2025 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. northern lights neuroscience symposium 2024 feel free to add comments by clicking these icons on the sidebar free neuropathology 5:30 (2024) meeting abstracts northern lights neuroscience symposium 2024 expanding spectrum of common dementia disorders meeting abstracts september 26–27, 2024   the northern lights neuroscience symposium 2024 “expanding spectrum of common dementia disorders” was held in hanasaari, helsinki (espoo), finland on september 26–27, 2024. the meeting was jointly organised by the scandinavian neuropathological society (chair olivera casar-borota) and university of helsinki. drs. liisa myllykangas (chair), olli tynninen, maria gardberg and tuomas rauramaa made up the organising committee. the event brought together neuropathologists, clinicians and neuroscientists from nine different counties. in total, 91 people had registered for the meeting, and there were 29 abstracts. the keynote lectures on neuropathological and clinical aspects of new dementia entities such as late and part as well as subtypes of ad and lbd, vascular and mixed patologies, were given by irina alafuzoff, peter nelson, david wolk, gabor kovacs, melissa murray, jacob vogel, liisa myllykangas, per borghammer, johannes attems and masafumi ihara. the patrick sourander lecture “investigating choroid plexus and cerebrospinal fluid roles and functions in neurologic disease“ was presented by maria lehtinen from harvard medical school. seven additional oral presentations (selected from submitted abstracts) were also given. the participants were highly appreciative of the meeting’s top level scientific content and of the networking event, which included a finnish sauna, a cruise in the helsinki archipelago and a dinner in the center of helsinki.   https://doi.org/10.17879/freeneuropathology-2024-6108 keywords: scandinavian neuropathological society, meeting abstracts, northern lights neuroscience symposium 2024 meeting abstracts irina alafuzoff expanding spectrum of dementia disorders remarh bsoul et al rt-quic with universal control fluid: standardized detection of alpha-synucleinopathies johannes attems mixed pathologies of the ageing brain emmilotta backman et al nigral neuroinflammation and dopaminergic neurons in pd, msa and psp: a comparative clinicopathological study per borghammer brain-first vs. body-first types of lewy body disease – clinical, imaging, and postmortem data emilia brandt et al gut microbiome changes in patients with idiopathic normal pressure hydrocephalus kia colangelo et al artificial intelligence algorithm for studying arteriolosclerosis in a finnish population-based study of the oldest-old (vantaa85+) henric ek olofsson et al microvascular brain raspberries: a clinicopathological study elisabet englund et al cardiac alpha-synuclein may promote sudden cardiac death in cases with lewy body disease benjamin englert et al ageing-related tau astrogliopathy in the oldest old population (vantaa 85+) maria gotkiewicz et al 3d microscopy unravels details in amyloid plaque-microglia interaction in app/ps1 mice masafumi ihara et al cadasil, the leading genetic cause of small vessel disease karri kaivola et al genetic subgroups in lewy body dementia: the influence of alzheimer's pathology ville kivistö et al association of cardiac sympathetic denervation with lewy pathology and its progression patterns eloise kok et al lewy-related pathology in the tampere sudden death study gabor g. kovacs update on tau-related conditions maria k. lehtinen investigating choroid plexus and cerebrospinal fluid roles and functions in neurologic disease sylwia libard et al neuropathologic change in olfactory bulb and nerve of subjects with covid19 infection elizaveta mikhailenko et al late-nc in the very elderly – the population-based vantaa 85+ study sara r. dunlop et al glial activation and tdp-43 differences among neuropathologic subtypes of alzheimer’s disease liisa myllykangas neuropathological subtypes of lewy body disease: evidence from a population-based study pete nelson disease-driving mechanisms of late-nc mithilesh prakash et al evaluating the impact of data harmonization on multi-site simulated neurodegeneration dataset analysis eino solje et al tdp-43 pathology in frontal cortical brain biopsies of normal pressure hydrocephalus patients pernilla syrjä et al cns amyloid patterns in aging canines – a pilotstudy regarding neuropathology in canine cognitive dysfunction matti tyrväinen et al common associations of transcranial magnetic stimulation (tms) parameters and behavioral symptoms in different neurocognitive diseases vladyslav vadymovych tkach et al digital morphological profiling reveals regionaland disease-specific differences of glial cells in neurodegenerative proteinopathies jacob vogel et al an update on alzheimer’s disease tau subtypes: clinical and biological insights david a. wolk clinical and imaging signatures of late: developing a diagnostic framework     free neuropathol 5:30:5 expanding spectrum of dementia disorders irina alafuzoff1 uppsala university hospital, uppsala, sweden during the last 40–50 years our knowledge regarding aging related cognitive impairment has evolved significantly. in 80s clinicians primarily referred to a senile dementia, a malady of the aged. the cause of this disease was mostly unknown. first international conference on alzheimer's disease and related disorders was organized by professors khalid iqbal, henry wisniewski and bengt winblad in 1988 in las vegas, usa. the total number of participants was quite sparse, most being clinicians and neuropathologist. at this time from the view of a neuropathologist the primary topics discussed were 1. how many plaques are needed for a diagnosis of alzheimer’s disease and 2. which is the primary lesion seen in the brain, amyloid β-protein or hyperphosphorylated τ? currently, several different conferences are organized yearly with thousands of participants from various fields of neuroscience. our knowledge regarding the neuropathology seen in subjects with aging related cognitive impairment has evolved significantly. this has been facilitated by the identification of proteins that are altered in the brain of aged. the insight that these protein alterations are initially seen in certain brain regions and progress following certain neuroanatomical connections has broadened our understanding. it has also been acknowledged that in some cases the disease is inherited based on genetic alterations leading to ample use of transgenic animals in research. sadly, we still are quite far from curative treatments of these maladies. recently it has become clear that in many aged, not few but rather several protein alterations are to be found in the brain and even this tendency seems to increase with age. a major obstacle today is limited number of human brain tissue available for research. the autopsy frequencies are extremely low and moreover the brain is seldom assessed postmortem. many studies can certainly be carried out in animal models. however, in line with the fruitful research carried out on tumors, where the use of surgical human samples has led to substantial results, also neurodegeneration needs to be studied in human setting. this is certainly needed to decrypt these complex diseases and hopefully find efficient treatment strategies improving the life quality of the affected subjects.   free neuropathol 5:30:6 rt-quic with universal control fluid: standardized detection of alpha-synucleinopathies remarh bsoul1, eva løbner lund1, kristian steen frederiksen1, sara brynhild winther bech2, anja hviid simonsen1, kirsten svenstrup2, aušrinė areškevičiūtė1 copenhagen university hospital, rigshospitalet, copenhagen, denmark bispebjerg and frederiksberg hospital, copenhagen, denmark we present a csf rt-quic-αsyn protocol that is relatively easy to adapt, performs stably, and enables a uniform preparation of both sample and control reactions. we adopted and modified rt-quic-αsyn method described by others. our protocol's sensitivity and specificity was estimated by testing csf samples from parkinson´s (pd) (n = 12), dementia with lewy-body (dlb) (n = 12), alzheimer´s (ad) (n = 12), motor neuron disease (mnd) (n = 15), multiple system atrophy (msa) (n = 18) and healthy controls (hc) (n = 12). we tested 3 volumes of each csf sample in quadruplicates to assess the correlation between added to the reaction csf volume and αsyn seeding efficiency. furthermore, we developed a universal control fluid (ucf) to standardize rt-quic reaction environment across all samples and controls by creating and testing various ucf compositions and sample/control reaction settings. our rt-quic-αsyn performs with > 95 % sensitivity for pd and dlb, > 95 % specificity when compared to ad and mnd, and 100 % specificity when compared to nc; whereas αsyn in msa samples was undetectable. we have defined a ucf composition that does not compromise rt-quic reactions' effectivity and gives 100 % sensitivity and specificity when comparing unseeded ucf and ucf seeded with patient material. the ucf has also been tested as a csf diluent and is suitable for broadening the seeding detection range while keeping the assay environment uniform. rt-quic-αsyn is a highly sensitive and specific clinical biomarker providing pd/dlb diagnosis within 48 hours, and distinguishing it from msa. rt-quic-αsyn with ucf has the potential for a standardized diagnostic protocol across laboratories.   free neuropathol 5:30:7 mixed pathologies of the ageing brain johannes attems1 translational and clinical research institute, newcastle university, newcastle upon tyne, united kingdom the defining neuropathological features of age associated neurodegenerative diseases are aggregations of misfolded proteins and the neuropathological diagnosis is based on the semiquantitative assessment of these misfolded proteins that constitute the neuropathological hallmark lesion for the respective disease: e.g. alzheimer's disease (ad), amyloid-β (aβ) hyperphosphorylated tau (tau); lewy body diseases, α-synuclein (α-syn); limbic-predominant age-related tdp-43 encephalopathy (late), tdp-43; frontotemporal lobar degeneration, tau or tdp-43 or ubiquitin or fus. in addition, cerebrovascular lesions are assessed for the diagnosis of cerebrovascular disease. however, in brains of elderly patients suffering from neurodegenerative diseases mixed pathologies are usually present and various amounts of neurodegenerative and cerebrovascular pathology are frequently seen even in brains of non-demented elderly. it does indeed become increasingly clear that the clinical picture of dementia in most aged patients results from mixed pathologies rather than from one single disease. importantly, these mixed pathologies are often not detected clinically, which has a detrimental impact on clinical studies since apparently homogeneous study cohorts (e.g. ad) are likely to be heterogeneous (e.g. ad only, ad and α-syn, ad and tdp-43), which in turn introduces a bias into therapeutic trials and biomarker/ imaging studies. hence, wherever possible clinical studies should ideally involve neuropathological post mortem assessment to correlate clinical with neuropathological data as this will enable a more accurate stratification of clinical cohorts according to the presence of multiple pathologies. this is crucial for a meaningful interpretation of clinical data on biomarkers and therapeutic effects.   free neuropathol 5:30:8 nigral neuroinflammation and dopaminergic neurons in pd, msa and psp: a comparative clinicopathological study emmilotta backman1,2, maria gardberg3, laura luntamo1,2, markus peurla4, tero vahlberg5, per borghammer6,7, nadia stefanova8, gregor wenning8, valtteri kaasinen1,2 clinical neurosciences, university of turku, turku, finland neurocenter, turku university hospital, turku, finland tyks laboratories, pathology, turku university hospital and institute of biomedicine, university of turku, turku, finland institute of biomedicine, university of turku, turku, finland department of biostatistics, university of turku and turku university hospital, turku, finland department of nuclear medicine and pet, aarhus university hospital, aarhus, denmark department of clinical medicine, aarhus university, aarhus, denmark division of neurobiology, department of neurology, medical university innsbruck, innsbruck, austria objective: to understand the complex interplay between neuroinflammation and clinical characteristics in parkinson’s disease (pd), multiple system atrophy (msa) and progressive supranuclear palsy (psp). materials and methods: postmortem neuropathological samples were obtained from 79 individuals (pd, n = 38; psp, n = 15; msa, n = 14; and controls, n = 12). the numbers and densities of snc tyrosine hydroxylase (th)-positive neurons and t cells (cd3+, cd4+ and cd8+) and iba1 expression (a microglial marker) were assessed in both the snc and crus cerebri. clinical data were collected from patient histories. results: psp patients had 89-212 % more nigral cd3+, cd4+, and cd8+ t cells than msa patients (p < 0.04), 125–178 % more cd3+ and cd4+ t cells than healthy controls (p < 0.002), and 95 % more cd4+ t cells than pd patients (p = 0.001). iba1 expression in the snc was significantly higher in pd patients than in msa patients (p = 0.004), while no significant variations were observed across other conditions. psp and msa patients had 58–67 % fewer snc th+ neurons than pd patients (p < 0.05) and 73–78 % fewer than healthy controls (p < 0.001). disease duration from symptom onset to death was negatively associated with snc cd3+ t-cell density across groups (p = 0.002). nigral dopaminergic neuronal density correlated positively with neuroinflammatory markers in pd. conclusions: our findings reveal significant and distinctive t-cell-mediated neuroinflammatory activity within the snc in psp patients, and substantial losses of snc dopaminergic neurons in msa and psp compared with pd patients. the results highlight distinct neuroinflammatory patterns in the snc among different parkinsonian disorders, indicating potential implications for disease progression and therapeutic strategies.   free neuropathol 5:30:9 brain-first vs. body-first types of lewy body disease – clinical, imaging, and postmortem data per borghammer1 aarhus university, aarhus, denmark within lewy body disorders (lbd), accumulating evidence support that aggregation and neuron-to-neuron propagation of alpha-synuclein aggregates may be a core pathogenic feature. the brain-first vs. body-first (bvb) model proposes that, in most lbd patients, the first alpha-synuclein aggregates initially form in a single location and then propagate through vulnerable parts of the connectome. the most common sites of origin are the olfactory system and the enteric nervous system. the bvb model suggests that when pathology begins in the gut, it results in a clinical body-first subtype marked by early autonomic symptoms and rem sleep behavior disorder. on the other hand, when pathology starts within the olfactory system, it leads to a brain-first subtype with fewer non-motor symptoms appearing before diagnosis. the bvb model also predicts that body-first lbd patients tend to be older, are more likely to experience symmetric dopaminergic degeneration, and face a higher risk of developing dementia compared to brain-first patients. thus, the bvb model proposes that most patients with dementia with lewy bodies are body-first, whereas most patients with parkinson’s disease are brain-first. this presentation will outline the theoretical framework underlying the bvb model and review supporting evidence from clinical, imaging, animal, and postmortem studies.   free neuropathol 5:30:10 gut microbiome changes in patients with idiopathic normal pressure hydrocephalus emilia brandt1,3, anne koivisto3,4,5, pedro pereira6, ella mustanoja7, petri auvinen7, toni saari8, juha-matti lehtola1,10, sanna hannonen1,3, minna rusanen1,3, ville leinonen2,3, filip scheperjans6,9, virve kärkkäinen2,3 neurology, institute of clinical medicine, school of medicine, university of eastern finland, kuopio, finland neurosurgery, institute of clinical medicine, school of medicine, university of eastern finland, kuopio, finland neurocenter, kuopio university hospital, kuopio, finland department of neurosciences, faculty of medicine, university of helsinki, helsinki, finland department of geriatrics, helsinki university hospital, helsinki, finland department of neurology, helsinki university hospital, helsinki, finland institute of biotechnology, university of helsinki, helsinki, finland institute for molecular medicine finland (fimm), helsinki institute of life sciences, university of helsinki, helsinki, finland clinicum, university of helsinki, helsinki, finland department of psychiatry, turku university hospital, turku, finland background: the gut microbiome is a complex system within the human gastrointestinal tract. the bacteria play a significant role in human health, and some can promote inflammation and pathologic processes through chemical interactions or metabolites. gut microbiome dysbiosis has been linked to some neurological and other diseases. here we aimed to examine microbiome differences between patients with a progressive neurological disorder, idiopathic normal pressure hydrocephalus (inph), compared with healthy controls (co). methods: we recruited 37 neurologically healthy co and 10 patients with shunted inph. we evaluated these participants’ cognition using the cerad-nb test battery and cdr test, and collected a variety of information, including about dietary habits and health. we also collected fecal samples, which were subjected to 16s amplicon sequencing to analyze differences in gut microbiome composition. results: we found that the inph group exhibited significantly different abundances of 10 bacterial genera compared with the co group. the escherichia/shigella and anaeromassilibacillus genera were most remarkably increased. other increased genera were butyrivibrio, duncaniella, and an unidentified genus. the decreased genera were agathobaculum, paramuribaculum, catenibacterium, and 2 unidentified genera. conclusions: here we report the first identified microbiome differences in inph patients compared with healthy controls.   free neuropathol 5:30:11 artificial intelligence algorithm for studying arteriolosclerosis in a finnish population-based study of the oldest-old (vantaa85+) kia colangelo1, eloise h. kok1, henri puttonen1,2, mikko i. mäyränpää1,2, olli tynninen1,2, tuomo polvikoski3, liisa myllykangas1,2 department of pathology, university of helsinki, helsinki, finland department of pathology, hus diagnostic center, helsinki university hospital, helsinki, finland translational and clinical research institute, newcastle university, newcastle upon tyne, united kingdom brain arteriolosclerosis, marked by thickening of the arteriolar walls, is a common finding in autopsies of elderly individuals and is known to be linked to cognitive impairment. the aim of this study was to create an artificial intelligence algorithm that measures the arteriolar thickness and to use this data to analyse associations between arteriolosclerosis and vascular risk factors, cognition parameters, and other neuropathologies. the vantaa 85+ study cohort, comprising 601 individuals aged > 85 years residing in vantaa, finland in 1991, forms the basis of our study. hematoxylin and eosin (he) staining of amygdala, hippocampus and frontal white matter tissue samples were used for the analysis. aiforia algorithms were used to detect vessels and to acquire sclerotic indexes (si) from each brain region. cognitive parameters and other neuropathologies have been assessed in previous studies. all three brain areas were found to have borderline moderate to severe arteriolosclerosis according to si values. we could not find strong associations between arteriolosclerosis and known vascular risk factors, such as hypertension, diabetes and cholesterol levels. however, we found significant associations between cognitive parameters (dementia and mmse scores) and arteriolosclerosis in all 3 brain areas. furthermore, adnc, late-nc and small brain infarcts were significantly associated with arteriolosclerosis in amygdala and frontal white matter. arteriolosclerosis may be involved in the pathogenesis of dementia, alzheimer’s disease and late-nc. vascular risk factors for arteriolosclerosis may be different from those of large vessel atherosclerosis in the very elderly age group.   free neuropathol 5:30:12 microvascular brain raspberries: a clinicopathological study henric ek olofsson1, elisabet englund1 division of pathology, clinical sciences lund, lund university, lund, sweden objectives: a raspberry is a microvascular formation of the human brain, defined histopathologically as three or more transversally sectioned vascular lumen within a common perivascular space. the aim of this project is to specify the clinicopathological context wherein raspberries occur. a hypothesis that has guided our study designs is that raspberries form in a setting of chronically recurrent hypoperfusion. materials and methods: this is a retrospective project based on archival brain tissue. the raspberries are quantified manually in haematoxylin and eosin-stained tissue sections from the cerebral cortex (mainly the frontal cortex, since raspberries have been encountered frequently in this region). we have examined whether the raspberry density varies in relation to age, large vessel disease, small vessel disease, vascular brain injury, vascular dementia, neurodegenerative disease, hypertension, and diabetes mellitus. results: the raspberry density increases with advancing age, independently of potential confounding factors. our findings further indicate an increased raspberry density in patients with cerebral atherosclerosis, hypertensive organ damage (exploratory), and vascular dementia. the raspberry density of the frontal cortex is increased in frontotemporal lobar degeneration, but not in alzheimer’s disease or lewy body disease. conclusion: raspberries are a partially age-related phenomenon that becomes more abundant in a setting of co-occurring vascular disease. the raspberry density of the frontal cortex is increased in frontotemporal lobar degeneration compared to alzheimer’s disease and lewy body disease. these findings can be applied to direct the focus of future research on the pathogenesis and consequences of raspberries, including transcriptomics, imaging, and animal models.   free neuropathol 5:30:13 cardiac alpha-synuclein may promote sudden cardiac death in cases with lewy body disease elisabet englund1, henric ek olofsson1, mattias haglund1, keivan javanshiri1 lund university, lund, sweden objectives: the hallmark pathology in lewy body disease (lbd) is the cns aggregates of alpha-synuclein (α-syn). these aggregates are seen in epicardial nerves, in clinical and preclinical stages of disease (1). their effects on cardiac function are not fully known, but they may trigger arrythmia and cardiac arrest (2). our aim was to investigate cause of death and prevalence of cardiovascular disease in neuropathologically confirmed lbd. method: the immediate cause of death was evaluated in 78 neuropathologically confirmed cases of lbd (brainstem, limbic or cortical). all cases exhibited cardiac α-syn. cardiovascular data (i.e. coronary atherosclerosis, cardiac hypertrophy, myocardial infarction) and clinical data on cardioand cerebrovascular disease (arrhythmias, congestive heart failure, elongated qtc, stroke), and risk factors (hypertension and t2 diabetes) were assessed. controls were 53 age-matched subjects with other major neurocognitive disease. results: the incidence of sudden cardiac death was 51.3 % in the lbd group compared to 22.6 % in the control group (p < 0.001). these cases had terminal cardiac failure without attributes of ischemic heart disease. no other differences were identified between the groups regarding other autopsy-reported cardiac findings or clinical disease or risk factors. conclusions: sudden cardiac death may be the leading cause of death in lbd, especially in the early phase, before clinically obvious neurological disease. there are possibly lethal alterations in cardiac function caused by α-synuclein aggregates. references: 1. javanshiri k et al. j park dis 2022;12:1125-1131. https://doi.org/10.3233/jpd-223161 2. javanshiri k et al. jnen 2023 feb 21;82(3):242-249. https://doi.org/10.1093/jnen/nlad001   free neuropathol 5:30:14 ageing-related tau astrogliopathy in the oldest old population (vantaa 85+) benjamin englert1, sara savola1, kia colangelo1, tuomo polvikoski2, liisa myllykangas1 hus helsinki university hospital, helsinki, finland translational and clinical research institute, newcastle university, newcastle upon tyne, united kingdom objectives: ageing-related tau astrogliopathy (artag), the occurrence of hyperphosphorylated tau in astrocytes, is a common finding in the ageing brain. however, its clinical significance remains elusive and quantitative studies are scarce. here we assessed the frequency and impact of artag in a general late-life aged population. methods: immunohistochemical staining using at8 tau antibody were carried out on the vantaa 85+ study, a population-based cohort containing over 300 brains of subjects older than 85 years with various levels of cognitive impairment and neurodegenerative pathologies. we assessed the appearance of pathognomonic astrocytic tau deposits (thorny-shaped and granular/fuzzy astrocytes) and their regional distribution in the limbic system (amygdala and hippocampus) of the oldest old in a quantitative manner using the aiforia digital pathology platform. we further evaluated the association of artag with various comorbid pathologies (alzheimer’s disease, lewy-related pathology, late, argyrophilic grains, vascular alterations, and hippocampal sclerosis). moreover, the artag burden was compared to clinical parameters such as age at death and sex, smoking, hypertension and type 2 diabetes mellitus medication, blood cholesterol and cognitive capacity (dementia according to dsm iii-r criteria and mmse scores), as well as genetic parameters (apoe genotype, tau h1/h2 haplotypes). expected results: we expect a detailed assessment of artag frequency in the general late-life aged population and its association with various clinicopathological parameters.   free neuropathol 5:30:15 3d microscopy unravels details in amyloid plaque-microglia interaction in app/ps1 mice maria gotkiewicz1, heikki tanila1 university of eastern finland, kuopio, finland objectives: microglia clustering around amyloid plaques is well established but how their contact the plaque with their processes is difficult to see in histological sections. we aimed to visualize details of amyloid plaque-microglia interaction in alzheimer model mice at different stages of the amyloid plaque formation through a 3d imaging approach. methods: we cross-bred amyloid plaque forming app/ps1 mice with cxcr1-gfp mice expressing gfp microglia which were examined at age of 13 months. we took 100 μm coronal sections at the level of dentate gyrus and stained them with antibodies against amyloid-β, galectin-3 and cd68 (marker of phagocytosis) protein. the mounting medium contained dapi which besides nuclei stains the amyloid plaque core. isolated clusters of amyloid plaque with the surrounding microglia were imaged in a 35 μm stack with zeiss lsm 800 confocal microscope and 3d rendered and analysed with imarisx64 10.0.1 results: we imaged 61 clusters from three mice. we observed that microglia processes were not attracted to diffuse amyloid of the plaque shell but made contacts with the dense dapi+ core. as shown earlier, homeostatic microglia in wildtype littermates were gal3while a subset of microglia in amyloid plaque expressing mice was gal3+. some microglia around amyloid plaques increased phagocytosis as evidenced cd68 that stains intracellular lysosomes. conclusions: combining endogenous microglia fluorescence, confocal 3d imaging of thick sections and digital reconstruction gave us an unprecedented visibility of microglia interactions with an amyloid plaque. this approach help understand the role of microglia at different stages of alzheimer pathology.   free neuropathol 5:30:16 cadasil, the leading genetic cause of small vessel disease masafumi ihara1 department of neurology, national cerebral and cardiovascular center, osaka, japan small vessel disease (svd) is a term that encompasses a variety of diseases and syndromes caused by pathological changes in the small blood vessels of the brain as a result of ageing, environmental and genetic factors. approximately 30 % of ischaemic strokes, almost all haemorrhagic strokes and almost half of vascular dementia are caused by cerebral small vessel disease. therefore, there are no comprehensive diagnostic criteria for all of them, but diagnostic criteria have been developed for some inherited svds. among these, cadasil is the most common inherited svd caused by notch3 mutations, and recent genetic analyses show that the frequency of notch3 mutations is 0.2 % in the uk general population and higher in east asia than in europe and the usa. cadasil is characterised by ischaemic changes including temporopolar white matter changes but some notch3 mutations are pro-haemorrhagic. for example, an east asia-specific notch3 p.r75p mutation underlies haemorrhagic presentations with milder and even absent temporopolar lesions, based on different properties of mutant notch3. pathologically, the p.r75p mutation results in less vascular notch3 extracellular domain accumulation, namely granular osmiophilic material, than the other conventional mutations. there are no disease-modifying treatments for cadasil, but we have recently completed a phase ii clinical trial, the amcad trial, of a peptide hormone, adrenomedullin, in cadasil patients (https://clinicaltrials.gov/study/nct06072118). an east asian cadasil registry has begun enrolling 1,000 cadasil patients to investigate the exact mechanisms of cadasil and identify effective treatments.   free neuropathol 5:30:17 genetic subgroups in lewy body dementia: the influence of alzheimer's pathology karri kaivola1,2, ruth chia3, zalak shah2, international lbd genomics consortium, jinhui ding4, raphael gibbs4, thomas beach5, sonja scholz2 university of helsinki. helsinki, finland neurodegenerative diseases research unit, national institute of neurological disorders and stroke, bethesda, usa neuromuscular diseases research section, laboratory of neurogenetics, national institute on aging, bethesda, usa computational biology group, laboratory of neurogenetics, national institute on aging, bethesda, usa banner sun health research institute, sun city, arizona, usa alzheimer’s disease (ad) pathology is common in lewy body dementia (lbd). how the genetic etiologies of ad and lbd overlap is still incompletely understood. we aimed to elucidate how ad pathology affects genetic associations in lbd. we used two whole-genome sequenced cohorts. the first cohort was a case-control cohort with 495 neuropathologically examined lbd patients and 2928 controls. the second was a brain bank cohort that consisted of 727 brains with alpha-synuclein, neuritic plaque and neurofibrillary tangle scores across regions of the brain. apoe e4 associated with lbd when there was strong (p = 1.29 x 10−32, or = 4.25, 95 % ci = 3.35–5.39) or intermediate ad pathology (p = 0.0011, or = 2.31, 95 % ci = 1.40–3.83) but not when there was no ad pathology (p = 0.31, or = 0.75, 95 % ci = 0.43–1.30). a regional analysis showed that the association of apoe e4 and ad polygenic risk score with alpha-synuclein pathology was not universal in the brain. for example, apoe e4 (p = 0.0016, or = 1.60, 95 % ci = 1.20–2.12) and ad polygenic risk score without apoe (p = 0.028, or = 1.21, 95 % ci = 1.02–1.44) predicted the presence of alpha-synuclein in the olfactory bulb/tract, but neither apoe e4 (p = 0.24) nor ad polygenic risk score without apoe (p = 0.12) predicted alpha-synuclein in the brainstem ix/x region. in conclusion, ad pathology affects the genetic associations of lbd and demonstrates putative lbd subgroups.   free neuropathol 5:30:18 association of cardiac sympathetic denervation with lewy pathology and its progression patterns ville kivistö1, eloise kok1, mikko mäyränpää1,2, tuomo polvikoski3, liisa myllykangas1,2 department of pathology, university of helsinki, helsinki, finland department of pathology, hus diagnostic center, helsinki university hospital, helsinki, finland translational and clinical research institute, newcastle university, newcastle upon tyne, united kingdom objectives: neuropathological and clinical data have led to hypotheses of multiple subtypes underlying lewy pathology (lp) in parkinson’s disease and dementia with lewy bodies. the main differentiating factor is whether the starting point is in the peripheral nervous system or the cerebrum, leading to a caudo-rostral or amygdala-based distribution of lp, respectively. a unique feature of the diseases is their peripheral manifestations, such as cardiac sympathetic denervation, seen as diminished tyrosine hydroxylase (th) staining. materials and methods: to quantify the effect of lp in the cns on cardiac sympathetic innervation and its relationship to the proposed caudo-rostral and amygdala-based progression patterns, we have developed an ai algorithm that quantifies th-positive staining in heart tissue and calculates its ratio to the analysed tissue area. preliminary data from left ventricular samples of 44 subjects from helsinki biobank and septal samples of 135 subjects from the vantaa 85+ -study were analysed. results: in the biobank subjects, more diffuse lp as defined by dlb consortium criteria, was inversely correlated with the th-positivity. in the vantaa 85+ -subjects, th-positivity was significantly lower in subjects with a caudo-rostral pattern compared to amygdala-based subjects and controls with no lp. th-positivity was not associated with myocardial infarcts nor with congo red-positivity in the heart, a marker for amyloidosis. conclusion: these results concur with previous research demonstrating the degeneration of cardiac sympathetic innervation associated with the progression of lp in the cns, and describe for the first time the difference in cardiac pathology manifestations between the proposed progression patterns.   free neuropathol 5:30:19 lewy-related pathology in the tampere sudden death study eloise kok1,2, anders paetau1,3, mika martiskainen2,4, leo-pekka lyytikäinen2,5, terho lehtimäki2,5, pekka karhunen2,5, liisa myllykangas1,3 university of helsinki, helsinki, finland tampere university, tampere, finland hus diagnostic center, helsinki, helsinki, finland finnish institute for health and welfare, helsinki, finland fimlab laboratories, tampere, finland objectives: when treatments against neurodegenerative diseases eventuate, the most successful time point to administer therapies will be the early stages, before symptoms begin to show. previous studies have found alpha-synuclein pathology in asymptomatic individuals aged 62 years and above, with prevalences from 8 to 17 %. materials and methods: we immunohistochemically stained alpha-synuclein (5g4 antibody) in brain tissue samples from the tampere sudden death study – a collection of forensic autopsies of individuals that lived outside hospitals in tampere and the surrounding regions, finland. representative paraffin-embedded brainstem tissue regions were available from 562 cases, aged 16 to 95 years, with a majority males (74 % vs 145 females). hippocampal regions were available in 476 cases (87 % of brainstem cases). results: alpha-synuclein pathology was seen in 46 individuals. the youngest (42y female) had clinical records of parkinsonism and neuropathologically determined as multiple system atrophy (msa). an additional 3 cases also showed msa-type glial cytoplasmic inclusions, without clinical history. 42 subjects showed lewy-related pathology (lrp) in brainstem. the youngest with lrp was 54 years with no reported neurodegenerative issues. 9 % of those aged 50 years or older had lrp with incidence increasing with age. conclusion: this is the first investigation of a non-hospitalised population across the adult life span showing the prevalence of lrp in individuals aged as young as their 50’s, suggesting lrp starts earlier than previously thought and is more common than expected in middle age.   free neuropathol 5:30:20 update on tau-related conditions gabor g. kovacs1 university of toronto and university health network, toronto, canada assemblies of the microtubule-associated protein tau characterise many neurodegenerative proteinopathies. tauopathies are clinically, morphologically and bio-chemically heterogeneous neurodegenerative diseases characterized by the deposition of abnormal tau protein in the brain. the neuropathological phenotypes are distinguished based on the involvement of different anatomical areas, cell types and presence of distinct isoforms of tau in the pathological deposits. electron cryo-microscopy has revealed the structures of tau protofilaments and the propagation of tau assemblies has been demonstrated. this is essential for the classification and diagnosis of tau-related conditions. mutations in the gene encoding the microtubule-associated protein tau are associated with frontotemporal dementia and parkinsonism. main tauopathy forms include pick’s disease, progressive supranuclear palsy, corticobasal degeneration, argyrophilic grain disease, primary age-related tauopathy, formerly called also as neurofibrillary tangle-only dementia, and globular glial tauopathy. a frequent form of tau pathology is ageing-related tau astrogliopathy (artag). in addition, further neurodegenerative conditions with diverse aetiologies may be associated with tau pathologies. thus, the spectrum of tau pathologies and tauopathy entities expands beyond the traditionally discussed disease forms. detailed multidisciplinary studies are still required to understand their significance.   free neuropathol 5:30:21 investigating choroid plexus and cerebrospinal fluid roles and functions in neurologic disease maria k. lehtinen1 department of pathology, boston children’s hospital and harvard medical school, boston, usa the choroid plexus (chp) is an epithelium located in each ventricle in the brain that produces cerebrospinal fluid (csf) and secretes important health and growth promoting factors for the brain throughout life. structural changes in human chp have been reported across various diseases in case reports and descriptive work. however, studies have yet to pin point the physiological relevance of these types of changes. for example, the chp forms a critical brain barrier implicated in coordinating inflammatory responses in a growing number of acute and chronic neurologic conditions ranging from multiple sclerosis to age-associated neurodegenerative diseases. we previously developed a comprehensive in vivo platform using two-photon imaging that enables real-time tracking of multiple genetically accessible chp cell types. pairing such live imaging with single cell transcriptomics analyses has uncovered a tightly choreographed program coordinated by chp epithelial cells to address and resolve cns infection. we anticipate shared mechanisms are engaged to varying degrees across neurologic diseases where inflammation plays an important role. our work suggests the chp acts as a key regulator of brain development and health throughout life.   free neuropathol 5:30:22 neuropathologic change in olfactory bulb and nerve of subjects with covid19 infection sylwia libard1,2, irina alafuzoff1 uppsala university hospital, uppsala, sweden uppsala university, uppsala, sweden objectives: to study neuropathological alterations in olfactory bulb/nerve (ob/n) of subjects deceased during ongoing covid19 infection materials and methods: post-mortem brains and ob/n from 32 subjects with ongoing covid19 infection and age, gender and brain neuropathology matched controls were assessed. the proteinopathies in the brain were assessed in line to the international consensus criteria. additionally, amyloid-β (aβ), hyperphosphorylated τ (hpτ), α-synuclein (α-syn), tar dna-binding protein 43 (tdp43) as well as inflammatory markers and an antibody against the sars virus were assessed within the ob/n. results: out of the 42 cases assessed 7 lacked proteinopathies in the ob/n whereas 81 % displayed hpτ, 41 % α-syn, 29 % tdp43 and 19 % aβ. there was no significant difference of incidence of various proteinopathies between the groups except for tdp43 that was more common in the covid19 group. the hpτ, α-syn and tdp43 in the brain correlated with hpτ, α-syn and aβ in ob/n, whereas brain aβ correlated with aβ and hpτ in ob/n. concomitant pathologies in the ob/n were seen in 52 % of cases. when studying inflammatory markers there was a pronounces microglial activation within the ob/n but lymphocytosis was sparse. no sars protein could be detected within our cohort. conclusion: all of the most common proteinopathies could be detected within the ob/n but according to our results subjects with covid19 are predisposed to tdp43 pathology within their ob/n. there is a substantial microgliosis within the ob/n independently of covid19 infection. the sars virus could not be detected within the ob/n.   free neuropathol 5:30:23 late-nc in the very elderly – the population-based vantaa 85+ study elizaveta mikhailenko1, kia colangelo1, jarno tuimala1, mia kero1,2, sara savola1,2, anna raunio1,2, eloise h. kok1, maarit tanskanen1, mira mäkelä1, henri puttonen1,2, mikko i. mäyränpää1,2, darshan kumar3, karri kaivola4,5, anders paetau1,2, pentti j. tienari4,5, tuomo polvikoski6, liisa myllykangas1,2 department of pathology, university of helsinki, helsinki, finland department of pathology, hus diagnostic center, helsinki university hospital, helsinki, finland aiforia technologies plc., helsinki, finland translational immunology, research programs unit, university of helsinki, helsinki, finland department of neurology, university of helsinki and helsinki university hospital, helsinki, finland translational and clinical research institute, newcastle university, newcastle upon tyne, united kingdom population-based cohort studies are essential for understanding the pathological basis of dementia in older populations. however, few studies have examined limbic-predominant age-related tdp-43 encephalopathy (late-nc) in a population-based setting. here we investigated late-nc frequency and its associations with other brain pathologies and cognition parameters in a population aged ≥ 85 years. the population-based vantaa 85+ study includes all 601 individuals aged ≥ 85 years who were living in vantaa, finland, in 1991. neuropathological examinations were performed on 304 subjects (50.5 %), with late-nc staging possible in 295. mmse scores were recorded at baseline and in three follow-ups (1994–99). late-nc stages were determined using tdp-43 immunohistochemistry. arteriolosclerosis was assessed digitally by calculating the average sclerotic index of five small arterioles in the amygdala, hippocampus, and frontal white matter. associations of late-nc with previously determined neuropathological variables such as alzheimer’s disease neuropathological change (adnc), lewy-related pathology (lrp), hippocampal sclerosis (hs), cerebral amyloid angiopathy (caa), and arteriolosclerosis, as well as cognitive variables were analyzed using fisher’s exact test and regression models. late-nc was found in 64.1 % of subjects, with stage 2 being the most common (28.5 %). stages 1a, 2, and 3 were significantly associated with dementia and lower mmse scores. late-nc was significantly associated with adnc, hs, diffuse neocortical lrp, and amygdala arteriolosclerosis. in most cases late-nc co-occurred with other neuropathological changes. in all multivariate models late-nc was among the strongest independent predictors of dementia. this study provides evidence that late-nc is common and a significant determinant of dementia in the very elderly population.   free neuropathol 5:30:24 glial activation and tdp-43 differences among neuropathologic subtypes of alzheimer’s disease sara r. dunlop1, zhongwei peng2, baayla d.c. boon1, christina m. moloney1, isabelle frankenhauser1,5, sydney a. labuzan1, cyril pottier1, daniel p. wickland2, kelsey caetano-anolles1, jessica f. tranovich1, ashley c. wood1, kelly m. hinkle1, sarah j. lincoln1, rain s. kwan2, owen a. ross1, christian lachner3,4, nilüfer ertekin-taner1,4, gregory s. day4, ranjan duara6, neill r. graff-radford4, dennis w. dickson1, rickey e. carter2, melissa e. murray1 department of neuroscience, mayo clinic, jacksonville, fl, usa department of quantitative health sciences, mayo clinic, jacksonville, fl, usa department of psychiatry and psychology, mayo clinic, jacksonville, fl, usa department of neurology, mayo clinic, jacksonville, fl, usa paracelsus medical private university, salzburg, austria wien center for alzheimer’s disease and memory disorders, mount sinai medical center, miami beach, fl, usa selective corticolimbic vulnerability to tau pathology in alzheimer’s disease (ad) underlies clinicopathologic heterogeneity. we aimed to re-express the ad subtype algorithm as a corticolimbic index (clix) to quantitatively examine heterogeneity and deeply phenotype corticolimbic patterns of ad pathology and glial activation in neuropathologically diagnosed ad. clix was developed (n = 1361 ad) to collapse spatial distribution of thioflavin-s tangle counts to assign a continuum: hippocampal sparing (< 10), representative/typical (≥ 10 to < 30), and limbic predominant (≥ 30). presence of atypical, non-amnestic clinical presentation and clinical progression on mmse were investigated. digital pathology measures of ad (tau [at8, gt-38], amyloid-β [6f/3d]) were performed in hippocampus and association cortices (n = 60), which were modeled with late-tdp-43 staging to predict glial activation burden (astroglial: gfap, microglial/macrophage: cd68). lower clix score correlated with younger age at onset (r = 0.39, p < 0.001), faster mmse decline (r = 0.27, p < 0.001), and associated with non-amnestic clinical presentations (median = 13 vs. 21, p < 0.001). advanced tangle maturity burden (gt-38) differed across all regions (p < 0.001). hippocampal sparing ad exhibited higher cortical tau pathology and reduced cortical cd68. regression modeling of glial activation (gfap [r2 = 0.21–0.56], cd68 [r2 = 0.23–0.60]) found tau and amyloid-β similarly associated with gfap and cd68, but tdp-43 stage associated with the greatest glial change. corticolimbic tangle distribution is correlated with ad clinical phenotype and progression. reduced cd68 in association cortices of cases with relative hippocampal sparing ad may account for higher tau burden compared to other subtypes. pending replication, corticolimbic vulnerability and tdp-43 co-pathology should be considered for incorporation into personalized combination therapies targeting chronic immune dysregulation.   free neuropathol 5:30:25 neuropathological subtypes of lewy body disease: evidence from a population-based study liisa myllykangas1 department of pathology, university of helsinki and hus diagnostic center and helsinki university hospital, helsinki, finland evidence from recent neuropathological, imaging and genetic studies has supported the existence of at least two anatomically distinct lewy-related pathology (lrp) subtypes consistent with the brain-first vs. body-first hypothesis. one reason why the anatomical heterogeneity of lrp has been overlooked for a long time, has been that research has focused on hypotheses based on selected materials, with very few studies conducted on unselected population-representative samples. recent studies have also revealed that synucleinopathies are one of the very few neurodegenerative diseases that show significant involvement of the peripheral autonomous nervous system. studying the spinal cord and peripheral lrp may therefore provide a unique window into the mechanisms for lewy body disorders and the origins of synuclein accumulation and spreading patterns. in this presentation, i review lrp studies in various brain, spinal cord and peripheral tissues in the unique population-based vantaa 85+ sample set (n = 304). taken together, the results provide support for the concept of at least two distinct lrp subtypes common in the general late-life population.   free neuropathol 5:30:26 disease-driving mechanisms of late-nc pete nelson1 university of kentucky, lexington, ky, usa limbic-predominant age-related tdp-43 encephalopathy neuropathologic change (late-nc) is present in ∼30 % of brains among people over age 85 at death. consensus-based classification of late-nc has catalyzed progress in worldwide efforts aimed at conceptualizing, studying, diagnosing, and ultimately treating the condition. whereas late-nc is associated strongly with cognitive impairment independent of other pathologies, a subset of other agingand dementia-related brain pathologies are seen with increased frequency in late-nc brains. more specifically, hippocampal sclerosis (hs), alzheimer’s disease neuropathologic changes (adnc), age-related tau astrogliopathy (artag), and brain arteriolosclerosis are each relatively common in brains with comorbid late-nc (and vice versa! in community-based autopsy cohorts, “pure late-nc” is unusual, and “pure adnc” also is unusual, because mixed pathologies are the norm.). analyses of high-quality autopsy cohorts’ data provide critical insights into underlying disease-associated biologic phenomena, including evidence of mechanistic interactions (“synergies”) between the different pathologic subtypes, while also suggesting common upstream influences. genes with variants associated with late-nc risk include tmem106by, grn, apoe, sorl1, abcc9, and tpcn1. among these, different genetic variants are specifically associated with (i.e., appear to influence biologically) distinct components of pathogenetic pathways, and may help to explain why some but not all late-nc+ brains have comorbid hs, and why some but not all late-nc+ brains have comorbid adnc. the association between late-nc and brain arteriolosclerosis also has intriguing implications. overall, salient late-nc-associated pathobiologic factors in aged brains include tau proteinopathy, endolysosomal pathways, and blood-brain barrier dysfunction. we conclude that the study of neuropathologic phenomenology provides clues about disease-driving mechanisms, and may also yield insights into diagnostic and therapeutic targets.   free neuropathol 5:30:27 evaluating the impact of data harmonization on multi-site simulated neurodegeneration dataset analysis mithilesh prakash1, jussi tohka1 a.i. virtanen institute for molecular sciences, university of eastern finland, kuopio, finland a study was conducted to evaluate the impact of raw and harmonized data on downstream analysis, employing a simulated multi-site neurodegeneration dataset. the synthetic dataset, created using a python function, included mri volumes, demographics, and groupings for 5000 subjects across four simulated sites. the data were modeled, with an xgboost classifier, as either pooled (raw) or harmonized using a combat variant called neuroharmonize. the models were evaluated using a “leave one site out” approach. two scenarios were examined. in scenario 1, certain volumes were atrophied where the label was 1. both raw and harmonized data overfit the training data (auc = 1.0), but harmonization more accurately reflected the classifier’s performance in the test folds (auc: 0.5 vs. 1.0 for raw). in scenario 2, with no atrophy, both data types overfit the training data (auc: ∼0.9), but the test data showed anticipated results (auc: ∼0.5), with harmonized data showing less variation. the study concluded that the effectiveness of harmonization compared to pooling of data is determined by the dataset. harmonization is preferable in multi-site data as it provides results closer to those anticipated in controlled settings. however, in certain circumstances, such as when the predictors and labels are highly correlated, raw data can outperform harmonized datasets. further research is needed to optimize the use of harmonization in multi-site neurodegeneration studies, particularly in the context of simulated atrophy.   free neuropathol 5:30:28 tdp-43 pathology in frontal cortical brain biopsies of normal pressure hydrocephalus patients eino solje1,2, ville korhonen3,4, anssi lipponen5, annakaisa haapasalo6, mikko hiltunen5, ville leinonen3,4, tuomas rauramaa7,8 institute of clinical medicine neurology, university of eastern finland, kuopio, finland neuro center, neurology, kuopio university hospital, kuopio, finland institute of clinical medicine neurosurgery, university of eastern finland, kuopio, finland department of neurosurgery, kuopio university hospital, kuopio, finland institute of biomedicine, faculty of health sciences, university of eastern finland, kuopio, finland a.i. virtanen institute for molecular sciences, university of eastern finland, kuopio, finland institute of clinical medicine pathology, university of eastern finland, kuopio, finland department of clinical pathology, kuopio university hospital, kuopio, finland objectives: alzheimer's disease (ad)-related pathologies are frequent comorbid findings in idiopathic normal pressure hydrocephalus (inph). frontotemporal lobar degeneration (ftld) is another important but less studied potential comorbidity, characterized by tdp-43 pathology in approximately half of the cases. to the best of our knowledge, tdp-43 pathology has not been systematically studied in brain biopsies of patients with inph.our aim was to evaluate the presence of tdp-43 pathology in brain biopsies obtained during shunt procedures due to inph. materials and methods: from 2015 to the end of 2023, 397 patients with inph underwent shunt surgery and right frontal brain biopsy at a mean age of 74 years. brain biopsies were stained for amyloid-β (6f/3d), hyperphosphorylated tau (at8), and tdp-43 (2e2-d3) and evaluated by a neuropathologist. results and conclusion: of the 397 patients, 16 (4 %) had tdp-43 positive neuronal cytoplasmic inclusions or dystrophic neurites, with a mean age of 74. of these, six patients had additional aβ pathology (aβ+tdp-43) and two had additional hyperphosphorylated tau pathology (tau+tdp-43). tdp-43 appears to be rather rare but potentially clinically highly relevant comorbid pathology in inph.   free neuropathol 5:30:29 cns amyloid patterns in aging canines – a pilotstudy regarding neuropathology in canine cognitive dysfunction pernilla syrjä1, linda rasmus1, tarja pääkkönen2 department of veterinary biosciences, veterinary pathology and parasitology, faculty of veterinary medicine, university of helsinki, helsinki, finland department of equine and small animal medicine, faculty of veterinary medicine, university of helsinki helsinki, finland the objective of this pilot study was to characterize cns amyloid deposit patterns in aging canines, to enable correlating them with canine cognitive dysfunction (ccd). the pilot starts a larger study investigating cns amyloid deposition, tauopathy, microglial changes, blood-derived macrophage response, microbleeds and extraneural amyloid of aging dogs with ccd in comparison to aging dogs without ccd. dogs share similar environment, basic neuropathological mechanisms, and amyloid type as man. therefore, insights into why dogs do not develop mature ad pathology would be of comparative value. the formalin-fixed frontal cortex and hippocampus from twenty dogs, average 11.2 years old (sd 2.2 years) were retrieved in retrospect from the patient archives of the section for pathology, faculty of veterinary medicine, university of helsinki and stained by ihc for amyloid beta. amyloid was present in the cortical neuropil, tunica externa and media of meningeal vessels, and in meningeal macrophages in 11/20 dogs. the hippocampal perforant pathways contained amyloid in eight of these eleven dogs. cortical capillary amyloid deposits were a main feature in 6/20 dogs. four dogs did not show cns amyloid. aging dogs develop amyloid deposits at specific locations in the brain, in patterns partially comparable to those of amyloid angiopathy in man. deposits reminiscent of dense core plaques were not detected in this cohort, consistent with what is currently known in dogs. this provides a starting point to assess if a distinct amyloid deposition pattern is associated with clinical ccd and to further elucidate the neuropathology of cognitive decline in dogs.   free neuropathol 5:30:30–31 common associations of transcranial magnetic stimulation (tms) parameters and behavioral symptoms in different neurocognitive diseases matti tyrväinen1, laura säisänen1,2,3, jelena hyppönen1,3, sara määttä3, kaarlo ylimäki2, kasper katisko2, johanna kruger4,5,6, noora-maria suhonen4,5,6, päivi hartikainen7, barbara borroni8, annakaisa haapasalo9, esa mervaala1,3, eino solje2,7 institute of clinical medicine – clinical neurophysiology, university of eastern finland, kuopio, finland institute of clinical medicine – neurology, university of eastern finland, kuopio, finland department of clinical neurophysiology, kuopio university hospital, kuopio, finland research unit of clinical medicine, neurology, university of oulu, oulu, finland mrc, oulu university hospital, oulu, finland neurocenter, neurology, oulu university hospital, oulu, finland neuro center – neurology, kuopio university hospital, kuopio, finland neurology unit, centre for neurodegenerative disorders, department of clinical and experimental sciences, university of brescia, brescia, italy a.i. virtanen institute for molecular sciences – university of eastern finland, kuopio, finland state of the art: behavioral changes are characteristic for frontotemporal dementia (ftd) and other forms of neurocognitive disorders. transcranial magnetic stimulation (tms) can be used to non-invasively assess disease-specific changes in inhibitory and excitatory processes. we determined the association of tms variables with ftd-like personality and behavioral changes, captured using the modified frontal behavior inventory (fbi-mod) questionnaire, independently from diagnostic classification. methodology: in total, 103 patients (35 % female, mean age 68 years, 28 ftd or ftd-like, 17 alzheimer’s continuum disease, 11 dementia with lewy bodies, 8 normal pressure hydrocephalus, 27 other or undetermined neurocognitive disease, and 12 controls) were included. for tms, short interval intracortical inhibition (sici), intracortical facilitation (icf), long-interval cortical inhibition (lici) and short-latency afferent inhibition (sai) were measured. the 22 items in fbi-mod were filled by a relative of the patient. the total score as well as the presence and severity of each symptom were analysed separately. results: multiple associations were found in the symptom-wise analysis. the presence of restlessness was associated with stronger inhibition in sai and sici tests, while impaired sici was associated with the presence of irritability, and impaired icf with the severity of inattention (p < 0.01 for all). the fbi-mod total and positive scores correlated with sai (rho = -0.2, p = 0.02 for both). conclusion: changes observed in the gabaergic (sici), glutamatergic (icf) and acetylcholinergic (sai) systems were found to correlate with specific behavioral symptoms, independently from the underlying diagnostic entity. these results and increasing dataset will enable more comprehensive understanding of pathophysiology behind distinct behavioral symptoms.   free neuropathol 5:30:32 digital morphological profiling reveals regionaland disease-specific differences of glial cells in neurodegenerative proteinopathies vladyslav vadymovych tkach1, ausrine areskeviciute1, remarh bsoul1, eva løbner lund1 danish reference center for prion diseases, department of pathology, copenhagen university hospital, copenhagen, denmark objectives: the importance of glial cells and neuroinflammation has been established in neurodegenerative proteinopathies. this study aims to characterize regional differences in microglial and astrocytic reactivity in two different neurodegenerative proteinopathies alzheimer’s disease (ad) and creutzfeldt-jakob’s disease (cjd). materials and methods: post-mortem brain tissue from both frontal and occipital neocortex was collected from patients diagnosed with either ad, cjd or with no neuropathological findings. the brain tissue was spatially divided into white matter, lower grey matter, and an upper grey matter area. a custom digital morphological profiling pipeline was applied on immunofluorescence images of the tissue slides, which were stained for both iba1 (microglia) and gfap (astrocytes). subsequent unsupervised clustering classified the segmented cells based on measured morphological features. protein expression data was obtained using nanostring protein assays and geomx spatial profiling. results and conclusion: predominant clusters representing reactive states (ameboid microglia and ramified astrocytes) were identified for both cell types respectively. the disease groups, and especially cjd patients, had increased presence of reactive microglia compared to controls, which was a specific finding found in grey matter but not in white matter. increased presence of reactive astrocytes was apparent for both diseases and across all cortex layers, including white matter. integration of the morphological data with protein expression data obtained from the same tissue slides showed correlation between cell morphology and expression level of different proteins. these results illustrate that morphological profiling can be applied in a digital pathology setting to study disease specific alterations of cell morphology.   free neuropathol 5:30:33 an update on alzheimer’s disease tau subtypes: clinical and biological insights jacob vogel1, lyduine collij2,3, sophie mastenbroek2,3, alexa pichet binette3, ruben smith3,4, sebastian palmqvist3,4, niklas mattsson-carlgren3,4, olof strandberg3,4, rik ossenkoppele3,5, oskar hansson3,4 department of clinical science malmö, scilifelab, lund university, lund, sweden amsterdam umc, department of radiology and nuclear medicine, amsterdam, the netherlands clinical memory research unit, department of clinical sciences malmö, lund university, lund, sweden memory clinic, skåne university hospital, malmö, sweden amsterdam umc, alzheimer center amsterdam, department of neurology, amsterdam, the netherlands objectives: using positron emission tomography (pet), we recently characterized four subtypes of tau accumulation within the ad population, each with distinct clinicopathological profiles. validation and further characterization of these subtypes is critical to better understand their clinical and therapeutic relevance. materials and methods: we applied the previously established subtype and stage inference (sustain) model to 1252 biofinder-2 (bf2) participants with available [18f]ro948 tau-pet. linear and mixed-effects models assessed subtype differences in csf-biomarkers regional, and brain atrophy and cognition over time, respectively. models included age, sex, and nuisance covariates and were fdr-corrected. exploratory analysis tested subtype differences in global and local functional mri (fmri) connectivity, as well as in prevalence of top ad risk genes results: participants classified to the limbic-subtype were older, while posterior-subtype participants were less often apoe-ε4 carriers. longitudinal regional gray matter volume reduction matched subtype-specific tau patterns. posterior and lateral-temporal-subtype participants had higher levels of nfl, ykl40, and ngn compared to limbic-subtype participants. limbic-subtype and lateral-temporal-subtype participants performed worse on adas-cog delayed recall and mmse compared to the mtl-sparing and limbic-subtype over time, respectively. subtype-specific patterns of local and global fmri connectivity were evident, but no genetic risk factors survived fdr correction. conclusions: in a large single-site dataset, we confirm and extend findings that spatiotemporal subtypes of tau burden are differentiated by demographics and risk-factors. cortical atrophy patterns are mostly consistent with the distribution of tau burden. the lateral-temporal-subtype showed higher tau burden and increased csf markers of neurodegeneration, in line with the volume loss and cognitive decline.   free neuropathol 5:30:34 clinical and imaging signatures of late: developing a diagnostic framework david a. wolk1–5 alzheimer’s disease research center, university of pennsylvania, philadelphia, pa, usa penn institute on aging, university of pennsylvania, philadelphia, pa, usa penn memory center, university of pennsylvania, philadelphia, pa, usa cognitive neurology, university of pennsylvania, philadelphia, pa, usa department of neurology, university of pennsylvania, philadelphia, pa, usa objective: the goal of this talk will be to provide a brief review of the literature focused on clinical and biomarker features of limbic-predominant age-related tdp-43 encephalopathy (late). this review will emphasize the clinical importance of this condition, particularly in the context of alzheimer’s disease immunotherapies, as well as a newly proposed framework for clinical identification. materials and methods: the speaker will review the existing literature focused primarily on clinical and neuroimaging studies in autopsy-confirmed cases with an emphasis on how these features overlap and differ from alzheimer’s disease, for which it is a frequent mimic. a particular focus will be on work from the speaker’s laboratory, including both ante-mortem and post-mortem mri imaging to determine signatures of late. results: based on literature review and consensus from experts in the field, newly proposed diagnostic criteria will be presented for in vivo detection of late in the context of it being the primary driver of clinical presentation and when it is concomitant with alzheimer’s disease pathology. supportive imaging features, including topography of medial temporal lobe atrophy and relationship to tau pathology (i.e. tau-neurodegeneration mismatch), will be highlighted. conclusion: emerging work on the recently codified pathological entity of late has matured to where a probabilistic in vivo diagnosis can be made, providing an important platform for future research. among other benefits, such clinical classification will help advance development of molecular biomarkers for tdp-43 and therapeutic trials specifically targeting this condition. copyright: © 2024 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. 35th australian and new zealand society for neuropathology (anzsnp) scientific meeting 2024 feel free to add comments by clicking these icons on the sidebar free neuropathology 5:29 (2024) meeting abstracts 35th australian and new zealand society for neuropathology (anzsnp) scientific meeting 2024 saturday 31 august sunday 1 september 2024 hotel crowne plaza queenstown, new zealand submitted: 24 october 2024 accepted: 25 october 2024 published: 18 november 2024   the anzsnp scientific meeting 2024 was held in the scenic city of queenstown, new zealand on 31 august and september 1. dr fouzia ziad, president of the anzsnp and dr laveniya satgunaseelan, secretary /treasurer of the anzsnp were the convenors of the meeting. the meeting was co-badged with the australasian winter conference on brain research (awcbr) 2024. there were 55 registrants from australia, new zealand, usa, europe and asia. the academic program covered a wide range of neuropathology topics ranging from neuro-trauma to neuro-oncology. the neuro-oncology sessions featured a series of lectures on updates in the classification and diagnosis of various cns tumours delivered by keynote speaker prof arie perry, ucsf. dr thomas robertson presented an approach to frozen sections in surgical neuropathology. an interactive session on the rcpa neuropathology quality assurance program (qap) was organised with the qap convenor, dr barbara koszyca. prof david capper delivered the plenary lecture on “dna methylation profiling and its practical implications” during the combined session with awcbr attendees. combined sessions also featured presentations on non-neoplastic conditions such as neurotrauma and neurodegeneration, comprising of presentations from invited faculty including prof michael buckland, dr helen murray and prof glenda halliday. four scientific research papers and 3 interesting cases were selected for oral presentation by the scientific committee. the meeting concluded with a plenary lecture delivered by prof arie perry on “biomarkers in neuro-oncology”. two of the research paper presentations were eligible for the bill evans memorial award for postgraduate students or trainees. the prize was awarded to dr monish maharaj for his presentation “dna methylation profiling to categorise meningioma recurrence risk: optimisation of resource allocation for patient selection”. the feedback from the participants suggested that the meeting was well organized and appreciated for its educational content.   https://doi.org/10.17879/freeneuropathology-2024-5984 keywords: anzsnp, 35th anzsnp scientific meeting 2024, australian and new zealand society for neuropathology (anzsnp), queenstown, new zealand table of contents research paper oral presentation abstracts abstract 1 saliva swabs: a game-changer for concussion diagnosis and management abstract 2: identification of recurrent genetic alterations in epigenetic pineoblastoma subtypes by high-resolution whole genome cytogenetics and dna panel sequencing abstract 3: dna-methylation profiling to categorise meningioma recurrence risk: optimisation of resource allocation for patient selection abstract 4: cell-free dna in adult diffuse glioma – clinical utility and idh mutation analysis interesting case presentation abstracts case presentation 1: cns neuroepithelial tumours with patz1 fusion case presentation 2: two interesting, rare cases of erdheim chester disease (ecd) case presentation 3: unusual cerebellar vermian lesions – a short case series   research paper oral presentation abstracts   abstract 1 free neuropathol 5:29:4 saliva swabs: a game-changer for concussion diagnosis and management tandia gooch1,2, ewan kennedy3, regis lamberts2, sarah appleby1 department of medicine, school of biomedical sciences, university of otago, christchurch, new zealand department of physiology, school of biomedical sciences, university of otago, dunedin, new zealand centre for health, activity, and rehabilitation research, school of physiotherapy, university of otago, dunedin, new zealand introduction: rugby is a physically demanding sport, as a result the injury of concussion is prevalent among players. concussions are highly individual injuries and challenging to diagnose, leading to underdiagnosis and inadequate management among community rugby players. the sport concussion assessment tool (scat6) is currently recommended for concussion diagnosis in rugby, alongside clinical evaluation. however, this assessment is not feasible during a typical 15-minute general practitioner appointment. additionally, scat6's reliability varies, with players often showing performance fluctuations even without a concussion. it also depends on players self-reporting symptoms, which often overlap with those experienced in the general population. therefore, a quick, simple test identifying physiological markers and indicating physiological recovery is needed. small non-coding rnas (sncrnas), which regulate gene expression, have shown promise as biomarkers for concussion. aims: this study aims to validate the ability of a panel of 14 sncrnas to diagnose concussions in community and adolescent rugby players. furthermore, it seeks to determine the right time for a player to return to play post-concussion using the a sncrna test. methods: data is collected at the otago concussion clinic. medical information and saliva swabs from players are obtained 48 hours and 19 days after injury. sncrna analysis of the saliva samples will be conducted by marker health. conclusion: this research has the potential to improve diagnosis of concussions in community rugby players using a simple saliva test. therefore, it might improve outcomes for players affected by concussions and reduce the risk of subsequent concussions, thereby enhancing overall player safety.   abstract 2 free neuropathol 5:29:5 identification of recurrent genetic alterations in epigenetic pineoblastoma subtypes by high-resolution whole genome cytogenetics and dna panel sequencing torsten pietsch1, evelyn dörner1, tobias goschzik1 department of neuropathology and dgnn brain tumor reference center, university of bonn medical center, bonn, germany genome-wide methylation analyses recently revealed distinct epigenetic pineoblastoma (pbl) subtypes. the aim of the study was to characterize genetic alterations underlying the pathogenesis of pineoblastoma subtypes. cytogenetic alterations of 83 ffpe materials from patients with pbl confirmed by central reference neuropathology and methylation-based subtyping were analyzed by high-resolution genome-wide molecular inversion probe (mip) analysis. mutational status was assessed by next-generation dna panel sequencing. clinical follow-up information of 54 patients was available. according to epigenetic consensus pbl subtypes (liu et al., 2021), our cohort consisted of 48 pbl-mirna1 (1a, 40; 1b, 8), 19 pbl-mirna2, 8 pbl-myc/foxr2, and 8 pbl-rb1 samples. pbl-mirna subtype tumors had characteristic, mutually exclusive alterations in microrna-processing genes; dicer1 mutations (n = 19) and homozygous deletions of the drosha locus (n = 17) were most abundant, followed by drosha (n = 12) and dgcr8 (n = 2) mutations. the most frequent cytogenetic aberrations in pbl-mirna were whole chromosome 7 gain (n = 31). pbl-mirna2 subtype was significantly associated with chromosome 14 loss and dicer1 mutation. in contrast, pbl-mirna1a/b showed frequent gains of chromosome 14 (n = 21) and also focal gains of the otx2 oncogene on chromosome 14q (n = 8). in the mirna subtypes 1a/b, 16 cases cases had polyploid cytogenetics. survival analysis did not show differences in the pbl-mirna subtypes 1a, 1b and 2. the epigenetically defined pbl-mirna subtypes are characterized by distinct cytogenetic alterations and mutational events. frequent gain of otx2 may indicate a role of this oncogene in the pathogenesis of pbl. a possible prognostic role of genetic alterations requires investigation in prospective clinical trials.   abstract 3 free neuropathol 5:29:6 dna-methylation profiling to categorise meningioma recurrence risk: optimisation of resource allocation for patient selection maharaj mm1, ziad f2, slatter t3, jones g4, saraiva a3, thotathil z5, bhat b3, gan p1, hung n3, jameson m6 neurosurgery, waikato hospital, hamilton, new zealand pathology, waikato hospital, hamilton, new zealand pathology, dunedin school of medicine, dunedin, new zealand surgical sciences, dunedin school of medicine, dunedin, new zealand radiation oncology, waikato hospital, hamilton, new zealand medical oncology, waikato hospital, hamilton, new zealand background: the emergence of an integrated classification including morphology, dna-methylation profiling and copy number changes is leading towards significant changes in meningioma patient management. global accessibility to methylation profiling remains costly and limited to the research setting in many countries. this requires the development of a strategy to optimise clinical utility. aims: (1) to determine histopathological factors that may influence discordant classification. (2) to examine patient factors that may influence benefit from further methylation testing. methods: dna extracted from ffpe samples of 71 patients with resected who cns grade 2 meningioma using the infinium hd ffpe restore protocol and methylation profiling with the illumina epic (850k) human bead chip (illumina, san diego, usa) was conducted. meningiomas were sub-classified using the integrated system published by mass sln et al (2021). factors including histopathological features, gross total resection, adjuvant therapy, mitotic activity and ethnicity were examined using standardised statistical methods. results: >50 % of the who cns 2 cohort (n = 71) was classified into the integrated low risk group. classification demonstrated good correlation with recurrence (p = 0.004). those without a gross total resection were more likely to recur (55 % vs 26 %). chordoid subtype was common in maori population but segregated across all risk groups. interestingly brain invasion correlated with a more benign methylation class (p = 0.05). conclusion: the implementation of integrated methylation testing carries significant implications on patient treatment and health economics. potential who cns 2 subgroups that may merit profiling include those with brain invasion, subtotal resection and certain histopathological subtypes.   abstract 4 free neuropathol 5:29:7 cell-free dna in adult diffuse glioma – clinical utility and idh mutation analysis nuzhat husain1, adil husain1,2, sridhar mishra1 department of pathology, dr. ram manohar lohia institute of medical sciences, lucknow, india department of biosciences, integral university, lucknow, india background: cell-free dna (cfdna) has found utility in diagnosis and management of malignancies with scant literature in adult diffuse gliomas (adg). aims: this study aimed to quantify baseline values and serial cfdna levels in the sera of patients with adg undergoing radiation therapy comparing responders vs non responders and further to detect idh1 mutation in cfdna. materials and methods: the study group comprised of histologically confirmed adg (n = 50), -grade 2 (n = 15), 3 (n = 08), 4 (n = 27) and controls (n = 15). cfdna was extracted using a chargeswitch gdna 1 ml serum kit (invitrogen) and quantified using sybr-based qpcr. idh1 mutation was detected using a castpcr assay, sanger’s sequencing and next generation sequencing. results: serum cfdna levels were considerably higher in patients with adg (median: 113.50 ng/ml; iqr: 44.66–243.20) than in healthy controls (median: 53.33 ng/ml; iqr: 40.67–73.87) (p = 0.02). cfdna values were highest in grade 4 (353.53 ± 601.22 ng/ml) followed by grade 2 (201.93 ± 245.35 ng/ml) and 3 (153.67 ± 162.83 ng/ml). the cfdna level was significantly higher in idh1wt cases (157.83 ng/ml; iqr: 92.55–667.65) than in idh1mt cases (69.55 ng/ml; iqr: 49.08–188.35) (p = 0.04). in non-responders, the cfdna level (median: 255.6 ng/ml) was significantly higher than that in responders (median: 66.70 ng/ml) (p = 0.005). in cfdna of idhmt adg, castpcr assay detected the idh1r132h mutation in 3/26, sanger’s sequencing failed to detect (0/20) while ngs was positive in 4/7 cases. conclusion: cfdna quantity in adg increases in proportion to the grade of the tumor, responders show higher levels, but the trend is inconsistent across cases. ngs is most sensitive for idh mutation detection in cfdna.   interesting case presentation abstracts   case presentation 1 free neuropathol 5:29:8 cns neuroepithelial tumours with patz1 fusion mike watson1 department of anatomical pathology, auckland city hospital, new zealand cns neuroepithelial tumours with patz1 fusions comprise a heterogenous group. we present two of these tumours, illustrating the diverse clinical presentation and morphology of these lesions. the first occurred in the cervical cord of an 8‑year-old and was thought initially to be an ependymoma. the second case was a left frontal tumour in a 54‑year-old, and the initial impression was of a high-grade glioma, possibly a gliosarcoma. the histology and genetics of this entity are discussed.   case presentation 2 free neuropathol 5:29:9 two interesting, rare cases of erdheim chester disease (ecd) mehar c sharma1, vaishali suri1, ajay garg2, ashish suri3 department of neuropathology, all india institute of medical sciences, new delhi, india department of neuroradiology, all india institute of medical sciences, new delhi, india department of neurosurgery, all india institute of medical sciences, new delhi, india case 1: this 51‑year-old lady presented with diabetes insipidus in 2001(at the age of 28 years) and diagnosed as case of empty sella syndrome. in 2008 developed pericardial effusion which was tapped thrice and was given a course of att without any improvement. in 2013 developed signs and symptoms of raised intracranial pressure and mri of brain revealed a sol in pineal region. subtotal excision of the lesion was done. in 2020 this mass increased in size and subtotal excision of lesion was done. this mass again increased in size and radiotherapy was given. currently patient is bed ridden and mass has grown into left lateral ventricle. case 2: a 38‑year-old man presented with signs and symptoms of raised intracranial pressure. mri brain showed multiple extra axial meningeal bases lesions and was suspected to be a case of nf2 with multiple meningiomas. one of the suprasellar lesion was excised and subjected for histopathological examination. pathological examination revealed similar features in both cases. microscopic examination revealed sheets of foamy cells admixed with lymphocytes, plasma cells and fibroblasts. emperipolesis of lymphoid cells was absent. an occasional tuoton giant cell was noted in second case. foamy cells were immunopositive for cd163 and cd68 but negative for cd1a, langerin & s100. ratio of igg4 to igg immunopositive cells was normal. foamy cells were immunopositive for brafv600. braf mutation was confirmed by sanger-sequencing. conclusion: ecd is a rare histiocytic disorder characterised by admixture of foamy histiocytes and inflammatory cells. involvement of central nervous system is extremely uncommon and poses a diagnostic dilemma. high index of suspicion is required for its diagnosis and differentiation from other close mimics.   case presentation 3 free neuropathol 5:29:10 unusual cerebellar vermian lesions – a short case series nandeesh bn1, vani santosh1, subhas konar2, netravathi m3, yasha tc1 department of neuropathology, nimhans, bengaluru, india department of neurosurgery, nimhans, bengaluru, india department of neurology, nimhans, bengaluru, india introduction: the central nervous system is known for its anatomical uniqueness and immune privileges. despite this, this organ becomes a host for many neoplastic and non–non-neoplastic lesions. some non-neoplastic lesions occur in such brain regions and present with clinicopathological features that mimic neoplastic conditions. the present study describes a short series (3 unique cases) of rare lesions involving the cerebellum and masquerading other lesions, including a neoplasm. clinical details: three cases aged 11, 50, and 67 years presented with headache, vomiting, and cerebellar signs of 2, 8, and one‑week duration. the 2nd case was a known case of seronegative myelopathy on oral steroids and mycophenolate; the 3rd case was a known case of b – cll (diagnosed two years back) and post-chemotherapy. the first case was negative for any investigated risk factors. the clinical/radiological diagnoses in all three cases included a tumor (medulloblastoma in the first case) and inflammatory/ infective/ deposits. the histology of the lesions revealed irregular confluent necrosis, including the blood vessels and mixed inflammatory cell infiltrate, along with histiocytic aggregates (ill-formed granulomas) and giant cells. all three cases showed typical amoebic trophocytes and cyst forms, indicating a diagnosis of granulomatous amoebic encephalitis (gae). discussion: gae, a rare protozoal infection of high mortality, is caused by acanthamoeba spp. and balamuthia mandrillaris. hence, it is essential to understand the clinicopathological characteristics of this rare infection and offer a timely and accurate diagnosis. thus, it becomes necessary to consider gae in the differential diagnoses of an atypical necrotic lesion and (even in an immunocompetent patient) search for the pathogen. copyright: © 2024 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. cryopreservation of brain cell structure: a review feel free to add comments by clicking these icons on the sidebar free neuropathology 5:35 (2024) review cryopreservation of brain cell structure: a review andrew t. mckenzie1, emma l. thorn2,3, oge nnadi4, borys wróbel5,6, emil kendziorra7, kurt farrell2,3, john f. crary2,3 apex neuroscience, salem, oregon, usa friedman brain institute, departments of pathology, neuroscience, and artificial intelligence & human health, icahn school of medicine at mount sinai, new york, new york, usa neuropathology brain bank & research core and ronald m. loeb center for alzheimer's disease, icahn school of medicine at mount sinai, new york, new york, usa brain preservation foundation, ashburn, virginia, usa european institute for brain research, amstelveen, the netherlands biopreservation institute, vancouver, washington, usa european biostasis foundation, riehen, canton of basel-stadt, switzerland corresponding authors: andrew t. mckenzie · apex neuroscience · 3265 marietta st se · salem · or 97317 · usa amckenzie@apexneuro.org john f. crary · friedman brain institute · departments of pathology, neuroscience, and artificial intelligence & human health, icahn school of medicine at mount sinai · 1 gustave l. levy place · box 1194 · new york · ny 10029 · usa john.crary@mountsinai.org additional resources and electronic supplementary material: supplementary material submitted: 11 september 2024 accepted: 19 october 2024 copyedited by: georg haase published: 11 december 2024 https://doi.org/10.17879/freeneuropathology-2024-5883 keywords: cryopreservation, vitrification, brain banking, cryoprotectant, cryofixation, ice artifact abstract cryopreservation, the preservation of tissues at subzero temperatures, is a mainstay of brain banking that allows for the storage of brain tissue without the use of chemical fixatives. this is particularly important for molecular studies that are incompatible with tissue fixation. however, brain tissue is vulnerable to various forms of damage during the cryopreservation process, in particular due to the phase transition of water from a liquid to a solid state with the formation of ice crystals, which can disrupt cellular morphology. there is a critical need to characterize the effects of cryopreservation on brain cell structure at the microscopic level. in this review, we conducted a comprehensive literature search, identifying 97 studies that yielded 146 distinct observations of the effects of cryopreservation on neurohistology. we classified the reviewed studies into three main categories: cryofixation, freezing, and cryopreservation with cryoprotectants. cryofixation techniques enable vitrification and excellent ultrastructural preservation of thin tissue samples but are limited in terms of the depth of tissue that can be preserved without ice artifacts. freezing methods, particularly when applied to brain slices, can achieve rapid cooling rates that result in minimal ice artifacts detectable by light microscopy. cryoprotectant-based approaches have the potential to reduce ice damage and achieve vitrification. for thin tissue samples, immersion in cryoprotectants has been found to be effective for structural preservation. however, for larger samples or the entire brain, perfusion of cryoprotectants is necessary to perform rapid distribution, and this has a more limited evidence base. in conclusion, while current cryopreservation methods can provide sufficient quality for some downstream applications, there is a need for improved techniques that enable the cryopreservation of larger brain tissue samples while maintaining excellent structural preservation. table of contents introduction methods realist synthesis eligibility criteria search methods and study selection data extraction qualitative data analysis example frozen brain tissue data characteristics of included studies mechanisms of brain cryopreservation physical aspects of cooling how ice damages biological tissue how ice damage can be prevented ice formation during freezing procedures eutectic crystallization cellular decomposition fractures summary cryofixation preservation process effects on tissue morphology ice damage damage by tissue cutting and handling summary freezing without cryoprotectants preservation process effects on tissue morphology comparison of methods damage during tissue rewarming tissue freezing in brain banking summary cryoprotectant-based cryopreservation preservation process cryoprotectant immersion cryoprotectant perfusion summary storage temperature and length relationship between ice crystal formation and postmortem decomposition effects of cryopreservation on biomolecular preservation comparison to other tissues comparison to previous reviews limitations of this review summary of cryopreservation methods discussed implications for brain banking areas for further research conclusions abbreviations acknowledgements data availability author contributions conflicts of interest statement funding statement supplementary files references introduction cryopreservation is a key technique in contemporary brain banking (mckee, 1999; vonsattel et al., 2008; ramirez et al., 2018). here, we define cryopreservation as the preservation of tissue at temperatures below 0 °c. this can allow for the long-term storage of preserved brain tissue for subsequent research, where it is frequently used for biomolecular profiling studies such as nucleic acid sequencing or metabolomics. cryopreservation preserves tissue by decreasing the rate of molecular motion across an entire biospecimen (wowk, 2010; weng et al., 2019). however, cryopreservation can cause several types of structural artifacts, including ice formation, dehydration, displacement of tissue structures, and local aggregation of biomolecules (rosene and rhodes, 1990; jain et al., 2021). these artifacts can be ameliorated by vitrification, wherein tissue is cooled to form a glass-like amorphous solid without ice crystal formation, although this can be difficult to achieve in the large tissue volumes typical of brain banking (wowk, 2010). advances in refrigeration, cryogen production, cryobiology, and their application in brain banking over the last several decades have allowed a set of best practices to emerge for how brain tissue should be cryopreserved. for example, many brain banks rapidly cool slabs of fresh brain tissue between cooled metal plates (jones et al., 1992; vonsattel et al., 2008). despite these advances, better cryopreservation solutions are clearly needed for many potential research applications. as a result, there is a critical need to examine the effects of cryopreservation on brain cell structure, to better understand what the state-of-the-art methods are and how they could potentially be improved upon. our review focuses on unfixed tissue. however, we note that there is an extensive literature showing that brain tissue, which is pre-fixed with chemicals can be effectively cryopreserved with the use of cryoprotectants (watson et al., 1986; meissner and schwarz, 1990; otubo et al., 2021). chemical fixation aids in the process of cryoprotection because it prevents decomposition during cryoprotectant diffusion, it mitigates damage due to dehydration, and it stabilizes biomolecules in place (paljärvi et al., 1979; robards and sleytr, 1985, p. 88; meissner and schwarz, 1990). perfusion with fixatives has also been shown to help with subsequent perfusion of cryoprotectant, possibly because fixation helps to stabilize blood vessels (mcintyre and fahy, 2015). with the use of sufficient cryoprotection, the tissue can be vitrified, entirely preventing ice formation and associated ice artifacts (mcintyre and fahy, 2015). chemical fixation prevents cellular damage during cryoprotection, aids with the membrane permeability of cryoprotectants, and may help stabilize membrane structure by “hardening” the lipids (moor et al., 1980). there are methods available in practical brain banking settings for fixation followed by cryopreservation and long-term storage (estrada et al., 2017; kumarasami et al., 2023; gasperoni and dworkin, 2024). however, for some research applications, fresh tissue is preferable to fixed tissue. therefore, in this review, we focus on the cryopreservation of unfixed brain tissue. in this review, we examine existing data on the cryopreservation of brain tissue, identifying previous methods used and their impacts on histologic integrity. in the studies we identified, the most common long-term storage temperatures range from ‑20 °c to ‑196 °c (liquid nitrogen temperature). we include approaches that attempt to freeze tissue while tolerating some degree of ice formation as well as approaches that attempt to vitrify tissue and thereby avoid ice formation. we focus on the effects of cryopreservation on brain structure as identified via microscopy, rather than the features of brain tissue identified by neuroimaging, electrophysiology, cell culture, or dissociated biomolecular profiling. we discuss the mechanisms of tissue damage in cryopreservation, the efficacy of different cooling methods, the use of chemical cryoprotectants to decrease ice crystal formation, and other factors that affect variability in cryopreservation outcomes between studies. through this review, we aim to illuminate the current landscape of cryopreservation techniques, identify gaps in knowledge, and discuss potential avenues for improvements. this review may be useful for individuals banking brain tissue via cryopreservation, by offering a summary of the upsides and downsides of the different methods available. in addition, researchers who analyze cryopreserved brain tissue may find this review valuable, as it summarizes our current knowledge about the expected burden of freezing artifacts, as well as which downstream research applications may be the most compatible with cryopreserved brain tissue. methods realist synthesis we carried out a realist synthesis review aimed at establishing a theoretical understanding of cryopreservation. this method integrates elements of a systematic review with an emphasis on theory and practical application (wong et al., 2013). we chose this approach due to the wide-ranging methods used in cryopreservation of brain tissue. we report our adherence to the rameses standards (supplementary file 1) (wong et al., 2013). before formalizing our search methodology, we first scoped the literature by ad hoc searches of pubmed, google, google scholar, and by holding discussions among the authors. the review protocol was preregistered here: https://osf.io/y6hv2. eligibility criteria to be included in this review, studies needed to report original empirical data relevant to the effects of unfixed brain tissue cryopreservation on histologic integrity. we included studies that met the following two criteria: (1) examined the cryopreservation of unfixed brain or other nervous system tissue. isolated cells, sub-cellular structures, clusters of cells, or organoids alone were not sufficient. (2) assessed histological and/or morphological preservation through microscopy or other structural analysis methods. through this criterion, we focused on structural and morphological integrity rather than functional or biomolecular preservation alone. both human and non-human animal studies were eligible for inclusion. if multiple publications report on the same underlying data, we only included the study that drew the most relevant conclusions about the effects on cryopreservation. when a single study reported multiple independent observations about the effects of cryopreservation, such as a different type of brain sample, cooling rate, or rewarming method, they were each extracted from the study as separate data points. search methods and study selection we searched pubmed to identify relevant studies with keywords related to cryopreservation of brain tissue. we searched the pubmed database from its inception to april 18th, 2024, using the following search query: «(freezing[tiab] or ice[tiab] or cryopreservation[tiab] or "cryopreservation"[mesh]) and (histology[tiab] or microscopy[tiab] or ultrastructure[tiab] or "electron microscopy"[tiab] or "ultrastructure"[subheading]) and (brain[tiab] or "central nervous system"[tiab] or "brain"[mesh])». one reviewer screened the titles and abstracts and identified studies for full-text review based on the eligibility criteria (supplementary file 2). next, one reviewer evaluated the full texts and selected studies to include (supplementary file 3). to find additional eligible studies, we also (a) hand-searched the reference lists and referencing studies of the most relevant papers and (b) performed additional ad hoc searches of the literature relevant to this topic. any studies meeting the inclusion criteria that were identified outside the formal search process, such as in the initial literature scoping process, were also included. data extraction for each included study, we extracted relevant data: species; specimen type; cryoprotectant, if any reported; cooling method; storage temperature; storage length; rewarming method; visualization method; any additional outcome specifier reported; the histological or morphological outcome(s) described; and any relevant quote or quotes. we treated each distinct cryopreservation condition, tissue type, visualization method, or other methodologic difference reported in a study as a separate “observation”. for example, if a study reported outcomes for tissue cooled at two different rates, these were recorded as two observations. however, minor variations in otherwise highly similar cryopreservation conditions or visualization methods from the same study were grouped into a single observation. the determination of whether two similar cryopreservation conditions or visualization methods should be grouped into one observation or separated was based on an assessment of the level of detail and independence reported for each in the text. for each study, one reviewer performed the data extraction. on randomly selected articles, chosen via https://www.random.org/, one other reviewer then evaluated the extracted data. this independent evaluation process was performed on n = 20 (21 %) of the studies and did not identify any major discrepancies in the extracted data. the extracted data table is available here: https://github.com/andymckenzie/cryopreservation_review. qualitative data analysis due to the wide range of cryopreservation techniques and outcomes reported, we performed a qualitative synthesis rather than attempting formal quantitative meta-analysis or grading of outcomes. we drew insights from the extracted data to categorize studies and identify key themes. we categorized the included studies into three partially overlapping types based on the cryopreservation approach used: (1) cryofixation, (2) freezing, and (3) cryopreservation with cryoprotectants. we focused on comparing histologic outcomes between different methods of cryopreserving brain tissue, as well as identifying mechanistic insights into the causes of these outcomes. example frozen brain tissue data the brain tissue used was obtained and de-identified at the icahn school of medicine at mount sinai in accordance with its policies, regulations, and institutional review board recommendations. all brain tissue processing was performed in the neuropathology brain bank & research core. the brain donor was female and was 76 years old at the time of death. the postmortem interval from time of death to freezing was 70.5 hours. the brain was removed from the skull and placed in a cooler for transport from the autopsy suite to the brain bank. the brain was bisected into two hemispheres. the right hemisphere was immersion fixed in toto in 10 % neutral buffered formalin, while the left hemisphere was sectioned fresh into approximately 0.5 cm coronal slices. these fresh slices were parcellated into approximately 2 x 2 cm blocks, and then cryopreserved between teflon-coated metal plates cooled at ‑80 °c. the tissue was stored at ‑80 °c for 5.3 years. one frozen tissue block of the lateral occipital cortex was then moved to a ‑20 °c freezer for 6 hours and 10 minutes. formalin fixative was also frozen at ‑20 °c and then moved to 4 °c at the same time as the tissue. the tissue was placed in the slushy consistency formalin at 4 °c for 18 hours before being moved to phosphate-buffered saline (pbs) containing 0.1 % sodium azide at room temperature for 4 hours and 45 minutes. it was then placed in room temperature formalin for 55 minutes before being processed for histology. frozen-thawed tissue from the lateral occipital cortex region was compared to immersion fixed tissue from the calcarine cortex of the contralateral hemisphere, which was fixed in toto for 37 days. the fixed tissue was paraffin embedded, and a microtome was used to prepare consecutive sections with a thickness of 5 µm, as previously described (mckenzie et al., 2022). these sections were then placed on glass slides, deparaffinized, and stained with hematoxylin and eosin, followed by imaging with a digital slide scanner. characteristics of included studies through our formal search procedure, we screened 402 abstracts, assessed 108 full-text articles, and identified 50 studies meeting our inclusion criteria (figure 1). an additional 47 studies were included based on citation analysis or supplementary ad hoc searches, yielding a total of 97 studies for this review. we categorized the included studies into three partially overlapping types based on the cryopreservation approach: (1) cryofixation studies, which used ultrarapid cooling methods like high-pressure freezing or plunge freezing to achieve vitrification of at least part of the tissue; (2) freezing studies, which cooled tissue without cryoprotectants at various rates, allowing some degree of ice crystal formation; and (3) cryopreservation with cryoprotectants studies, which used substances like dmso or glycerol to reduce ice crystal formation during the cooling process. the studies were performed on tissue from various species, including humans, rats, and mice. before discussing the outcomes reported in these studies, we first review the mechanistic aspects of cryopreservation that provide important context for interpreting the results. figure 1. study selection flow diagram. studies were screened and selected using the web-based software rayyan. mechanisms of brain cryopreservation physical aspects of cooling to understand the effects of cryopreservation on brain cell structure, it is helpful to build up a basic understanding of what happens during cryopreservation of brain tissue (mcgrath, 1985; karlsson and toner, 1996; wowk, 2010). note that this section is not meant to be a comprehensive summary of this complex topic, but rather to provide a context for the observed phenomena and outcomes in the empirical studies we reviewed. the reason that cooling can act as a preservation method is that as temperature decreases, the kinetic energy of molecules is reduced, leading to slower molecular motion and chemical reactions. at low enough temperatures, a specimen, or parts of a specimen, will convert from a viscous liquid to a glass-like state, in the process of vitrification (fahy and wowk, 2021). this effectively “pauses” biological processes, including those that lead to cell death and tissue decay (wowk, 2010). however, as the temperature decreases below the freezing point of water, water molecules also have a thermodynamic tendency to spontaneously rearrange into ice crystals, which cause damage to the tissue. in biological tissues, there is a further complication, which is the potential for non-water substances to crystallize, such as in the process of eutectic crystallization. we will discuss the effects of ice formation first. how ice damages biological tissue ice formation leads to damage to brain cell structure through several mechanisms. these forms of damage can collectivity be called “freezing injury”. first, formed ice crystals can cause mechanical damage to local structures as they expand, such as causing the displacement or rupture of cell and organelle membranes (fahy and wowk, 2021). second, as water is drawn towards growing ice crystals, this causes large stresses to build up near the ice-water interface, which is a phenomenon known as cryosuction (gerber et al., 2022). this stress is associated with high local pressures of around 1 mpa per degree of temperature below the freezing point, leading to potential alterations of biomolecular distributions in the local area. third, as extracellular and intracellular water is removed and cells become dehydrated, high solute concentrations in the cells lead to structural damage (fahy and wowk, 2015). damage due to high solute concentrations has been called “solution effects” (mazur et al., 1972). for example, solution effects have been reported to cause the denaturation and aggregation of biomolecules (li et al., 2023). the formation of ice during freezing can be used to permeabilize cells, which has been attributed to both mechanical disruption of membranes by ice and osmotic forces (zhang et al., 2016). smaller ice crystals may be particularly likely to cause the membrane damage that contributes to cell permeabilization (zhang et al., 2016). as cell membranes are permeabilized, this allows soluble biomolecules to leak out of cells and potentially diffuse away from their original locations, which is another form of damage to brain cell structure during cryopreservation (rossner et al., 2006; notter et al., 2014; gellhaar et al., 2017). the location of ice formation across tissues is not random, but rather can be predicted to some degree. in many organs, ice formation tends to propagate along the vascular system, likely because there is no barrier to crystal growth in these areas (rubinsky and pegg, 1988). when these tissues are examined under a microscope, the structural changes caused by large ice crystal formation also follow predictable patterns. upon rewarming, areas of tissue that previously underwent ice formation tend to have anisotropic spaces empty of tissue, indicative of the crystalline structures of ice when visualized under a microscope. these ice damage artifacts have been described as vacuoles, “swiss cheese holes,” or as having a “sponge-like” appearance (chambers et al., 1932; taqi et al., 2018). in addition to generating open spaces, ice formation can cause other structural changes, such as disrupting membrane structures or creating a web-like pattern (reticulation) in the nucleus and cytoplasm (möbius et al., 2010). this reticulation can look like irregularly shaped dark strands when viewed under an electron microscope. after severe ice damage, the tissue can be dramatically modified. it has been reported that large ice crystals can distort cell membranes and organelles to such an extent that the pre-existing structural configuration is unrecognizable and the underlying structure of the specimen appears to be destroyed (ryan, 1991). how ice damage can be prevented one method available to entirely prevent ice formation is the use of an extremely rapid cooling rate. if the temperature of the biospecimen is reduced very quickly, ice does not have the time to form or grow to a meaningful degree. the minimum rate of cooling necessary to cool a sample fast enough to “outrun” the speed of ice formation and achieve vitrification is called the critical cooling rate (fahy and wowk, 2021). because the required cooling rate is very rapid, this generally requires exposing very small samples of cells or tissues to extremely cold temperatures. in larger samples, it is not possible to perform this, because the slow rate of dissipation of heat by convection in biological tissue prevents it. for example, one source reports that an infinitely fast cooling rate at the surface of an aqueous sample would lead to a cooling rate of around 5000 k/s at the depth of 100 µm, which is not sufficiently fast for vitrification (studer et al., 2014). another method to prevent ice formation is to add cryoprotectants to the tissue. cryoprotectants are chemicals that decrease or prevent freezing injury during cryopreservation (fahy and wowk, 2015). cryoprotectants can aid in vitrification by directly forming hydrogen bonds with water molecules (lee and baiz, 2022), causing freezing point depression as a colligative effect and/or increasing the viscosity of the tissue. although there are endogenous chemicals in tissues that act as cryoprotectants, the term “cryoprotectant” usually refers to an external chemical that is added to a tissue for this purpose. these substances often replace water as the tissue solvent. examples of cryoprotectants include small molecules such as glycerol, ethylene glycol, dimethyl sulfoxide (dmso), or ice blockers such as antifreeze proteins (fahy and wowk, 2015). notably, cryoprotectants and faster cooling rates can work together to protect a tissue from ice damage during cryopreservation. with a higher concentration of cryoprotectants, a lower cooling rate is necessary to prevent ice damage, and vice versa (wowk, 2010). by preventing ice formation, both cryoprotectants and faster cooling rates are expected to improve structural preservation quality during cryopreservation. however, the introduction of cryoprotectants can also alter cellular and molecular structures (robards and sleytr, 1985, p. 77). most commonly, the high concentration of cryoprotectants needed to prevent ice formation leads to cell shrinkage and/or osmotic stress, particularly if the addition of cryoprotectants is not performed in a graded manner. to minimize damage from osmotic stress, cryoprotectants are typically added slowly, allowing cells time to equilibrate and adjust to the changing osmotic environment. in larger samples, the slow addition of cryoprotectants via immersion also renders the tissue vulnerable to decomposition during this process. one way to minimize the time of cellular decomposition is to perfuse cryoprotectants through the blood vessels of the brain, which allows the cryoprotectants to extravasate into the tissue. however, perfusion of the postmortem brain is a challenging problem, especially in the setting of extended periods without blood flow (mcfadden et al., 2019). ice formation during freezing procedures in the absence of cryoprotectants, complete vitrification is not achievable for larger samples. therefore, ice crystals will form. the total amount of ice formed is primarily dependent on the water content of the tissue and the final temperature reached, rather than on the cooling rate. for example, in one study of frozen sponge cake, the total ice volume fraction – about 60 % – was the same in samples frozen with fast (17.2 °c/min) and slow (0.3 °c/min) cooling rates (zennoune et al., 2022). however, the distribution and size of ice crystals can differ substantially based on the cooling rate, which can have important effects on microstructural preservation. relatively faster cooling results in the formation of numerous but smaller ice crystals, which are less disruptive to cellular structures (pérez-bermúdez et al., 2023). this is because rapid cooling reduces the time available for ice crystal growth and rearrangement. smaller, more numerous ice crystals that form during rapid cooling are generally found to be less damaging to tissue morphology than larger crystals that form during slow cooling. notably, sufficiently small ice crystals are not expected to be visible via light microscopy (szabo and burg, 2024). it is also helpful to understand mechanistically what occurs during slower cooling processes without a sufficient concentration of cryoprotectants to vitrify the entire tissue. in this situation, if the temperature is lowered to below the glass transition temperature, then vitrification is still expected to occur in localized areas between pockets of ice crystals. this is because as ice formation progresses during cooling, the concentration of solutes in the remaining unfrozen areas eventually becomes high enough to cause these areas to vitrify when the temperature reaches the glass transition temperature (wowk, 2010). localized vitrification within confined spaces during slow cooling is particularly relevant for intracellular spaces, which are densely packed with proteins and other biomolecules (clarke et al., 2013). cryoprotectants can protect cells from morphological damage during this process by limiting ice formation and preventing harmful, elevated salt concentrations. for example, in a study of slowly cooling jurkat cells (an immortalized t cell line) in the presence of dmso, an endothermic event was observed around ‑47 °c, which was attributed to a colloidal-like glass transition in the cytoplasm resulting from cellular dehydration and increased intracellular macromolecular crowding (meneghel et al., 2019). the specific temperature of any intracellular glass transition will vary depending on many factors, such as cell type, protein content, solute concentration, and the concentration of any cryoprotectants. in the context of brain tissue, even if some degree of intracellular vitrification occurs, the fine cell processes or cell-cell connections may be mechanically damaged if extracellular ice is formed. eutectic crystallization although not commonly discussed, we note for completeness that eutectic crystallization can occur during cryopreservation in addition to the crystallization of water forming ice. a eutectic system is a mixture of substances that has a melting point below either of the original substances. when the melting point of this mixture is reached, the eutectic system can solidify. in practical terms, sodium chloride tends to be the main relevant electrolyte in biological tissues like the brain (han and bischof, 2004). as ice forms during cryopreservation, the water-sodium chloride solution increases in concentration and it can form a eutectic system because the mixture has a melting point lower than either of the individual components. in one study, eutectic crystallization of water-sodium chloride solutions – forming hydrohalite – occurred at approximately ‑30 °c, but this may differ based on the cooling rate (han and bischof, 2004). in yeast cells, hydrohalite has been found primarily around cell membranes (okotrub and surovtsev, 2013). such eutectic crystallization can cause damage to cellular structures beyond that caused by ice formation alone (han and bischof, 2004; klbik et al., 2022). it is unclear how much eutectic crystallization occurs in brain tissue during cryopreservation. however, it theoretically could pose additional challenges for maintaining cellular morphology. notably, the use of cryoprotectants or the application of very rapid freezing techniques can potentially prevent eutectic crystallization. cellular decomposition another issue during cryopreservation is cellular decomposition by autolysis (self-digestion by cellular enzymes) and putrefaction (bacterial breakdown of tissues). these processes can occur during cooling and thawing of large brain tissue samples because they do not cool instantaneously, due to the limits of convective heat transfer (pontén et al., 1973). autolysis begins immediately after death when cellular enzymes are released, while putrefaction typically starts days later when bacteria proliferate. importantly, cellular decomposition is dramatically inhibited by lower temperatures (krassner et al., 2023). this is another reason that swift cooling is critical in cryopreservation protocols. fractures one downside of cooling to very low temperatures is the possibility of fractures. fracturing can occur in tissues or organs that are vitrified or frozen. it has been attributed to thermal gradients – i.e. differences in temperature in different parts of the organ (woods and mullen, 2016). it seems to be more common in larger specimens. one study found that brain tissue blocks of larger than 2.5 to 3.0 cm tend to fragment when immersed in liquid nitrogen-cooled liquid isopentane (vonsattel et al., 2008). fracturing has also been reported when whole rodent brains are cooled in liquid nitrogen (peters, 2010). fractures are more likely to occur at lower temperatures, as thermal stress accumulates. one study found that thermal stress in a cryoprotective solution is negligible until a temperature of around 5–10 °c above the glass transition temperature, depending on the viscosity of the solution (rabin et al., 2006). slower cooling rates allow more time for temperature to equilibrate across different areas of the organ or tissue, lowering the magnitude of thermal gradients. while slower cooling rates are associated with lower fracture rates, this effect appears to be relatively modest. for example, in one study, increasing the cooling rate by three orders of magnitude, from 0.5 °c/min to 500 °c/min, decreased the temperature at which thermal stress began to build up and fractures began to occur by only 7.2 °c (rabin et al., 2006). the formation of brittle ice crystals in tissue can not only enhance fracturing during cooling or storage, but also during subsequent sectioning, posing a challenge for sample preparation of cryopreserved tissue (marshall, 1940). summary most of the structural damage that occurs during cryopreservation of brain tissue is related to ice formation (table 1). in addition, there are other forms of structural damage, such as eutectic crystallization, cellular decomposition, and fractures that can be independent of ice formation. mechanism description mechanical ice damage ice crystals can cause displacement or rupture of cell and organelle membranes cryosuction water drawn towards growing ice crystals causes high local pressures, leading to potential alterations of biomolecular distributions dehydration-associated damage rapid removal of water, leads to osmotic stress, cell dehydration, and potentially anisotropic shrinkage solution effects high solute concentrations during freezing can cause biomolecular denaturation and aggregation, as well as other forms of damage cell membrane permeabilization if cell membranes are damaged during cryopreservation, they can be permeabilized, leaking intracellular biomolecules into the local area cryoprotectant toxicity cryoprotectants, especially if used at high concentrations and/or introduced rapidly, can cause osmotic stress and potentially other forms of toxicity eutectic crystallization formation of eutectic crystals, such as those formed by water-sodium chloride, can cause damage to cellular structures cellular decomposition regions of the tissue that have not yet cooled to sufficiently low temperatures may undergo autolysis or putrefaction fractures susceptibility to fractures during cooling and storage table 1. summary of mechanisms of damage during cryopreservation of brain tissue. cryofixation preservation process cryofixation refers to the ultrarapid cooling of tissue that is fast enough to prevent ice formation during cryopreservation (gilkey and staehelin, 1986). major types include (a) plunge freezing, which directly immerses a sample into a liquid cryogen, (b) propane jet freezing, which sprays a liquid cryogen onto a sample, (c) cold metal block freezing, which rapidly brings the sample into contact with a pre-cooled metal surface, and (d) high-pressure freezing, which performs the cooling in a high pressure system to help suppress ice formation (gilkey and staehelin, 1986). one source reports that, in tissues without any cryoprotectants added, vitrification will result when it is cooled at a rate faster than 200,000 k/s (studer et al., 2014). by increasing the pressure to around 2000 bar, high-pressure freezing lowers the freezing point of water to ‑22 °c, which reduces the critical cooling rate for ice formation by a factor of 100 (studer et al., 2014). the vitrification depth of a cryofixation procedure is the distance from the surface of the tissue that can be vitrified. with the use of plunge freezing or cold metal block freezing, the vitrification depth is usually approximately 10–20 µm, but this can be increased 10-fold to samples around 200 µm thick with the use of high-pressure freezing (studer et al., 2014). while increased pressure to this degree can be lethal, it can be kept to a very short period of less than 0.1 seconds during the high-pressure freezing procedure (moor et al., 1980). as a result, the increased pressure is expected to have minimal effect on the tissue, although it has been reported to affect the structure of some biomolecules (vanhecke et al., 2010). occasionally, cryoprotectants are also used to aid in the process of cryofixation. this helps to prevent ice damage, but can lead to structural alterations of their own. as a result, cryoprotectants are only sometimes used in cryofixation, depending on the goals of the experiment. notably, potential damage during rewarming is often avoided entirely for cryofixation studies because investigators frequently use freeze substitution, or less commonly freeze fracture, to avoid this issue. effects on tissue morphology the majority of studies we identified found that cryofixation, and especially high-pressure freezing, can enable pristine ultrastructural preservation of brain tissue in the areas that are vitrified (table 2; supplementary file 4) (benson et al., 1985; guo et al., 2020). cryofixation preserves membranes, organelles, synapses, and other neural structures with life-like morphology. indeed, vitrification by cryofixation is considered the best method for structural preservation, superior to chemical fixation, because it can preserve tissue within milliseconds, rather than chemical fixation, which takes at least seconds to minutes (sosinsky et al., 2008). sample type (study) cooling method visualization method key findings slabs of mouse cerebellar cortex (heuser et al., 1976) pressing tissue against copper block cooled to liquid helium temperature freeze-fracture electron microscopy first 20 µm has good ultrastructure; deeper than 20 µm, cell processes are distorted and large ice crystals appear present 3 mm mouse cerebellar hemisphere slices (landis and reese, 1983) freezing with a copper block cooled with liquid helium freeze substitution, electron microscopy well preserved ultrastructure in surface areas with ability to detect filaments; deeper regions have ice damage 1 mm rat cerebral cortex blocks (linner et al., 1986) frozen against copper block cooled with liquid nitrogen vacuum drying, osmication, electron microscopy excellent preservation with no ice damage in first 10 µm; deeper, ice crystals cause damage by physically compressing tissue rat neurointermediate lobe (tian et al., 1991) falling onto copper plug cooled with liquid nitrogen freeze substitution, electron microscopy good morphology in the first 10–12 µm; deeper, ice crystals make membranes indistinct, and less material in extracellular space 200 µm thick sections of mouse cortex (korogod et al., 2015) high-pressure freezing, storage at liquid nitrogen temperature freeze substitution, electron microscopy at 10 µm into the sample, high quality ultrastructure; at 50 µm, cellular elements disrupted in a lattice pattern due to ice crystals 200 µm thick sections of mouse cortex (dumoux et al., 2023) cryoprotected in 20 % bovine serum albumin, then high-pressure freezing cryo-plasma focused ion beam scanning electron microscopy high-quality preservation via vitrification, with structural preservation of synapses, myelin, and mitochondria table 2. reported histologic effects in representative observations from cryofixation studies. there is a large literature comparing vitrification via cryofixation to chemical fixation, which are two of the major ways currently available to ascertain native tissue structure. in general, most studies comparing the two methods have found that the ultrastructural appearance is largely similar, with cryofixation sometimes showing subtle improvements in the preservation of certain features (landis and reese, 1981, 1983; reger and escaig, 1988; zhao et al., 2012; korogod et al., 2015). for example, one study found that cryofixation of samples by placing them against a metal surface cooled to ‑196 °c better maintained the naturally smooth contour of cell processes and organelles (hirokawa and kirino, 1980). the authors also suggested that cryofixation appears to better preserve the extracellular spaces and better retain water-soluble components. better maintenance of water-soluble components, may in turn lead to a more natural density of the cytoplasm and the mitochondrial matrix (hirokawa and kirino, 1980). it has been reported that microtubules are better preserved after cryofixation (moor et al., 1980). cryofixation also leads to better preservation of antigenicity (hisano et al., 1986). finally, cryofixation likely preserves a more natural distribution of water and ions between intracellular and extracellular compartments (harreveld and fifkova, 1975; moor et al., 1980). however, despite these benefits, the differences between cryofixation and chemical fixation are generally subtle when both techniques are optimally performed. indeed, tissue that is chemically fixed and cryoprotected with glycerol has been used as a control to ensure that the cryofixation procedure adequately preserves the structure of the tissue (heuser et al., 1976). many of the chemical fixation artifacts that have been described, such as cell shrinkage and membrane discontinuities, appear to be largely related to the tissue processing steps after fixation rather than due to the initial fixation itself (sosinsky et al., 2008). ice damage cryofixation can enable excellent ultrastructural preservation in successfully vitrified regions. however, it is expected that in sufficiently deep areas of the tissue, where the cooling rate is insufficient to achieve vitrification, there will be ice crystal formation. the effects of ice crystals on tissue morphology manifest depending on the depth of the tissue and the size of the crystals. in the areas that border vitrified tissue, where the cooling rate becomes too slow to vitrify the tissue, small ice crystals and more subtle signs of suboptimal preservation are expected. for example, there may be blebbing of the nuclear envelope or alterations of the mitochondria (möbius et al., 2010). membranes may not show their clear expected structure (senda et al., 2005). in deeper areas, the ice crystals are expected to be larger and to increasingly distort cellular structures, pressing the latter into one another, and causing large areas to be entirely devoid of structure (heuser et al., 1976; senda et al., 2005). several studies also describe ice artifacts in deeper regions as causing reticulation of intracellular areas (linner et al., 1986; siksou et al., 2007; möbius et al., 2010). damage by tissue cutting and handling to isolate a sample that is sufficiently thin for cryofixation, cutting the tissue is generally required. this in turn leads to compression artifacts near the cut surface of the tissue, which can be severe (zuber et al., 2005). one study omitted the first 20 µm of the sample from their analysis as a result of these artifacts (borges-merjane et al., 2020). another study noted that dissection led to ultrastructural artifacts within 100 µm from the cutting site (siksou et al., 2007). handling artifacts that occur during preparation for cryofixation have also been reported to cause detachment of myelin lamellae or separation of axons from the myelin sheath (möbius et al., 2010). summary the major limitation of cryofixation methods is their inability to preserve large tissue volumes without ice damage in the sample core. although regions close to the surface of a tissue sample can be exquisitely preserved in a vitrified state, deeper regions suffer from progressively more severe freezing artifacts due to slower cooling rates. increasing the pressure during freezing or using cryoprotectants can reduce the critical cooling rate required for vitrification and thereby increase the vitrification depth. the thinness of the samples required for cryofixation is also a challenge for long-term brain banking, as only small pieces of tissue can be stored. therefore, while cryofixation enables optimal ultrastructural preservation of brain tissue, it is currently limited to relatively small samples, precluding its widespread use in neuroscience research requiring whole brain or thick tissue preservation. freezing without cryoprotectants preservation process in the absence of exogenous cryoprotectants or sufficient cooling rates for cryofixation, ice formation during freezing of brain tissue is inevitable. the critical question becomes how much damage to the tissue morphology will occur because of this ice formation. slower cooling rates allow water to leave cells during the freezing process, causing ice to form predominantly in the extracellular space. some studies have suggested that more rapid cooling rates are preferred when the goal is to preserve tissue morphology, particularly in specimens with complex microanatomical structures like brain tissue. this is because extracellular ice, while less damaging to cell viability because it helps avoid intracellular ice, can severely disrupt tissue structures, such as cellular processes and synaptic connections (pegg, 2007). rapid cooling minimizes the time available for ice crystal nucleation, growth, and rearrangement, which may cause less damage to cellular morphology (rosene et al., 1986). therefore, in the context of freezing brain tissue without cryoprotectants, the most common approach used by investigators with the goal of morphological preservation has been to maximize the cooling rate. the main challenge lies in achieving sufficiently rapid cooling throughout the entire tissue volume, particularly for larger specimens. effects on tissue morphology there were numerous methods described for freezing brain and other nervous tissue in the studies we identified. these included (a) placing the specimen in a freezer, such as one set to ‑80 °c or ‑20 °c (vonsattel et al., 1995; kagan and viner, 2022), (b) placing the tissue in or between a form of dry ice at ‑78.5 °c (beach et al., 2008), (c) immersing the specimen in liquid nitrogen (ulfig et al., 1998), (d) immersing the specimen in liquid nitrogen vapor (davis et al., 2019), (e) immersing the specimen in isopentane that has been pre-cooled with a different cryogen, such as liquid nitrogen or dry ice (priemer et al., 2023), or (f) freezing the specimen against metal surfaces that have been pre-cooled with a different method, such as liquid nitrogen vapor or a ‑80 °c freezer (vonsattel et al., 2008). the efficacy of these freezing methods varied considerably in the studies we identified (table 3; supplementary file 4) (rosene and rhodes, 1990). one study found that freezing human brain slices either with dry ice snow, isopentane pre-cooled in liquid nitrogen, copper plates pre-cooled to ‑80 °c, or placing it in a ‑20 °c freezer after embedding the tissue in a cryo-conductive mixture of chemicals called optimal cutting temperature (oct) all provided good results, with well-preserved brain tissue architecture and cell morphology as seen on light microscopy. in contrast, samples directly frozen in a ‑80 °c freezer or immersed in liquid nitrogen showed high levels of ice crystal artifacts (meyronet et al., 2015). a particularly important clinical application of freezing methods is the use of frozen sections. one study found that snap freezing biopsy samples in dry ice-cooled isopentane led to considerably better histology preservation and less ice formation compared to freezing samples by placing them on the freezing shelf within the cryostat at a temperature of around ‑20 °c (priemer et al., 2023). sample type (study) cooling method visualization method key findings human frontal white matter samples (ansari et al., 1976) frozen in a dry ice-acetone bath, stored at ‑80 °c thawed in fixative, electron microscopy myelin has separation of lamellae and numerous regions with disruption of the lamellae mouse sciatic nerves (kirschner et al., 1979) frozen in a freon 22 slush cooled with liquid nitrogen x-ray diffraction, freeze-fracture electron microscopy most membrane arrays in myelin sheaths are distorted, but a few regions that are normally ordered remain 1 cm thick dog hemisphere sections (baraibar and schoning, 1985) frozen to ‑18 °c, stored for 2 days fixed at room temperature, light microscopy with h&e staining large vacuoles suggestive of ice formation, lack of detail in cell outlines, hemolysis, increased eosin staining 1 cm thick human brain sections (nochlin et al., 1993) frozen between aluminum plates cooled with dry ice immersion fixed, electron microscopy high quality ultrastructure without evidence of ice crystals found in most images half human brains (vonsattel et al., 1995) frozen en bloc in a freezer or on dry ice entire specimen warmed, cut prior to microscopy severe freezing artifacts so that it is difficult for experienced people to distinguish cells 10 to 12 mm thick slices of human brain (meyronet et al., 2015) surrounded by large pulverized dry ice, at least 2x the volume of the tissue cryostat sections, light microscopy high quality preservation of brain tissue architecture and cellular morphology surgical biopsies of neoplastic brain tissue (priemer et al., 2023) covered in oct compound and frozen in a cryostat at ‑18 to ‑21 °c cryostat sections, light microscopy, h&e staining high ice crystal and vacuole formation, lower preservation quality than faster cooled samples in the same study table 3. reported histologic effects in representative observations from freezing studies. h&e: hematoxylin and eosin, oct: optimal cutting temperature. comparison of methods the differences in histologic outcomes between these freezing methods is thought to largely stem from their different cooling rates, which in turn is related to the properties of the cooling media. freezer-based methods, while practical for long-term storage, typically offer slow cooling rates because they rely on convective heat transfer via the air, which is a poor thermal conductor. as a result, the cooling process is gradual and less efficient. immersion of brain slices in liquid nitrogen vapor, although also a gas, has been reported to result in freezing with good morphological preservation (vonsattel et al., 1995; davis et al., 2019). this is likely in part because liquid nitrogen vapor is much colder (often between ‑150 °c and ‑196 °c) than a typical ultracold freezer (‑80 °c). the greater temperature differential between the liquid nitrogen vapor and the tissue allows for more rapid heat transfer and cooling, even though nitrogen vapor is still a relatively poor thermal conductor compared to liquids. dry ice methods provide faster cooling than the use of freezers, but the cooling rate can be inconsistent depending on how well the tissue contacts the dry ice. otherwise, these methods face the same issue of inefficient heat transfer through gas. freezing on pre-cooled metal surfaces can achieve very high cooling rates at and close to the freezing surface. one study measured the efficacy of aluminum sheets pre-cooled with dry ice in cooling 1 cm thick fresh brain slices (nochlin et al., 1993). using a thermocouple probe, the authors found that a core temperature in the slices of ‑70 °c was reached within 10 minutes. they found no ice crystals in most light micrographs. this result suggests that a sufficiently fast cooling rate was achieved, so that only relatively small ice crystals were formed. however, the rate of freezing when using cooled surfaces is expected to decrease with depth. as a result, methods relying on physical contact with cooled surfaces are more effective for preserving the morphology of surface structures, such as brain slices, but will be less effective for larger specimens, especially if the tissue is not flat. when comparing isopentane and liquid nitrogen immersion, isopentane is generally preferred (wieser et al., 2022). the key reason is that liquid nitrogen has an extremely low boiling point of ‑196 °c. this means that even a slight degree of warming due to the specimen being plunged into it causes a rapid vaporization that leads to an insulating layer of vapor around the specimen that impedes effective heat transfer out of the specimen. this is known as the leidenfrost effect. on the other hand, isopentane cooled by a different cryogen remains liquid at temperatures well below its boiling point of 28 °c. this large gap between its cooled temperature and boiling point allows isopentane to maintain direct contact with the specimen without forming a vapor barrier, thus enabling more efficient heat transfer and rapid cooling. in other words, cooled isopentane is not susceptible to the leidenfrost effect. for this reason, pre-cooled isopentane allows for better thermal contact as well as the potential for very low temperature, making it a commonly preferred option in biobanking. one study measured the rate of cooling using dry ice and dry ice-cooled isopentane in a albumin-gelatin block meant to simulate brain tissue (rosene et al., 1986). they found that dry ice led to cooling rates of ‑1.5 to ‑1.7 °c/min, while dry-ice cooled isopentane led to cooling rates of ‑3.2 to ‑3.6 °c/min, consistent with isopentane acting as a very effective cryo-conductor. one reported downside of immersion in both isopentane and liquid nitrogen is the higher rate of fractures in brain tissue, as compared to freezing in liquid nitrogen vapor (vonsattel et al., 1995). as previously discussed, the fractures may be caused by mechanical stress due to sudden temperature changes (adam et al., 1990). pulverized dry ice has been reported to be able to cryopreserve brain hemispheres without causing deep crevasses due to opposed temperature fronts in the specimen (annese, 2004). damage during tissue rewarming the rewarming phase can significantly impact the final tissue morphology and is often thought to cause more damage than the initial freezing. this is related to the physics of ice formation. at higher subzero temperatures, the probability of forming new ice nuclei is relatively low, while the rate of ice growth is relatively high (fahy and wowk, 2015). on the other hand, at low subzero temperatures that are still above the glass transition temperature, the probability of forming new ice nuclei is relatively high, while the rate of ice growth is relatively low. as a result, as the temperature increases during tissue warming, smaller ice nuclei have the opportunity to recrystallize into larger ice crystals (fahy and wowk, 2015). larger ice crystals are expected to cause morphological structural damage. to mitigate ice damage during rewarming, several approaches have been developed. some sources have reported that more rapid thawing of tissue leads to better outcomes. for example, one study found better neural transplantation outcomes after thawing in a hot water bath at 37 °c for 30–40 seconds, compared with thawing by leaving the neural tissue at room temperature for 5–7 minutes (das et al., 1983). another source found high-quality ultrastructural preservation of several non-brain tissue types when blocks were frozen in liquid nitrogen, sectioned into around 5 mm3 blocks on dry ice, and then rapidly thawed at 37 °c in fixative (galhuber et al., 2021). alternatively, one protocol involved transferring frozen tissue directly into chilled fixative solutions (ramirez et al., 2018). this study reported minimal freezing artifacts and well-preserved cell morphology when transferring frozen brain tissue to 1 °c chilled formalin for 72 hours before further processing. the approach of breaking frozen tissue into pieces and immersing it into fixative while the fragments are still frozen, prior to fixation at refrigerator temperature, has also been reported to be effective in rewarming frozen pancreatic tissue (fortunato et al., 2016). as an additional complication, rewarming frozen tissue may actually decrease the observed burden of histologic alterations in some circumstances. for example, one study using slow freezing of rat cutaneous nerves at ‑10 °c to ‑15 °c with subsequent freeze substitution followed by electron microscopy found that the tissue showed severe ice damage and ultrastructural artifacts (menz, 1971). only some ultrastructural features could be recognized, which was attributed to severe dehydration of other structures such as neurofilaments and mitochondria. however, when the slowly frozen tissue was rewarmed via rapid thawing to 40 °c prior to fixation, electron microscopy showed an improved preservation of neurofilaments and mitochondria. notably, there were still numerous artifacts; for example, the myelin layers were disturbed, and the mitochondria were empty and lacked clearly defined membranes. mechanistically, the dehydration that prevented the visualization of structures in the freeze-substituted state may have been reversed by thawing. a separate study examined electron microscopy in 1 cm thick sections of human brain tissue that were frozen between dry ice-cooled aluminum plates and immersion-fixed during thawing (nochlin et al., 1993). the study found that the preservation quality in most images was similar to that in non-frozen brain tissue, with the different cell types and processes easily able to be identified. this finding contradicts the literature using cryofixation and freeze substitution followed by electron microscopy, which, as discussed above, generally finds severe histologic damage in regions deep enough to be affected by ice crystals. one source discusses ultrastructural preservation quality after the cryopreservation of several different cell types, especially hepatic cells, but also with some discussion of brain cells (trump et al., 1965). the authors note that slow freezing causes significant extracellular ice damage, but that there is a significant amount of what they call “structural restitution” on thawing when cells are preserved with slow freezing. they primarily discuss intracellular features such as mitochondria, which are more likely to be protected by the predominantly extracellular ice formation that will occur during slow freezing. also, the extent to which any such structural restitution yields the same original morphology present prior to freezing is not entirely clear. taken together, the issue of histologic damage during rewarming, and how to best decrease or prevent it, appears to be an unresolved one. there is a need for more research comparing different cooling and rewarming methods and for the development of more objective histologic metrics to compare across methods and studies. tissue freezing in brain banking perhaps the simplest way of freezing tissue in brain banking is to isolate the whole brain or a brain hemisphere and place it in a freezer. this method was used early in the history of brain banking (bird, 1980). however, this method has been found to lead to severe ice artifacts on histology, consistent with the slow cooling and rewarming rates expected when freezing and thawing a large brain specimen in this manner (tourtellotte et al., 1993; vonsattel et al., 1995, 2008). cryopreserving very large samples is also impractical for most research applications, because accessing small parts of the brain for study would require several freeze-thaw cycles, leading to even more severe morphological damage. instead, what appears to be the most common method in modern brain banking is to section fresh brain tissue and use pre-cooled metal plates to freeze the slices (vonsattel et al., 2008). to assess the degree of morphological preservation in a routinely banked brain preserved via freezing in such a manner, we examined images from cortical tissue that was initially preserved by freezing between metal plates pre-cooled in a ‑80 °c freezer and then rewarmed in cold fixative. we found that in this sample, ice artifacts are readily apparent in the frozen tissue compared to the immersion-fixed tissue (figure 2). the cells, especially those in the grey matter, are shrunken and mechanically squashed into the pockets of non-frozen tissue between large areas of empty space indicative of ice formation. in this example, the degree of ice artifact in white matter appears to be relatively less severe than in the grey matter. notably, myelin has previously been reported to be relatively resistant to freezing damage (möbius et al., 2010). figure 2. example images showing ice artifacts in frozen-thawed brain tissue. this brain tissue is from a 76-year-old female brain donor with a postmortem interval of 70.5 hours prior to preservation. the frozen tissue was from approximately 0.5 cm fresh sections frozen between teflon-coated metal plates cooled at ‑80 °c. the frozen tissue was stored at ‑80 °c for over 5 years before being thawed in cold formalin. ice artifacts in frozen-thawed tissue (b, d, f, h) can be observed when compared with immersion fixed tissue (a, c, e, g) even at low magnification (a, b). higher magnification makes the ice artifacts seen in the frozen but not the immersion fixed tissue more apparent, particularly in the grey matter (c, d), but also to a lesser degree in the white matter (e, f) and subpial area (g, h), with shrunken, distorted cells and large empty spaces indicative of ice crystals. scale bars: a, b: 1000 μm. c–h: 100 μm. summary freezing methods are the most common approach for cryopreserving relatively large brain tissue samples. the efficacy of freezing methods varies considerably based on the specific technique used. placing large tissue samples directly in a freezer leads in general to severe ice artifacts due to the relatively slow cooling rates. on the other hand, freezing brain slices using either pre-cooled metal plates, embedding in dry ice, immersing in cooled isopentane, or freezing in liquid nitrogen vapor, have all been reported to achieve relatively rapid cooling and to maintain morphology with relatively few ice artifacts detectable by light microscopy. cryoprotectant-based cryopreservation preservation process cryoprotectants are substances that can be added to tissues to prevent ice formation during the cryopreservation process. the most frequently used cryoprotectants reported in the studies we identified include dimethyl sulfoxide (dmso), glycerol, sucrose, dextran, bovine serum albumin, and polyvinylpyrrolidone (pvp). these substances act by forming hydrogen bonds with water molecules, causing freezing point depression, and increasing tissue viscosity. to achieve complete vitrification and prevent ice formation entirely, a high concentration of cryoprotectants is needed. for example, a solution containing 61 % w/v ethylene glycol has been used to vitrify mouse brain slices alongside cooling in liquid nitrogen (german and akdaş, 2024). lower concentrations of cryoprotectant have been used in various studies but are not likely to achieve complete vitrification. notably, cryoprotectants can also cause structural damage. for example, dmso and glycerol have both been reported to cause ultrastructural alterations to the cell membrane, with dmso being more damaging than glycerol (mcintyre et al., 1974). furthermore, cell organelles can be disrupted and myelin-like figures can occur within the cytoplasm. cryoprotectant immersion several studies that we identified reported immersion of cryoprotectants into brain tissue prior to cryopreservation (table 4; supplementary file 4). among these, multiple studies found that areas with less take-up of cryoprotectant tend to have more freezing artifacts, but that in areas where an adequate concentration of cryoprotectant was successfully taken up, cells are undamaged (seilhean et al., 1996; pichugin et al., 2006). another study demonstrated a potential issue with cryoprotectant immersion, as cellular necrosis occurred during the time necessary for the immersion procedure to be completed (german and akdaş, 2024). sample type (study) cooling method visualization method key findings mouse sciatic nerves (kirschner et al., 1979) immersion in 20 % dmso, frozen in a freon 22 slush cooled with liquid nitrogen x-ray diffraction, freeze-fracture electron microscopy well-ordered myelin membranes, slightly larger myelin sheaths compared to control tissue rat embryonic neural structures (das et al., 1983) immersion in 10 % dmso, frozen in dry ice vapors light microscopy, cresyl violet staining tissue and cells appear histologically normal human or mouse fetal cns, about 1 mm3 (seilhean et al., 1996) immersion in 10 % dmso at 4 °c, kept at ‑80 °c overnight, then frozen in liquid nitrogen rewarming at 37 °c, then electron microscopy cells in the periphery of the sample are intact, but disrupted membranes in center areas, possibly due to ice damage 475 µm thick rat hippocampal slices (pichugin et al., 2006) immersion in vm3 vitrification solution, exposed to aluminum block cooled to ‑130 °c rewarming to 35 °c, fixation, then electron microscopy with adequate uptake of cryoprotectant, ultrastructure is high-quality, effectively equivalent to that of controls 400 μm thick mouse cortico-hippocampal slices (german and akdaş, 2024) stepwise immersion in ethylene glycol, up to 61 % w/v ethylene glycol, then transferred into liquid nitrogen stepwise removal of cryoprotectant, electrophysiology, fixation, then microscopy light and electron microscopy shows cell swelling, vacuolization, and cell membrane damage after electrophysiological recordings table 4. reported histologic effects in representative observations from studies using cryopreservation with cryoprotectant immersion. dmso: dimethyl sulfoxide; cns: central nervous system; vm3: vitrification solution. cryoprotectant perfusion perfusion is an alternative method for delivering cryoprotectants through the cerebrovascular system that can potentially achieve more uniform distribution. several studies that we identified used perfusion of cryoprotectants through the blood vessels of the brain in laboratory animals (table 5; supplementary file 4). one study tested perfusion of rat brains with either a vitrification solution called vm1 or a solution containing 13 % dmso and 13 % glycerol (canatelli-mallat et al., 2020). while no ice formation was observed in either group on light microscopy, significant shrinkage of neurons occurred. another study used perfusion of mouse brains with 30 % sucrose for 15 minutes, followed by overnight immersion in 30 % sucrose under refrigeration before freezing in isopentane/2-methylbutane cooled with liquid nitrogen (comba et al., 2020). this protocol was found to preserve morphology in tumor tissue within the brain as seen on light microscopy, with no histologically detectable ice crystals. the extended immersion period makes sense because perfusion of the brain is not completely reliable, even in ideal, controlled laboratory conditions (cahill et al., 2012). although the latter procedure also raises concerns about potential tissue decomposition, the authors did not report that the extended immersion period affected tissue morphology, consistent with previous literature suggesting that the cellular effects of postmortem decomposition have a relatively slow onset, especially in refrigerated tissue (krassner et al., 2023). however, cryoprotectant perfusion is expected to be more challenging in a brain banking context, largely as a result of agonal and postmortem changes (mcfadden et al., 2019; mckenzie et al., 2022). sample type (study) cooling method visualization method key findings whole cat brain within cranium (suda et al., 1966) perfusion with 15 % glycerol, then placed in a ‑20 °c freezer light microscopy with nissl staining reported to have "almost normal cell arrangements" whole rat brain (canatelli-mallat et al., 2020) intracardial perfusion at 4 °c with increasing cpa concentration, up to vm1 vitrification solution immersed in fixative at ‑20 °c, light microscopy with immuno-histochemistry no histologic evidence of ice formation, well preserved synaptic immunostaining, fainter neun staining, shrinkage of neurons whole mouse brain (comba et al., 2020) perfusion with 30 % sucrose, then overnight immersion in 30 % sucrose, embedded in oct compound, and frozen in isopentane/2-methylbutane cooled with liquid nitrogen cryostat sectioning at ‑20 to ‑24 °c, ethanol fixation, light microscopy after h&e staining no ice crystals observed on light microscopy, morphology and rna integrity of tissue reported to be preserved table 5. reported histologic effects in representative observations from studies using cryopreservation with cryoprotectant perfusion. vm1: vitrification solution; neun: neuronal nuclei, a neuronal marker protein; oct: optimal cutting temperature; h&e: hematoxylin and eosin; cpa: cryoprotective agent. summary cryoprotectants like dmso, glycerol, and sucrose can reduce ice formation during cryopreservation and potentially enable vitrification. high cryoprotectant concentrations are needed for complete vitrification, which can cause osmotic stress if the procedure is not performed in a properly graded manner. immersion of brain tissue in cryoprotectants has been reported to preserve ultrastructure, but certain areas can still have freezing artifacts if cryoprotectant diffusion or uptake by cells are incomplete. perfusion of cryoprotectants through the cerebrovascular system may enable more uniform cryoprotectant distribution and better tissue preservation in larger brain samples, but this approach is challenging to perform, especially in postmortem brain banking settings, and the evidence for its efficacy in the published literature is currently limited. storage temperature and length one previous source has comprehensively reviewed the literature on cryostorage (hubel et al., 2014). the authors report that unfixed biospecimens kept above the tissue’s glass transition temperature are expected to show degradation, even at the ultra-low freezer temperature of ‑80 °c. there are several reasons for this. first, the presence of residual liquid water allows for ongoing molecular motion. second, degradative biomolecules, such as proteases, nucleases, and lipases, may still have activity, which can lead to biochemical alterations. although the activity of degradative enzymes is expected to be greatly reduced, it is not necessarily completely halted at ‑80 °c, and this can add up over long periods of storage. third, ice crystals will become larger during long-term storage, leading to more damage to tissue morphology (choi et al., 2015; zennoune et al., 2022). such ice recrystallization is expected to be especially common as a result of temperature fluctuations (petzold and aguilera, 2009). all these factors add up to potentially damage tissue during storage at temperatures above the glass transition temperature. this has been borne out empirically in some, but not all studies. one study found that human brain tissue stored at ‑70 °c for more than one year developed irregular spaces and variations in section thickness that hindered comparative analyses (itoyama et al., 1980). another study of dog brain samples held at ‑18 °c found that this led to increased damage to neurons and to increased large, irregular, clear areas under the microscope after 7 days, as compared to 2 days of storage (baraibar and schoning, 1985). notably, in this study, hemolysis was already complete by 2 days, so it did not worsen by 7 days of storage. however, other studies have found that histomorphological features were not altered after years of storage of brain tissue at ‑80 °c (zilles et al., 2002; andreasson et al., 2013). taken together, these findings underscore the importance of considering storage length as a potential confounding variable when analyzing banked specimens, although whether it makes a large difference to the morphology in practice is still an open question and likely depends on the variable of interest. storage below the glass transition temperature, on the other hand, is expected to cause fewer changes. for example, one study found that while liver cell spheroids stored at ‑80 °c had a loss of viable cells starting at one month and progressing substantially up until one year, storage in liquid nitrogen vapor at approximately ‑170 °c led to no changes in viability (massie et al., 2013). another source using storage at ‑180 °c found that morphology did not vary over the storage period of a few days to up to more than 5 years (seilhean et al., 1996). however, as previously discussed, storage at this temperature can cause a buildup of fractures (hunt et al., 1994). perhaps more importantly, storage at temperatures below ‑80 °c would incur a significant cost that may not be justified for many brain banks. it is also worthwhile to point out that any freeze-thaw cycle is likely to be extremely damaging to tissue morphology. one study found that a freeze-thaw cycle softened brain tissue to such a degree that it was too difficult to process it for histologic study at all (schäfer and kaufmann, 1999). the authors noted that this phenomenon was common in tissues like the brain that have a relatively high extracellular water content and relatively weak physical connections between cells. this is one of the reasons that brain banks typically break up fresh tissue into smaller blocks prior to the initial freezing procedure (shepherd et al., 2019). relationship between ice crystal formation and postmortem decomposition it is mechanistically plausible that a longer postmortem interval could increase ice artifacts, for two reasons. first, the levels of free water have been found to increase in the brain after death (ansari et al., 1976; leonard et al., 2016). the increased water content may make tissue more susceptible to ice crystal formation during the freezing process. second, the molecular links maintaining tissue structures break down during the postmortem interval (krassner et al., 2023). this may render tissue structures more vulnerable to the mechanical stresses induced by ice crystal growth. this relationship is also consistent with the findings in frozen sections that tissue edema tends to worsen ice artifacts (tofte et al., 2014; priemer et al., 2023). notably, astrocytes tend to accumulate water in the postmortem interval, and electron microscopy has found that astrocyte membranes are the most vulnerable to freezing damage, which is particularly perceptible around neurons, oligodendrocytes, and capillaries (walder and vrensen, 1972). one study found that the method of tissue preservation affected how a 20 hour postmortem delay affects myelin basic protein immunostaining (itoyama et al., 1980). when using immersion fixation, the authors found that some myelin sheaths became vacuolated after this postmortem delay, but the overall preservation was satisfactory. on the other hand, tissue that was frozen after the same postmortem delay showed more severe changes, including few visible oligodendrocyte processes and only faintly stained myelin sheaths. although the hypothesis of a relationship between postmortem interval and ice damage is interesting, it has not been widely investigated and it is worthy of more research before too much confidence is placed into these preliminary observations. another relevant factor is that some structural changes observed in frozen tissue can overlap with those seen in postmortem autolysis. mechanistically, both freezing and postmortem decomposition would be expected to cause cell membrane damage. for example, one study noted that hemolysis, characterized by the loss of erythrocyte cytoplasm, was present in both frozen-thawed and decomposed fish brain tissue (kagan and viner, 2022). another study, comparing the ultrastructural alterations in larval fly brains exposed to either lethal freezing or anoxia, found similarities such as nuclear membrane expansions, nuclear chromatin clumping, and rough endoplasmic reticulum swelling (lee et al., 1997). these findings suggest that some of the damage observed in frozen brain tissue may be exacerbated by – or difficult to distinguish from – the changes that occur during the postmortem interval. effects of cryopreservation on biomolecular preservation modern brain banking aims to preserve multiple tissue properties, and many tissue requests are for molecular analyses, making this aspect of preservation quality also critical to consider. from a mechanistic perspective, cryopreservation is not thought to cause the formation or breakdown of covalent bonds in biomolecules (fahy and wowk, 2015), which is an obvious advantage over chemical fixation by aldehydes for the purpose of biomolecular assays. however, cryopreservation can cause numerous other effects on biomolecules, including phase changes in membrane lipids, conformational changes due to loss of the hydration shell, hydrophobic aggregation of biomolecules, and protein inactivation, for example of the sodium–potassium pump (jain et al., 2021; pegg, 1976). some biomolecular changes occur during the cryopreservation process because metabolism does not fully stop until very low temperatures are reached. for this reason, faster cooling processes might be expected to yield superior biomolecular preservation. indeed, for tumor tissues, snap freezing of tumor biopsies has been reported to be the gold standard for maintenance of biomolecular properties of tissues, such as their content of nucleic acids and phosphoproteins (van der wijngaart et al., 2023). however, other data have suggested that slow freezing may be preferable for maintaining biomolecular properties, because fast freezing yields small ice crystals with an increased contact area between ice and liquid, which may increase the net exposure of proteins to the damaging effects of ice (cao et al., 2003). one potential solution to the problem of biomolecular damage due to ice formation would be to use cryoprotectants, since a sufficient concentration of cryoprotectants could prevent ice damage. cryoprotectants could also allow for better maintenance of cellular integrity, which could be useful for the molecular profiling of single cells. however, if cryoprotectants are used, they also have the potential to affect the molecular properties of the tissue itself, for instance by causing protein denaturation (fahy and wowk, 2015). for some molecular assays, cryoprotectants used at potentially variable concentrations within or between samples could also create batch effects that would confound subsequent analyses. in practical brain banking settings, cryopreservation of small tissue blocks in isopentane cooled by liquid nitrogen has been reported to be a preferred method for obtaining high-quality rna and protein preservation (durrenberger et al., 2010). however, different freezing methods have been found not to have a significant effect on the quality of rna harvested from brain tissue (durrenberger et al., 2010). additionally, one study found that there was no difference between three different freezing methods in the protein profiles detected by mass spectrometry (meyronet et al., 2015), even though there were differences in the degrees of morphological preservation. it seems that differences in cryopreservation protocols have smaller effects on many biomolecular features than on histology. for nucleic acids in particular, studies have found that proxies of a prolonged and severe agonal state, such as hospitalization prior to death or decreased brain ph, are the strongest correlate of worsened rna quality (li et al., 2004, 2007; durrenberger et al., 2010; hagenauer et al., 2018). comparison to other tissues several studies have noted that the brain appears to be more vulnerable to freezing damage than other organs and tissues. one source found that while kidney tissue did not show substantial changes after freezing, brain tissue softened significantly, making it difficult to process the brain tissue for evaluation (schäfer and kaufmann, 1999). these authors hypothesized that tissue softening after freezing occurs in tissues with high extracellular water content and with relatively weak physical connections holding the tissue together. they also noted that during slow freezing, extracellular ice formation can cause cells to shrink significantly, potentially tearing apart cell-cell connections, which will not redevelop upon thawing. another source observed that freezing damage in the brain seemed to be worse compared to other tissues (lee et al., 1997). the authors noted that the brain appears to be the “weak link” with the most alterations following freezing in the fly larvae they were studying. finally, one cryofixation study noted that nervous system tissue is more difficult to vitrify than any other tissue they had studied (zuber et al., 2005). the authors reported that while tissues like cartilage or skin could be vitrified without cryoprotectants, this was not possible for nervous tissue, which required the use of cryoprotectants for successful vitrification via cryofixation. the authors attributed this difficulty to the brain's high content of water and lower intrinsic effect of cryoprotectants. collectively, these findings suggest that the unique composition and structure of brain tissue makes it particularly challenging to cryopreserve brain tissue effectively as compared to many other tissue types. comparison to previous reviews to the best of our knowledge, there has not been a previous comprehensive review primarily on the cryopreservation of brain tissue. there have been many previous reviews on cryobiology in general, several of which can be highlighted (bojic et al., 2021; fahy and wowk, 2021; murray and gibson, 2022). in recent years there has also been an increased focus on cryopreserving complex tissues or organs (giwa et al., 2017). however, these reviews have not focused on cryopreservation of brain tissue, which is not currently considered to be as important as other tissue types for clinical applications such as transplantation. the reviews on brain tissue cryopreservation have tended to focus on aspects other than histology, such as biobanking in general (shabihkhani et al., 2014) or cellular survival (paynter, 2008). notably, one review notes that most techniques on nerve cell cryopreservation have been derived from those applied to cells of other organs, rather than being optimized for nerve cells in particular (paynter, 2008). one review article primarily focusing on biochemical and biomolecular studies in banked brain tissue also reports that frozen brain tissue can been used in detailed morphological studies (hynd et al., 2003). finally, one review on the practice of cryonics discusses the cryopreservation of the brain (best, 2008). the author primarily discusses articles describing successful preservation of brain tissue when cryoprotectants are used. compared to our review, the aforementioned review does not extensively discuss studies on the histological outcomes of freezing without cryoprotectants, which will occur if the cryoprotectant solution does not reach all areas of the brain. limitations of this review this review has a number of limitations that should be considered. first, the data reported in some articles were occasionally ambiguous, making it difficult to tell whether the observations were based on first-hand experience or on second-hand reports. while we attempted to include only primary data, this ambiguity may have introduced some second-hand data into our findings. second, our review focused primarily on brain tissue cryopreservation, with limited consideration of other tissue types. given the potential similarities across tissues, a more comprehensive review incorporating the broader literature could provide additional insights. third, the “grey” literature on this technical topic, such as information shared on mailing lists and platforms like researchgate, was not included in our review. future reviews may benefit from carefully considering these unpublished data, as they may contain valuable insights from researchers who have addressed the topic in practice without formally publishing their findings. fourth, the distinctions between the different categories of cryopreservation techniques of cryofixation, freezing, and cryopreservation with cryoprotectants can be somewhat blurred, as some studies may employ methods that overlap or share similarities across these categories. for example, some cryofixation studies use cryoprotectants. this potential ambiguity in categorization may have influenced the interpretation and synthesis of the reviewed studies. fifth, our assessment of preservation quality relies heavily on the verbal reports and descriptions provided by the authors of the reviewed studies. the lack of standardized, quantitative metrics for evaluating preservation quality across studies limits the objectivity and comparability of these assessments. overall, the literature on morphological outcomes following cryopreservation of unfixed brain tissue is relatively limited compared to studies using fixed tissue, particularly outside of the cryofixation domain. the majority of the reviewed studies have small sample sizes and limited replication, which may affect the generalizability of their findings. as a result, the conclusions drawn in this review should be interpreted with caution, as they are based on a relatively small number of studies. further research with larger sample sizes and standardized evaluation methods is needed to corroborate and extend the findings of this review. summary of cryopreservation methods discussed we can summarize several of the cryopreservation methods discussed in this article (table 6). for smaller specimens such as tissue biopsy samples, cryofixation or cryoprotectant-based methods likely offer the best histologic preservation, especially as viewed under the electron microscope. however, for the larger whole brain or hemisphere slices that are typically preserved in brain banking, freezing methods are the only published methods that have been reported to be reliably effective. among the cryopreservation methods for brain slices, freezing between cooled metal plates, embedding in dry ice, or immersion in cooled isopentane appear to be the most effective for preserving relatively large samples while minimizing ice artifacts as seen on light microscopy (vonsattel et al., 2008; meyronet et al., 2015). notably, many investigators do not focus on detailed morphological preservation for their frozen tissue, focusing instead on preservation of biochemical properties, making the histologic considerations less important. method benefits drawbacks cryofixation • excellent ultrastructural preservation • minimal ice artifacts in vitrified regions • limited to very thin samples, typically ˜20–200 μm or less • requires specialized equipment direct placement in freezer • simple, accessible, and low-cost • easily integrates with long-term storage • slow cooling rate due to poor thermal conductivity leads to severe ice artifacts and poor histologic preservation freezing in dry ice • relatively accessible, and low cost • relatively fast cooling, can handle large samples • can still produce significant ice artifacts • cooling rate inconsistent based on contact with dry ice freezing between cooled metal plates • practical for large but relatively thin brain slices • relatively rapid cooling, and widely used in brain banks • cooling rate decreases with depth • damage at cut interfaces • not suitable for preservation of whole brains of larger mammals freezing in liquid nitrogen vapor • relatively fast cooling rate and very low storage temperature achievable • less likely to cause tissue cracking than liquid immersion • cooling rate may still be too slow for optimal preservation • requires specialized equipment • potential safety hazards with liquid nitrogen handling freezing in cooled isopentane • rapid, uniform cooling along surface inwards • avoids leidenfrost effect seen with liquid nitrogen immersion • excellent for preservation of small brain samples, as seen on light microscopy • requires careful handling of isopentane • may cause tissue cracking in large samples • not widely tested on large samples, still limited by depth cryopreservation by immersion in cryoprotectants • reduces ice crystal formation and improves preservation of cellular structures • potential for vitrification in thin samples • slow penetration rate makes larger samples challenging • cryoprotectants can cause osmotic stress, which may alter tissue ultrastructure cryopreservation by perfusion with cryoprotectants • more uniform distribution of cryoprotectants • potential for better preservation in larger samples, with vitrification being also theoretically possible • challenging to perform, especially postmortem. not widely performed • risk of incomplete perfusion • time-consuming process with risk of cellular decomposition table 6. summary of methods for cryopreserving brain tissue. several approaches for cryopreserving brain tissue are described, alongside their relative benefits and drawbacks. implications for brain banking brain banks may have different needs for their cryopreservation methods depending upon the intended use of their tissue samples (table 7). for whole brain slices, which are commonly used in brain banking, preservation of overall tissue architecture is often a primary concern. in this case, freezing the slices between cooled metal plates offers a relatively simple and effective approach. for smaller tissue fragments, where preservation of cell membrane morphology as seen on light microscopy is desired, rapid freezing methods such as immersion in liquid nitrogen vapor or liquid nitrogen-cooled isopentane can be used. however, the effectiveness of these methods for preserving delicate cellular structures can vary depending on factors such as tissue type and freezing rate. if one has access to specialized equipment and expertise, then cryofixation could potentially be used on very thin sections from small biopsy specimens to achieve high-quality preservation of subcellular structures such as synaptic vesicles. for those more interested in preserving the biomolecular properties of the tissue and not as much in detailed histological preservation, the choice of freezing method is less critical. in this case, simpler methods, such as placement in dry ice or in an ultra-low temperature (‑80 °c) freezer, might be sufficient. finally, for specialized research applications, such as those interested in the preservation of electrophysiological features of fresh tissue slices (german and akdaş, 2024), the use of cryoprotective agents, while a relatively complex procedure, might be justified. because cryopreservation can be expensive and highly labor-intensive, it is essential to be pragmatic and to choose the simplest method that adequately meets the specific preservation needs of the brain bank and its research community. this approach ensures efficient use of resources while still maintaining the quality necessary for intended downstream applications. tissue type preservation goal recommended approach whole brain slices overall tissue architecture freezing between cooled metal plates small tissue fragments cell membrane morphology on light microscopy snap freezing in liquid nitrogen-cooled isopentane very thin sections subcellular structural features on electron microscopy cryofixation, for example via high-pressure freezing any tissue type biomolecular properties (e.g., rna, proteins) multiple options, including placement in dry ice, or placement in a ‑80 °c freezer fresh tissue slices electrophysiological properties immersion of cryoprotective agents, or perfusion prior to sectioning table 7. recommended cryopreservation methods for several different brain banking goals and tissue types. we can also consider the perspective of researchers using existing tissue banked at brain banks. samples that are frozen may have more variable histologic qualities than commonly appreciated. for researchers interested in histological features, it may be worthwhile to seek tissue that has been frozen in a rapid, well-controlled manner, rather than assuming that the morphological quality of all frozen tissue will be identical. years of storage at ‑80 °c also has the potential to affect tissue quality. using rapid thawing techniques and/or thawing in fixative may facilitate morphological preservation in research applications, although the best methods for tissue rewarming are still not well established. notably, preservation of histologic features is generally expected to be superior in banked tissue that has been chemically fixed. therefore, there is currently a trade-off between the types of tissue generally available from brain banks, with frozen samples allowing certain molecular biology studies to be more easily performed, and fixed samples expected to have better morphological preservation. areas for further research some research applications, such as long-distance circuit mapping and also biochemical assays, could potentially benefit from the ability to cryopreserve an entire brain while maintaining its structural integrity. however, current methods struggle with cryopreserving large tissue volumes without ice damage. future studies could explore improved methods for cryoprotectant perfusion of the brain, which has received relatively little attention in the published literature over the years (suda et al., 1966; lemler et al., 2004; canatelli-mallat et al., 2020; comba et al., 2020). corroborating and potentially improving such efforts could provide insights into the feasibility and the limitations of whole brain cryopreservation via cryoprotectant perfusion. several open questions also remain regarding the basic mechanisms and limits of ice damage during cryopreservation. research is needed to better characterize the ice damage caused both during the initial cooling of brain tissue and during the rewarming of large brain samples. technological approaches to increase the speed of rewarming, such as dielectric warming (wowk et al., 2024) or ultrasound rewarming (alcalá et al., 2023), may have the potential to decrease damage in cryopreserved brain tissue further, although this requires more study. finally, the use of computational methods to infer the most probable original state of ice-damaged tissue is an area for future work. some progress has already been made in this direction for frozen sections (ozyoruk et al., 2022). however, in this study, the algorithm could not reverse the alterations of cellular positions when severe ice artifacts were present. there is a need for a better theoretical understanding of the limits of such computational inference methods to reverse ice damage. conclusions this review highlights the complex landscape of brain tissue cryopreservation, revealing both the potential and limitations of current methods. key themes that emerged from our literature review included the relationship between cooling rate and freezing injury, the effects of different cryoprotectants, and the impact of tissue characteristics on cryopreservation outcomes. of the study types identified, cryofixation offers exceptional ultrastructural preservation but is limited to very thin samples, while freezing approaches can achieve minimal ice artifacts as seen on light microscopy in relatively larger specimens, particularly when rapid cooling methods are employed. the use of cryoprotectants shows promise for high-quality preservation, but remains challenging for achieving consistently even distribution, especially in larger samples and postmortem settings. notably, brain tissue appears to be particularly vulnerable to freezing damage as compared to other tissues, underscoring the need for more research in this area. while current cryopreservation techniques are sufficient for many research applications, there remains an unmet need for methods capable of preserving larger brain samples with minimal structural disruption. future research may benefit from a focus on improving cryoprotectant delivery, optimizing cooling and rewarming protocols, and exploring computational methods to infer the original state of the tissue despite the presence of freezing artifacts. abbreviations dmso dimethyl sulfoxide; oct optimal cutting temperature compound; pmi postmortem interval; tem transmission electron microscopy; vm1 and vm3 vitrification solutions composed of multiple cryoprotectants. acknowledgements we would like to thank brian wowk and kenneth hayworth for helpful personal communications regarding this topic. data availability this article was primarily built upon publicly available data. the relevant data and analyses are presented in the article or in the supplementary materials. author contributions a.t.m., e.f.k., k.f., and j.c. conceptualized the article. a.t.m. performed article searching and screening. a.t.m., o.n., and b.w. performed data extraction and review of data extraction. e.t. performed experimental studies and imaging of frozen-thawed brain tissue. a.t.m. wrote the initial draft of the manuscript. all authors reviewed the manuscript. all authors approved the final manuscript. conflicts of interest statement a.t.m. is an employee of oregon brain preservation, a non-profit brain preservation organization, and a director of apex neuroscience, a non-profit research organization. b.w. is affiliated with the european institute for brain research, a non-profit research foundation, and the biopreservation institute, a non-profit research organization that works together with nectome inc., which plans to offer aldehyde-based biostasis. e.f.k. is a shareholder and ceo of tomorrow biostasis, a biostasis provider, and president of the board of the european biostasis foundation, a non-profit research foundation. funding statement borys wróbel’s research is supported by a grant from the biomedical research and longevity society. otherwise, the authors received no specific funding for this work. supplementary files supplementary file 1 [download] rameses checklist [pdf format]. supplementary file 2 [download] abstract screening results [csv format]. 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hiv and covid-19: two pandemics with significant (but different) central nervous system complications feel free to add comments by clicking these icons on the sidebar free neuropathology 5:5 (2024) review hiv and covid-19: two pandemics with significant (but different) central nervous system complications shino magaki1, ting zhang1, karam han1a, hilda mirbaha1, william h. yong2, cristian achim3, gregory fishbein4, michael c. fishbein4, omai garner4, noriko salamon5, christopher k. williams1, miguel a. valdes-sueiras6, jeffrey j. hsu7, theodoros kelesidis8b, glenn e. mathisen9, helen lavretsky10, elyse j. singer6, harry v. vinters1,6,11 section of neuropathology, department of pathology and laboratory medicine, david geffen school of medicine, university of california los angeles, los angeles ca, usa department of pathology and laboratory medicine, university of california-irvine school of medicine, irvine, ca, usa department of psychiatry, university of california san diego, la jolla, san diego, ca, usa department of pathology and laboratory medicine, david geffen school of medicine, university of california los angeles, los angeles, ca, usa department of radiological sciences, david geffen school of medicine, university of california los angeles, los angeles, ca, usa department of neurology, david geffen school of medicine, university of california los angeles, los angeles, ca, usa division of cardiology, david geffen school of medicine, university of california los angeles, los angeles, ca, usa department of medicine, division of infectious diseases, david geffen school of medicine, university of california los angeles, los angeles, ca, usa department of infectious diseases, olive view-university of california los angeles medical center, sylmar, ca, usa department of psychiatry and biobehavioral sciences, david geffen school of medicine, university of california los angeles, los angeles, ca, usa brain research institute, david geffen school of medicine, university of california los angeles, los angeles, ca, usa a current affiliation: department of pathology and laboratory medicine, school of medicine and public health, university of wisconsin-madison, madison, wi, usa b current affiliation: division of infectious diseases and geographic medicine, department of internal medicine, university of texas southwestern medical center, dallas, tx, usa corresponding author: shino magaki · department of pathology & laboratory medicine · ronald reagan ucla medical center and david geffen school of medicine · center for health sciences, rm 1p-250 · 10833 le conte ave, los angeles, ca 90095-1732 · usa smagaki@mednet.ucla.edu submitted: 05 february 2024 accepted: 02 march 2024 copyedited by: shafiq qaadri published: 05 march 2024 https://doi.org/10.17879/freeneuropathology-2024-5343 keywords: hiv, covid-19, hiv-associated neurocognitive disorders, long covid, post-covid conditions, neuropathology abstract human immunodeficiency virus (hiv) and severe acute respiratory syndrome coronavirus 2 (sars-cov-2) cause significant neurologic disease. central nervous system (cns) involvement of hiv has been extensively studied, with well-documented invasion of hiv into the brain in the initial stage of infection, while the acute effects of sars-cov-2 in the brain are unclear. neuropathologic features of active hiv infection in the brain are well characterized whereas neuropathologic findings in acute covid-19 are largely non-specific. on the other hand, neuropathologic substrates of chronic dysfunction in both infections, as hiv-associated neurocognitive disorders (hand) and post-covid conditions (pcc)/long covid are unknown. thus far, neuropathologic studies on patients with hand in the era of combined antiretroviral therapy have been inconclusive, and autopsy studies on patients diagnosed with pcc have yet to be published. further longitudinal, multidisciplinary studies on patients with hand and pcc and neuropathologic studies in comparison to controls are warranted to help elucidate the mechanisms of cns dysfunction in both conditions. introduction human immunodeficiency virus (hiv) and severe acute respiratory syndrome coronavirus 2 (sars-cov-2) are both human viruses that cause neurologic disease. hiv is a retrovirus and is clearly neurotropic, with tropism for microglial cells in the central nervous system (cns), infection of which generates downstream effects that injure brain parenchyma. its clinical and pathologic manifestations are fairly well characterized. by contrast, coronavirus disease 2019 (covid-19) caused by the novel coronavirus sars-cov-2 leads to neuropsychiatric morbidity in a significant percentage of infected patients, but the mechanisms for this are heterogeneous and may include indirect effects on the brain through activation of inflammatory cascades, possible microangiopathic changes and disruption of the neurovascular unit. however, knowledge of hiv neuropathogenesis may be instructive for understanding many aspects of covid-19 neuropathogenesis. the brain research institute at ucla sponsored two half-day "neurovirology affinity group" meetings in the spring of 2022 and 2023 at which a multidisciplinary team of representatives from neuropathology, cardiovascular pathology, microbiology, radiology, infectious disease, cardiology, neurology, and psychiatry convened to address the cns complications of covid-19, considering what we have learned from the hiv pandemic over the past several decades. the meeting aims were to identify gaps in current knowledge on cns disease in covid-19 and hiv and potential directions for future study. this selective review summarizes the main topics discussed and conclusions reached. human immunodeficiency virus (hiv) an immunodeficiency disease, later named acquired immunodeficiency syndrome (aids), started to become recognized in the summer of 1981 in the united states [1,2]. this discovery was followed soon after by the identification of human immunodeficiency virus-1 (hiv-1) as the causative agent [3–5]. at the time, nearly all patients infected with hiv progressed to end stage aids with fatal opportunistic infections and malignancies, many involving the cns [6]. a few years later, hiv-2, primarily restricted to west africa, was discovered [7–9]. with the advent of combined antiretroviral therapy (cart) in the mid 1990s, hiv has become a chronic disease with near normal life expectancies, at least in industrialized countries [6,10]. there have been approximately 40 million deaths since the beginning of the pandemic and currently 38 million infected worldwide according to the joint united nations programme on hiv/aids (unaids) [11]. hiv-1 and hiv-2 are lentiviruses of the retrovirus family and predominantly infect monocytes/macrophages, as with all lentiviruses, but also cd4 t-cells by binding to cd4 and the chemokine co-receptors cxcr4 and ccr5 [12,13]. other co-receptors such as ccr2 and ccr3 have also been reported to mediate infection in vitro [12,13]. infection of macrophages (and microglia) is primarily through ccr5 [14,15]. the entrance of virus into the cell may result in productive infection or latent infection [7,12,16,17]. hiv is neuroinvasive, characteristic of lentiviruses, and enters the cns at the initial stage of infection [13,18–20]. neurologic manifestations hiv causes neurologic complications, due to opportunistic infections and hiv infection itself, in over half of patients not receiving cart [21,22]. with the advent of cart, the rates of opportunistic infections of the cns such as by cytomegalovirus, toxoplasma, cryptococcus and progressive multifocal leukoencephalopathy caused by jc virus, as well as primary cns lymphoma, have decreased in some series [21,23]. hiv-associated neurocognitive disorders (hand), with revised consensus nomenclature and criteria in 2007 [24], previously referred to as aids dementia complex and hiv-1 associated cognitive/motor complex [25–28], comprise a spectrum of cognitive dysfunction associated with hiv infection [24,25]. hand includes, with increasing severity, asymptomatic neurocognitive impairment (ani), mild neurocognitive disorder (mnd) and hiv-associated dementia (had), and continues to affect approximately 33-50 % of treated hiv-positive individuals [24,29–31]. had is the most severe and was the most common form of hand in the pre-cart era but has decreased with the advent of cart, now comprising less than 10 % of those with hand [6]. however, the overall proportion of individuals with hand has not changed in the cart era due to a relative increase in the milder forms of hand, the pathogenesis and neuropathologic substrates of which are unclear [6,31]. the clinical features of hand have also changed before and after cart [32]. in the pre-cart era, patients commonly presented with extrapyramidal signs (bradykinesia, rigidity and tremor) and more frequent subcortical features with motor dysfunction and speed of processing deficits [26,32–35]. in the cart era, extrapyramidal signs are less common with more cortical features with deficits in learning, memory and executive functioning [32–34,36]. risk factors for hand include older age [37], cerebrovascular disease risk factors [38,39], duration of hiv infection and history of aids defining illness [40], lower cd4 nadir [40,41], and in treatment naïve patients, burden of hiv dna in monocyte-enriched peripheral blood cells [42]. currently, the only treatment for hand is cart, but cart is effective in only a subset of patients with hand [32,43]. after the advent of cart, the median survival of had patients increased to 38 months compared to 5 months pre-cart [44], and most individuals with hand on cart remained stable [29,32]. however, even in patients on cart, hand is associated with shorter survival and is an important cause of morbidity and mortality in the aging hiv-positive population [45–47]. a recent study showed that cognitive decline in hiv positive individuals was not associated with age or markers of hiv disease but rather comorbidities such as hypertension and diabetes [48]. before cart, brain magnetic resonance imaging (mri) studies showed accelerated global white matter atrophy and cerebrospinal fluid (csf) volume increase (corresponding to tissue loss) as well as atrophy of the caudate in hiv-positive individuals [49,50]. postmortem mri studies demonstrated that increase in volume of abnormal white matter and decrease in volume of deep gray matter correlated with increasing viral burden as assessed by immunohistochemistry for hiv protein [51]. even after cart, compared to hiv-negative individuals, hiv-positive individuals show higher rates of white matter atrophy, which correlate with lower cd4 counts [52]. mri changes can be seen in all degrees of hand, with individuals with hand showing greater abnormality in cerebral white matter compared to neurocognitively intact hiv-positive individuals [53]. utilizing dynamic contrast enhanced perfusion (dce p) mri, a marker of capillary permeability, to assess for blood-brain barrier (bbb) disruption, chaganti et al. showed impaired bbb in the frontal white matter and basal ganglia in virally suppressed patients with hand compared to controls [54]. neuropathologic findings neuropathologic studies have made important contributions to understanding hiv infection in the cns [7,12,55–57]. hiv has been isolated from the csf, brain including frontal and temporal lobes, caudate, and cerebellum, spinal cord, and sural nerve from aids patients with meningitis, encephalopathy, myelopathy, and peripheral neuropathy [58-60]. even in asymptomatic patients, hiv specific antibodies can be detected and hiv isolated from the csf [19,61]. hiv-1 has also been isolated from the brain 15 days after a patient accidentally inoculated himself with hiv-1 infected white blood cells [62]. hiv is thought to enter the cns predominantly through a trojan horse-like mechanism in which hiv-1 infected monocytes cross the blood-brain barrier (bbb) and release viral particles in the brain parenchyma [16,19,63] although entry may also be facilitated through a disrupted bbb [64]. hiv-1 has been shown to productively infect brain microglia/macrophages [65–69]. although hiv-1 dna and proteins have been detected in other cns cell types, such as astrocytes and endothelial cells in postmortem brain [17,18,70,71] and shown to infect other cell types in vitro, their role in propagation of cns infection is unclear [12]. neuropathologic changes can be seen early in hiv infection [20,72]. in asymptomatic hiv-positive patients who died from other causes, lymphocytic leptomeningitis, perivascular mononuclear cell infiltrates, microgliosis and astrocytic gliosis, which is more prominent in white matter compared to gray matter, myelin pallor, and elevated cytokines, have been seen along with hiv-1 proviral dna (but no hiv-1 protein) in a subset [20,72,73]. before the advent of cart, most hiv-positive individuals progressed to aids [6], and the majority 70-90 % demonstrated neuropathologic abnormalities at autopsy [27,55,56,68,74–77]. in addition to frequent opportunistic infections and lymphomas seen in the context of immunodeficiency, there are characteristic neuropathologic findings attributed to hiv infection itself [19,27,68,74]. these were summarized in consensus guidelines in 1991 [28] and include hiv encephalitis (hive), hiv leukoencephalopathy, lymphocytic (aseptic) leptomeningitis, and diffuse microgliosis and astrogliosis in cerebral gray matter ("diffuse poliodystrophy") [7,56,78]. hiv encephalitis, seen in 10-63 % of autopsies, is characterized by microglial nodules and multinucleated giant cells (figure 1) and/or evidence of hiv in the brain [57,79,80] typically affecting the white matter most severely, followed by the deep gray matter and then cortex [27,28,65,80]. multinucleated giant cells, often seen in a perivascular distribution and more commonly in the subcortical white matter or deep gray matter, are the hallmark finding in hive and result from virus-induced fusion of macrophages which have been shown to contain hiv protein [65,81–83]. figure 1. neuropathologic findings in covid-19 and hiv infection. a) acute/subacute microinfarct (arrows) in the internal capsule and b) sparse leptomeningeal (arrow) and parenchymal (arrowhead) t lymphocytes highlighted by cd3 immunohistochemistry in patients with covid-19. c) hiv encephalitis with multinucleated giant cells (arrows) and d) microglial activation (arrowheads) highlighted by immunohistochemistry for iba-1 in patients with hiv infection. scale bars: a = 50 μm, b,d = 100 μm, c = 20 μm in the post cart era, the majority of hiv positive individuals show no hiv related neuropathology at autopsy, although hive has been reported in 8-25 % of case series with equal or even increased frequency post cart in some series [84–87]. opportunistic infections, albeit decreased in frequency after cart, are still seen [84–92]. hive indicates active viral replication and is an important pathologic substrate of had [93], but not all subjects with cognitive impairment have hive and not all patients with hive have cognitive impairment, which was seen before but especially after the advent of cart [56,68,81,84,88,94]. in the post cart era, hiv associated neuropathologic findings have not correlated with cognitive impairment [84]. aids patients demonstrate cortical atrophy and neuronal apoptosis [95,96], but neither neuronal apoptosis [97] nor neuronal loss in the frontal and temporal cortices of patients with aids [98] correlate with dementia. on the other hand, decreased cortical synaptic density and dendritic complexity were demonstrated even with mild cognitive impairment and have been shown to correlate with severity of cognitive impairment [99–101]. disruption of the bbb has been seen in patients both with aids with and without dementia as well as with and without hive [64,102]. several gene expression profiling studies performed on the brains of hiv positive individuals have found upregulation of endothelial cell type transcripts [103] and dysregulation of microglial transcripts [104] in patients with hand but without hive and increased expression of a subset of cytokines in white matter [103–107]. although hiv antigen can be detected throughout the cns including the cerebral hemispheres, cerebellum, brainstem, and spinal cord using various methods such as immunohistochemistry, in situ hybridization (ish), pcr, and electron microscopy, the highest levels of hiv proteins, rna and dna, primarily in macrophages, microglia and multinucleated giant cells, have been seen in deep gray matter and hippocampus [67,81,108,109]. even with viral suppression on cart, low levels of hiv-1 rna and anti-hiv antibodies can be detected in the csf [110,111]. hiv-1 dna, including intact proviruses, and rna have been found in the brains of virally suppressed hiv-positive individuals in microglia/macrophages [112–114]. these reservoirs have been postulated to cause chronic inflammation and neuronal dysfunction [114,115], and microglial activation and increased cytokine levels in the brain have been shown to correlate with hand [94,116–119]. however, although levels of hiv-1 dna are on average higher in brains from patients with aids compared to patients without aids, there is overlap [18,117]. brain viral load correlates poorly with neuropathologic changes or cognitive impairment [94,117]. furthermore, gelman et al. have shown that subjects with hand and hive have higher brain hiv rna and dna levels than patients without hand, but individuals with hand without hive show no difference compared to patients without hand [88]. thus, complex mechanisms beyond viral replication in the brain likely underlie hand, especially in the post cart era (figure 2) [56,84,120,121]. figure 2. potential mechanisms of neurologic disease in acute/chronic hiv infection and covid-19/pcc. hand, hiv-associated neurocognitive disorders; pcc, post-covid conditions. figure was created using biorender. severe acute respiratory syndrome coronavirus 2 (sars-cov-2) a few weeks after a cluster of viral pneumonia cases, later called covid-19, was first described in december of 2019 in china [122–124], the causative agent was determined to be a new human pathogen, the coronavirus later named severe acute respiratory syndrome coronavirus 2 (sars-cov-2) [123]. several vaccines were rapidly developed within a year and a significant proportion of the population of many countries, mostly high-income, have been vaccinated, albeit with large differences among countries [122,125,126]. however, there have been approximately 6.9 million deaths and over 750 million infected worldwide according to the world health organization (who) [127]. coronaviruses are classified into four distinct genera: alpha, beta, gamma and deltacoronavirus [128]. sars-cov-2 and sars-cov belong to the same species, severe acute respiratory syndrome-related coronavirus (sarsr-cov), which is of the subgenus sarbecovirus, genus betacoronavirus which also includes middle east respiratory syndrome coronavirus (mers-cov), of the subfamily orthocoronaviridae [128,129]. during its spread several sars-cov-2 variants have emerged, termed "variants of concern", including alpha, gamma, delta, and omicron, that have caused disease of similar severity except for omicron which has been associated with lower rates of hospitalization [128,130]. sars-cov-2 predominantly infects nasal or upper respiratory epithelium and sustentacular cells of the olfactory mucosa [131–134] with a decreasing gradient of infection from the proximal to distal respiratory tract [135]. as with sars-cov, sars-cov-2 uses angiotensin converting enzyme 2 (ace2) as the obligate receptor for host cell entry through binding of the viral spike (s) protein, proteolytically activated by transmembrane protease/serine subfamily member 2 (tmprss2), although ace2-bound virus can also enter the cell via clathrin-mediated endocytosis without tmprss2 [129,136–140]. ace2 is a human homologue of ace, and both ace and ace2 play important roles in the renin-angiotensin system [141]. ace2 mrna has been reported in many different organs including, at least in some studies, the brain [142–145]. however, mrna and protein levels of ace2 have been shown to be discordant [146]. ace2 protein has been detected in several cell types including ciliated cells of the nasal mucosa and bronchus, cardiomyocytes, enterocytes of the gastrointestinal tract, gallbladder epithelium, kidney proximal tubule epithelium, and sertoli and leydig cells of the testis [143,146,147]. in the brain, ace2 protein expression has been seen by immunohistochemistry in vascular smooth muscle cells and/or endothelial cells/pericytes in some studies but not others [143,147–151]. few studies have reported ace2 immunopositivity in choroid plexus, ependymal cells, meningothelial cells, and neurons in the medulla [151,152]. neurologic manifestations approximately a third of patients with acute covid-19 exhibit neurologic and psychiatric manifestations, most commonly fatigue, alterations in consciousness, impairment in smell and taste, seizures, myalgia, anxiety, and stroke [153–156]. ischemic stroke is seen in approximately 2 % of covid-19 patients [153–156], and occurs in younger individuals compared to ischemic stroke in non-covid-19 patients [157]. moreover, patients with covid-19 have increased risk for ischemic and hemorrhagic stroke compared to patients with influenza or other upper respiratory tract infections [155,158], and increased risk for ischemic stroke and myocardial infarct compared to the background population in sweden [159]. neurologic manifestations have been associated with increased mortality, especially in older individuals [154,156]. neurologic and psychiatric complications are also seen as part of the constellation of post-covid conditions (pcc), also known as post-acute sequelae of covid-19 (pasc) or long covid, defined by the who as persistent symptoms usually 3 months from onset of covid-19 with symptoms lasting at least 2 months with no alternative diagnosis [160–162]. most frequent neurologic manifestations of pcc include fatigue and cognitive impairment, impaired concentration or "brain fog", which has been shown to correlate with abnormal brain activation on task-activated blood oxygenation level-dependent functional mri (bold-fmri) [163], as well as psychiatric disorders such as post-traumatic stress disorder, anxiety and depression [164,165]. similar complications were seen in the sars 2003 pandemic [166], and post-viral syndromes occur in other viruses [167]. cognitive decline and psychiatric symptoms have also been described after serious illness requiring admission to the intensive care unit such as sepsis [168,169]. however, examining the electronic health records of the us department of veterans affairs, al-aly et al. found a higher burden of both pulmonary and extrapulmonary disease, including neuropsychiatric and cardiovascular disorders, in patients who had been hospitalized for covid-19 and survived for at least 30 days after admission compared to individuals hospitalized for influenza [170]. a different study using electronic health records of health care organizations mostly in the us found that neurologic and psychiatric diagnoses are more common in the 6 months following the diagnosis of covid-19 compared to other respiratory infections such as influenza [155]. pcc can occur regardless of the severity of acute disease but is more severe in hospitalized patients, and those with comorbidities appear to be at increased risk not only for acute disease but also for pcc [160,164,171]. vaccination may be less effective at preventing pcc than severe acute covid-19 [172]. further clinical phenotyping of pcc, as with hand in hiv, may be helpful in further defining research nosology [24]. brain mri findings in patients with acute covid-19 presenting with neurologic symptoms include acute/subacute infarct, which is the most common finding, abnormalities of the olfactory bulb, white matter abnormalities, cerebral microbleeds, gray matter abnormalities, leptomeningeal enhancement, acute disseminated encephalomyelitis (adem) and adem-like lesions, intracerebral hemorrhage, and posterior reversible encephalopathy syndrome (pres) [173,174]. in 18f-fdg pet studies, subacute covid-19 patients have shown hypometabolism in the frontoparietal cortex, which correlated with cognitive performance, with reduction in hypometabolism along with improvement in cognitive performance approximately 6 months after onset of symptoms [175,176]. in a large longitudinal study examining participants in the uk biobank who had undergone repeat imaging, a decrease in gray matter thickness in the orbitofrontal cortex and parahippocampal gyrus as well as decrease in global brain size were seen between two scans in those who had been infected by sars-cov-2, likely with different variants and averaging 4-5 months after infection, compared to those who had not been infected by sars-cov-2 [177]. greater cognitive decline was also seen in the sars-cov-2 positive group [177]. subsequently, du et al. found that male patients demonstrated reduced gray matter thickness in the parietal and occipital cortices and hippocampal volume after omicron infection compared to before infection [178]. in patients diagnosed with pcc, gray matter volume loss has been associated with cognitive dysfunction [179]. neuropathologic findings brain autopsy examination of patients who died from covid-19 have shown a variety of neuropathologic findings, although many are non-specific [180–184]. most commonly seen are acute hypoxic-ischemic changes with neuronal eosinophilia, acute/subacute infarcts (figure 1) with macrophage infiltration and neovascularization, hemorrhage [149,185–191], microthrombi [192–194], astrogliosis and microglial activation occasionally with microglial nodules and neuronophagia especially in the cerebellum and brainstem [185,195–199], and t-lymphocytic infiltration, predominantly sparse in a perivascular distribution [180,182,185,186,193, 195,200,201]. similar features including hypoxic-ischemic injury, microinfarcts, hemorrhage, and sparse lymphocytic infiltrate as well as fibrinogen leakage were seen between delta, omicron and non-delta/non-omicron variants [202]. other findings described in patients who died from acute covid-19 infection include perivascular hemosiderin deposition/leakage [182,193], though perivascular hemosiderin deposition is a common finding in autopsy brains, especially in older individuals, and can be seen in both brains with and without significant vascular disease [182,203–206]. they have also been shown to correspond to a subset of cerebral microbleeds on mri [207]. megakaryocytes in cortical capillaries and in an infarct were also reported in acute covid-19, but capillary megakaryocytes can been seen in the setting of lung injury due to a variety of causes [208–210]. uncommon findings include multifocal necrotizing leukoencephalopathy [185], acute encephalitis with lymphohistiocytic infiltrate and hemorrhage [200], and adem/acute hemorrhagic leukoencephalopathy (ahle) and adem/ahle-like pathology [149,195,211–213]. some comorbid neuropathologic findings described include other infections such as hsv-1 encephalitis [185] and bacterial infection [193], neurodegenerative diseases including alzheimer disease and lewy body disease [185,195], and cerebrovascular disease such as cerebral amyloid angiopathy [210], not surprising given the older patient age in some series [185,195,200,214]. few studies have compared brains from covid-19 patients to controls [152,197,198,200]. no differences in frequencies of hypoxic-ischemic changes, infarcts or hemorrhages were seen between covid-19 and non-covid-19 patients in one study [200]. in another study, perivascular and leptomeningeal t-lymphocytic infiltrates were seen in both covid-19 patients and in controls with sepsis /systemic inflammation, and microglial, activation in the pons in covid-19 patients was greater as compared to controls without sepsis but similar to controls with sepsis [152]. however, lee et al. showed that the brains of covid-19 patients demonstrate increased leakage of fibrinogen, consistent with bbb disruption, complement activation and microthrombi compared to controls, supportive of neurovascular injury [198,215]. leakage of fibrinogen and other serum proteins in the brain in covid-19 have also been reported [150,202]. several gene expression profiling studies on various regions of the brain from covid-19 patients have shown downregulation of neuronal and synaptic pathways in the olfactory bulb and amygdala [200], dysregulation that overlap with aging and neurodegenerative diseases in the frontal cortex [145,216], metabolic dysregulation in the brainstem [198], and inflammation in the choroid plexus [145,217]. very few autopsy studies have examined patients who recovered from covid-19 prior to death; these have also shown microglial activation/macrophages [195,197]. one of the studies included two patients with mild covid-19 symptoms who died 4-5 and 10 weeks after testing positive for covid-19 from causes unrelated to the infection [197]. in these subjects microglial activation/macrophages were also seen, similar to findings in patients with acute covid-19 in the brainstem and cerebellum but to a milder degree in the cerebral white matter [197]. so far there have been no autopsy studies of patients diagnosed with pcc (long covid). in a brief look at other pandemics, hypoxicischemic changes, infarcts, hemorrhage, microglial activation, and adem-like lesions were also seen in autopsies of patients who died from the novel influenza a h1n1 virus in 2009 [218]. the few autopsies reported on sars-cov, the etiologic agent responsible for the sars pandemic which emerged in 2002 [219], described hypoxic-ischemic changes and gliosis in the brain [220,221]. there are only rare reports of sars-cov detected in the csf [222,223] and in brain using pcr, ish, immunohistochemistry, and electron microscopy [220–222,224] with isolation of sars-cov reported from the brain of one patient [221]. the "brain isolation" may have reflected isolation of virus from brain vasculature. although several studies have detected sars-cov-2 rna in the brain using pcr or transcriptomic analysis [152,185,193,200,225–227], including in the affected area of a case of hemorrhagic encephalitis possibly attributed to covid-19 [200], other studies have not detected viral rna or protein in the brains of patients with covid-19 using a variety of methods [145,182,186,192,195,197,198,201,216,217], and sars-cov-2 has never been cultured from brain [131,180]. few studies have performed cellular localization of viral rna using ish or viral protein using immunohistochemistry and most have shown that they are restricted to vessels/near vessels such as in endothelium, within macrophages or near capillaries [148,182,225,228]. there are rare reports of viral protein or rna in brain tissue or cranial nerves [148,182,185,229–231], including a report by thakur et al. which found low levels of viral rna by rt-pcr but no viral rna or protein by rnascope and immunohistochemistry [185]. viral rna was seen in the adventitia of a meningeal blood vessel outside the medulla in one case [185]. because of frequent anosmia/hyposmia and olfactory dysfunction in covid-19, the suspicion is that the virus may enter the brain via the olfactory route through the cribriform plate, similar to neurotropic viruses [232–234]. several studies have examined the olfactory bulbs [185,193,201,230,231,235]. most showed variable degrees of t-lymphocytic infiltration and gliosis without presence of viral protein, but variability in presence of viral rna in human tissue [185,193,201,230,231,235]. these discrepancies may partly be due to differences in patient cohorts and methods employed including the usage of different commercial antibodies. some antibodies, including a widely used spike antibody (abcam 3a2), have subsequently been shown to label vessels and neurons in both covid-19 and control patients or have high background [145,180,197,236]. viral nucleocapsid antigen and rna have only rarely been detected in csf in acute disease [237–240]. csf analysis often shows slightly increased white blood cell counts as well as elevated albumin and protein levels and an elevated albumin quotient, which suggests disruption of the bbb [238]. similar to the brain, sars-cov-2 has never been cultured from csf even when virus could be grown from paired nasopharyngeal swabs [131]. in electron microscopy studies, virions have mainly been identified in the respiratory tract, and reports of ultrastructural detection of virus outside the respiratory tract are controversial due to potential misinterpretation of virus-like particles [236,241–243]. thus, it is unclear whether sars-cov-2 is neurotropic or neuroinvasive, and mechanisms other than direct infection likely contribute to both acute and chronic forms of the disease (figure 2) [167,232]. conclusions and next steps hiv and sars-cov-2 are associated with significant neurologic morbidity but with differing pathogenesis underlying acute and chronic disease. in acute disease, hiv directly invades the cns while sars-cov-2 may exert its effects more indirectly through systemic inflammation. in chronic disease, hiv persists in the brain despite cart, and sequelae of low-level infection may contribute to hand [115]. pcc may also be associated with sequelae of inflammation, but in both diseases, complex interactions with comorbidities likely underlie neurologic manifestations [48,160]. furthermore, both pcc and hand have significant components of psychiatric disease in which functional abnormalities are thought to predominate over structural abnormalities [244,245]. mechanisms of aging related neurodegeneration have also been implicated. brain lysates from covid-19 patients have shown activation of tgf-β signaling and increased oxidative stress as well as activation of pathways leading to tau hyperphosphorylation associated with alzheimer disease [246]. one promising avenue of investigation is the cerebral vasculature, as vascular disease and bbb dysfunction have been implicated to play a significant role in hand and acute covid-19 [54,64,102,198]. furthermore, it is well established that vascular disease plays a significant role in aging and neurodegenerative diseases [247], pathways which have been implicated in covid-19 as well as hand [145,216,248,249]. the following are potential "next steps" to elucidate the mechanisms underlying the neurologic manifestations in hand and covid-19/pcc discussed at the two meetings: longitudinal studies of patients with hand and pcc incorporating clinical, microbiology and pathologic findings using a multidisciplinary approach are warranted [250]. consensus on more sensitive and specific cognitive assessment tools for pcc will help in characterizing the clinical spectrum of pcc and guiding further investigation [251,252]. neuropathologic studies of patients with pcc in comparison to control groups using standardized guidelines and quantitative morphometric approaches, as has been done for hiv, may be helpful [251,253,254]. as routine light microscopy may not be sensitive enough to detect subtle changes, more in depth molecular investigation in conjunction with histopathologic examination, as has been pioneered by several groups showing transcriptomic changes in both hiv and covid-19, holds promise in shedding light on their neuropathogenesis [56,103,145]. greater funding is needed to perform labor and resource intensive rapid autopsies on patients with hand and pcc as mrna 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2024 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. histologic and molecular characterization of a maz::ncoa2 fusion-positive intracranial neoplasm feel free to add comments by clicking these icons on the sidebar free neuropathology 6:21 (2025) letter histologic and molecular characterization of a maz::ncoa2 fusion-positive intracranial neoplasm elliot stalter1, claire voyles1, leonardo f. freitas2, martha m. quezado3, brian j. dlouhy4, andrew groves5, osorio lopes abath neto1,6 university of iowa carver college of medicine, iowa city, usa florida international university, herbert wertheim college of medicine, miami, usa laboratory of pathology, center for cancer research, national cancer institute, national institutes of health, bethesda, usa department of neurosurgery, university of iowa health care, iowa city, usa department of pediatrics, university of iowa health care, iowa city, usa department of pathology, neuropathology, university of iowa health care, iowa city, usa corresponding author: osorio lopes abath neto · department of pathology, neuropathology · university of iowa health care · 200 hawkins dr · iowa city · ia 52242 · usa osorio-lopesabathneto@uiowa.edu additional resources and electronic supplementary material: supplementary material submitted: 3 october 2025 accepted: 02 november 2025 copyedited by: joão gama published: 11 november 2025 https://doi.org/10.17879/freeneuropathology-2025-9012 keywords: infantile cns tumor, gene fusion, maz::ncoa2, glioma, case report gene fusions have emerged as one of the primary drivers of oncogenesis in various tumor types, functioning through activation of signaling pathways or dysregulation of transcription. more than one third of soft tissue tumor types harbor gene fusions, over half of which are recurrent, and in the central nervous system (cns) fusions are uniquely associated with infantile tumors1. identification of novel fusions is essential to map the landscape of diagnostic tumor types and to develop targeted therapies. here we describe the first reported case of an intracranial neoplasm harboring a maz::ncoa2 fusion, highlighting a novel genetic alteration in the central nervous system and expanding the molecular spectrum of infantile fusion-driven neoplasms. a previously healthy 12-month-old female presented with a 3-week history of developmental regression, intermittent seizures, and truncal ataxia. physical examination revealed hypotonia, generalized hyporeflexia, and a head circumference greater than the 99th percentile for age. brain imaging demonstrated a large left parieto-occipital intraparenchymal mass with cystic and solid components, heterogeneous contrast enhancement, and signs of hydrocephalus with uncal herniation secondary to mass effect (figure 1a). there were small areas of meningeal contact in the mesial left parieto-occipital regions, but no meningeal thickening or enhancement were present. urgent craniotomy was performed with successful gross total resection of the mass (figure 1b). staging procedures were performed, and tumor was determined to be localized based on negative spine mri and clear csf on lumbar puncture (performed > 14 days after resection). figure 1 a. brain mri (post-contrast axial t1-weighted) revealed a contrast-enhancing left parieto-occipital mass (11.5 x 7.4 x 8.2 cm) with mixed cystic and solid components. the tumor abuts the leptomeninges but there is no meningeal thickening or enhancement. there is secondary hydrocephalus and midline shift resulting from the mass effect. b. post-operative imaging (post-contrast axial t1-weighted) showing gross total resection of the mass. histologically, the neoplasm was well-circumscribed (figure 2a, 2b) and showed variable cellularity. regions of lower cellularity predominantly consisted of spindle cells with a fibrillary cytoplasm (figure 2c) embedded in a heterogeneous myxoid stroma, which included frequent cystic spaces containing basophilic material (figure 2d). regions of higher cellularity (figure 2e) were located at the interface with brain parenchyma and displayed elevated mitotic activity, focal necrosis, and ki-67 proliferative rates of up to 30 % (figure 2l). the immunophenotype of the neoplastic cells followed a regional distribution, with diffuse strong gfap positivity (figure 2f) but mostly absent olig2 expression (figure 2h) in the lower-cellularity regions, but loss of gfap expression (figure 2g) and focal strong olig2 positivity (figure 2i) in the higher-cellularity regions. sox10 expression was strong and diffuse (figure 2j), and cd99 was weak but diffuse (figure 2k). neurofilament decorated rare cells within the tumor. markers that were notably negative in the neoplastic population included synaptophysin, cd34, ema, desmin, myogenin, nkx2.2, pan-keratin, smooth muscle actin, p63, p40, alk, ros1, braf v600e, hey1, and bcor. nuclear expressions of ini1, brg1, atrx, and h3 k27me3 were retained, and p53 had a non-clonal staining pattern. beta-catenin showed cytoplasmic positivity but only equivocal weak nuclear staining in rare cells. figure 2 morphologic and molecular features of the maz::nco2 fusion-positive tumor. a–b. the neoplasm (a; h&e, 100x) is well demarcated from the surrounding reactive brain parenchyma (asterisks), which contains numerous gemistocytic astrocytes and a rim of gliosis indicated by arrows (b; h&e, 200x). c–l. an intraoperative smear preparation (c; h&e, 400x) shows that tumor cells are spindled, with hyperchromatic elongated nuclei and processes. the tumor architecture varies from areas of relatively lower cellularity with a loose myxoid and cystic background (d; h&e, 400x) to areas of marked hypercellularity and mitotic activity (e; h&e, 400x). gfap staining (f, g; 400x) shows positivity in tumor cells in looser areas (f) but loss of expression in more cellular areas (g, top), in contrast to the reactive brain parenchyma (bottom). olig2 (h, i; 400x), by contrast, is mostly negative in looser areas (h) but positive in areas of higher cellularity (i). notable markers for which tumor cells are positive include sox10 (j, 400x) and cd99 (k, 400x). the ki-67 proliferative rate (l; 400x) is elevated in hypercellular areas, up to 30 %. m. in-frame maz::ncoa2 fusion including exon 4 of maz and exon 12 of ncoa2. n. copy number plot extracted from dna methylation array showed a flat profile. clicking into the picture will lead you to the full virtual slide https://doi.org/10.57860/min_dts_000025 molecular testing was performed using customized targeted next-generation sequencing (ngs) panels for mutations and fusions using an illumina nextseq platform at university of iowa. the dna panel covers the full coding sequence of 93 and hotspot mutation regions of 109 cancer-related genes, while the rna fusion panel can interrogate known and novel fusions involving 146 cancer-related genes. testing detected an in-frame maz::ncoa2 fusion involving the dna-binding domain of maz (enst00000545521.5_1) at exon 4 and the transactivation domain of ncoa2 (enst00000452400.7_4) at exon 12 (figure 2m). a pathogenic missense mutation in ctnnb1 (c.134c>t, p.ser45phe) was also detected, despite the inconclusive beta-catenin immunostain. dna methylation profiling did not result in a match using the german cancer research center (dkfz) cns tumor classifier versions 11b6 and 12.8, dkfz sarcoma classifier version 12b6, or national cancer institute bethesda classifiers versions 2 and 3. highest scoring methylation classes were divergent for the various classifiers and all resulted in very low scores: "cns tumor with patz1 fusion" (score 0.229) and "ewing sarcoma" (score 0.222) for bethesda v3; "chordoma" (score 0.108) and "hemangioblastoma" (score 0.04) for dkfz cns v12.8; and "osteosarcoma" (score 0.163) and "chondrosarcoma" (score 0.07) for dkfz sarcoma v12b6. dimensionality reduction techniques (umap and t-sne) likewise did not show consistent clustering in the embedding. copy number plots derived from methylation data revealed a flat profile, consistent with a stable genome (figure 2n). these findings supported a maz::ncoa2 fusion-positive neoplasm, not elsewhere classified. the patient was treated as per pog9233 ("baby pog") with multi-agent chemotherapy including vincristine, cyclophosphamide, cisplatin, and etoposide for 72 weeks, without radiation therapy2. she developed cisplatin-related hearing loss but remains without evidence of disease 22 months after resection. the ncoa2 gene is a transcriptional coactivator in the p160 steroid receptor coactivator family, playing a role in the regulation of muscle differentiation3. ncoa2 rearrangements have been described in several soft tissue tumor types, including congenital spindle cell rhabdomyosarcoma, mesenchymal chondrosarcoma, angiofibroma of soft tissue, myoepithelioma, and vascular tumors. maz encodes the myc-associated zinc finger protein, a broadly expressed transcription factor implicated in multiple cell programs that binds to gc-rich dna motifs and regulates the myc promoter and other regions4,5. a maz::ncoa2 fusion has been previously reported in a case of intraorbital myoepithelioma6, and recently it has been identified in a subcutaneous round cell sarcoma in a 10-month-old7. however, it has never been described in cns tumors, expanding the spectrum of ncoa2-rearranged tumors. the morphology of our case is neither reminiscent of myoepithelioma nor small round cell sarcoma, but there is some immunophenotypic overlap, particularly the positivity for sox10. the absence of a match in dna methylation classifiers suggests maz::ncoa2 fusion-positive tumors may represent an as yet unrecognized cns tumor type. comparisons of methylation profiling of reported cases and identification of additional examples are important next steps to further characterize these tumors. from a clinical standpoint, the significance of the fusion is unclear. ncoa2 fusion-positive tumors in pediatric soft tissues can show variable outcomes, with biologic behaviors that vary from indolent to aggressive. our patient remains disease free at 22 months after gross total resection and chemotherapy, but longer follow-up and additional reported cases will be necessary to firmly establish outcomes. conflict of interest statement the authors report no conflicts of interest. funding statement the authors report no funding related this manuscript. supplementary material material 1–6 (.pdf-file; 1270 kb): supplementary figure 1: beta-catenin stain supplementary figure 2: p40 stain supplementary figure 3: pan-keratin stain supplementary figure 4: sma stain supplementary figure 5: t-sne bethesda classifier v31 supplementary figure 6: umap bethesda classifier references 1. mertens f, antonescu cr, mitelman f. gene fusions in soft tissue tumors: recurrent and overlapping pathogenetic themes. genes chromosomes cancer 2016;55(4):291-310. https://doi.org/10.1002/gcc.22335 2. strother dr, lafay-cousin l, boyett jm, burger p, aronin p, constine l, et al. benefit from prolonged dose-intensive chemotherapy for infants with malignant brain tumors is restricted to patients with ependymoma: a report of the pediatric oncology group randomized controlled trial 9233/34. neuro oncol 2014 mar;16(3):457-65. https://doi.org/10.1093/neuonc/not163 3. mosquera jm, sboner a, zhang l, kitabayashi n, chen cl, sung ys, et al. recurrent ncoa2 gene rearrangements in congenital/infantile spindle cell rhabdomyosarcoma. genes chromosomes cancer 2013;52(6):538-50. https://doi.org/10.1002/gcc.22050. 4. bossone sa, asselin c, patel aj, marcu kb. maz, a zinc finger protein, binds to c-myc and c2 gene sequences regulating transcriptional initiation and termination. proc natl acad sci u s a 1992;89(16):7452-6. https://doi.org/10.1073/pnas.89.16.7452 5. song j, murakami h, tsutsui h, tang x, matsumura m, itakura k, et al. genomic organization and expression of a human gene for myc-associated zinc finger protein (maz). j biol chem 1998;273(32):20603-14. https://doi.org/10.1074/jbc.273.32.20603 6. an s, koh hh, chang es, choi j, song jy, lee ms, et al. unearthing novel fusions as therapeutic targets in solid tumors using targeted rna sequencing. front oncol 2022;12:892918. https://doi.org/10.3389/fonc.2022.892918 7. chen h, zhang p, zhou l. a maz::ncoa2 subcutaneous small round cell sarcoma of infancy with diffuse s100/sox10 positivity: a novel entity. genes chromosomes cancer 2025;64(2):e70034. https://doi.org/10.1002/gcc.70034 copyright: © 2025 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. multiple sclerosis: 2024 update feel free to add comments by clicking these icons on the sidebar free neuropathology 6:14 (2025) review multiple sclerosis: 2024 update luisa klotz1, maija saraste2,3,4,5, laura airas2,3,4,5, tanja kuhlmann6 department of neurology, university hospital münster, münster, germany turku pet centre, turku university hospital, turku, finland neurocenter, turku university hospital, turku, finland clinical neurosciences, university of turku, turku, finland inflames research flagship, university of turku, turku, finland institute of neuropathology, university hospital münster, münster, germany corresponding author: tanja kuhlmann · institute of neuropathology · university hospital münster · pottkamp 2, 48149 · münster · germany tanja.kuhlmann@ukmuenster.de submitted: 11 may 2025 accepted: 24 june 2025 copyedited by: joão gama published: 08 july 2025 https://doi.org/10.17879/freeneuropathology-2025-6762 keywords: multiple sclerosis, genetics, disease course, diagnostic criteria, biomarkers, imaging, treatment abstract multiple sclerosis (ms) is a complex immune-mediated disease that leads to neurological disability, with ongoing challenges in understanding its initiation, predicting progression, and optimizing personalized treatment. this review article summarizes key research findings from 2024, covering advances in diagnostic criteria, understanding of pathophysiology, and treatment strategies. new studies reinforce the strong link between epstein-barr virus (ebv) and ms, while recent data point towards a role of genetics in ms disease progression. the 2024 mcdonald criteria revision enhances diagnostic specificity and includes novel mri markers and facilitates measurement of cerebrospinal fluid biomarkers. additionally, recent genetic discoveries, advanced imaging techniques, and emerging biomarkers are refining disease monitoring and prognosis. finally, we highlight promising therapeutic developments, including bruton tyrosine kinase (btk) inhibitors and car t-cell therapies, with the former representing a paradigm shift in the potential of targeting ms progression beyond focal inflammation. introduction multiple sclerosis (ms) is a complex immune-mediated disease of the central nervous system and a leading cause of permanent neurological disability in young adults. despite progress in reducing focal inflammatory activity through high-efficacy immunomodulatory treatments, major challenges remain in ms research. our incomplete understanding of disease initiation in susceptible individuals hinders the development of preventive treatments. additionally, the variability in disease course makes it difficult to predict outcomes and individualize therapy for optimal efficacy and minimal side effects ("treat-to-target"). effective monitoring of this heterogeneous disease and its treatment requires a personalized approach using reliable imaging and biomarkers that offer insight into individual disease biology. furthermore, recent discoveries on myeloid and b cells in disease progression have driven the development of novel therapies. based on these considerations, we have selected publications from 2024 that provide new insights into ms pathophysiology, evolving diagnostics, novel biomarkers, imaging techniques, and emerging treatment strategies (fig. 1). monozygotic twin studies revealed a heritability risk of approximately 25–30 %. so far, studies have identified up to 233 genetic variants linked to ms susceptibility, most expressed in immune cells1. in 2023, the first study focused on ms severity and identified one significant risk allele and 11 suggestive loci, all encoding for genes expressed in the cns2. further 2024 research has explored correlations between these snps and aspects of disease severity. figure 1 schematic illustration depicting the ms disease course and established and explorative tools to diagnose ms and monitor or predict the disease course. a role of ebv in ms pathogenesis has long been suspected and several studies provide compelling evidence supporting this connection. nearly all ms patients have prior ebv infection, and a landmark study showed ebv increases ms risk more than 30-fold3. in 2024, research confirmed that pediatric-onset ms is strongly associated with ebv, differentiating it from mog antibody-associated disease (mogad). ms patients exhibit dysregulated immune responses to ebv, suggesting impaired control of latent infection, raising the potential for antiviral therapies. accurate, early ms diagnosis remains crucial for improved long-term treatment outcomes. the 2024 mcdonald criteria revision aims at enhancing specificity while maintaining sensitivity. key updates include recognizing optic nerve involvement as part of dissemination in space (dis), incorporating mri markers like the central vein sign (cvs) and paramagnetic rim lesions (prl), and using kappa-free light chains (kflc) as an alternative to oligoclonal bands (ocbs) in cerebrospinal fluid analysis. furthermore, accumulating evidence indicates that ms pathology begins long before clinical onset. consequently, radiologically isolated syndrome (ris), identified via incidental mri findings, is now recognized as a preclinical ms stage allowing ms diagnosis even in the absence of clinical manifestation, with disease-modifying therapies shown to delay conversion to ms. we also highlight recent findings on the ms prodromal stage, characterized by early nonspecific symptoms such as fatigue, depression, and sleep disturbances. biomarkers are essential for individual disease prognosis and prediction but remain underutilized in ms. serum neurofilament light (nfl) is the most advanced biomarker, correlating with disease activity and long-term disability risk. glial fibrillary acidic protein (gfap) has emerged as a complementary biomarker, and findings from 2024 suggest their combination might help to distinguish inflammatory damage from neurodegeneration. furthermore, promising new data point towards novel approaches to improve patient stratification and predict treatment responses. novel imaging markers like cvs and prl enhance specificity of ms diagnosis, and in particular prls are considered as novel biomarker of disease progression. besides novel imaging approaches, the implementation of ai-driven analysis of imaging and clinical data facilitates both diagnosis and prognosis, potentially enabling earlier intervention and personalized therapy in the future. recent findings challenge the traditional view that ms disability is primarily relapse-driven, as progression independent of relapse activity (pira) significantly contributes to long-term disability, even in early ms. reflecting this conceptual shift, we discuss recent clinical trial outcomes on bruton tyrosine kinase (btk) inhibitors as the most appreciated clinical highlight in ms research in 2024. furthermore, we discuss novel cellular therapies such as car t cells, which target deep tissue b-cell depletion, offering a new frontier in ms treatment in the future. pathogenesis of ms ms and genetics based on family and twin studies, the heritability of ms is estimated to be approximately 30 %4. hla-drb1*15:01 is the strongest ms risk factor, which increases the ms risk threefold in individuals carrying at least one copy of the allele4. barrie and colleagues now demonstrated that the genetic risk of ms rose among the pastoralists in the pontic steppe and was brought to europe by the yamnaya-related migration approximately 5000 years ago. the authors analyzed datasets from the mesolithic, medieval and post-medieval periods and found a positive selection of ms associated immunogenetic variant risk genes. interestingly, most of the alleles under positive selection were associated with protective effects against specific pathogens and/or infectious diseases, suggesting that transmission of pathogens drove the selection of immune gene variants, which are now associated with an increased risk of autoimmune diseases4. in 2019, the international ms genetic consortium provided a detailed genetic and genomic map of multiple sclerosis1. this study identified 200 autosomal susceptibility variants outside the major histocompatibility complex (mhc), one chromosome x variant, and 32 within the extended mhc. these genes associated with ms susceptibility are implicated in multiple innate and adaptive pathways (e.g. tnfa, and type 1 interferons) and cells of the immune system including microglia, thus strongly supporting the immune-driven nature of disease onset. the authors of these studies estimated that their results could explain 48 % of ms heritability. however, only in 2023, the first snp associated with disease severity (reflected by the age-related ms severity score) was identified in a genome-wide association study including data from 12,584 cases and replicated in an additional study comprising further 9,805 cases2. the authors found a significant association with rs10191329 in the dysf–znf638 locus. dysf is well known for its function in muscle membrane repair and calcium dependent membrane fusion, whereas znf638 is a transcriptional co-activator, which is involved in cell differentiation and proliferation5. both genes are expressed by neurons and glia cells, suggesting that disease severity may be influenced by central nervous system (cns) intrinsic mechanisms. the authors could also show that rs10191329 was linked to higher lesion load in brain stem and cortex in a large ms autopsy cohort, however the molecular pathways and relevant cell types mediating this phenotype are unknown2. maybe as important than identifying gene variants associated with ms severity, the authors observed a significant heritability enrichment in cns tissue, further suggesting that cns resident cells determine severity and outcome of the disease2. interestingly, in 2024, an independent imaging study including a discovery cohort of 748 and a replication study of 360 people with relapsing remitting ms observed an association with 28 % more brain atrophy per rs10191329*a allele, further corroborating its role in pathophysiological processes underlying disease progression. the authors therefore encourage stratification for rs10191329 in clinical trials6. in contrast, other studies were not able to detect a correlation between rs10191329 and longitudinal binary disease severity or other clinically relevant outcomes7,8. however, the sample size of the aforementioned studies was modest compared to the initial study and the estimated effect size of rs10191329 rather low, further strengthen the necessity for collaborative research approaches to maximize sample sizes to further disentangle the genetic background of ms. ms and ebv ebv has been implicated in the pathogenesis of ms for a long time as reviewed and summarized in a number of well-written and informative reviews9,10. approximately 90 % of the population are infected by ebv within the first two decades in life; the virus is transmitted by saliva or infectious b cells. after infection, the virus establishes latency resulting in its lifelong persistence. primary infection during childhood is usually asymptomatic, but the majority of individuals infected during adolescence or adulthood will develop infectious mononucleosis. a seminal longitudinal study published in science 2022 studying a cohort of more than 10 million young adults on active duty in the us military, among them 955 who were diagnosed with ms during their period of service, demonstrated that ebv infection increases the risk to develop ms more than 30-fold3. in contrast, previous studies reported lower rates of ebv infection among children with pediatric onset of ms raising questions about whether ebv infection is indeed pre-requisite across the age spectrum11. however, those studies were conducted before mog antibody tests were broadly available and therefore these studies could not differentiate reliably between pediatric onset-ms and mog antibody-associated disease (mogad). in this line, a recent study differentiating between mogad and pediatric onset ms now demonstrated that 96 % of the children with pediatric onset ms had antibodies directed against the viral capsid of ebv and 90 % had antibodies directed against ebna1 (a marker of a remote ebv infection) further supporting the notion that ebv infection is required to trigger ms across the whole age spectrum12. interestingly, children with mogad had similar rates of ebv seropositivity as healthy children, indicating that ebv infection is not a risk factor for mogad. the mechanisms underlying the increased ms risk associated with ebv infection are still poorly understood. results from previous studies suggest that the ebv infection is less well controlled in people with ms13,14. this concept is further supported by a number of findings published during the last year: 1.) the ebv antibody response is not limited to ebna1, suggesting a larger dysregulation of ebv-specific antibody responses than previously recognized in pwms15. 2.) pwms, but not individuals with other neuroinflammatory diseases including neuromyelitis optica, mogad or susac’s syndrome display an aberrant mhc-i-restricted t cell response directed against ebv16 and 3.) the frequencies of cxcr3+ memory b cells are reduced in the blood of genetically identical twins with ms compared to their unaffected siblings. based on the latter finding, the authors propose that these memory b cells migrate into the cns, mature into antibody secreting cells in the cns and drive the disease17. 4.) spontaneous lymphoblastoid cell lines (slcls) isolated from pwms with active disease had higher ebv lytic gene expression than slcls from ms patients with stable disease or hcs. furthermore, lcls from patients with active disease displayed activation of selected inflammatory pathways and of genes associated with the lytic gene expression of ebv indicating that dysregulation of ebv gene expression by b cells drives a pro-inflammatory, pathogenic b cell phenotype18. interestingly, the authors provide also evidence that antiviral approaches targeting ebv replication decreased cytokine production and autologous cd4+ t cell responses. the identification of ebv as an important contributing factor of ms raises the questions whether anti-viral treatment approaches could either prevent or slow down ms disease progression and first clinical trials are currently under way10,19. novel insight into ms pathophysiology using new technologies there are ongoing efforts to characterize molecular signatures associated with ms lesion types, remyelination failure or neurodegeneration using modern sequencing technologies, such as sc/snrna sequencing or spatial transcriptomics (st) to identify new pharmacological treatment targets20–22. spatial transcriptomics enables spatially resolved analysis of gene expression within intact tissue sections in contrast to bulk or single-cell rna sequencing (scrna-seq), which requires the dissociation of tissues and loses spatial context. lerma-martin and colleagues analyzed 12 subcortical ms lesions from six donors and seven controls by combining spatial transcriptomics (10x genomics visium spatial gene expression platform) with snrna sequencing. the comparison between histologically annotated areas and unsupervised molecularly defined niches revealed a significant overlap both at the cluster and tissue section level validating the reliability of the method20. alemsa and colleagues performed a similar study using two different spatial transcriptomic platforms (10x genomics visium spatial gene expression platform, nanostring geomx )21. both publications provide further insights into potential cell-cell communication via receptor ligand interactions, which will contribute to the disentanglement of the dynamic cellular and molecular changes occurring in ms lesions. furthermore, they compared the transcriptional profiles in perilesional wm and nawm and observed lesion-type dependent alterations, further supporting the notion that perilesional white matter directly adjacent to a lesion differs from nawm, which is in line with observations from histopathology and imaging23–25. together, these data suggest, that the inflammatory infiltrates in active and mixed active/inactive lesions affect the perilesional tissue environment. however, although spatial transcriptomics is a valuable new tool for the identification of disease associated molecular pathways in ms, the techniques used in the above mentioned manuscript do not reach single cell resolution. a new method, named in situ sequencing allows the identification of transcriptomic patterns with single cell resolution. kukanja and colleagues used this method to disentangle molecular mechanisms underlying lesion formation in experimental autoimmune encephalitis, an animal model of ms26. they also identified disease-associated glia cells, which were detected outside of eae lesions, and which were dynamically induced and resolved during the eae course in line with the observation of disease associated changes also outside of ms lesions. the authors could also provide evidence that the method is suitable to analyze human ms tissue samples. a limitation of these techniques are the limited number of genes that can be analyzed (in this study 239 and 266 genes in mouse and human samples, respectively); however there are ongoing efforts to enable whole transcriptome single cell scale analysis in in formalin fixed paraffin embedded tissue sections27. preclinical stages of ms – expanding the ms disease continuume historically, ms diagnosis required a clinically defined event, but recent evidence indicates that disease processes begin well before symptoms emerge. mri studies have shown that asymptomatic individuals can have lesions suggestive of inflammatory demyelination, with some later developing clinical ms – a condition termed radiologically isolated syndrome (ris), first described by okuda et al. in 200928. ris is a condition in which asymptomatic individuals exhibit mri lesions in characteristic locations that are highly suggestive for ms. in 2023, the ris consortium refined the diagnostic criteria for ris: individuals with lesions in at least three key cns locations (periventricular, juxtacortical, infratentorial, or spinal cord) fulfill ris imaging criteria.29 alternatively, those having lesions in only one or two of these areas but additionally exhibiting with two of the following criteria – spinal cord lesion, csf-restricted oligoclonal bands, or new demyelinating lesions on follow-up mri – fulfill the ris definition. this revision improves prognostic stratification, as studies report conversion rates to clinical ms of 34 % at five years and 51 % at ten years. additional risk factors for conversion into ms include younger age (30 based on these considerations, some ris constellations now fulfill the new diagnostic criteria of ms, which are discussed below, which illustrates that biological rather than purely clinical considerations are now implemented in our current concept of ms diagnosis. beyond imaging, a range of nonspecific clinical symptoms – including depression, anxiety, fatigue, sleep disturbances, and headache – have been identified as part of the so-called ms prodrome31. recently, studies in both children and adults confirm that these early signs, along with elevated serum neurofilament light chain levels up to nine years before clinical onset, support the concept that ms pathophysiology initiates long before overt clinical symptoms32–34. however, due to the unspecific nature of these symptoms and a high overlap with other immune-mediated diseases, it is currently not possible to provide a clear ms prodrome definition that may guide further diagnostic workup to facilitate early ms diagnosis. new diagnostic framework of ms diagnosis of ms – the 2024 update of the mcdonald criteria diagnosing multiple sclerosis requires balancing early detection with minimizing misdiagnosis. over two decades, diagnostic criteria have evolved with mri and csf biomarkers, enabling earlier and more accurate diagnosis. after the last revision of diagnostic criteria in 201735, another update has been proposed in 2024. this update is based on novel data highlighting the role of the optic nerve, the relevance of ris, the necessity to differentiate ms from other autoimmune conditions like nmosd and mogad, acknowledgement of the concept that disease progression is a key feature of relapsing ms, and finally, the diagnostic challenge of ms in older individuals and those with comorbidities. based on these emerging concepts, the following changes have been presented at the ectrims congress in 2024 by x. montalban on behalf of the international advisory committee on clinical trials in ms36,37, however the diagnostic criteria have not been published yet. first, the optic nerve is now included as a diagnostic region providing evidence for the dissemination in space (dis), and besides clinical manifestation as optic neuritis, optic nerve involvement can be illustrated by optical coherence tomography, visual evoked potential and mri. second, ms diagnosis requires documentation of dis in at least two of five cns regions (optic nerve, cortical/juxtacortical, periventricular, infratentorial, spinal cord), if this is supplemented with either dissemination in time, or detection of oligoclonal bands or kappa free light chains in the csf. notably, the dissemination in time (dit) is no longer mandatory for diagnosis, as this is not exclusive to ms and imaging variability can affect its interpretation. with regard to imaging, two new imaging criteria have newly been implemented that are not mandatory but may facilitate diagnosis based on their high specificity for ms: the central vein sign (cvs), caused by inflammatory lesions forming around central veins, can be visualized by t2* mr imaging, and detection of at least 6 cvs can confirm ms in a situation when only two topographies are affected38. paramagnetic rim lesions (prl) indicate chronic active ms lesions and can be detected by susceptibility-weighted mri sequences, which are sensitive for detection of iron accumulation in a rim of myeloid cells around chronic ms lesions38. these prls are not exclusively found in ms but display high specificity; therefore, detection of at least one prl in the presence of either dit or csf positivity can now confirm ms in cases with so far only one topography affected. for csf analysis in the context of ms workup, the kappa-free light chain (kflc) index can now replace ocb detection, both reflecting intrathecal immunoglobulin production39. this may facilitate diagnostic procedures as this is a cost-effective and rater-independent method based on nephelometry or turbidimetry. furthermore, as described in the previous section, evidence supports ris as part of the ms continuum, with over half of ris cases developing clinical ms within 10 years40. therefore, the diagnostic criteria now allow for ms diagnosis in those ris patients exhibiting lesions in at least two topographies plus dit or csf positivity. this represents a real conceptual change as this accepts ms as a biological diagnosis even in the absence of any clinical manifestation. regarding the former separation of diagnostic criteria for relapsing versus progressive ms, the common biological mechanisms of these disease courses have been acknowledged, and therefore, the newly revised diagnostic criteria can be applied to both disease courses. finally, criteria have been adapted in individuals over 50 or with comorbidities acknowledging the increased risk of ms misdiagnosis in these conditions due to small vessel disease, migraine or other inflammatory disorders, which can also present with t2 lesions. in these cases, additional criteria should be fulfilled, such as detection of at least one spinal cord lesion, positive csf and/or detection of cvs. together, this 2024 revision of the ms diagnostic criteria aims to facilitate the diagnosis of ms in individuals based on biological considerations without compromising specificity, which will ultimately improve clinical outcomes of people with ms globally. monitoring of disease activity biomarkers and their use for personalized medicine – where do we stand? biomarkers are key to advancing personalized medicine by enabling precise diagnosis, risk stratification, and guidance in treatment responses. in oncology, the use of biomarkers for personalized medicine approaches is already firmly established due to the unique genetic and molecular profiles of a patient’s tumor. in autoimmune diseases, personalized medicine based on biomarkers is still in its infancy, primarily due to the complexity of their pathophysiology and the heterogeneity in clinical presentation. in the field of ms, recent publications illustrate both advances and limitations in this area of research. besides mri, serum neurofilament light chain (nfl) levels represent the most advanced biomarker for facilitating the assessment of a patient’s individual prognosis41. although nfl is a non-specific marker of neuronal injury and therefore not limited to ms, elevated serum nfl (snfl) levels have been associated with acute relapse activity and responses to highly active treatments42. recently, the relevance of nfl in predicting disability accumulation has been further explored, demonstrating that elevated snfl levels after a first demyelinating event are associated with an increased risk of future disability accumulation43. furthermore, glial fibrillary acidic protein (gfap) has emerged as another serum biomarker in ms. one study proposed that the combined assessment of gfap and nfl may help distinguish between acute focal inflammatory damage – reflected by nfl elevation – and relapse-independent progression, as indicated by gfap elevation44. in contrast, a more recent study by monreal and colleagues suggested that higher nfl levels were associated with an increased risk of both relapse-associated worsening (raw) and pira, confirming its prognostic value at disease onset. higher gfap levels were linked only to a higher risk of reaching an edss score of 3, but not to raw or pira. however, in a subset of patients with low nfl levels, gfap was also associated with pira. in individuals with low levels of both markers, the risk for all outcomes was lowest. these findings suggest that gfap may indicate progression only in a subset of patients, potentially driven by a distinct pathophysiology, and that combining both markers may enhance prediction accuracy45. additional experimental approaches have been published, highlighting the potential of more pathophysiology-driven biomarkers to improve patient stratification in ms. one study, based on the concept that ms has a strong genetic component, used a genetic risk score (grs) in individuals with optic neuritis (on) to predict the future development of ms46. the study demonstrated that combining genetic data with demographic factors significantly improved the prediction of ms in individuals with undifferentiated on, with results replicated in an independent cohort. another study analyzed whole-proteome autoantibody profiles in individuals both before and after ms onset. notably, around 10 % of ms patients displayed a unique signature that could already be detected several years before disease onset47. in line with other evidence pointing toward the relevance of certain virus-immune interactions as prerequisites for ms development in susceptible individuals, this antibody signature included a common motif observed in several human infectious pathogens, including ebv. although present in only a smaller subset of ms patients, these findings raise the possibility that at least a fraction of individuals at high risk of developing ms could be identified before clinical disease onset, potentially facilitating the implementation of preventive strategies in the future. a german study employed high-dimensional immunological profiling of peripheral blood in untreated early ms patients to identify subgroups based on distinct immunological characteristics47. this approach enabled the identification of three distinct subgroups with unique immune patterns – one associated with high inflammatory activity based on clinical and imaging measures, and another characterized by early signs of tissue destruction and neurodegeneration. notably, these subgroups not only exhibited differences in clinical disease trajectories but also in response to immune treatments, highlighting that a better characterization of distinct immunobiological patterns may help predict individual treatment responses in the future. similarly, analyzing the so far largest ms brain tissue collection comprising normal appearing white matter as well as grey and white matter lesions revealed different cellular compositions between the lesions but surprisingly similar cell-type gene expression patterns both within and across patients, suggesting patient-dependent global changes. based on these observations, the authors stratified the patients into different molecular subgroups suggesting that different molecular mechanisms may drive pathophysiology and predict response to treatments targeting cns intrinsic disease mechanism. however, the correlation of these molecular subtypes with pathological or clinical disease trajectories has not yet been established22. novel advances in imaging to detect disease progression conventional mri detects focal lesions with great sensitivity, but it does not perform well in detecting the diffuse pathology responsible for pira. the imminent need of treatments for slowing down disease progression in ms has prompted wide interest in the application of advanced imaging methods to better understand and assess the progression-promoting pathological processes within the cns. a number of comprehensive review articles on imaging of focal and diffuse compartmentalized inflammatory and neurodegenerative processes were published in 202448–53. potential imaging biomarkers of ms progression include detection of paramagnetic lesions (prls) using iron-sensitive mri sequences, identification of cortical lesions using double inversion recovery (dir), assessment of grey matter damage, and measurement of choroid plexus volume. in advanced clinical imaging, prls, slowly expanding lesions (sels) and tspo-rim-active lesions are considered to represent chronic active lesions (cals) (also termed mixed active/inactive lesions)50, which are characterized by a hypocellular lesion center and a rim of macrophages/microglia (fig. 2). however, which of these different methods is the best predictor of disease progression has yet to be determined. notably, recent work suggests that there is only partial overlap in cal-detection using these imaging methods. here, numbers of sels were shown to be higher than those of prls (616 vs 80), and the correlation between lesion counts was quite moderate (ρ = 0.28, p = 0.03)54. this suggests that sel and prl may capture distinct pathophysiological features of chronic active lesions. similarly, based on a recent publication, tspo-pet has a higher sensitivity to detect more cals compared to susceptibility weighted mri, although there was a correlation between the number of 11c-pbr28 active lesions and prls in 7t phase images55. moreover, this study found that tspo-pet whole active lesion volume had the strongest association with the edss score in a cohort of 30 study patients including equal numbers of patients with rrms and spms55. prls have been particularly widely studied during the past years and are now considered a predictive imaging biomarker for greater disease severity and progression and correlates with brain and spinal cord atrophy53. a consensus statement developed by the north american imaging in multiple sclerosis (naims) cooperative published in 2024 provides guidance for the definition and measurement of prls to promote their clinical translation51. this also prompted their inclusion in the new diagnostic criteria for ms as described above. figure 2: histological and imaging detection of chronic active lesions. histological characterization of a chronic active lesions (= mixed active/inactive lesions) (a to d). the lesion is completely demyelinated and has a sharp border to the adjacent normal appearing white matter (luxol-fast blue staining) (a). chronic active lesions are characterized by a rim of myeloid cells expressing cd68 and tspo (b and c). a subset of chronic active lesions displays a dense rim of myeloid cells with cytoplasmic iron depositions (turnbull staining) (d). different imaging techniques, such as tspo-pet and quantitative susceptibility mapping (qsm) are currently used to identify this lesion type in individuals with ms (e to g). the red circle in indicates a hypointense t1 lesions (e), that displays a tspo positive rim in tspo pet (ral: rim active lesion) (f) and a paramagnetic rim in qsm (prl: paramagnetic rim lesion) (g). following this line, several articles published in 2024 provided further evidence regarding the association of prls with disease progression: .1) patients with pira had significantly more prls, also when analysis was restricted to patients with rrms56. 2) prls associated with pira over the 2 years after study entry demonstrating their predictive power for pira 38. 3) a longitudinal study with median follow-up time of 5.6 years showed that the appearance of new prls was associated with increased rates of pira57. on the other hand, prl disappearance was associated with reduced rates of confirmed disability progression, suggesting the need of additional studies to understand the predictive value of these dynamic changes. imaging tools to assess neurodegeneration are another approach to predict disease progression. higher baseline cortical lesion load, but not white matter lesion load or new cortical lesions during the observation period of three years, predicted disability worsening suggesting that cortical lesions forming earlier during the disease course contribute to disability progression rather than new cortical lesions58. however, an alternative explanation could be that the detrimental effects of cortical lesions on disease progression require time to manifest clinically. thalamic atrophy is well-known to be associated with disease progression. the study by cagol and colleagues now provides further insight into the underlying mechanisms of thalamic atrophy. using advanced quantitative mri, they demonstrated that microstructural thalamic changes linked to demyelination, neuroaxonal loss, and disturbances in iron homeostasis correlated well with clinical disability, cognitive impairment, and mri measures of disease burden59. interestingly, a recent longitudinal study provided further evidence about the gradual increase of choroid plexus volume (1.4 % per year) and its association with brain atrophy60. in this study from sydney, 57 patients with rrms underwent annual mri scans during a minimum follow-up of four years. interestingly, they showed that the annual change in choroid plexus volume correlated with chronic lesion expansion (r = 0.46, p < 0.001), further supporting the notion that plexus enlargement is at least partly induced by cellular or molecular inflammatory mediators. an association between choroid plexus volume, cognitive impairment, and fatigue was also recently demonstrated61. there are also several other advanced imaging methods that have shown promise for identification of disease progression, such as the diffusion mri based neurite orientation dispersion and density imaging (noddi) that provides specific measures of tissue microstructure, soma and neurite density imaging (sandi), and leptomeningeal enhancement that can be visualized using delayed post-contrast flair, demonstrating meningeal b cell aggregates53. use of ai the potential of artificial intelligence (ai) and machine learning continues to be explored in the context of ms as reviewed by collorone et al62. ai has been used in explorative studies to guide ms diagnosis, prediction, lesion segmentation, and investigation of disease mechanisms. the authors conclude that although there are several challenges regarding the quality of input data and ethical issues, the use of ai has made significant progress in recent years in the ms field. however, the reproducibility and validation of the results, which is important for the integration of ai based methods into clinical practice, warrants further studies. in 2024, noteboom et al63 studied how various machine learning models were capable of determining clinical impairment at baseline and of predicting future clinical worsening in two cohorts (n = 123 and 330, respectively). support vector machine classifier was the best ai tool to identify higher disability (edss ≥ 4) and impaired cognition (sdmt z-score ≤ −1.5) when clinical factors and global or regional mri volumes were used as input. however, the machine learning models were not able to predict clinical worsening after two or five years. on the other hand, andorra et al showed that random forest algorithms predicted neda (no evidence of disease activity) with auc 0.80 and confirmed disability accumulation with aucs 0.62, 0,63 and 0.61 for edss, sdmt and 9hpt, respectively64. algorithms were first tested using a prospective multi-centric cohort including 322 patients with ms and 98 healthy controls, and then using a prospective cohort of 271 patients with ms. their findings suggest, that combining clinical, and imaging and in some instances also omics data with machine learning may help identifying ms patients at risk of disability worsening. proper identification of focal ms lesions is the basis for successful diagnosis, treatment and disease monitoring. furthermore, reliable identification of chronic active lesions gives valuable information on the risk of later progression as described above. manual lesion segmentation is time-consuming, but thus far it has proven more reliable than existing automated lesion segmentation methods. this is now being challenged by recent lesion-detection work using deep learning models. pasquale de rosa et al applied a consensus-based framework, which combines five publicly available deep learning models, to improve lesion segmentation65. they used two datasets, including 131 and 30 patients with ms to compare it with lesions masks segmented manually. their method showed good agreement with the volume and numbers of lesions identified through manual segmentation (ρ = 0.92 and ρ = 0.97, and ρ = 0.83 and ρ = 0.94 for datasets i and ii, respectively). automated methods for prl assessment are also urgently needed. now the naims cooperative have tested an automated paramagnetic rim lesion (aprl) algorithm in a multi-center setting and reported that this automated segmentation method successfully captured 115 (78 %) of manually identified prls66. this gives promise for facilitated prl-detection in large datasets; an improvement that could enable routine prl detection in clinical settings and in the context of treatment trials. rethinking ms progression and treatment strategies the concept of progression independent from relapse activity and emerging treatments targeting the pathophysiology of progression while it has long been accepted that disability accrual in ms is primarily driven by focal inflammation as the pathophysiological correlate of relapses, recent research has identified significant disease progression independent of relapse activity, termed pira. identification of pira became possible through careful re-evaluation of clinical trial data from high-efficacy treatments – particularly ocrelizumab – where progression was observed despite an almost complete absence of relapses67. in 2023, data from a large cis / early ms cohort revealed that pira can occur even in early relapsing ms and is associated with an unfavorable long-term prognosis68. although several studies have shown that current treatments partially influence pira69, none of the approved therapies are sufficient to control it, and its exact pathophysiology remains elusive, hampering the development of targeted treatments. in this context, a novel class of drugs – bruton’s tyrosine kinase (btk) inhibitors – has gained attention in ms treatment. btk inhibitors block btk, an enzyme essential for intracellular signaling during b cell receptor activation and for activating myeloid cells, without directly affecting t cells70. several btk inhibitors have already been approved for hematologic malignancies such as chronic lymphocytic leukemia and mantle cell lymphoma; their dual action on b cells and myeloid cells has sparked interest for autoimmune diseases70. btk inhibition modulates both adaptive and innate immune responses by suppressing proinflammatory cytokine production, antigen presentation, and cell survival71. the recognized role of b cells in ms pathophysiology, along with the contribution of myeloid cells to chronic lesion formation and progression, has led to multiple trials in both relapsing and progressive ms70. data from several recent trials investigating evobrutinib and tolebrutinib have been presented in 2024. notably, evobrutinib failed to demonstrate superiority over teriflunomide (which interferes with lymphocyte proliferation and metabolic activity) in controlling focal inflammatory activity and confirmed accumulation of disability72, whereas tolebrutinib showed efficacy in reducing confirmed disability progression in three clinical trials studying its efficacy in people with rrms and spms (gemini i and ii and hercules trials)73,74. recently, new data have been presented demonstrating that the effect of tolebrutinib on disability progression was only seen in patients with prls at baseline75. this is particularly remarkable as tolebrutinib exerted only modest effects on markers of acute focal inflammation, therefore providing first evidence that progression-related pathology can be modulated independently of acute focal activity and this is centered around modulation of b cells and/or myeloid cells73,74. emerging cellular therapies – car t cells in other autoimmune diseases, novel cellular therapies are increasingly being explored for their potential applications. one of the most promising candidates already in clinical use in oncology are chimeric antigen receptor (car) t cells76. these are t cells that have been genetically modified to express a construct enabling high-affinity antigen recognition and downstream signaling, thereby conferring proliferative capacity, effector function, and persistence in the recipient for a durable therapeutic effect77. importantly, the car t receptor is not restricted by mhc for antigen recognition. in most cases, this is done autologously – t cells are obtained from the patient, engineered ex vivo with car constructs, expanded, and then reinfused. the concept currently under evaluation in neuroimmunology, including ms, involves car t cells targeting b cells (via cd19 or bcma) to achieve thorough b cell depletion even in tissues, as these cells have demonstrated good deep tissue penetration, including the cns78,79. indeed, several case series have recently been published illustrating profound effects of this therapeutic approach in several patients with refractory myasthenia gravis and stiff person syndrome, however results from clinical trials are not yet available78,80,81. our current understanding of ms progression centers on a compartmentalized inflammatory process that limits the efficacy of primarily peripherally acting dmts and car t cells may represent an emerging technology to tackle this treatment challenge. published 2024 data from two progressive ms patients treated with a single dose of fully humanized second-generation cd19 car t cells showed persistence of car t cells in both the peripheral blood and csf82. notably, car t cell treatment resulted in peripheral b cell depletion and a sustained reduction of csf oligoclonal bands in one patient, providing indirect evidence of effective cns plasma cell depletion. although this small case series with a limited observation time precludes definitive efficacy evaluation, the treatment was well tolerated, with only mild cytokine release syndrome and no icans. several clinical trials in relapsing and progressive ms are underway to further explore this promising approach and assess potential side effects, including secondary malignancies, which would strongly limit use of this approach in the context of autoimmunity. preclinical studies the search for neuroprotective and remyelination promoting therapies continues, but is hampered by the limited understanding of the molecular and cellular mechanisms driving neurodegeneration and remyelination failure in ms. oligodendrocytes and neurons are not passive targets of the immune response, as extensive previous research suggest that oligodendrocytes as well as neurons can launch an inflammatory response83–87. the group led by manuel friese discovered, in detailed and elaborate in vitro and in vivo studies, the stimulator of interferon genes (sting) as an important regulator in neurons, which is essential for the homeostasis of the neuronal red-ox system and inflammation-induced ferroptosis. sting is upregulated in neurons in eae and in people with ms and most interesting both, pharmacological and genetic ablation of sting protected against inflammation-induced neurodegeneration in eae mice. sting1-cko eae mice displayed ameliorated eae disease course and increased numbers of surviving neurons, but no differences in the extent of inflammatory infiltrates suggesting that pharmacological targeting of sting may represent a direct neuroprotective treatment approach88. another focus in preclinical ms research is to unravel the underlying molecular mechanism for remyelination failure. current concepts suggest that not only a differentiation block of opc into mature myelinating oligodendrocytes but also an impaired myelin sheath formation by mature oligodendrocytes may contribute to remyelination failure in ms89. a potential explanation for the latter might be that mature oligodendrocytes are epigenetically silenced, as suggested by the work of liu and colleagues90. they screened an epigenetic compound library and identified the small molecule esi1 that promoted the maturation and/or in vitro myelination of primary mouse and human ipsc derived oligodendrocytes, promoted remyelination in different animal models and improved clinical signs in the eae model. in summary, this study identifies epigenetic silencing of mature oligodendrocytes as a new pathomechanism contributing to remyelination failure in ms and provides evidence that targeting the epigenetic machinery might be a promising new pharmacological target to overcome remyelination failure in ms. summary conceptually, the identification of the first snp associated with disease severity represents a major breakthrough, since it suggests a genetic component in the pathophysiology of disease progression independent from the immune system. the accumulating evidence of ebv as important trigger of ms disease onset raises the fascinating possibility of an anti-viral treatment which may either stop or prevent ms. the new diagnostic framework of ms establishes ms as a disease continuum and allows diagnosis based on purely biologic considerations in the absence of any clinical manifestation and new imaging and biomarkers as well as ai approaches may facilitate the prediction of the disease course and treatment responses in individual pwms in the not too far future. a conceptual game changer from a therapeutic perspective are the results from the tolebrutinib study program, as they demonstrate for the first time that tackling clinical outcomes of disease progression is feasible beyond targeting focal inflammation. the ultimate challenge for the future will be the translation of our continuously increasing understanding of ms pathophysiology into new treatment approaches to successfully stop or prevent ms. acknowledgements this work was supported by the dfg, crc tr128 project a08 to lk and b07 to t.k., trr332 project b2 to l.k, ku1477/13-1 to t.k., by the national ms society (rfa180-2202-39141 to lk and tk), the interdisciplinary center for clinical research (kut3/010/24 to t.k. and l.k.), by the inflames flagship program of the research council of finland (decision numbers: 337530, 357910 and 358823), research council of finland clinical investigator grant program (decision number: 330902), us national ms society (rfa-2203-39281) and jane and aatos erkko foundation to l.a. dr. marjo nylund is warmly acknowledged for preparation of the pet and mri images. conflict of interest and funding statement l.k. receives research support from the german research foundation (dfg), the interdisciplinary center for clinical research (izkf) münster, national ms society, biogen, novartis and merck serono. she received compensation for serving on scientific advisory boards and speaker honoraria from alexion, amgen, argenx, bayer, biogen, bristol-myers squibb, grifols, hexal, horizon, janssen, merck serono, novartis, roche, sandoz, sanofi, santhera, teva and viatris. l.a. receives grants from the research council of finland, aatos erkko foundation, us national ms society, merckserono and sanofi. she received speaker and advising honoraria from sanofi, biogen, novartis, kiniksa, continuum therapeutics and merck. m.s. declares no conflicts of interest. t.k. receives research funding from the german research foundation, interdisciplinary center for clinical research (izkf) münster, national ms society, german ms society and novartis. she received compensation for serving on scientific advisory boards from novartis, sanofi and merck and speaker honoraria from novartis, biogen, sanofi and roche. references 1. international multiple sclerosis genetics, c. multiple sclerosis genomic map implicates peripheral immune cells and microglia 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author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. assessment of intraepidermal nerve fiber densities in 5 µm sections from arm and leg – a search for normative age-related values feel free to add comments by clicking these icons on the sidebar free neuropathology 5:24 (2024) original paper assessment of intraepidermal nerve fiber densities in 5 µm sections from arm and leg – a search for normative age-related values linnéa ekman1, lars b. dahlin1, 2, 3, elisabet englund4 department of translational medicine, hand surgery, lund university, malmö, sweden department of hand surgery, skåne university hospital, malmö, sweden department of biomedical and clinical sciences, linköping university, linköping, sweden division of pathology, department of clinical sciences, lund university, lund, sweden corresponding author: linnéa ekman · department of translational medicine · hand surgery · lund university · jan waldenströms gata 5 · 205 02 malmö · sweden linnea.ekman@med.lu.se submitted: 19 august 2024 accepted: 23 september 2024 copyedited by: aivi nguyen published: 11 october 2024 https://doi.org/10.17879/freeneuropathology-2024-5815 keywords: intraepidermal nerve fiber density, skin biopsy, peripheral neuropathy, small nerve fiber neuropathy abstract background and aims: normative values are lacking regarding intraepidermal nerve fiber density (ienfd) in thin sections of 5 µm. thus, we aimed to assess ienfd in thin sections in a healthy adult population as well as to investigate whether ienfd is related to age, sex, and site of excision. methods: archival skin biopsies or excisions at the department of pathology, lund, sweden, from arm and leg were collected, re-sectioned, and immunohistochemically stained for protein gene product 9.5 during 2020–2023. nerve fibers were manually quantified in the 5 µm thin sections, and ienfd was compared between age groups, sex, and excision sites. results: ienfds were evaluated in 602 samples from 591 healthy adults aged 18 to 97 years (295 women, 296 men). median ienfd values are presented, stratified by age groups, sex, and excision sites. higher ienfd was observed in the arm compared to the leg, as well as in the proximal compared to the distal leg, however not across all age groups. levels of ienfd were lower among older adults, compared to all younger groups. conclusion: we have presented data on ienfd in thin 5 µm sections from a healthy adult population. despite differences in ienfd observed across age groups, sexes, and excision sites, no strong conclusions regarding affecting factors could be drawn except that individuals > 65 years present with lower ienfd. additional research and development of the method are warranted. introduction the use of skin biopsy with subsequent assessment of the intraepidermal nerve fiber density (ienfd) has been developed over the last three decades.1 the assessment can reveal loss of small a and unmyelinated c fibers in the epidermal skin layer. an early study used ienfd to demonstrate small fiber neuropathy (sfn), where no underlying cause was known and nerve conduction was normal.2 another study reported a high sensitivity (88 %) in diagnosing sfn through ienfd.3 beyond the area of sfn, studies have highlighted the utility of evaluating ienfd at the distal leg in peripheral neuropathies associated with conditions such as diabetes mellitus,4,5 systemic lupus erythematosus,6 alcohol dependency,7 and hereditary transthyretin amyloidosis polyneuropathy.8 guidelines on the use of skin biopsy and ienfd for diagnosing peripheral neuropathy were published in 2005, as requested by the european federation of neurological societies.9 the recommended methodology entails obtaining a 3 mm punch skin biopsy at the distal leg, immunostaining with the protein gene product (pgp) 9.5 antibody, and quantification of ienf in at least three 50 µm thick sections through either bright-field or immunofluorescence microscopy. pgp 9.5 is a ubiquitin hydrolase that is highly expressed in neurons and serves as an effective marker for the finest nerves, such as skin innervation, outperforming other markers such as neuron-specific enolase and neurofilament10,11 two large multi-center studies have established reference values for ienfd in 50 µm sections obtained from the distal leg based on compiled data from healthy individuals, one for each microscopy method.12,13 both studies found similar ageand sex-related effects on ienfd values, where ienfd declined with age and women have slightly higher densities than men. however, to our knowledge, handling 50 µm sections to perform immunohistochemical staining is not a routine procedure in most clinical pathology laboratories, thereby not applicable in clinical practice, and hence mostly used for research purposes or in commercial laboratories. modifications of the method have been proposed where e.g. koskinen et al measured ienf per epidermal area instead of length in 10 µm sections.14 thereto, dabby et al. studied the dermal, and not epidermal, autonomic nerve fibers in 5 µm sections.15 a third modification of the method was developed within the department of pathology in lund, sweden, where ienfd is assessed in the epidermal layer of 5 µm sections.16,17 the method is possible to employ in an ordinary diagnostic laboratory situation since it requires only standard laboratory equipment and techniques.16,18,19 through our previous studies, we have learned that ienfds in the distal leg are lower in people with type 2 diabetes compared to healthy controls, but also that a temporal decline can be found in both populations.20 at the wrist level, however, no impairment in ienfds could be found within either type 1 or type 2 diabetes in comparison to controls, despite signs of neuropathy in biopsies from a nearby nerve, i.e. the posterior interosseous nerve.16 other findings of that study included significantly higher densities in women compared to men as well as in hairy skin compared to glabrous skin. we have also shown that the nerve fiber assessment in 5 µm sections has a high interand intra-rater reliability.18,20 however, reference values are lacking for our modified method, which is a crucial step towards enabling clinical evaluation and distinction between health and illness. the overall aim of this study was to assess ienfd in thin 5 µm skin sections in a healthy population with the attempt to establish normative data for potential future diagnostic use. additionally, we aimed to explore any variations in ienfd across different ages, sexes, and excision sites, to enhance the understanding of skin innervation throughout the body. methods and materials ethics statement the study was approved by the swedish ethical review authority as ethical permission no. 2020-03597. the study was conducted in accordance with the declaration of helsinki. study population and skin tissue sampling samples were manually selected and collected from archival tissue material in the clinical department of pathology in lund-malmö, sweden, between october 2020 and july 2023. all the archival tissue had been previously examined as the primary sampling was based on clinical diagnostic issues, such as nodules or pigmented lesions requiring histopathologic examination, and stored according to the swedish biobank act. the pathology database system was searched for tissue material comprising excisions or diagnostic biopsies obtained in the arm or leg of adult individuals aged 18 years or above in considerable health. each specimen’s distal ends, around a central abnormality in the ovoid-shaped excision, were checked for morphologically normal appearance and could be included if accepted. causes for exclusion were inflammation, signs of itching or scratching, hyperplastic epithelium, or scarred tissue. skin samples from both arm and leg were further divided into two categories: the proximal or the distal part of the limb. the proximal arm was defined as the region extending from the level of the glenohumeral joint to just proximal to the elbow, while the distal arm included the elbow and reached down to the wrist. correspondingly, the proximal leg referred to the thigh (distal to the inguinal ligament), whereas the distal leg included the knee and continued down to the ankle (fig. 1). samples were thus included if the site of excision (or biopsy) was anatomically defined, and the presence of healthy tissue could be confirmed in either the whole or a part of the specimen. correlating paraffin-embedded tissue blocks were retrieved from the archive to be re-sectioned and immunohistochemically stained. figure 1. anatomical classification of skin tissue samples included in the study ventral (panel a) and dorsal view (panel b) of the four anatomical areas that comprised the excision sites: proximal arm (blue), distal arm (green), proximal leg (purple), and distal leg (orange). the web-based medical record system melior, used in the health care system in region skåne, sweden, was searched for information on prevalent diseases and potential causes, or symptoms, of neuropathy. exclusion criteria were diabetes mellitus, lyme disease, parkinson’s disease, uremia, multiple sclerosis, amyloidosis, scleroderma, dysregulated thyroid function, alcoholism, b12 deficiency, cytostatic treatment, diagnosed neuropathy, or nerve damage affecting the specific limb. other causes for exclusion from the study were insufficient amount of tissue as well as suboptimal histochemical quality of the sections. immunohistochemistry all skin biopsies and excisions were uniformly short-time fixed in a 4 % buffered formaldehyde solution following the standard procedures of the clinical diagnostic laboratory. after being in fixation for at least 24 hours, the samples were dehydrated and embedded in paraffin. upon inclusion in the study, the paraffin-embedded tissue blocks were re-sectioned at 5 µm and mounted on glass for immunohistochemical staining with, generally, two serial sections on each glass. the sections were dried at 60° c for one hour, de-waxed, rehydrated, and microwave-pre-treated in 10 mm citrate buffer (ph 6.0) for 19 minutes at 750 w. the automated immunostainer (techmate 500 plus; dako) was implemented for immunohistochemical staining and the rabbit polyclonal protein gene product (pgp) 9.5 antibody (cell marque, rocklin, usa) was used as the primary antibody, in a 1 : 3000 dilution. all samples were sequentially sectioned and stained batch-wise, using the same procedure, in the laboratories of lund and malmö, sweden. assessment of ienfd assessment of ienfd was performed manually in sectra ids7, where the scanned tissue sections could be viewed and examined microscopically. the epidermal layer was digitally measured and intraepidermal nerve fibers (ienf) were identified. the number of individual pgp 9.5-positive fibers was counted according to our previously published criteria, however with a new criterion for the minimal length of fibers to be counted, i.e. the fiber was counted if the length measured ≥ 15 µm (fig. 2).16, 20 fibers that branched within the epidermal layer were counted as a single fiber. the highest number obtained from any of the serial sections was recorded. to obtain ienfd, the number of nerve fibers was related to the length of the epidermal layer of each sample and expressed as fibers/mm. figure 2. assessment of ienfd in a 5 µm section intraepidermal nerve fiber density assessment illustrated in a 5 µm section stained with the pgp 9.5-antibody (overview to the left, close-up of the box-indicated portion to the right). each counted nerve fiber (≥ 15 µm, see scale bar in the bottom right corner) is indicated by an arrow. epidermal length was measured with an in-program device, see the green line (left). statistical analyses the data on ienfd were not normally distributed and thus presented as a median with 25 and 75 percentiles. data was stratified by age, with the first group comprising individuals aged 18–29 years, followed by groups in 10-year intervals, and the final group including participants aged 80 years and older. the ienfds were compared between age groups, excision sites, and sex using the kruskal-wallis test and dunn’s post-hoc testing with bonferroni correction. an additional group comparison was made between young (18–44 years), middle-aged (45–65 years), and older adults (> 65 years). quantile regression analyses were applied to investigate the effect of age and sex on ienfd across deciles (0.1–0.9) on each of the excision sites. the model was adjusted with a quadratic term for age. p-values < 0.05 were considered statistically significant. statistical analyses were performed using spss statistics version 27. results a total of 602 samples were collected from 591 unique subjects, all considered healthy, ranging in age from 18 to 97 years (median 55 years; 295 women and 296 men). values of ienfd ranged between 0 and 8.6 fibers/mm in the entire cohort. a complete absence of nerve fibers was noted in 34 samples. median ienfd values at the four excision sites (proximal arm, distal arm, proximal leg, and distal leg as defined in methods) are presented for men and women as well as for each age group in table 1. table 1. intraepidermal nerve fiber densities (ienfd) in 5 µm sections from upper and lower limb in healthy adults proximal arm distal arm proximal leg distal leg   age (y) n ienfd n ienfd n ienfd n ienfd men 18-29 11 2.75 (1.59-3.60) 11 1.62 (1.16-3.69) 11 1.39 (0.82-2.53) 11 0.73 (0.56-2.55)   30-39 10 2.12 (1.02-3.73) 13 1.58 (1.40-2.61) 10 1.56 (0.96-2.61) 12 0.85 (0.31-1.11) 40-49 10 2.40 (1.24-3.27) 10 2.18 (1.06-3.04) 10 1.53 (0.87-3.01) 11 0.45 (0.20-1.06) 50-59 10 1.85 (0.96-2.35) 10 0.89 (0.53-2.33) 10 1.40 (1.04-3.08) 10 1.10 (0.60-1.55) 60-69 10 0.90 (0.55-2.20) 10 0.89 (0.50-1.26) 10 0.82 (0.58-2.26) 10 0.55 (0.23-0.86) 70-79 10 0.93 (0.48-1.76) 10 0.83 (0.53-1.49) 10 0.57 (0.22-1.30) 11 0.15 (0.00-0.82) ≥ 80 14 1.03 (0.32-1.77) 11 0.39 (0.27-0.65) 12 0.48 (0.20-0.75) 14 0.00 (0.22-0.54) women 18-29 11 2.31 (1.81-5.37) 10 2.21 (1.34-3.08) 10 1.60 (1.27-2.09) 11 0.98 (0.63-1.32)   30-39 10 2.36 (1.81-5.21) 11 2.12 (1.13-2.56) 10 2.46 (2.01-3.76) 11 2.28 (0.61-2.97) 40-49 10 2.19 (1.53-5.27) 10 2.38 (1.79-4.19) 10 2.32 (1.82-3.60) 10 1.72 (1.15-3.08) 50-59 10 1.34 (0.82-2.70) 10 2.39 (1.52-2.94) 12 2.10 (1.57-2.81) 11 0.82 (0.42-1.11) 60-69 11 1.98 (0.89-3.09) 10 1.69 (0.63-2.17) 10 0.80 (0.21-1.98) 10 0.78 (0.18-1.15) 70-79 10 1.17 (0.37-2.14) 10 1.49 (0.73-2.24) 10 1.06 (0.63-1.61) 10 0.40 (0.08-1.50) ≥ 80 13 0.59 (0.33-1.70) 13 0.70 (0.43-1.32) 11 0.90 (0.28-2.07) 15 0.13 (0.00-0.48) data are presented as median (25th–75th percentiles) or numbers. ienfd: intraepidermal nerve fiber density (fibers/mm). when comparing the four excision sites, overall differences were found among the three lower age groups for men (p < 0.019), and within the groups aged 18–29, 50–59, and > 80 years for women (p < 0.013). post-hoc testing showed generally higher ienfd in the arm compared to the leg (p < 0.032). differences between proximal and distal portions of the leg were only found for women aged 50–59 years (p = 0.024). between the proximal and distal arm, however, no significant differences were observed. regarding age, statistically significant differences in ienfd were observed when testing across all age groups at all four excision sites for both men and women (p < 0.031; fig. 3). post-hoc testing revealed that these differences were primarily between the eldest group (> 80 years) and the four younger groups (18–29, 30–39, 40–49, and 50–59 years), though the pattern was rather inconsistent. when the cohort was divided into three broader age categories – young (18–44 years), middle-aged (45–65 years), and older adults (> 65 years) – the differences became more pronounced (p < 0.005). post-hoc analyses indicated that ienfds were lower at all excision sites in both men and women aged > 65 compared to the young group (p < 0.015). the ienfds were also lower among the older adults in comparison to the middle-aged adults at the proximal (p < 0.029) and distal leg (p < 0.018), as well as at the distal arm (p < 0.045). notably, no differences in ienfd were found between the young and middle-aged groups (fig. 4). figure 3. levels of ienfd at four anatomical skin excision sites stratified in age deciles intraepidermal nerve fiber densities (ienfd) in the proximal arm (a) and leg (b), as well as the distal arm (c) and leg (d), in healthy subjects of different ages, stratified in deciles. each dot demonstrates an individual data point. bars indicate the median values for each group. figure 4. levels of ienfd at four anatomical skin excision sites in young, middle-aged, and older adults intraepidermal nerve fiber densities (ienfd) in the proximal arm (a) and leg (b), as well as the distal arm (c) and leg (d), in healthy subjects of different ages, stratified in three groups of young (18–44 years), middle-aged (45–65 years), and older adults (> 65 years). the median values for each group are indicated by the lines. age and sex differences were further investigated using quantile regression analysis, which indicated an age impact for the first and second quantiles at the proximal arm (decrease by 0.04 to 0.05 fibers/mm per year, p < 0.021), as well as in the ninth quantile at the proximal leg (increase of 0.25 fibers/mm per year, p = 0.011). impact of sex was found within quantiles 3 to 6 for the distal arm and quantiles 4 to 6 at the proximal leg, with higher predicted ienfd in women compared to men (0.47–0.68 fibers/mm, p < 0.014). discussion the primary objective of this present study was to establish normative values for ienfd assessment in thin 5 µm sections, with the additional goal of investigating potential affecting factors in terms of age, sex, and anatomical site of excision. given our previous findings regarding a temporal decline in ienfd for both people with and without type 2 diabetes mellitus,20 we hypothesized an age-dependency for ienfd where densities decrease with advancing age. this hypothesis was further supported by the previous ienfd reference materials, although assessed in thicker sections of 50 µm.12,13 while we did not identify a clear pattern when comparing ienfd across age deciles, we found significantly lower ienfd levels in individuals aged 65 years and older, compared to the groups of young and middle-aged adults. this could suggest that an age-related nerve decline may not be linear but rather occurs later in life. a non-linear pattern of age impact on nerve conduction velocities has been demonstrated in rat models where levels remained stable until the last third of life, after which an impairment was observed 21. a comparable trend has also been suggested in humans where nerve conduction velocities and amplitudes appear to worsen with increasing age.22 however, the quantile regression analysis performed in our study did not identify any definitive non-linear relationships between ienfd and age. moreover, the quantile regression implied sex differences corresponding to the ones found in prior studies,12,13,16,23 but again, not in a conclusive pattern. interestingly, all studies that found sex differences have consistently observed higher densities among women, implicating e.g. hormonal status as a potential explanatory factor. on the other hand, one study reported no sex-related effects, while another revealed that the initially noted higher ienfd in women lost statistical significance when adjusting for confounding variables, including height and weight.24,25 in this study, ienfd was also compared between the proximal and distal excision sites, as well as between arm and leg, to explore potential variations in nerve fiber distribution across the body. this approach extends previous research on thicker sections, which primarily have been focused on the distal leg.12,13,23 our findings revealed higher ienfds in the arm compared to the leg. higher ienfds were also found at the proximal portion of the leg, as compared to the distal part, though this was only statistically significant for women aged 50–59 years. this observation aligns with the concept of a proximal-distal gradient, as described by mcarthur et al., who reported a 60 % higher density at the thigh compared to the distal leg.24 umapathi et al. referred to this as the ienfd ratio and used it as a measure of the length-dependent dying-back phenomenon observed in axonal neuropathies.25,26 although skin innervation in the arms has been investigated priorly, we are not aware of any studies that compare e.g. the density between the proximal and distal arm. a study of pain and touch acuity across different body sites showed better two-point discrimination in the distal arm compared to the shoulder region, but an opposite pattern was observed regarding pain sensitivity to heat.27 this suggests that the mechanoreceptor density is higher in the distal parts, whereas the thermal nociceptors and thus epidermal c-fibers are more abundant in the proximal parts. hence, a similar pattern as in the legs, i.e. a proximal-distal pattern, could be expected, but we did not find a significant difference between the sites in our study. a substantial proportion of the samples included in our study exhibited markedly low densities, some even as low as zero fibers. the data generally demonstrated greater variability in ienfd than anticipated, leading to inconclusive results. a contributing factor may be the specimen’s anatomical locations, which were roughly categorized into either proximal or distal portions of the arm and leg. it is reasonable to assume variability even within each of these categories. for instance, a mildly traumatized lateral side of the calf probably differs from the protected skin just medial to the tibia, and comparing an area near the wrist with one closer to the elbow may potentially also reveal variations. previous research has also highlighted distinctions between hairy and glabrous skin at the wrist level.16 the hand and foot were deliberately excluded from the study due to highly area-specific densities (e.g. finger pulp compared to palm of hand) and thus the anticipation of even greater variabilities. the innervation of the hand and foot is unevenly distributed in an intricate pattern across the palm and sole, as demonstrated by corniani and saal.28 although a more nuanced categorization for our study would have been ideal, sparse referral texts necessitated adherence to broader areas. it may also be postulated that the challenge lies in the selection of sufficiently healthy material, as the samples were sourced from specimens submitted for skin pathological examination. despite efforts to ensure peripheral tissue health and a meticulous review of medical records to exclude nerve-affecting disorders, the possibility of inadvertently including individuals with undetected underlying illnesses cannot be entirely ruled out. the proximity and potential impact of pathology should also be considered, as the skin sections used in the present study were all harvested from tissue blocks comprising excisions of skin lesions. the impact of skin lesions on fiber density might be different depending on the type. for example, in a study by bröcker et al., the number of nerve fibers was shown to be higher within melanocytic nevi compared to the surrounding tissue, whereas no differences could be observed within cutaneous melanoma metastases.29 additionally, nerve fiber counts were found to be influenced by tumor properties, such as increasing tumor thickness in cutaneous melanomas leading to decreasing nerve counts.in our study, we focused on obtaining samples from the macroscopically intact outer ends of excisions, where the radicality of the excision could be ensured. in cases where such end snippets were not available, ienfd assessment was only performed if the skin section was sufficiently long to provide a substantial distance from the skin lesion, and the surrounding skin appeared normal and healthy. these precautions were taken to minimize the potential influence of the lesion on nerve fiber density and ensure that the assessment was conducted on skin resembling normal skin. after analysis of a substantial number of sections in this study, it became evident that nerve fibers were being excluded under the preexisting criteria of our method. consequently, we explored and implemented a modification: counting fibers ≥ 15 µm in length to capture a higher number of solid fibers, while still excluding debris. this modification, differing from our previous studies, proved to be more inclusive. despite our efforts to adapt the method for thinner sections, we still obtained very low counts in some sections, posing potential challenges in clinical applications where distinguishing between healthy and pathological samples is crucial. traditional approaches, presenting the 5 percentile as a cut-off threshold for normative values, are deemed impractical for our methodology as this percentile reaches an ienfd of zero fibers/mm for some locations and age spans.12,23,24 in this study, absolute cut-offs could not be established. a recently published article by aspegren and pourhamidi has demonstrated promising results using a new method, called estimated ienfd (eienfd) assessment, in thin sections, produced comparable numbers to the 50-micrometer method.30 this approach utilizes the same thin sectioning and immunohistochemical procedure as in our study. however, the eienfd method involves counting even smaller fragments than in our proposed method, which may present additional challenges. despite these challenges, as well as those identified in our study, it is essential to develop a method that is applicable within standard laboratory settings for diagnostic suitability, particularly in sweden. therefore, further development of ienfd assessment in thin sections is necessary. during the work, we have also acknowledged some qualitative differences within the samples, emphasizing a shift towards exploring this aspect more comprehensively in forthcoming studies. we are motivated to delve further into this area, as the assessment of ienfd offers an objective investigation of nerve fibers directly related to the region where symptoms manifest. limitations as previously mentioned, the variable anatomical excision sites are this study’s most important limitation. standardized sampling on well-described anatomical sites might have yielded a clearer result. incomplete access to participants’ full medical records poses another limitation. the system melior encompasses data from all public specialist health care in region skåne, yet information about patients residing elsewhere or receiving treatment solely from general practitioners was not available. conclusion this study provides ienfd values obtained in thin 5 µm sections from both the proximal and distal parts of the arm and leg in a healthy adult population, in a search for potentially normative ienfd values. differences in ienfd levels were observed across excision sites and age groups, showing notably lower levels among the older groups. however, the data exhibited a greater variability than expected, preventing the identification of clear influencing factors or cut-off values. further research and continued method development are warranted. acknowledgments we extend our gratitude to our colleagues at the pathology laboratories in lund and malmö for their invaluable assistance in sectioning and staining of all the specimens, and to the dermatopathologists for being patient and helpful. funding statement the work was supported by grants from the swedish research council (2021-01942), skåne university hospital (2022-974), alf (2022-projekt0129), the swedish diabetes foundation (dia2020-492), elly olsson’s foundation for scientific research, and the hans-gabriel and alice trolle-wachtmeister foundation for medical research. conflicts of interest statement the authors declare no conflict of interest. references 1. lauria g, faber cg, cornblath dr. skin biopsy and small fibre neuropathies: facts and thoughts 30 years later. j neurol neurosurg psychiatry. 2022;93(9):915-8. https://doi.org/10.1136/jnnp-2021-327742 2. holland nr, crawford to, hauer p, cornblath dr, 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https://doi.org/10.1002/ana.24179 28. corniani g, saal hp. tactile innervation densities across the whole body. j neurophysiol. 2020;124(4):1229-40. https://doi.org/10.1152/jn.00313.2020 29. bröcker eb, magiera h, herlyn m. nerve growth and expression of receptors for nerve growth factor in tumors of melanocyte origin. j invest dermatol. 1991;96(5):662-5. https://doi.org/10.1111/1523-1747.ep12470585 30. aspegren o, pourhamidi k. reliable method for estimating nerve fiber density in epidermis using routine histopathologic tissue preparation: a promising diagnostic tool for small fiber neuropathy. appl immunohistochem mol morphol. 2024;32(5):215-21. https://doi.org/10.1097/pai.0000000000001193 copyright: © 2024 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. morphological substrate of chronic schizophrenia in elderly patients: a clinicopathological study feel free to add comments by clicking these icons on the sidebar free neuropathology 6:9 (2025) original paper morphological substrate of chronic schizophrenia in elderly patients: a clinicopathological study kurt a. jellinger institute of clinical neurobiology, vienna, austria corresponding author: kurt a. jellinger · institute of clinical neurobiology · alberichgasse 5/13 · 1150 vienna · austria kurt.jellinger@univie.ac.at submitted: 21 february 2025 accepted: 06 march 2025 copyedited by: cinthya aguero published: 13 march 2025 https://doi.org/10.17879/freeneuropathology-2025-6315 keywords: schizophrenia; cognitive impairment; neuropathology; alzheimer disease; concomitant pathologies abstract objective: patients with schizophrenia are at a higher risk of developing dementia, but the basis of cognitive impairment is a matter of discussion. conflicting results regarding the association of schizophrenia with alzheimer disease (ad) may partly be attributable to the inclusion of non-ad lesions, which few clinicopathological studies have considered. therefore, a re-evaluation of an autopsy cohort of elderly schizophrenics published previously [1] was performed. material & methods: among 99 consecutive autopsy cases of patients who met the dsm-5 and icd.10 criteria for schizophrenia (mean age 69.5 ± 8.25 years), 56 showed moderate to severe dementia. all brains were blindly examined using the current criteria for ad and looking for concomitant lesions. they were compared with the frequency of ad in an autopsy series of 1.750 aged demented individuals results: four cases revealed the features of definite ad, five probable ad, and three aged 82–89 years were classified as primary age-related tauopathy (part). two cases were a cortical type of dementia with lewy bodies (dlb), one lewy body disease of brainstem type; six showed hippocampal sclerosis, 14 argyrophilic grain disease (agd), and one progressive supranuclear palsy (psp). other co-pathologies were frequent lacunes in basal ganglia, moderate cerebral amyloid angiopathy, minor development anomalies in the entorhinal cortex, fahr's disease, metastatic tumors, and acute or old cerebral infarctions (n = 4 each). definite ad was seen in 48 % of the age-matched demented control group. conclusions: in this cohort of elderly schizophrenic patients, only 7.6 % fulfilled the neuropathological criteria of definite or probable ad and 3.6 % of part compared to 6 % to 13.7 % typical and atypical ad in the literature, whereas a considerable number of cases showed non-ad co-pathologies. this is in line with other studies showing that the frequency of ad in elderly schizophrenics may be equal to or less than in age-matched controls. further studies are needed to elucidate the mechanisms of cognitive decline in schizophrenia. abbreviations ad alzheimer disease, adnc alzheimer disease neuropathological changes, agd argyrophilic grain disease, caa cerebral amyloid angiopathy, ci cognitive impairment, dlb dementia with lewy bodies, nft neurofibrillary tangle, part primary age-related tauopathy, scz schizophrenia introduction current literature suggests that the pathology of schizophrenia (scz) has mainly developmental origins based on genetic factors, perinatal abnormalities, and other factors [2], but this cannot explain the evidence of accelerated cognitive decline and increased risk of dementia in elderly patients with scz [3–19]. the prevalence of dementia among us adults with scz at 66 years of age was 23.2–27.9 % compared with 1.3 % in the group without serious mental illness and at age 80 increased to 70.2 % in the group of schizophrenia and 11.3 % in the control group (6.2 times higher) [19]. among new zealanders with scz aged 65 years or older, 23 % had a dementia diagnosis [16], while in a population-based study in taiwan, scz patients exhibited a 1.8-fold risk of dementia compared to controls, with a higher risk of developing alzheimer disease (ad) [15]. up to now, no clear relationship between cognitive decline/dementia and scz has been found, and the cause of this impairment is still unclear [6,15,18–23]. mri studies revealed brain structural abnormalities with reduced gray matter volumes in the right orbitofrontal cortex and cingulate gyrus [4], prefrontal cortex, hippocampus, and subcortical structures [22], hippocampus [24,25], hippocampus and amygdala [26,27], and in the mediodorsal thalamus and pulvinar, which was confirmed by postmortem assessment [28]. scz patients showed a higher brain-predicted age difference compared to ageand sex-matched controls [11]. recent studies identified disconnectivity in corticocortical and corticostriatal circuits, each with specific clinical symptoms [29,30]. several reports have provided conflicting results regarding the association between scz and alzheimer disease (ad). however, both disorders partially overlap in terms of psychiatric symptoms and some aspects of cognitive impairment (ci) and interference with each other through amyloid evolvability [23]. in both, the left hippocampus and amygdala volume are significantly smaller. still, the hippocampus/amygdala ratio was significantly lower in scz compared to ad and age-matched controls, which correlated with the age at disease onset [26]. on the other hand, behavioral and social dysfunction in both disorders has been linked to hyperactivity within the fronto-parieto-limbic brain systems [31]. earlier studies suggesting that the frequency of ad pathology (alzheimer disease neuropathologic changes adnc) is increased in elderly schizophrenics [32–34] are questionable. in the largest postmortem series of 544 people with schizophrenia (mean age 77 years), with an overall frequency of ad of 28 %, it increased from 6 % among those under age 65 to 45 % in those over age 85 years [35]. although no control group was included and the diagnostic criteria of ad were not clearly defined, its frequency was more common than in age-related populations [36]. among 100 autopsy cases of scz (mean age 76.5 years), 72 % showed cognitive impairment, but only 9 % were diagnosed with ad and 4 % with other dementing disorders [8]. several postmortem studies showed that the frequency of ad in scz is not different from that in age-matched controls [37,38] or in the general population [1,7,39–41] or showed even less incidence than expected [39,42]. no differences in β-amyloid (aβ) deposition between scz and comparison groups indicated that the mechanisms of dementia development in scz may not share the neuropathology of ad [43] and that the aβ levels in scz patients are not different from elderly controls [3]. neurofibrillary tangle (nft) densities in the hippocampus, entorhinal, and inferior temporal cortex in elderly patients with late-onset scz were not significantly different from controls [44]. a recent meta-analysis reported lower severity for both plaques and nfts in the scz group versus ad and that no higher rates of adnc were found in cognitively impaired scz patients compared with age-matched controls [10]. on the other hand, adnc and argyrophilic grain pathology have been observed in elderly scz patients with cognitive decline [45,46]. a recent study distinguished two types of scz patients with incident dementia: those with coexisting neurodegenerative diseases (ad, argyrophilic grain disease /agd/, etc.) and those without such co-pathologies [47]. given the current controversy about the morphological basis of ci in elderly schizophrenics, the postmortem findings in a cohort of elderly patients with chronic scz (published by [1]) were re-examined and compared with a large autopsy series of demented elderly subjects. material and methods the study included 99 patients (mean age at death 69.5 ± 8.25, range 60–89 years; 68 older than 65 years) who met the dsm-5 and icd-10 criteria of schizophrenia and had a complete neuropathological examination. they died between 1980 and 1999. the clinical data were retrospectively derived from hospital records evaluated by an experienced psychiatrist. for each case, information was obtained on the age at onset of psychiatric disease, age at first hospitalization, family, medical, and neurological history, alcohol and drug abuse, drug and, in particular, antipsychotic medication, clinical signs and symptoms including extrapyramidal side effects of neuroleptic treatment, other motor, sensory and cognitive impairment, and the clinical status immediately before death. based on these data, each case was psychiatrically classified [48]. most had residual scz, some paranoid or undifferentiated forms. the mean duration of illness was 35.15 ± 10.17, with a range of 14–60 years. exclusion criteria were severe neurological disease (e.g., huntington or parkinson disease), acute stroke, and brain tumors. a neuropathological examination was performed according to standard protocols and a macroscopic examination of the brain. histological examination of multiple paraffin blocks was performed using routine stains (h&e, cresyl violet, klüver-barrera or heidenhain stain, bodian silver impregnation, holzer stain for fibrillary gliosis), immunohistochemistry for glial fibrillary protein (gfap), β-amyloid (clone 4g8), tau (antibody at-8) and α-synuclein. tdp-43 pathology was not examined since no antibodies were available at that time. braak nft stages, thal aβ phases, and the cerad criteria [49] were evaluated, and the diagnosis of ad was made according to the nia-aa criteria [50]. the diagnosis of other neurodegenerative disorders (dementia with lewy bodies /dlb, agd, progressive supranuclear palsy, etc.) was performed according to current criteria. furthermore, large and small cerebral infarcts [51], cerebral amyloid angiopathy (caa), and other co-pathologies (e.g., hippocampal sclerosis, fahr's disease, cerebral metastases, or minimal developmental anomalies) were described. results of the 99 patients who met dsm-5 and icd-10 criteria for scz, 56 showed moderate to severe dementia (mean mmse 15.4 ± 8,3; range 6–22), often more involving sensory than motor behavior and/or various domains of cognitive functions, in particular executive and attentional dysfunctions, short term memory and behavioral disorders. patients with ci were significantly older (mean age 73.36 ± 6.85 years) than non-demented ones (mean 64.02 ± 6.54 years) (p < 0.001) and had a longer duration of illness (mean 41.1 vs 28.9 years) (p < 0.001). the brain weight of the demented was significantly lower than that of the non-demented (1119.5 ± 106.1 g vs 1216.3 ± 136.2 g) (p < 0.001). only two patients (aged 60 and 67 years, respectively) met the neuropathological criteria of definite ad or typical adnc (braak stage v–vi, abc 3/3/3), i.e., 2.1 % of the total, and 1.4 % (1/68) of those over age 65 years). probable ad (braak stages iv–v, cerad b, abc 2/2/2) was diagnosed in five (aged 71–89, mean 79 years) or intermediate adnc, i.e., 7.6 % of the total and 7.3 % of those older than 65. two females aged 63 and 65 years fulfilled the diagnosis of diffuse dlb, and another one showed nigral degeneration with subcortical lewy bodies without cortical involvement, thus meeting the criteria of lewy body (lb) disease of the brainstem type. three females, aged between 82 and 89 years, met the criteria of primary age-related tauopathy (part) (braak stage iv, nia, and cerad negative with only minimal aβ deposits and caa) [52], all three had apoe ε2/3 genotype. in summary, definite/ probable ad accounted for 7.6 % of the total cohort and 10.3 % of those over age 65. further, 3 % were part, compared to 48 % ad in an autopsy cohort of 1,750 age-matched demented patients [53]. the other brains of scz patients with mild to moderate ci showed braak states ii–iv (mean 2.47), thal aβ stages 1–2, occasional lewy bodies and agd (14 each), and many with mild to moderate caa, and more than a half, lacunes in basal ganglia and cerebral white matter. other neuropathological changes were sequelae of frontal lobotomies (n = 8), minor developmental anomalies (less neuronal clustering in layer ii of the entorhinal cortex, n = 7), hippocampal sclerosis (n = 6), fahr's disease, cerebral metastases (four each), recent and old cerebral infarcts (four), and one with the diagnosis of progressive supranuclear palsy. the essential clinical and neuropathological data are given in table 1. table 1: demographic and essential neuropathological data demented non-demented total number of patients 56 43 99 age at death (years) 73.3 ± 6.85 64.25 ± 6.45 69.5 ± 8.25 age at onset (years) 31.7 ± 4.3 32.4 ± 5.6 32.0 ± 5.0 duration of illness (years, mean) 41.1 28.9 35 ± 0 mmse score 15.4 ± 8.3 27.3 ± 9.8 21.5 ± 8.9 brain weight (g) 1119.5 ± 106.15 1216.3 ± 136.25 typical adnc (braak v–vi 2 (3.6 %) 0 2 (2.02 %) intermediate adnc (braak iii–iv) 5 (8.9 %) 0 5 (5.05 %) part (braak iv) 3 (5.4 %) 0 3 (3.03 %) dlb 2 (3.6 %) 0 2 (2.02 %) lbd (brainstem) 0 1 (2.3 %) 1 (1.01 %) lewy bodies 9 (16.1 %) 5 (11.6 %) 14 (14.14 %) agd 7 (12.5 %) 7 (16.3 %) 14 (14.14 %) psp 0 1 (2.3 %) 1 (1.01 %) fahr's disease 2 (3.6 %) 2 (4.7 %) 4 (4.04 %) hippocampal sclerosis 4 (7.1 %) 2 (4.7 %) 6 (6.06 %) microcerebrovascular lesions (lacunes) 35 (62.5 %) 16 (37.2 %) 51 (51.52 %) old infarcts 2 (3.6 %) 2 (4.7 %) 4 (4.04 %) developmental anomalies 4 (7.1 %) 3 (7.0 %) 7 (7.07 %) caa severity range 1–3 (2.5 ± 0.3) 0–2 (0.5 ± 0.1) 0–3 (1.5) thal aβ plaques 2–4 (2.3 ± 0.5) 0–2 (1.0 ± 0.5) 0–4 (1.6) mmse: mini-mental state examination; adnc: alzheimer disease neuropathologic changes; part: primary age-related tauopathy; lbd: lewy body disease; dlb: dementia with lewy bodies; agd: argyrophilic grain disease; psp: progressive supranuclear palsy; caa: cerebral amyloid angiopathy discussion in the present sample of 99 subjects older than 60 years with chronic scz, 56 % showed moderate to severe ci/dementia, only seven met the neuropathological criteria of probable or definite ad, and three that of part, a rare form of ad-like changes but braak stages up to iv and minimal aβ pathology [52]. thus, only 10.9 % of the present sample were classified as having the likelihood of adnc or ad-related pathology, whereas the majority of the cases presented non-ad changes. these data are in accordance with other studies, like 9 % in a slightly older us cohort [38] 6.3 % or 13.7 % when including mild or atypical ad lesions [42]. neuritic braak stages iii–iv were seen in 36.7 % of early-onset, 58.8 % of late-onset scz, and 11.1 % of controls. the restricted limbic tauopathy in both earlyand late-onset schizophrenic patients was associated with sparse aβ deposits [54], which would suggest the diagnosis of part in this scz cohort. while of the subjects with scz, 68 % had definite ci, only 8 % fulfilled the criteria for ad [12]; according to others, all patients with early or late onset scz showed neuritic braak stages of iii or less, which may correspond to normal aging [44]. in a recent study, 12.5 % had an intermediate and none had a high likelihood of adnc [47]. all these values are at the lower end of the range of ad prevalence in the general population reported in various epidemiological studies [55,56] or among cognitively intact age-related subjects, where the incidence of definite ad was 11.6 % [57]. among 100 non-demented subjects aged over 65 years, 18 % fulfilled the criteria of probable but none that of definite ad [58]. in an older study, elderly patients with late-onset scz showed nft densities in the hippocampal ca1 field, entorhinal, and inferior temporal cortex corresponding to braak stage iii or less that were not significantly different from controls [44]. these and other results support the notion that there is no increased frequency of adnc in elderly patients with chronic scz, which were derived from retrospective [8,36,42,59,60] and prospective studies [61]. they are at variance with previous studies showing an increased incidence of ad in elderly schizophrenics [32,35], which are difficult to interpret due to methodological shortcomings. although neuritic plaques and hippocampal nfts were related to dementia severity in schizophrenic patients, none of them were pathologically diagnosed as ad due to the lack of significant aβ deposition [62], suggesting that these findings correspond to part [52]. biomarker studies in patients with scz also showed heterogeneous results. while one study reported significantly lower levels of serum total tau and phosphorylated tau than in healthy controls [63], an 83-year-old female with late-onset scz had slightly elevated cerebrospinal fluid total tau and slightly decreased aβ-42 indicating ad [64]. a recent study showed both positive and negative cerebrospinal fluid ad biomarkers in a patient with very late-onset scz, but no significant differences in mmse scores and psychotic symptoms between the groups [65]. the claim that antipsychotic drug treatment resulted in increased frequency of neurofibrillary adnc, while schizophrenic patients who did not receive neuroleptics, showed similar changes in age-matched controls [66], was not confirmed by others [36,37,67]. in the present cohort, all patients had a long history of neuroleptic drug use, and a large proportion showed extrapyramidal side effects. the low frequency of ad in the present and other studies [42] does not support the assumption that long-term antipsychotic/neuroleptic medication promotes the development of neuritic adnc, which is in line with japanese studies [37,59]. a possible explanation of ci in chronic scz without considerable adnc could be the sequelae of frontal lobotomy, which was performed in 8 patients of the present cohort. sparse neuropathological studies of lobotomized schizophrenics revealed loss of neurons in the dorsomedial thalamus as a probable consequence of disconnection of the prefrontal cortex to central regions of the brain since the dorsomedial thalamus represents a major efferent projection to the prefrontal cortex [68]. a 69-year-old patient with scz who died 32 years after prefrontal lobotomy showed severe demyelination of the frontal lobe and marked retrograde degeneration and gliosis in the thalamic nuclei [69]. these data suggest an explanation of cognitive impairment in lobotomized patients with scz, not only by neuronal loss in specific brain areas but also by disconnectivity of cortico-cortical and cortico-thalamic circuits. unfortunately, in the present cohort, no such studies were performed in order to find an alternative explanation for cognitive changes in chronic scz in the absence of neurodegenerative proteinopathies. there is little data regarding non-adnc in patients with scz. in our cohort, three patients at autopsy presented lb disorders and six agd, which is much less than in other studies. in an earlier study, 7.3 % of patients with scz showed αsyn-positive structures, which were consistent with dlb and incidental lb disease, which did not differ from controls [70]. another study reported an incidence rate of lb pathology and agd of 26.1 % and 21.7 %, respectively [45], while among 196 older patients with scz, 12.7 % presented dlb disease at autopsy (21 dlb and 4 parkinson disease) [62]. other neurodegenerative changes, such as tdp-43 pathology, were reported in 29 % of patients with scz [71] or even in 56.3 % [47]. limitations of the present study are due to the fact that this is a re-evaluation of the neuropathology of archival material that does not allow any correlation between the neuropathological diagnoses and clinical correlates, as well as the specific implications of neuroleptic therapy on neuropathological changes. moreover, tdp-43 pathology could not be assessed since respective antibodies were unavailable at the time of the study. however, the goal was to emphasize the relatively minor importance of ad and part compared to other co-pathologies in patients with chronic scz and ci. furthermore, no studies of the sequelae of prefrontal lobotomy were performed that could have demonstrated secondary degenerative changes in relevant brain areas. conclusion and further aspects older patients with chronic and ci/dementia show a heterogeneous neuropathological substrate: a comparatively small group with ad and related pathologies (part and late) and those with other coexisting pathologies. in older schizophrenic patients with ad-related pathologies, there are no or only little differences between those with and without incident dementia or with age-related controls. unfortunately, there are currently only very few clinico-pathological studies considering non-ad pathologies in chronic scz with and without cognitive and behavioral impairment and their impact on the clinical aspects and course of the disease, as well as the impact of antipsychotic treatment. in particular, the effects of antipsychotics and other therapies on brain circuitry should be examined using serial brain sections. to understand the neurobiological and pathogenic aspects of incident ci/dementia in older patients with early and late-onset scz, further clinico-pathological studies in well-documented cohorts are needed. acknowledgments the author thanks mr. e. mitter-ferstl for his secretarial and editorial work. funding statement the study was funded by the society for the promotion of research in experimental neurology, vienna, austria. ethics approval this study adheres to all relevant international, national and/or institutional guidelines for ethical standards. conflict of interest statement the author 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(https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. 69th annual meeting of the german society of neuropathology and neuroanatomy (dgnn) meeting abstracts free neuropathology 6:17 (2025) meeting abstracts 69th annual meeting of the german society of neuropathology and neuroanatomy (dgnn) meeting abstracts september 25–27, 2025, frankfurt am main, germany german society of neuropathology and neuroanatomy (dgnn) submitted: 15 september 2025 accepted: 15 september 2025 published: 18 september 2025 https://doi.org/10.17879/freeneuropathology-2025-8974 keywords: german society of neuropathology and neuroanatomy, dgnn, meeting abstracts sehr geehrte damen und herren, liebe kolleginnen und kollegen, im namen der deutschen gesellschaft für neuropathologie und neuroanatomie heiße ich sie herzlich willkommen zur 69. jahrestagung unserer fachgesellschaft, die in diesem jahr zudem das 75-jährige jubiläum der dgnn feiert. dieses besondere jubiläum gibt uns anlass, auf die wurzeln und die beeindruckende entwicklung unserer fachgesellschaft zurückzublicken. die frankfurter schule mit persönlichkeiten wie carl weigert und ludwig edinger legte wesentliche grundlagen für die neurowissenschaften, deren bedeutung bis heute kaum zu überschätzen ist. ein entscheidender schritt in der geschichte unseres faches war der zusammenschluss der neuropathologen in der „arbeitsgemeinschaft morphologisch arbeitender neurologen und psychiater“. unterstützt durch den pathologen prof. lauche und die frankfurter edinger-stiftung fanden im mai 1950 vorgespräche statt, die schließlich am 7. oktober 1950 zur gründungsversammlung der „vereinigung deutscher neuropathologen“ führten. diese gründung legte den grundstein für die heutige dgnn, die seit 75 jahren als wissenschaftliche plattform für austausch und fortschritt im bereich der neuropathologischen forschung dient. besonders hervorheben möchte ich den hohen stellenwert der nachwuchsförderung in unserer gesellschaft. seit jeher ist es ein zentrales anliegen der dgnn, junge wissenschaftlerinnen und wissenschaftler zu unterstützen, zu fördern und ihnen eine plattform für ihre wissenschaftliche entwicklung zu bieten. nur durch die gezielte förderung des wissenschaftlichen nachwuchses können wir die zukunft unseres fachgebiets sichern und kontinuierlich neue impulse für die neuropathologie setzen. der kongress 2025 in frankfurt am main bietet uns erneut die gelegenheit, in vorträgen und diskussionen die neuesten wissenschaftlichen erkenntnisse zu erörtern sowie den kollegialen austausch zu pflegen. besonders freue ich mich auf das breite spektrum an themen und den angeregten dialog, der unsere fachgesellschaft lebendig hält. mein dank gilt allen mitgliedern und unterstützern, die die dgnn mit ihrem engagement und ihrer leidenschaft prägen und voranbringen. ich wünsche uns allen einen erfolgreichen und inspirierenden kongress sowie ein bedeutungsvolles jubiläumsjahr. mit herzlichen grüßen karl heinz plate frankfurt am main, im august 2025 contents i. artificial intelligence p01 accurate, inexpensive & fast – computational pathology for ependymoma subtyping p02 three dimensional reconstruction of neurons and connectivity patterns in the human spinal cord ii. digital pathology p03 reliable and instant slice-free intraoperative histology in neurooncology: updated study results with multiphoton microscopy p04 longitudinal cell state screening through rapid deconvolution of intra-tumor heterogeneity in glioblastoma p05 foundation models encode technical biases: a hidden risk for multi-institutional histopathology ai iii. epilepsy p06 investigating neurological deficits in meis2-related syndrome using mescs p07 comparison of neuroinflammatory cellular composition of tumors and peritumoral tissue in leat patients with and without post-surgical persistence of epilepsy iv. muscle & nerve p08 diagnosis of immune-mediated myopathies on formalin-fixed, paraffin-embedded muscle tissue samples v. neurodegeneration p09 the interaction between kallikrein-8 and its inhibitory network affects alzheimer’s disease p10 associations between age, sex and apoe with amyloid-beta, tau and α-synuclein loads in alzheimer’s disease p11 white matter granular astrocytic tau inclusions are a highly consistent feature in multiple system atrophy p12 impact of the gut microbiome on microglia and alzheimer’s disease in different mouse models p13 first report of clinico-pathological features associated with tardbp p.m311v mutation vi. neuroinflammation p14 borna disease virus 1 infection in organotypic hippocampal slide cultures from adult rats p15 immune profile and clinical characteristics of central nervous system graft-versus-host-disease p16 hpgv-1 is infecting glial cells in immunosuppressed patients p17 t cell infiltration patterns and clinical correlates in natalizumab-related pml lesions p18 multiplexed viral detection in brain tissue – methodological challenges and opportunities p19 neuropathological characteristics of anti-gaba-b-receptor-encephalitis p20 impact of myelin phagocytosis on human blood-derived macrophages and microglia and its effect on human oligodendrocytes p21 remyelination in multiple sclerosis depends on the mechanism of lesion development p22 il-12 drives neuroinflammation-linked lipid dysregulation in a mouse model of alzheimer’s disease p23 development and validation of a multiplex microarray for pathogen detection in paraffin-embedded cns tissue vii. single-cell technologies p24 investigation of intratumour heterogeneity in primary and recurrent glioblastoma via single cell whole exome sequencing p25 exploring interand intratumoral heterogeneity of choroid plexus tumors on a single cell level viii. tumor p26 analysis of histological, molecular and preanalytic features of unclassifiable cns specimens: comparison of the heidelberg v12.8e, bethesda v3 methylation-based brain tumor classifiers and the hetairos h&e histology-based classifier p27 immunothrombosis and endothelial priming in early brain metastasis p28 molecular and histological analyses of at/rt-tyr suggest the choroid plexus of the fourth ventricle as cellular origin p29 pediatric and aya meningiomas exhibit distinct molecular and clinical features not reflected in adult-based classification systems p30 case of a myxopapillary ependymoma with multiple relapses and pulmonary metastasis p31 unravelling ependymoma heterogeneity using integrated proteomic analyses p32 identification of a novel type of pineal region tumors with distinct global dna methylation and sh3tc2 fusions p33 ttf-1 expression is associated with a hypomethylation signature in schwannomas p34 expansion of the spectrum of tumors diagnosed as myxopapillary ependymomas p35 addressing mgmt activity in glial cell lines p36 loss of global dna methylation is prognostic in oligodendrogliomas p37 leveraging peptide-level proteomics to detect brain cancer specific proteoforms p38 glioblastoma-derived secreted proteins drive invasion and affect neuronal integrity p39 establishment of droplet digital™ pcr (ddpcr™)-based assays for the diagnostic detection of mgmt promoter methylation in malignant gliomas p40 exploring glioblastoma through cpg site-specific methylation and transcription factor occupancy p41 integrated analyses reveal four distinct molecular subgroups in corticotroph pituitary neuroendocrine tumors/adenomas p42 harnessing stress adaptive response mechanisms for pharmacological targeting of cancer p43 mechanisms of transformation from subependymoma to ependymoma p44 leveraging off-target reads for genome-wide copy number profiling in ngs panels p45 establishing ultra low-input spatial proteomics for glioblastoma analysis p46 comparison of diffuse glioma types with gliomatosis cerebri growth pattern in adult patients p47 immunohistochemical expression and differential methylation of hoxb13 reliably distinguishes myxopapillary ependymoma from spinal ependymoma p48 spatial mapping of immunogenic niches in melanoma brain metastases p49 targeting phosphorylation events in glioblastoma: a semi-spatial (phospho)proteomic workflow for diffuse glioma ffpe samples p50 meningioma sanity check: increasing classifier credibility through prototypical feature statistics congress ids abstract id p name abstract id p name 18 p28 lea altendorf, hamburg 72 p40 kimia kafi cheraghi, frankfurt 27 p29 natalie berghaus, heidelberg 73 p41 matthias dottermusch, hamburg 37 p04 paul kerbs, heidelberg 74 p42 laura hruby, düsseldorf 38 p15 ida vogt, frankfurt 75 p19 valérie quinot, wien 39 p30 catena kresbach, hamburg 82 p43 hannah jessner, münster 43 p16 helena radbruch, berlin 84 p44 christian thomas, münster 44 p17 imke metz, göttingen 85 p25 anna klötergens, münster 45 p24 natalie schoebe, heidelberg 86 p45 rhaissa ribeiro da silva, heidelberg 46 p31 antonia gocke, hamburg 87 p46 marco münzberg, frankfurt 47 p32 helena bode, hamburg 88 p47 suvendu purkait, düsseldorf 52 p07 dorothea münch, frankfurt 89 p20 laura schmitz-gielsdorf, münster 53 p10 antonia neubauer, münchen 90 p21 lidia stork, göttingen 56 p11 viktoria ruf, münchen 91 p05 jannik sehring, gießen 57 p33 max braune, leipzig 93 p22 frank heppner, berlin 58 p34 fuat aras, heidelberg 95 p48 josefine radke, greifswald 60 p12 marina jendrach, berlin 96 p23 imke metz, göttingen 62 p01 antonia gocke, hamburg 97 p02 anika simonovska serra, wien 63 p35 helene blatt, homburg 98 p49 gianluca sigismondo, heidelberg 64 p36 felix hinz, heidelberg 99 p50 kai schmid, gießen 65 p37 ian fichtner, heidelberg 100 p26 charlotte brandenburg, frankfurt 66 p08 selin kanarya, düsseldorf 101 p06 kiana kafi cheraghi, frankfurt 67 p38 tanja buhlmann, frankfurt 102 p09 paula pereira de almeida, essen 68 p39 marietta wolter, düsseldorf 105 p14 maximilien lépine, gießen 69 p18 raphael raspe, berlin 107 p27 willy hube, münchen 70 p13 alberto grassini, tübingen 108 p03 jan philip kolb, hamburg i. artificial intelligence p01 free neuropathol 6:17:6 accurate, inexpensive & fast – computational pathology for ependymoma subtyping yannis schumann1, maximilian middelkamp2, antonia gocke3,4, jette alfken5, sarah becker5, tasja lempertz3, jennifer ahrens3, karoline hack6, markus glatzel7, claire ertelt-delbridge8, ulrich schüller6,7,9, tim salditt5, julia neumann3,7 1 deutsches elektronen-synchrotron desy, it department, data management and analysis, hamburg, germany 2department of neurosurgery, university medical center hamburg-eppendorf, hamburg, germany 3 center for molecular neurobiology (zmnh), university medical center hamburg-eppendorf, hamburg, germany 4 section of mass spectrometric proteomics, university medical center hamburg-eppendorf, hamburg, germany 5 institute for x-ray physics, university of göttingen, göttingen, germany 6 children’s cancer research center hamburg, hamburg, germany 7 institute of neuropathology, university medical center hamburg-eppendorf, hamburg, germany 8 institute of pathology, school of medicine and health, technical university of munich, munich, germany 9 department of pediatric hematology and oncology, university medical center hamburg-eppendorf, hamburg, germany background: ependymal tumours (epns) are clinically heterogeneous neoplasms and can occur in all three compartments of the central nervous system. the 2021 who classification defines 10 clinically distinct types of epns based on molecular, anatomical and immunohistochemical criteria. objective(s): precise diagnoses, such as from dna methylation analyses, are crucial for optimal therapy and patient outcome. yet, these analyses are time consuming and expensive. we discuss multiple machine-learning methods for computational pathology of ependymomas and highlight how such approaches may act as an inexpensive and fast surrogate for molecular analysis. method(s): whole-slide images of tissues with hematoxylin and eosin stain were acquired and matched with dna methylation profiles. interpretable machine-learning methods and multi-scale approaches were used to predict dna-methylation types from image data. stacking was used to incorporate basic clinical information (e.g. sex, age, location). result(s): we acquired a cohort of sample-matched h&e wsis and dna-methylation analyses of epn (n = 597). while both clam (clustering-constrained attention multiple instance learning) and hipt (hierarchical image pyramid transformers) showed near perfect classification results when incorporating clinical variables, our results indicate that the hipt method performed consistently better in predicting the molecular epn types from the image data. conclusion(s): this study aims to leverage the prospects of computational pathology for cns tumour diagnostics, driven by the increasing digitization of pathology workflows. selecting the clinically relevant epns as use case, we demonstrate accurate predictions of molecular epn types based on inexpensive, fast to acquire image data. p02 free neuropathol 6:17:8 three dimensional reconstruction of neurons and connectivity patterns in the human spinal cord anika simonovska serra1, philipp jauk2, sonja klement2, andreas hainfellner3, romana höftberger1, karen minassian2 1 medical university of vienna, division of neuropathology and neurochemistry, department of neurology, vienna, austria 2 medical university of vienna, center for medical physics and biomedical engineering, vienna, austria 3 medical university of vienna, center for anatomy and cell biology, vienna, austria background: traditional histopathological staining methods are performed on specimens with a thickness of 2–5 μm, providing a two-dimensional cross-sectional view of neurons that is largely limited to the soma and proximal dendrites. a staining method that enables a three-dimensional visualization of whole neurons is the golgi-cox impregnation technique, which can be combined with immunofluorescent antibodies targeting specific proteins. recent advancements in microscopy imaging and computational postprocessing techniques have further facilitated the acquisition of high-resolution images of neurons. objective(s): to provide a three-dimensional visualization of single neurons in the spinal cord and their connectivity patterns, as well as the dendritic spine morphology and density that are crucial for synaptic plasticity. question(s): to investigate differences in synaptic density in the spinal cord in the context of neurodegenerative diseases. method(s): in this study, human post-mortem spinal cord tissue will be used for the golgi-cox impregnation of spinal neurons in combination with tissue clearing and immunofluorescence staining of catecholaminergic receptors and synaptic proteins. images will be acquired using confocal microscopy. result(s): single-impregnated neurons including their soma and dendritic architecture will be segmented and subsequently reconstructed in three dimensions using aiand deep learning-based models. the resulting images will be compiled into a comprehensive cytoand dendroarchitectonic atlas of the lumbar spinal cord. conclusion(s): this staining technique can be applied to various regions of cns and offers numerous applications in basic research, providing a deeper understanding of healthy neuronal function and potentially offering new insights into neurological disorders associated with neurodegeneration and synaptopathy. ii. digital pathology p03 free neuropathol 6:17:9 reliable and instant slice-free intraoperative histology in neurooncology: updated study results with multiphoton microscopy jessica kren1, jan philip kolb2, matthias strauch2, niloofar khosravi2, álvaro vega pérez2, matteo m. bonsanto1, julia e. neumann3,4 1 uksh lübeck, klinik für neurochirurgie, lübeck, germany 2 histolution gmbh, lübeck, germany 3 uke hamburg, institut für neuropathologie, hamburg, germany 4 uke hamburg, zentrum für molekulare neurobiologie hamburg (zmnh), hamburg, germany multiphoton microscopy (mpm) enables rapid, high-quality histological assessment of tissue samples without the need for paraffin or frozen sectioning. this technology can be integrated into the operating room workflow, providing results within few minutes. the aim of this study was to evaluate the diagnostic accuracy and practical applicability of mpm for intraoperative assessment of neurosurgical tumor samples. the central question was whether mpm can reliably reproduce routine histological diagnoses and thus support fast intraoperative decision-making in neurosurgery. a total of 76 neurosurgical tumor samples were examined using mpm and compared to standard paraffin section histology. after a brief 2.5-minute staining, tissue blocks were scanned at 4 min/cm², with potential acceleration to 30 s/cm². the resulting digital images were assessed in a blinded manner by a board-certified neuropathologist with no prior mpm experience. for 43 of the 76 samples, the neuropathologist also received the suspected clinical diagnosis in addition to age, sex, and localization. in 72 of 76 cases (94.7 %), mpm results were fully consistent with routine histology. notably, in all 43 cases where the suspected diagnosis was provided, mpm findings matched the final diagnosis. the digital images showed excellent visualization of cell morphology and tissue architecture, closely resembling standard h&e slides. these findings highlight the reliability of mpm, even when used by neuropathologists without specific training. mpm offers a significant advance for intraoperative diagnostics, with the potential to improve patient outcomes by enabling faster and more accurate decision-making. ongoing research will further optimize technical parameters and validate broad applicability in neuro-oncology. p04 free neuropathol 6:17:10 longitudinal cell state screening through rapid deconvolution of intra-tumor heterogeneity in glioblastoma jakob ito1,2, dana silverbush3, wolfgang wick1,2,4, andreas von deimling5,6, tobias kessler1,2,4,5, felix sahm5,6, paul kerbs5,6 1 clinical cooperation unit neurooncology, german cancer research center (dkfz), german cancer consortium (dktk), heidelberg, germany 2 department of neurology, university hospital heidelberg, heidelberg, germany 3 department of cancer biology, perelman school of medicine, university of pennsylvania, philadelphia, united states 4 national center for tumor diseases (nct), a partnership between dkfz and heidelberg university hospital, heidelberg, germany 5 german consortium for translational cancer research (dktk), german cancer research center (dkfz), heidelberg, germany 6 department of neuropathology, university hospital heidelberg, heidelberg, germany background: glioblastoma (gb) is characterized by intra-tumor heterogeneity (ith), a major factor contributing to tumor progression and therapy resistance. the limited efficacy of precision therapies in gb targeting oncogenic drivers highlights the need to address ith and its plasticity in treatment strategies. integration of multi-omics single-cell data enabled the resolution of ith in gb and the development of a deconvolution method which can accurately predict cell type and malignant cell state compositions from bulk methylation array data. previous analyses revealed distinct vulnerabilities in deconvolved cell states, identifying them as potentially targetable biomarkers. we believe cell state monitoring holds significant potential for improving therapy guidance but requires rapid, scalable, and cost-effective screening methods. objective(s): establishing a functional precision medicine platform modelling cell state responses in patient-derived cell lines. enabling cell state deconvolution from sparse methylation data like liquid biopsies revolutionizing the ability to longitudinally monitor gb patients throughout their treatment. method(s): methylation-based cell state deconvolution from oxford nanopore technologies (ont) sequencing data. result(s): we adapted cell state deconvolution to ont sequencing data and subsequently evaluated cell state predictions in a cohort of 59 ont sequencing samples from fresh-frozen tissue with corresponding methylation array data. comparative analysis revealed 100 % concordance in the identification of the predominant cell state. we established the first standardized pipeline–csgo (cellular state resolution for glioblastoma oncology)–to rapidly resolve the cellular states of gb patients. conclusion(s): our pipeline lays the foundation for the integration of ith in clinical decision making and drug response monitoring. p05 free neuropathol 6:17:12 foundation models encode technical biases: a hidden risk for multi-institutional histopathology ai jannik sehring1, hildergard dohmen1, kai schmid1, gudrun schmidt1, friederike liesche-starnecker2, jörg felsberg3, guido reifenberger3, moritz armbrust4, katharina j. weber4, karl heinz plate4, christian mawrin5, markus j. riemenschneider6, david capper7, frank heppner7, stefan hamelmann8, felix sahm8, daniel amsel1, till acker1 1 justus-liebig-university giessen, institute of neuropathology, giessen, germany 2 university of augsburg, medical faculty, department of neuropathology, augsburg, germany 3 university hospital düsseldorf, institute of neuropathology, düsseldorf, germany 4 university hospital frankfurt, neurological institute (edinger institute), frankfurt am main, germany 5 otto-von-guericke university magdeburg, institute of neuropathology, magdeburg, germany 6 regensburg university hospital, department of neuropathology, regensburg, germany 7 charité berlin, department of neuropathology, berlin, germany 8 heidelberg university hospital, department of neuropathology, heidelberg, germany background: foundation models are widely used in histopathological ai due to their capacity to encode complex morphological features. however, these models may inadvertently capture uninformative variation, including technical disparities in histological staining and digitization, potentially compromising model generalizability. this is particularly relevant for multi-institutional studies and downstream clinical applications, which require both robust cross-site performance and reliable transferability to individual laboratories. objective(s): determine whether pathological foundation models encode technical biases. method(s): a large multi-institutional dataset of h&e-stained meningioma sections (n = 180) was compiled, with each case stained at three different institutes and digitized using three different scanners. after image registration, the feature embeddings of 100 patches per slide were encoded using seven different foundation models. to quantify the encoding of technical variables, we trained a multi-layer perceptron to classify either the scanner or the staining institute from the embeddings, keeping the other variables constant. result(s): classification accuracies were evaluated across a five-fold cross-validation for each foundation model. scanner prediction yielded very high accuracy (0.98–0.99), while institute prediction ranged from 0.73 to 0.91, depending on the foundation model used. conclusion(s): foundation models encode substantial technical variation, even in controlled multi-center datasets. this raises concerns regarding interpretability and robustness, particularly when confounders correlate with clinical labels. these biases must be considered in computational pathology workflows to avoid misleading associations and to ensure that models can be both generalized across sites and reliably deployed in individual diagnostic settings. iii. epilepsy p06 free neuropathol 6:17:14 investigating neurological deficits in meis2-related syndrome using mescs tamara müller1, kiana kafi cheraghi1, shripriya bhat1,2, ian donaldson3, stefan günther4, miguel torres5, nicoletta bobola6, dorothea schulte1 1 universitätsmedizin frankfurt, goethe universität; neurologisches institut, edinger institut, frankfurt am main, germany 2 university of bern, faculty of medicine, department for biomedical research (dbmr), bern, switzerland 3 university of manchester, faculty of biology, medicine and health, bioinformatics core facility, manchester, united kingdom 4 max planck institute for heart and lung research, bioinformatics and deep sequencing platform, bad nauheim, germany 5 centro nacional de investigaciones cardiovasculares (cnic), madrid, spain 6 university of manchester, faculty of biology, medicine and health, manchester, united kingdom background: meis2-related syndrome, resulting from heterozygous mutation of the transcription factor meis2, is a severe congenital disorder characterized by a triad of symptoms of cleft palate, heart malformations, and neurological deficits. the neurological side of this disorder is complex but consistently includes developmental delay with intellectual disability, severe speech and varying motor delay, autistic behavioral abnormalities, disturbed sleep patterns, and epileptic seizures. objective(s): explore the molecular basis of neurological deficits in meis2-related syndrome use an in vitro mesc neurogenic differentiation model to mimic early neurodevelopmental aspects question(s): which molecular mechanisms drive the neurological symptoms of meis2-related syndrome? how well does mesc neurogenic differentiation recapitulate early neural development in this syndrome? method(s): mescs with or without crispr-generated meis2 mutation subjected to neurogenic-gabaergic differentiation chromatin immunoprecipitation and chip-seq for meis2 at early differentiation stages transcriptome analysis and qpcr of differentiating mescs rna-sequencing of later neuronally differentiated mescs luciferase reporter assays on meis2-bound genomic regions result(s): at early differentiation stages: identification of a key mammalian brain development regulator as a direct meis2 target meis2-bound regions function as transcriptional enhancers in later neuronally differentiated mescs: mutant neurons exhibit aberrant expression of specific synaptic markers dysregulation of genes involved in neurotransmitter metabolism and catabolism conclusion(s): collectively, these results provide a conceptional framework for understanding the neurological deficits seen in meis2-related syndrome patients and show an unexpected link between two neurodevelopmental disorders previously considered to be unrelated. p07 free neuropathol 6:17:16 comparison of neuroinflammatory cellular composition of tumors and peritumoral tissue in leat patients with and without post-surgical persistence of epilepsy dorothea münch1, ricardo kienitz2, andrea spyrantis3, jürgen konczalla3, ida vogt4, pia s. zeiner4,5,6,7,8, karl heinz plate1,6,7,8, felix rosenow2, catrin mann2, katharina j. weber1,6,7,8 1 goethe university frankfurt, university hospital, neurological institute (edinger institute), frankfurt am main, germany 2 goethe university frankfurt, epilepsy center frankfurt rhine-main, department for neurology, university medical center frankfurt, frankfurt am main, germany 3 goethe university frankfurt, department of neurosurgery, center of neurology and neurosurgery, frankfurt am main, germany 4 goethe university frankfurt, university hospital, dr. senckenberg institute of neurooncology, germany, frankfurt am main, germany 5 goethe university frankfurt, university hospital, department of neurology, frankfurt am main, germany 6 goethe university frankfurt, frankfurt cancer institute (fci), frankfurt am main, germany 7 german cancer consortium (dktk), partner site frankfurt, and german cancer research center (dkfz), heidelberg, germany 8 goethe university frankfurt, university hospital, university cancer center (uct), frankfurt am main, germany background: low-grade epilepsy-associated tumors (leat) are primary brain tumours, usually classified as cns who grade 1 lesions. leats are clinically associated with drug-resistant epilepsy. early epilepsy surgery is recommended in pharmacorefractory patients. given that surgical outcomes improve significantly when both the tumor is excised early in the disease course, and adjacent epileptogenic tissue is removed, it is assumed the perilesional tissue alterations play a vital role in the establishment of a pro-convulsive environment. aim: this study aims to investigate the cellular composition of leat and peritumoral tissue with a focus on the peritumoral zone and its potential epileptogenic role. methods: 27 leat patients were included. in each patient the cellular composition of the vital tumor and adjacent tissue were analysed. adaptive and myeloid immune cells, astrocytes and neurons were analysed by immunohistochemistry and assessed semi-quantitatively using an image analysis software. epigenetic alterations potentially contributing to epileptogenesis were examined in five representative tumors. the results were correlated with clinical parameters. results: our preliminary results from the immunohistochemical staining show that the highest levels of lymphocytes, macrophages and microglia were found in the tumors. the peritumoral zone showed increased levels in comparison with distant tissue. additionally, we found correlations between increased levels of immune cell infiltration and a longer duration of epilepsy. conclusion: the peritumoral zone constitutes a distinct microenvironment, displaying characteristic neuroinflammatory alterations that differentiate it from both the tumor and the more distant surrounding tissue. these alterations may result from seizures but could also contribute to the progression of epileptogenesis. iv. muscle & nerve p08 free neuropathol 6:17:18 diagnosis of immune-mediated myopathies on formalin-fixed, paraffin-embedded muscle tissue samples anna brunn1, selin kanarya1, wolfgang merkt2 1 institute of neuropathology, university hospital düsseldorf and medical faculty, heinrich heine university, düsseldorf, germany 2 department of rheumatology, university hospital düsseldorf, heinrich heine university, düsseldorf, germany background: immune-mediated myopathies (imm) are the most common indication for a muscle biopsy. according to the guidelines for processing muscle biopsies, cryopreserved tissue is required for immunohistochemistry of the canonical hla-abc molecule of the major histocompatibility-complex (mhc) class i, the well-established biomarker of imm. objective: muscle biopsies with suspected imm are occasionally submitted as formalin-fixed and paraffin-embedded (ffpe) specimen, in which mhc-i testing is less accurate. question: we tested the hypothesis that an antibody targeting the non-canonical mhc-i molecule hla-e may overcome this limitation and can be used on cryoand ffpe tissue. method: we examined hla-e expression on fifty frozen muscle biopsies, clinically and histologically diagnosed as imm, and ten frozen samples with morphological hallmarks of neurogenic muscular atrophy without signs of inflammation. subsequently, we tested the same hla-e antibody on twenty ffpe muscle biopsies with confirmed inflammation in which mhc-i expression could not be determined up to now, as well as in ten ffpe samples without inflammation as controls. result: immunoreaction of hla-e antibody successfully detected sarcolemmal and/or sarcoplasmatic upregulation associated with inflammation. this was true for both frozen and ffpe samples in specimens in which the diagnosis of imm has been established based on frozen samples. in contrast, in samples with neurogenic muscular atrophy both canonical mhc-i and hla-e expression were absent. conclusion: immunostaining for hla-e enables the diagnosis of imm using ffpe muscle samples when frozen tissue is unavailable, offering a valuable diagnostic alternative in cases in which tissue handling did not adhere to the guidelines. v. neurodegeneration p09 free neuropathol 6:17:19 the interaction between kallikrein-8 and its inhibitory network affects alzheimer’s disease paula pereira de almeida1, nicole macha1, arne herring1, michael wissoly2, ulf brockmeier3, olga shevchuk4, stephanie schaefer-tautges4, hannah voss4, daniel engel4, syed ahsan raza1, kathy keyvani1 1 university hospital essen, institute of neuropathology, essen, germany 2 university hospital essen, institute of pathology, essen, germany 3 university hospital essen, institute of neurology, essen, germany 4 university hospital essen, institute of experimental immunology and imaging, essen, germany background: increased kallikrein-8 (klk8) level seems to contribute to alzheimer’s disease (ad) pathology, but it remains unknown whether a disruption of its inhibitory network is involved. serpin a3, serpin b6, alpha-2-macroglobulin (α2m), and the acetylcholine (ach)producing protein pebp1 are the putative inhibitors of klk8. objective: to examine klk8↔inhibitor interactions and their functional consequence in ad pathology. methods: we combined in silico modeling and enzymatic assays to assess binding and inhibition. complex formation was validated by co-immunoprecipitation, western blot and mass spectrometry, and was quantified in the ad pathology affected human and murine hippocampus. cellular models (sh-sy5y, primary glia) tested klk8 inhibition effects on aβ phagocytosis, neurite outgrowth, and ach production. results: a protein-protein complexation between klk8 and its putative inhibitors was predicted by alphafold artificial intelligence (ai). all four inhibitors bound and inhibited klk8 in vitro, and klk8 inhibition restored neurite outgrowth and boosted aβ clearance in cellular models. klk8 complexes with serpin a3, serpin b6 and α2m were significantly reduced in human (but not murine) hippocampus when affected by ad pathology. the reduced ach levels upon klk8 exposure in sh-sy5y cells were countered when klk8 was pre-incubated with pebp1. conclusion: our results indicate that reduced klk8 complexation with its inhibitors α2m serpin a6 and b6 may contribute to higher klk8 activity as seen in ad brain, while increased klk8↔pebp1 interaction might explain the compromised cholinergic signaling in ad brain. these results underscore the therapeutic potential of targeting klk8's regulatory network in ad. p10 free neuropathol 6:17:20 associations between age, sex and apoe with amyloid-beta, tau and α-synuclein loads in alzheimer’s disease antonia neubauer1,2, doris weissenbrunner1,2, susanna pekrun1,2, sigrun roeber1,2, viktoria ruf1,2, thomas arzberger1,3, paul feyen1,2, felix strübing1,2, jochen herms1,2,4 1ludwig maximilians university of munich, center for neuropathology and prion research, munich, germany 2 german center for neurodegenerative diseases (dzne), munich, germany 3 department of psychiatry and psychotherapy, university hospital, ludwig maximilians university of munich, munich, germany 4 munich cluster for systems neurology (synergy), munich, germany background: age, apoe-genotype and sex influence the risk for alzheimer’s disease (ad) and lewy body dementia. the relationship with mixed pathologies requires further elucidation. objectives: we aim to assess the association between age, apoe4, and sex with α-synuclein (α-syn), amyloid-beta (aβ) and tau load in ad and how these factors are distributed with variable α-syn deposit distributions. methods: 72 advanced ad cases of the neurobiobank munich were included. α-syn, aβ, and tau deposits were automatically quantified with random forest pixel classifiers on diaminobenzidine stainings for α-syn42, 4g8, and at8 in up to 28 brain regions per case. associations between deposit loads and age at death, sex, and apoe4 were analyzed with multiple linear regression models. additionally, age, sex, and apoe4 carriage were compared between α-syn co-pathology distributions. results: while female cases showed a significantly higher cortical and hippocampal aβ load, males presented with a higher hippocampal and amygdala-entorhinal tau load. patients with a younger age at death showed focally higher aβ and tau loads. apoe4 carriers had higher cortical aβ loads and an increased hippocampal α-syn load. although not significantly, there was a trend towards more female cases and more apoe4 carriers presenting with α-syn co-pathology. ad cases with cortically disseminated α-syn deposits tended to have a lower age at death. conclusion: despite a limited cohort selection, age, sex, and apoe-genotype showed associations with aβ and tau load, and α-syn co-pathology in ad. therefore, these factors should be considered in patient stratification for therapeutic trials. p11 free neuropathol 6:17:21 white matter granular astrocytic tau inclusions are a highly consistent feature in multiple system atrophy viktoria ruf1, doris weissenbrunner1,2, sigrun roeber1, thomas arzberger1,2, camelia-maria monoranu3, paul feyen1,2, jochen herms1,2,4 1 center for neuropathology and prion research, faculty of medicine, lmu munich, munich, germany 2 german center for neurodegenerative diseases (dzne) e. v., munich, germany 3 department of neuropathology, institute of pathology, university of würzburg, würzburg, germany 4 munich cluster of systems neurology (synergy), munich, germany background: multiple system atrophy (msa) is a fatal neurodegenerative disorder characterized by alpha-synuclein aggregates in oligodendrocytes (glial cytoplasmic inclusions; gcis). furthermore, small granular tau inclusions in white matter have occasionally been described, however, these were controversially discussed. aim: based on our own observations and our snrna-seq data showing significant upregulation of mapt in astrocytes from msa patients, we aimed to characterize their presence more systematically. methods: immunohistochemistry and immunofluorescence were applied to archived ffpe tissue from the neurobiobank munich. results: we confirmed the presence of granular tau inclusions, which differ from previously described tau pathologies, and excluded tissue processing or staining artifacts as their cause. the inclusions, also positive for rd3, rd4, ptau-thr231 and ptau-ser396, were closely associated with astrocytes but not with oligodendrocytes or microglia. importantly, there was no apparent colocalization of tau inclusions and gcis. in parallel, we reviewed archival msa cases along with cases with other neurodegenerative diseases and healthy controls. while we detected granular astrocytic tau inclusions (gatis), though to different extents, in all msa cases examined (n = 45), they were absent in other neurodegenerative diseases or healthy controls. gatis were distributed throughout the white matter of the entire brain and spinal cord. the regional distribution closely mirrored that of gcis and we also found a positive correlation of the burden of gcis and gatis. conclusions: gatis appear to represent a highly consistent msa-specific tau pathology, distinct from previously known tau pathologies, with their distribution strongly paralleling alpha-synuclein pathology. their pathological significance, however, remains to be clarified. p12 free neuropathol 6:17:22 impact of the gut microbiome on microglia and alzheimer’s disease in different mouse models katharina janvier1, amelie weber2, vadim farztdinov3, stefan jordan2, michael mülleder3, marina jendrach4, frank l. heppner4 1 department der neuropathology, charité, berlin, germany 2 department of microbiology and infection immunology, charité – universitätsmedizin berlin, berlin, germany 3 core facility high-throughput mass spectrometry, charité universitätsmedizin berlin, berlin, germany 4 department of neuropathology, charité – universitätsmedizin berlin, berlin, germany background: the gut microbiome plays a crucial role in shaping the immune response, which is closely linked to the development and progression of alzheimer's disease (ad). standard laboratory mice, known as spf mice, have a limited and less diverse microbiome than mice living in the wild or humans, resulting in an immune system that may not fully reflect the immune response of adult human. this difference may potentially reduce their translational relevance. objective(s): wildling mice, are laboratory mice that have a natural microbiome, comparable to a mouse in the wild. we are part of the framework of the "charité 3r wildling mice in health and disease" consortium, where the wildling mouse is validated in different pathological settings. question(s): do wildling mice provide a more translationally relevant model for studying alzheimer’s disease? method(s): ad pathology was assessed in spf and wildling mice using electrochemiluminescence assays, mass spectrometry, and immunohistochemistry. gut microbiota composition was analysed, and immune profiling was performed by flow cytometry. result(s): we examined wildtype wildling mice and transgenic appps1.wildling mice in direct comparison to their spf counterparts. wildling mice displayed a significantly different gut microbiome composition and immune cell population. notably, appps1.wildling mice showed signs of ad-related pathology already at early disease stages, earlier and more pronounced than in spf mice. conclusion(s): our results suggest that wild-type mice more accurately reflect important aspects of human ad pathology, particularly regarding gut-brain interactions. p13 free neuropathol 6:17:23 first report of clinico-pathological features associated with tardbp p.m311v mutation alberto grassini1, johannes prudlo2,3, hermann andreas2,4, manuela neumann5,6 1 uniklinikum tübingen, neuropathology, tübingen, germany 2 university medical center rostock, rostock, germany 3 german center for neurodegenerative diseases, rostock, germany 4 center for transdisciplinary neurosciences rostock, rostock, germany 5 uniklinikum tübingen, tübingen, germany 6 german center for neurodegenerative diseases, tübingen, germany tdp-43 is the aggregating disease protein in most patients with als and in ~50 % of patients with ftd. mutations in the encoding gene tardbp are usually associated with clinical als. few clinical reports describe combined als and ftd or pure ftd with tardbp mutations; however, detailed clinico-pathological reports from tardbp mutation carriers are rare. we report a 55-year-old man with a tardbp p.m311v mutation. symptoms started at age 54 with impaired naming and single-word comprehension, fulfilling diagnostic criteria for semantic variant of primary progressive aphasia (svppa). about a year later symptoms were accompanied by behavioral alterations and signs of motor neuron disease. the patient deceased ~18 months after disease onset. neuropathological examination revealed a severe atrophy of the temporal lobe and amygdala with marked asymmetry (left > right). degeneration of the primary motor system was moderate. pronounced tdp-43 pathology was observed in these regions predominantly as compact neuronal cytoplasmic inclusions; however, their morphology and laminar distribution pattern did not correspond to any of the known ftld-tdp subtypes. the observed svppa due to predominant temporal atrophy in this p.m311v patient is comparable to reports of two patients with a p.i383v mutation. it remains to be seen, if this is a consistent feature of the p.m311v mutation; however, our findings imply that some tardbp mutations are associated with a higher toxicity to neurons in the temporal lobe than to motor neurons. identification of factors modifying the consequences of tardbp mutations will be crucial for better understanding of the pathogenesis underlying tdp-43 proteinopathies. vi. neuroinflammation p14 free neuropathol 6:17:24 borna disease virus 1 infection in organotypic hippocampal slide cultures from adult rats maximilien lépine1, christiane herden1 1 institut für veterinär-pathologie, justus-liebig-university giessen, giessen, germany background: borna disease virus 1 (bodv 1) is a zoonotic and neurotropic virus that causes fatal non-suppurative encephalitis in humans, horses, sheep, and alpacas. the white-toothed shrew (crocidura leucodon) has been identified as a natural reservoir host, harboring a persistent infection without developing neurological impairments. for infection studies, the adult rat serves as a suitable model for studying inflammation in dead-end hosts. method(s): viability of organotypic hippocampal slice cultures of adult rats was analyzed by lve/dead immunofluoresence staining, ldh assay, pcr of housekeeping genes and morphologic integrity. in a second step, viral spread, infection patterns, and the local innate immune response were analyzed using immunofluorescence and qpcr to assess viral load. result(s): ohcs were susceptible to infection while maintaining tissue integrity for up to 28 days in culture. viral distribution was uniform across hippocampal regions, but viral load decreased between days 3 and 7 post-infection (p.i.) before increasing between days 7 and 21 p.i. conclusion(s): these results demonstrate that ohcs provide a valuable model for studying viral persistence, distribution, and load in adult rats. p15 free neuropathol 6:17:25 immune profile and clinical characteristics of central nervous system graft-versus-host-disease ida vogt1, benedikt sauer1, dorothea münch2, salem ajib3, katharina wenger-alakmeh4,5,6,7, svenja klinsing1, patrick n. harter8, hildegard dohmen9, till acker9, martina deckert10, karl heinz plate2,4,5, christine stadelmann-nessler11, joachim p. steinbach1,4,5, werner stenzel12, katharina j. weber2,4,5,6, pia zeiner1,4,5,6 1 department of neurology, dr. senckenberg institute of neurooncology, university hospital frankfurt, goethe university, frankfurt am main, germany 2 edinger institute, university hospital frankfurt, goethe university, frankfurt am main, germany 3 department of medicine, hematology and oncology, university hospital frankfurt, goethe university, frankfurt am main, germany 4 german cancer research center (dkfz) heidelberg, germany; german cancer consortium (dktk), frankfurt am main, germany 5 frankfurt cancer institute (fci), goethe university frankfurt, frankfurt am main, germany 6 mildred-scheelnachwuchszentrum, goethe university, frankfurt am main, germany 7 institute of neuroradiology, university hospital frankfurt, goethe university, frankfurt am main, germany 8 center for neuropathology and prion research, faculty of medicine, lmu munich, munich, germany 9 institute of neuropathology, justus.liebig-university giessen, giessen, germany 10 institute of neuropathology, university hospital düsseldorf and medical faculty, heinrich heine university düsseldorf, germany 11 insitute of neuropathology, university medical center, georg august university, göttingen, germany 12 department of neuropathology, charité – universitätsmedizin berlin, corporate member of freie universität berlin und humboldt-universität zu berlin, berlin, germany background: central nervous system graft-versus-host disease (cns-gvhd) is a rare complication following allogeneic hematopoietic stem cell transplantation (allo-hsct). early diagnosis and treatment are essential to halt disease progression. data on risk factors, disease mechanisms and treatment is scarce. while clinical criteria have been suggested, histopathological criteria are lacking. objective(s): this project aims to assess frequency, clinical features and histopathological findings to improve our understanding of its immune microenvironment. question(s): 1. is cns-gvhd a clinically significant complication in allo-hsct patients? 2. what insights do cns-gvhd biopsies reveal? 3. can cns-gvhd diagnosis criteria be improved? method(s): 1436 patients treated with hsct at the university hospital frankfurt between 01.01.2014 and 31.07.2024 were retrospectively examined for cns-gvhd, risk factors and outcomes. in an additional multicenter cns-gvhd cohort of 7 biopsied patients, intensity, localization, cellular composition of inflammation, necrosis and gliosis were assessed. result(s): of 1436 hsct patients, 786 received their first allo-hsct. among those, 5 patients were diagnosed with cns-gvhd, corresponding to a prevalence of 0.6 %. cns-gvhd manifested shortly after discontinuation of immunosuppression in all patients. under high-dose glucocorticoid therapy, clinical response was varying. histologically, cns-gvhd displayed perivascular and parenchymal infiltrates, predominantly consisting of cd8-positive t-lymphocytes. we identified two inflammation patterns: one adaptive-immune-response pattern dominated by lymphocytic infiltration (4 patients) and one innate-immune-response pattern dominated by macrophage infiltration and phagocytosis (3 patients). conclusion(s): cns-gvhd is uncommon but clinically significant. its manifestation shortly after discontinuation of immunosuppression supports a gvhd-related pathophysiology. histopathological findings suggest distinct immune response patterns that may reflect different stages of cns-gvhd. p16 free neuropathol 6:17:27 hpgv-1 is infecting glial cells in immunosuppressed patients helena radbruch1, jenny meinhardt1, raphael raspe1, julia melchert1, victor corman1, emanuel wyler2, klemens ruprecht1, franziska scheibe1, eberhard siebert1 1 charité universitätsmedizin berlin, institut für neuropathologie, berlin, germany 2 max delbrück center für molekulare medizin, berlin, germany background: pegivirus hominis (hpgv-1) is a single-stranded rna flavivirus previously considered non-pathogenic in humans, although it has been reported in association with leukoencephalitis and myelitis in e.g. the context of hiv. objective(s): we want to investigate the neuropathological manifestation of pegivirus-associated encephalomyelitis (paem). question: is paem a distinct encephalomyelitis in immunocompromised patients? method(s): here, we report a neuropathological work up of two chronically immunosuppressed patients presenting with an encephalomyelitis associated with hpgv-1 detection in the central nervous system (cns). result(s): autopsies revealed infiltration by macrophages and t cells, upregulation of hla-dr, tissue destruction — findings compatible with viral encephalomyelitis in areas where we could detect hpgv-1 rna by pcr and in-situ hybridization (ish). pgv-1 rna viral loads were significantly higher in central and peripheral nervous system compared to peripheral organs, with the highest viral loads in the optic nerves and cervical spinal cord, corresponding to the affected clinical and radiologic regions. ish showed viral rna mainly in glial cells of the white matter. analysis of complete hpgv-1 genome sequences obtained from tissue samples revealed single nucleotide polymorphisms, amino acid substitutions, and deletions within the cns. conclusion(s): paem is a distinct and severe form of encephalomyelitis in chronically immunosuppressed patients, predominantly affecting the optic tracts as well as the motor and sensory pathways of the brain and spinal cord. the detection of viral rna in glial cells, genetically distinct viral populations and evidence of independent viral replication in the cns supports a causative role of hpgv-1 in paem. p17 free neuropathol 6:17:28 t cell infiltration patterns and clinical correlates in natalizumab-related pml lesions benita raabe1, adriane kutllovci1, lidia stork1, imke metz1 1 institut für neuropathologie, universitätsmedizin göttingen, göttingen, germany background: progressive multifocal leukoencephalopathy (pml) is an opportunistic viral demyelinating brain disease caused by the jc virus that may occur during natalizumab treatment for multiple sclerosis. tissue resident memory (trm) cd8+ t cells are long-lived stationary immune cells that provide key frontline defense against viral reactivation. cd4+ and cd8+ t cell populations are reported to be essential for antiviral immunity in pml. method(s): we investigated t cell infiltration patterns (trm cd8+ t cells, cd4+ t cells, cd8+ t cells), viral clearance, and clinical outcomes in natalizumab-associated pml, and compared them with classic pml lesions (< 500 t cells/mm²) and inflammatory pml lesions (> 500 t cells/mm²) from patients without prior natalizumab exposure. clinical outcomes were assessed using the expanded disability status scale (edss) and modified rankin scale (mrs). result(s): pml lesions following natalizumab treatment showed high t cell infiltration including trm cd8+ t cells (164.2 cells/mm²), comparable to inflammatory pml lesions without natalizumab pretreatment. elevated t cell numbers persisted until 234 days after the last natalizumab infusion. approximately four months after natalizumab withdrawal, profound reduction of virally infected cells was evident (> 120 days: 1.8 cells/mm²). negative correlation between cd8+ cytotoxic t cells and trm cd8+ t cells with clinical disability scores was observed (edss: p = 0.05, p = 0.03; mrs: p = 0.004, p = 0.03). conclusion(s): after natalizumab withdrawal in pml, immune system reconstitution occurs with profound viral clearance at approximately four months. cd8+ cytotoxic t cells, including trm cd8+ t cells, seem to play crucial roles in viral control and are associated with improved clinical outcomes. p18 free neuropathol 6:17:29 multiplexed viral detection in brain tissue – methodological challenges and opportunities raphael raspe1, emanuel wyler2, viktor horst1, kristin hartmann3, osama mohamed3, julia melchert4, jenny meinhardt1, fabian heinrich5, julia berger6, susanne pfefferle6, shui-fen cheng3, leona kawelke1, nikolaus deigendesch7, stephan frank7, christel bonnas8, jakob matschke3, benjamin ondruschka5, marc luetgehetmann6, izabela plumbom2, janine altmüller2, thomas conrad2, artür manukyan2, altuna akalin2, markus landthaler2, victor corman4, frank heppner1, markus glatzel3, helena radbruch1, susanne krasemann3 1 charité university medicine berlin, department of neuropathology, berlin, germany 2 max-delbrück-center for molecular medicine in the helmholtz association, berlin, germany 3 university medical center hamburg-eppendorf, institute of neuropathology, hamburg, germany 4 charité university medicine berlin, institute of virology, berlin, germany 5 university medical center hamburg-eppendorf, institute of legal medicine, hamburg, germany 6 university medical center hamburg-eppendorf, institute for medical microbiology, hamburg, germany 7 basel university hospital, institute of pathology, basel, switzerland 8 synaptic systems gmbh, göttingen, germany background: following initial claims for viral detection in cns tissue during the sars-cov-2 pandemic, subsequent studies revealed reduced specificity of immunohistological stains and common misidentification of cellular structures as viral in studies using electron microscopy. objective(s): the resulting scientific debate on viral presence in the cns highlights the utmost importance of robust methods for specific detection of viral products. method(s): in a multicenter approach, we generated tissue microarrays containing 380 thalamic and cerebellar samples from donors deceased of covid-19 (n = 134) and age-matched prepandemic controls (n = 71). sars-cov-2 transcriptional abundance and interferon-related gene expression were analyzed using 10x xenium probe-based spatial transcriptomics, with pulmonary tissue of covid-19 donors serving as control for active infection and three hsv1 probes serving as control for neurotropism. qpcr and immunohistochemistry on a subset of samples served as orthogonal validation. result(s): pulmonary controls contained sars-cov-2 rna transcripts, and brain samples from one systemically hsv1-infected but not previously diagnosed donor showed cells positive for all three hsv1 probes. among all other samples, significant but weak signal for both sars-cov-2 and hsv1 probes was restricted to granule cell areas of few pcr-negative cerebellar samples and correlated closely with negative control background. interferon-related gene expression in early covid-19 brain samples was elevated in comparison to controls and late covid-19. conclusion(s): our results point to an acute bystander activation of the cns during systemic covid-19 infection, rather than true neurotropism of sars-cov-2. they highlight the importance of using orthogonal methods for viral detection. p19 free neuropathol 6:17:31 neuropathological characteristics of anti-gaba-b-receptor-encephalitis valérie quinot1,2, verena endmayr1,2, jordi estela-herrero3, mar guasp4,5,6, ellen gelpi1,2, jan bauer7, michael winklehner8, serge weiss9, romana höftberger1,2 1 division of neuropathology and neurochemistry, department of neurology, medical university of vienna, vienna, austria 2 comprehensive center for clinical neurosciences and mental health, medical university of vienna, vienna, austria 3 neurology service, hospital parc taulí, sabadell, spain 4 hospital clínic de barcelona, university of barcelona, barcelona, neuroimmunology unit, department of neurology, barcelona, spain 5 institut d’investigacions biomèdiques august pi i sunyer (idibaps)-caixa research institute, barcelona, spain 6 centro de investigación biomédica en red, enfermedades raras (ciberer), madrid, spain 7 department of neuroimmunology, center for brain research, medical university of vienna, vienna, austria 8 johannes kepler university linz, kepler university hospital, department of neurology, linz, austria 9 kepler university hospital, johannes kepler university, department of pathology and molecular pathology, neuromed campus, division of neuropathology, linz, austria background: antibodies (ab) targeting the γ-aminobutyric acid type b receptor (gababr) lead to symptoms associated with limbic encephalitis, including seizures, memory loss and confusion. 50 % of patients have underlying small-cell lung cancer (sclc). recently, ab directed against the potassium channel tetramerization domain-containing (kctd) protein 16, accessory to the gababr-complex, have been associated with paraneoplastic anti-gababr-encephalitis with sclc. objective(s): to characterize the type and topographic distribution of cellular and humoral inflammatory response in post-mortem brains and to correlate the findings with clinical data. method(s): histological and immunohistochemical evaluation of 4 archival brain autopsies from patients with anti-gababr-encephalitis. determination of serum-anti-kctd16-ab-status by cba. result(s): neurological symptoms of the 4 patients (4 males, mean age of 74a) included epileptic seizures (n = 2), dysarthria (n = 1), dysphagia (n = 1), and vertigo (n = 1). 1 patient had sclc. on histology, mild to moderate cd8+ t-cell-dominated parenchymal and perivascular inflammation in the patient with positive anti-kctd16-ab-status, and a smaller fraction of cd79+ b-cells/plasma cells was observed in amygdala, hippocampus, and cingulate gyrus compatible with predominantly limbic encephalitis, followed by inflammation in brainstem and basal ganglia in all patients. microglia profile was consistently proinflammatory (hla-dr+, p22phox+). one patient showed bilateral hippocampal sclerosis, while unilateral hippocampal sclerosis was identified in 2/4 patients. two patients showed combined neurodegenerative proteinopathies. conclusion(s): our results indicate a t-cell-inflammatory component in addition to an ab-mediated pathophysiology in anti-gababr-encephalitis, which may be more pronounced in the paraneoplastic form associated with sclc and anti-kctd16-ab. p20 free neuropathol 6:17:33 impact of myelin phagocytosis on human blood-derived macrophages and microglia and its effect on human oligodendrocytes laura schmitz-gielsdorf1, katharina heß1, marie-france dorion2, jack antel2, inge huitinga3, luisa klotz4, tanja kuhlmann1 1 institute of neuropathology, university hospital münster, münster, germany 2 montreal neurological institute, neuroimmunology unit, quebec, canada 3 netherlands institute for neuroscience, department of neuroimmunology , amsterdam, netherlands 4 institute of translational neurology, university hospital münster, münster, germany background: myelin-debris clearance by myeloid cells in multiple sclerosis (ms)-lesions is essential for remyelination, as myelin-debris itself can impede this process. however, the effects of myelin-uptake on macrophages remain controversial, with studies reporting both pro-inflammatory and disease-resolving phenotypes that could either hinder or promote remyelination. notably, human-based data supporting the latter hypothesis and investigating subsequent effects on human oligodendrocytes are lacking. objective(s): here we investigate phenotypic changes in myeloid-cells due to myelin-uptake at different stages of myelin-processing and their impact on human oligodendrocyte-differentiation and (re)myelination. method(s): we analysed oligodendrocyte numbers and remyelination in 28 lesions from 25 ms-patients, comparing lesions with and without lipid-droplet (ld)-forming myeloid cells. myeloid-cells from controls and ms-patients were exposed to control or ms-derived myelin for various periods. inflammatory profiles were assessed, and conditioned media were applied to ipsc-derived oligodendrocytes (hiol) to evaluate effects on differentiation. result(s): no correlation was found between ld-forming macrophages and oligodendrocyte numbers or remyelination in ms-lesions. in vitro, myelin-uptake led to a proto anti-inflammatory shift in healthy myeloid-cells, yet hiol differentiation was inhibited at both stages. unexpectedly, in ms-macrophages, control myelin had minimal effect, while ms-myelin induced polarization and again inhibited hiol-differentiation. conclusion(s): although myelin-uptake promotes an anti-inflammatory shift in myeloid-cells, this did not support oligodendrocyte-differentiation. furthermore, ld-formation did not associate with remyelination in ms-lesions. concluding: contrary to published animal studies, our findings, using human oligodendrocyte lineage cells, show no evidence for a role of ld-formation in macrophages on remyelination failure and no evidence that an anti-inflammatory phenotype promotes oligodendrocyte-differentiation. p21 free neuropathol 6:17:34 remyelination in multiple sclerosis depends on the mechanism of lesion development lidia stork1, theresa klossok1, imke metz1 1 university medical center göttingen, institute of neuropathology, göttingen, germany immunopathological patterns are histological signatures reflecting the heterogeneity of multiple sclerosis (ms), encompassing clinical symptoms and treatment response. patterns suggest different mechanisms of lesion development: pattern i and ii with immune-mediated demyelination, and pattern iii reflecting primary oligodendrocyte damage. we hypothesized that these mechanisms also influence remyelination in ms. using the bcas1 marker for remyelinating oligodendrocytes, we analyzed the number and morphological characteristics of bcas1+ cells across the three patterns. additionally, we analyzed oligodendrocyte precursor cells (strong olig2+) and mature nogoa+ cells. for oligodendrocyte precursor cells, no significant difference was observed between patterns (p = 0.4), but mature nogoa+ oligodendrocytes were slightly higher in pattern iii. the number of bcas1+ cells exhibited considerable variability among patients, with some showing low (median: 4.5 cells/mm²), moderate (median: 15.5 cells/mm²), or high (median: 44.1 cells/mm²) numbers. no significant differences were found between the immunopathological patterns for bcas1+ cells (p = 0.9). to assess active myelination, we used double immunofluorescence with myelin-associated glycoprotein (mag). the ratio of bcas1+mag+ to all bcas1+ cells was similar across patterns, but pattern iii was characterized by a significantly higher number of ramified bcas1+mag+ oligodendrocytes (median: pattern i: 0.2, pattern ii: 0.2, pattern iii: 0.5 cells; p = 0.04). in conclusion, these findings indicate that lesions of pattern iii, despite being characterized by primary oligodendrocyte dystrophy, paradoxically demonstrate a significantly higher number of mature oligodendrocytes and ramified, active myelinating bcas1+ cells in areas of tissue response, potentially reflecting an enhanced reactive regenerative capacity and superior remyelination potential compared to other lesional patterns. p22 free neuropathol 6:17:35 il-12 drives neuroinflammation-linked lipid dysregulation in a mouse model of alzheimer’s disease maria geesdorf1, marina jendrach1, andreas hülsmeier2, thorsten hornemann2, frank heppner1 1 charité universitätsmedizin berlin, institut für neuropathologie, berlin, germany 2 universitätsspital zürich, institut für klinische chemie, zürich, switzerland background: neuroinflammation is central to alzheimer’s disease (ad) pathology. microglia-derived interleukin (il)-12 promotes inflammation and disease progression in ad patients and appps1 mice, which recapitulate the first stages of ad, with amyloid beta (a β) accumulation, neuroinflammation, and myelin disruption. objective(s): lipid metabolism seems to be altered in ad, but the interplay with neuroinflammation is yet unclear. therefore, we aimed to characterize lipidomic alterations and metabolic changes in key brain cells during ad progression in the appps1 mouse model and clarify il-12’s role in these processes. question(s): 1. how is the lipidomic profile, especially sphingolipids and myelin-related lipids, affected at different disease stages? 2. how do a β and myelin pathology impact metabolic function in diverse cell types? 3. does il-12 play a role in modulating these pathological features? method(s): we performed sphingolipid-targeted lipidomics by lc-ms on gray and white matter from wild type, appps1, and appps1.il12b-/mice at 4 and 8 months and used immunohistochemistry to evaluate lipid metabolism-related proteins. result(s): 4-month-old appps1 mice show distinct lipid profile changes vs. wild type mice that diminish with disease progression. parvalbumin-positive (pv+) interneurons display reduced lamp+ lysosomes, signaling impaired metabolism, while plin3+ lipid droplet levels remain stable. microglia accumulate more, smaller bodipy+ lipid droplets in appps1, whereas wild type microglia have fewer, larger droplets. il12b deletion partially restores these phenotypes. conclusion(s): our findings support il-12’s role in neuroinflammation-driven lipid metabolism disruption and support il-12 inhibition as a potential ad therapy. p23 free neuropathol 6:17:36 development and validation of a multiplex microarray for pathogen detection in paraffin-embedded cns tissue imke metz1, lidia stork1, hannah bernauer1, selina rehländer2, sandra ehser2, andreas richter2 1 institut für neuropathologie, universitätsmedizin göttingen, göttingen, germany 2 zytovision, bremerhaven, germany background: inflammatory cns diseases can be caused by different pathogens, including viruses, bacteria, fungi, and protozoa. differential diagnoses include autoimmune diseases that require different therapeutic approaches. histopathology may not always detect pathogens, and specific antibodies or stainings are not available for all pathogens. objective(s): to develop a comprehensive, practical test to detect pathogens in paraffin-embedded cns tissue. method(s): a new cns multiplex microarray chip based on the visionarray® system is developed in collaboration between zytovision gmbh (a zytomics company), and the institute of neuropathology, university medical center göttingen, encompassing the most important pathogens for cns infections in europe. result(s): high sensitivity was found for toxoplasmosis and progressive multifocal leukoencephalopathy (9/9 and 13/13 cases positive, respectively). four of five cases with inflammatory necrotic lesions in immunosuppressed patients suspicious for toxoplasmosis but negative on immunohistochemistry were also positive. for progressive multifocal leukoencephalopathy, 3/4 cases suspicious for pml but negative on conventional testing were positive. no false positive results were found. tests for other pathogens still require optimization. conclusion(s): this cns microarray shows promise for detecting certain pathogens with high sensitivity and specificity, providing clear added value compared to histopathology alone. vii. single-cell technologies p24 free neuropathol 6:17:37 investigation of intratumour heterogeneity in primary and recurrent glioblastoma via single cell whole exome sequencing natalie schoebe1,2, isai gonzález3, franziska maria ippen4, paul kerbs1,2, philipp sievers1,2, stefan pfister5,6, sandro krieg7, wolfgang wick4, andreas von deimling1,2, christel herold-mende7, supat thongjuea6,8, natalie berghaus1,2, felix sahm1,2 1 university hospital heidelberg, department of neuropathology, heidelberg, germany 2 german cancer research center (dkfz), german consortium for translational cancer research (dktk), clinical cooperation unit neuropathology, heidelberg, germany 3 bioskryb genomics, durham, united states 4 university hospital heidelberg, department of neurology, heidelberg, germany 5 university hospital heidelberg, department of pediatric hematology and oncology, heidelberg, germany 6 hopp children's cancer center heidelberg (kitz), heidelberg, germany 7 university hospital heidelberg, department of neurosurgery, heidelberg, germany 8 german cancer research center (dkfz), neurooncology (b062), heidelberg, germany background: glioblastoma (gbm) are aggressive brain tumours with high rates of recurrence, likely driven by a high level of intratumour heterogeneity. yet, standard dna sequencing methods do not allow for single cell resolution. objective(s): to characterize subclonal dynamics in the transition from primary to recurrent tumour and uncover potential mechanisms of therapy resistance. question(s): which subclones persist, evolve or emerge under therapeutic pressure and through which molecular mechanisms do they gain this advantage? method(s): primary and recurrent gbm samples from two patients who underwent standard of care treatment were analysed. fresh frozen tumour tissue was dissociated, and single nuclei were sorted into 384-well plates for primary template-directed amplification (pta), a single-cell whole genome amplification method enabling single nuclei genome (snwgs) and exome sequencing (snwes) while ensuring uniform genome coverage and low amplification bias. result(s): snwgs from pair 1 revealed canonical chr7 gain and chr10 loss, with additional alterations such as chr3 loss in the recurrent tumour. pair 2 showed a shift from partial to a full 7/10 signature, with other unique changes between states. for example, the primary tumour harboured a chr22 loss that was not present in the recurrent sample which, in turn, presented a chr11q loss. conclusion(s): pta snwgs cnv profiling detects subtle genomic changes: overall, the primary tumours showed more subclonal alterations than the recurrent ones, suggesting an evolutionary selection for certain treatment resistant subclones after recurrence. additional snwes will provide more insight into treatment-resistant tumour clones and their mutations and mutational signatures. p25 free neuropathol 6:17:39 exploring interand intratumoral heterogeneity of choroid plexus tumors on a single cell level anna klötergens1, flavia de faria1, clara singla2, archana verma1, thomas albert1, i-na lu1, uwe kordes3, denise obrecht-sturm3, christian thomas4, kornelius kerl1 1 department of pediatric hematology and oncology, university children’s hospital münster, münster, germany 2 institute of medical informatics, university of münster, münster, germany 3 department of pediatric hematology and oncology, university medical center hamburg-eppendorf, hamburg, germany 4 institute of neuropathology, university hospital münster, münster, germany background: choroid plexus tumors (cpts) are rare intraventricular neoplasms arising from choroid plexus (chp) epithelial cells, predominantly affecting children. they include three histological subtypes: choroid plexus papilloma (cpp), atypical cpp (acpp), and choroid plexus carcinoma (cpc), and cluster into three epigenetic groups: pediatric a, pediatric b, and adult. objectives: we aimed to elucidate the cellular composition and molecular underpinnings of cpts across histological and epigenetic subtypes. methods: single-cell transcriptomics was applied to 43 cpts spanning all histological and epigenetic subtypes, alongside 12 normal chp samples. spatial transcriptomics was conducted on 15 tumors and 2 non-neoplastic chp samples. integration with fetal, pediatric, and adult chp tissue enabled the creation of a comprehensive single-cell atlas of human chp cells, illuminating developmental and tumor-related transcriptional programs. results: our analyses revealed distinct molecular profiles by subtype and treatment status. notably, pediatric b papillomas resembled carcinomas more than other papillomas, sharing signaling pathway enrichment, loss of motile cilia, and similar cnv patterns. spatial transcriptomics uncovered organized myeloid populations with subtype-specific gene signatures, implicating them in tumor-stroma interactions. notably, both cpc-associated myeloid and tumor cells exhibited enhanced interferon signaling, which decreased following treatment. this reduction aligns with our observation that treated cpc cells cease cycling and regain ciliary function, suggesting a profound molecular reprogramming. conclusion: this study reveals previously unexplored aspects of cpt biology, providing novel insights into the cellular and spatial heterogeneity of cpt subtypes, thereby paving the way toward more personalized and effective treatment strategies for patients with cpt. viii. tumor p26 free neuropathol 6:17:40 analysis of histological, molecular and preanalytic features of unclassifiable cns specimens: comparison of the heidelberg v12.8e, bethesda v3 methylation-based brain tumor classifiers and the hetairos h&e histology-based classifier charlotte brandenburg1, akash kumar1, tatjana starzetz1, patricia amlung2, antje habel3, merten bohn1, helena radbruch4, anna luger5, omkar singh6, artem shmatko7, moritz gerstung7, kenneth d aldape7, leonille schweizer1,3 1 institute of neurology (edinger institute), university hospital frankfurt, goethe university, frankfurt am main, germany 2 german cancer consortium (dktk), partner site frankfurt am mainz, german cancer research center (dkfz), heidelberg, germany 3 department of neuropathology, university hospital heidelberg, heidelberg, germany 4 department of neuropathology, charité-universitätsmedizin berlin, berlin, germany 5 dr. senckenberg institute of neurooncology, university hospital frankfurt, goethe university, frankfurt am main, germany 6 laboratory of pathology, center for cancer research, national cancer institute, national institutes of health, bethesda, maryland, united states 7 division of artificial intelligence in oncology, german cancer research center, heidelberg, germany dna methylation profiling has become an important tool in neuropathological diagnostics, often enabling precise who classification of cns tumors. however, a subset of cases remains unclassifiable using current methylation-based brain tumor classifiers. we retrospectively analyzed a cohort of 1,853 cns tissue samples diagnosed between 2017 and 2024 using genome-wide dna methylation profiling and identified a cohort of 393 cases (21 %) unclassifiable by the heidelberg brain tumor classifier v12.8e. for a representative subset of 252 cases, we compared the classification results of the bethesda classifier v3 (bv3) and hetairos, a novel classifier predicting methylation classes from digital h&e-stained slides, and examined preanalytic, histological, and molecular features. among 252 cases that were unclassifiable by v12.8e, 107/252 cases (42 %) were correctly classified using the bv3 (unclassifiable cases: 118/252 47 %; alternative diagnostic suggestion: in 27/252 cases 11 %). 93/252 cases (37 %) were correctly classified by hetairos (unclassifiable cases: 145/252 58 %; alternative diagnostic suggestion: in 14/252 cases 6 %). the bv3 correctly identified 74/112 glioblastomas, idh-wildtype (gbm; 66 %), including 76 % histological (61/80) and 41 % molecular (13/32) gbm. hetairos identified 78/112 gbm (70 %), including 75 % of the histological (60/80) and 56 % of the molecular gbm (18/32). in both cases, the rf_purify-estimated tumor cell content of correctly classified gbm samples was significantly lower compared to 458 gbm cases correctly classified by v12.8e (p = 0.01086 and p = 0.01966, respectively). these results highlight the complementary value of bv3 and hetairos in reliably classifying diagnostically challenging cases, especially in samples with low tumor cell content. p27 free neuropathol 6:17:42 immunothrombosis and endothelial priming in early brain metastasis katharina müller1, willy hube2, ferdinand schallerer3, linda bergmayr4, robert piecyk5, thomas eska2, edis jardim3, elene nicolaishvili3, lisa sevenich6, kristian unger5, philipp jurmeister4, louisa von baumgarten3, patrick n. harter2 1 department of neurology, university hospital, ludwig-maximilians-university munich, munich, germany 2 center for neuropathology and prion research, faculty of medicine, ludwig-maximilians-university munich, munich, germany 3 department of neurosurgery, university hospital, ludwig-maximilians-university munich, munich, germany 4 department of pathology, faculty of medicine, ludwig-maximilians-university munich, munich, germany 5 department of radiation oncology, university hospital, ludwig-maximilians-university munich, munich, germany 6 university of tübingen m3 institute, tübingen, germany background: the incidence of brain metastases is rising among patients with systemic malignancies, yet preventive strategies targeting the earliest phases of metastatic seeding into the cns remain lacking. emerging evidence points to the critical role of vascular and thromboinflammatory responses during the initial arrest of circulating tumor cells in the brain microvasculature—a process that mirrors key aspects of ischemic stroke. methods: using syngeneic mouse models, we injected tumor cells into the internal carotid artery to induce brain metastases and analyzed early host responses via high-resolution spatial transcriptomics. transcriptional profiling was performed at days 1 and 4 post-injection, focusing on the perivascular microenvironment surrounding arrested ctcs. operating under the hypothesis that early thrombotic events are critical for brain metastasis seeding, we investigated the use of rivaroxaban as a preventive anticoagulant therapy. results: endothelial cells exhibited the most pronounced early transcriptional response, with upregulation of vascular remodeling genes. this gene expression was spatially restricted to areas in direct proximity to tumor cells. glial cells near these sites expressed angiogenic factors as well, indicating a supportive role of the glial compartment in pre-metastatic niche formation. notably, we observed lymphocyte-rich thrombi containing intravascular tumor cells. treatment with the anticoagulant rivaroxaban significantly reduced tumor clot formation, and the development of parenchymal brain metastases. conclusion: our findings identify endothelial activation and thromboinflammatory signaling as key early events in brain metastasis formation, paralleling mechanisms seen in ischemic stroke. these insights suggest that combinatory antithrombotic therapies may offer a viable preventive strategy against metastatic brain colonization. p28 free neuropathol 6:17:43 molecular and histological analyses of at/rt-tyr suggest the choroid plexus of the fourth ventricle as cellular origin lea altendorf1,2, levke-sophie peter1,2, karoline hack1,2, matthias dottermusch3, svenja tonn1,2, rajanya roy4, michael j holtzman5, christian thomas6, kornelius kerl4, melanie schoof1,2, martin hasselblatt6, ulrich schüller1,2,3 1 department of pediatric hematology and oncology, university medical center hamburg-eppendorf, hamburg, germany 2 research institute children’s cancer, university medical center hamburg-eppendorf, hamburg, germany 3 institute of neuropathology, university medical center hamburg-eppendorf, hamburg, germany 4 department of pediatric hematology and oncology, university medical center münster, münster, germany 5 pulmonary and critical care medicine, department of medicine, washington university, st. louis, united states 6 institute of neuropathology, university hospital münster, münster, germany atypical teratoid/rhabdoid tumors (at/rt) belong to the most common tumors of the central nervous system (cns) during infancy. they split into four major dna methylation subtypes: at/rt-tyr, at/rt-shh, at/rt-myc, and at/rt-smarca4. each of these types demonstrates distinct clinical characteristics and gene expression profiles, with at/rt-tyr most frequently occurring in the fourth ventricle. however, details on the cellular origins and tumor initiation of at/rt-tyr remain largely unknown and mouse models providing insights into tumor development are completely lacking. therefore, we aim to identify and characterize the cellular origin of at/rt-tyr. we performed histopathological examination and bulkand single-nucleus rna sequencing analyses across various brain tumor entities. genetically engineered mouse models with a conditional, cre/loxp-induced smarcb1 loss were generated. here, we show that at/rt-tyr very often appear intermingled with fourth ventricle choroid plexus (cp) tissue and that tumor cells heavily express cp markers. analyses of bulkand single-cell rna sequencing data reveal a clear resemblance of the at/rt-tyr to the cp of the fourth ventricle. finally, foxj1-cre::smarcb1fl/fl mice showing loss of smarcb1 in early cp progenitors gave rise to large, atypical, monociliary cp cells with gene expression most similar to human at/rt-tyr. in conclusion, analyses of human at/rt-tyr as well as murine cp cells lacking smarcb1 point towards the cp of the fourth ventricle as a potential cellular origin of at/rt-tyr. p29 free neuropathol 6:17:44 pediatric and aya meningiomas exhibit distinct molecular and clinical features not reflected in adult-based classification systems natalie berghaus1, arnault tauziède-espariat2, thomas hielscher3, christian mawrin4, miriam ratliff5, christel herold-mende6, sandro m. krieg7, wolfgang wick8, stefan m. pfister9, pieter wesseling10, andreas von deimling1, pascale varlet2, felix sahm1, philipp sievers1 1 department of neuropathology, institute of pathology, university hospital heidelberg, heidelberg, germany 2 department of neuropathology, ghu paris psychiatry and neurosciences, sainte-anne hospital, paris, france 3 division of biostatistics, german cancer research center (dkfz), heidelberg, germany 4 department of neuropathology, otto-von-guericke-university, magdeburg, germany 5 neurosurgery clinic, university hospital mannheim, mannheim, germany 6 division of experimental neurosurgery, department of neurosurgery, university hospital heidelberg, heidelberg, germany 7 department of neurosurgery, heidelberg university hospital, heidelberg, germany 8 department of neurology and neurooncology program, national center for tumor diseases, heidelberg university hospital, heidelberg, germany 9 hopp children’s cancer center heidelberg (kitz), heidelberg, germany 10 princess máxima center for pediatric oncology, utrecht, netherlands background: meningiomas are the most common primary intracranial tumors in adults but are rare in children and adolescents/young adults (aya), comprising only 0.4–2.5 % of all cases. pediatric and aya meningiomas differ significantly from adult counterparts in epidemiology, anatomical location, histopathology, molecular alterations, and clinical behavior. this study aimed to define the molecular and clinical landscape of meningiomas in patients aged 0–39 years and to evaluate the applicability of adult-derived prognostic frameworks in this population. methods: we analyzed 294 meningiomas from patients aged 0–39 years, with 115 patients aged 0–14 and 179 aged 15–39. integrated analyses included histopathological evaluation, dna methylation profiling, copy-number analysis, targeted next-generation sequencing (ngs), and clinical outcome assessment. results: the cohort showed a high prevalence of nf2-driven and smarce1-altered tumors, often in a hereditary context. in contrast, high-risk alterations common in adult meningiomas, such as tert promoter mutations and homozygous cdkn2a/b deletions, were nearly absent. established grading parameters based on histology, methylation class, and copy-number alterations failed to reliably predict clinical outcomes. the tumor microenvironment resembled that of adult meningiomas but did not correlate with prognosis. instead, favorable outcomes were primarily linked to extent of resection and specific chromosomal gains. conclusions: pediatric and aya meningiomas constitute a biologically distinct subgroup with unique molecular features and clinical behavior. current adult-based classification and risk stratification systems are inadequate for this population, underscoring the need for age-specific diagnostic and prognostic models. p30 free neuropathol 6:17:46 case of a myxopapillary ependymoma with multiple relapses and pulmonary metastasis catena kresbach1,2, karoline hack2, ulrich schüller1,2 1 institut für neuropathologie, universitätsklinikum hamburg-eppendorf, hamburg, germany 2 forschungsinstitut kinderkrebs-zentrum hamburg, hamburg, germany background: myxopapillary ependymoma (mpe) is a clinically heterogenous disease. while histopathological diagnosis is straightforward, combining histomorphology with hoxb13 immunohistochemistry, predicting the clinical course of mpe is challenging. approximately half of the patients experience local or even distant recurrences. objectives: investigate prognostic and therapeutic molecular characteristics in mpe in an exemplary case and a small mpe cohort. results: we report the case of a 30 year old female patient diagnosed with a presacral mpe which was surgically resected. she suffered from three local relapses 3, 15, and 17 years after primary resection and was treated with repeated resections and adjuvant irradiation. recently, at the age of 53, the patient was diagnosed with mpe metastasis to mediastinal lymph nodes, pleura and lung. methylation profiling indicated a strong methylation of the mgmt promotor. treatment with temozolomide was initiated. the methylation profile matched the published subgroup mpe-a which is correlated with a high 10-year relapse rate of 85 % (bockmayr et al., 2022). additionally, we detected high expression of pdgfra in the lung metastasis and the relapses. our preliminary analyses in a cohort of 25 mpe indicate that pdgfra is overexpressed in mpe with elevated risk for relapse. conclusion: in summary, we present an unusual case of mpe with multiple local recurrences and pulmonary metastasis. its epigenetic profile of mpe-a is in line with the poor prognosis of this subgroup. first data indicate that expression of pdgfra in mpe might be an additional marker of a more aggressive biology and a new therapeutic target. p31 free neuropathol 6:17:47 unravelling ependymoma heterogeneity using integrated proteomic analyses antonia gocke1,2, shweta godbole1, lisa siemers1, bente siebels2, claire delbridge3, christian thomas4, martin mynarek5,6, denise obrecht-sturm6, lan kluwe7,8, said farschtschi8, mario dorostkar9,10, viktoria ruf9, stefan rutkowski6, hartmut schlüter2, ulrich schüller6,11,12, julia neumann1,11 1 center for molecular neurobiology (zmnh), university medical center hamburg-eppendorf, hamburg, germany 2 section of mass spectrometric proteomics, university medical center hamburg-eppendorf, hamburg, germany 3 institute of pathology, school of medicine and health, technical university of munich, munich, germany 4 institute of neuropathology, university hospital münster, münster, germany 5 mildred scheel cancer career center hatrics4, university medical center hamburg-eppendorf, hamburg, germany 6 department of pediatric hematology and oncology, university medical center hamburg-eppendorf, hamburg, germany 7 department of oral and maxillofacial surgery, university medical center hamburg-eppendorf, hamburg, germany 8 department of neurology, university medical center hamburg-eppendorf, hamburg, germany 9 center for neuropathology and prion research, ludwig-maximilians-university, munich, germany 10 karl landsteiner privatuniversität für gesundheitswissenschaften, st. pölten, austria 11 institute of neuropathology, university medical center hamburg-eppendorf, hamburg, germany 12 children’s cancer research center hamburg, hamburg, germany background: ependymomas (epn) are heterogeneous tumours occurring across all different ages and the three major compartments of the central nervous system (spine (sp), posterior fossa (pf), supratentorial (st)), being subdivided into 10 molecular types (who 2021), which display distinct clinical characteristics. objective(s): despite advancements in diagnostics and tumour characterisation, the prognosis remains variable and largely dependent on the extent of tumour resection. thus, there is a high need for targeted adjuvant therapy. through integrated proteomic analyses, the goal was to identify targetable proteins and putative biomarkers for epn types. method(s): histomorphology, dna-methylation-, proteomeand phosphoproteome data was assessed from formalin-fixed paraffin-embedded (ffpe) samples of primary human epn. molecular diagnosis was verified based on dna-methylation data using the brain tumour classifier (v12.8, classifier score > 0.8). proteome and phosphoproteome data were generated using mass spectrometry. established epigenomic epn types were reflected in our main cohort (n = 197 epn with mpe:n = 40, sp-epn:n = 31, sp-epn-mycn:n = 9, sp-se:n =  11, st-se:n = 16, pfa1:n = 14, pfa2:n = 5, pfb:n = 19, pf-se:n = 24, epn-yap:n = 6, epn-zfta:n = 22). result(s): snf clustering integrating methylome and proteome data revealed stable clustering of epn types across data modalities. epn types displayed distinct protein patterns allowing for detection of putative markers and targetable proteins. results were confirmed in a validation cohort (n = 73). in-depth integrative analyses are ongoing and will help to identify epn type specific dysregulated biological pathways, biomarkers and treatment targets. conclusion(s): epn types displayed distinct protein patterns allowing for detection of putative markers and targetable proteins. preliminary findings of the phosphoproteome analysis showed high epn heterogeneity and overlap with known subtypes. p32 free neuropathol 6:17:49 identification of a novel type of pineal region tumors with distinct global dna methylation and sh3tc2 fusions abigail suwala1,2, helena bode3,4,5,6, lea altendorf3,4, karoline hack3,4, alicia fürst3,4, neal geisemeyer5, alexandre vasiljevic7, caterina giannini8,9, ziedulla abdullaev10, sebastian brandner11, jan beckervordersandforth12,13, matija snuderl14, josefine radke15,16, carlos rodríguez antolín17,18, lindsey lowder19, aaron mammoser20, tyler j. kenning21, christian thomas22, martin hasselblatt22, kay hertel23, michael brodhun23,24, quynh nhu nguyen1,2, andreas von deimling1,2, stefan pfister5,6,25,26, brian gudenas27, paul northcott27, felix sahm15, marcel kool5,28, ulrich schüller3,4,29 1 department of neuropathology, institute of pathology, university of heidelberg, heidelberg, germany 2 clinical cooperation unit neuropathology, german cancer research center (dkfz), german consortium for translational cancer research (dktk), heidelberg, germany 3 research institute children’s cancer center hamburg, hamburg, germany 4 pediatric hematology and oncology, university medical center hamburg-eppendorf, hamburg, germany 5 hopp children’s cancer center heidelberg (kitz), heidelberg, germany 6 division of pediatric neurooncology, german cancer consortium (dktk) and german cancer research center (dkfz), heidelberg, germany 7 centre de pathologie et neuropathologie est, centre de biologie et pathologie est, groupement hospitalier est, hospices civils de lyon, bron, france 8 department of laboratory medicine and pathology, mayo clinic, rochester, minnesota, united states 9 department of biomedical and neuromotor sciences (dibinem), alma mater studiorum, bologna, italy 10 laboratory of pathology clinical methylation unit, center for cancer research, national cancer institute, bethesda, maryland, united states 11 department of neurodegenerative disease, ucl queen square institute of neurology and division of neuropathology, university college london hospitals nhs foundation trust, london, united kingdom 12 grow-school for oncology and reproduction, maastricht university, maastricht, netherlands 13 department of pathology, maastricht university medical center, maastricht, netherlands 14 department of pathology, new york university langone health, new york, united states 15 department of pathology, university medicine greifswald, greifswald, germany 16 institute of molecular genomics, university medicine greifswald, greifswald, germany 17 cancer epigenetics laboratory, ingemm, la paz university hospital, biomarkers and experimental therapeutics in cancer, idipaz, madrid, spain 18 bioinformatics unit, ingemm, la paz university hospital, madrid, spain 19 department of pathology and laboratory medicine, emory university school of medicine, atlanta, georgia, united states 20 department of neuroscience and oncology, piedmont brain tumor center, piedmont atlanta hospital, atlanta, georgia, united states 21 neurosurgery department, piedmont atlanta hospital, atlanta, georgia, united states 22 institute of neuropathology, university hospital münster, münster, germany 23 department of pathology and neuropathology, helios clinic erfurt, health and medical university, erfurt, germany 24 department of neurosurgery, helios clinic erfurt, health and medical university, erfurt, germany 25 department of pediatric hematology and oncology, heidelberg university hospital, heidelberg, germany 26 national center for tumor diseases (nct), heidelberg, germany 27 department of developmental neurobiology, st. jude children's research hospital, memphis, tennessee, united states 28 princess máxima center for pediatric oncology, utrecht, netherlands 29 institute of neuropathology, university medical center hamburg-eppendorf, hamburg, germany pineal tumors are rare neoplasms, accounting for less than 1 % of all central nervous system (cns) tumors and occurring primarily in adults. the 2021 world health organization (who) classification recognizes five distinct tumor entities of primary pineal origin: pineocytoma; pineal parenchymal tumor of intermediate differentiation (pptid, with two molecular subgroups); pineoblastoma (including four molecular subgroups); papillary tumor of the pineal region (ptpr, with three molecular subgroups); and desmoplastic myxoid tumor of the pineal region, smarcb1-mutant. here, we describe a previously unrecognized tumor type of the pineal region (n = 27) characterized by a unique and consistent dna methylation profile that does not match any known cns tumor class and is distinct from all currently recognized pineal tumor entities. the cohort displayed a balanced sex distribution, with a mean patient age of 35 years (range: 7–74 years). histopathological analysis revealed features reminiscent of other pineal tumors, including papillary architecture and expression of cytokeratin, synaptophysin, and otx2. a subset of our tumors also focally expressed desmin, whereas all tumors lacked crx expression. notably, 10 of the 13 tumors analyzed harbored a novel rp11-331k21.1:sh3tc2 gene fusion, which has not previously been reported in any cns tumor entity. taken together, our findings define a novel molecular subgroup of pineal region tumors characterized by sh3tc2 fusions and a unique dna methylation signature. recognition of this group is important for accurate diagnosis and may have implications for future tumor classification and treatment strategies. p33 free neuropathol 6:17:51 ttf-1 expression is associated with a hypomethylation signature in schwannomas max braune1, alim e. basaran2, peter kuzman1, johannes wach2, erdem güresir2, ruth m. stassart1, wolf müller1, alonso barrantes-freer1 1 paul-flechsig-institute of neuropathology, university hospital leipzig, leipzig, germany 2 department of neurosurgery, university hospital leipzig, leipzig, germany background: thyroid transcription factor-1 (ttf-1/ nkx2.1) is a well-known transcription factor, most notably associated with adenocarcinoma of the lung. recently, ttf-1 expression has been reported in a subgroup of schwannomas, though its biological and clinical significance remains unclear. objectives: this study aims to characterize the epigenetic and clinical features of ttf-1-positive schwannomas. questions: do ttf-1-positive schwannomas exhibit a distinct epigenetic signature? how is this signature characterized? could ttf-1 expression define a biologically distinct schwannoma subgroup? is ttf-1 expression associated with clinical features such as localization or growth behavior? methods: we performed immunohistochemical, and clinical analyses on 67 schwannomas, including vestibular, spinal, and peripheral schwannomas. ttf-1 expression was assessed by immunohistochemistry. epigenetic profiling of 24 schwannomas was conducted using the illumina epic 850k array. bioinformatic analysis was performed using established r packages (champ, limma). results: unsupervised dimensionality reduction using t-sne and umap revealed distinct clustering of ttf-1-positive schwannomas, based on the 1,000 most variable cpg sites. differential methylation analysis revealed 2,234 cpg sites, predominantly hypomethylated (n = 1,924). enrichment analysis (kegg, go) indicated enrichment of cancer-related pathways (pi3k/akt/mtor, mapk, hippo) in differentially methylated cpg sites of ttf-1-positive schwannomas. the ttf-1/nkx2.1 locus itself showed no differential methylation but exhibited increased copy number variation, particularly in spinal schwannomas. mib1 staining suggested higher proliferative activity in ttf-1-positive spinal schwannomas. limited follow-up data indicated a tendency toward increased facial nerve involvement and proximity to the brainstem. conclusions: ttf-1 expression defines a schwannoma subgroup with distinct epigenetic alterations. further studies are needed to clarify its clinical implications. p34 free neuropathol 6:17:52 expansion of the spectrum of tumors diagnosed as myxopapillary ependymomas fuat aras1, dennis friedel1, david reuss1, gianluca sigismondo1, andreas von deimling1 1 universitätsklinikum heidelberg, neuropathologie, heidelberg, germany background: for a significant portion of spinal ependymomas (spe), pathologists reported conflicting results between morphologic diagnosis and the dna methylation-based classification. a study by the german glioma network reported that nearly one third of the histologically diagnosed spinal ependymomas were assigned by methylation to the myxopapillary ependymoma (mpe) class. objective(s): we address this topic and focus on spe cases exhibiting a methylation profile of mpe. method(s): we performed immunohistochemical, ai-assisted morphological and methylation analyses on 100 mpes and spes. mass spectrometry-based proteomic analysis was conducted on 54 of these tumors. result(s): pearson correlation matrix showed higher similarity between discrepant cases and mpes. principal component analysis revealed predominant clustering of the discrepant cases with mpes. proteomic analysis identified hoxb13 as the most differentially expressed protein between mpe and spe. immunohistochemical staining of 100 tumor samples demonstrated 100 % sensitivity and 100 % specificity. hoxb13 showed positive staining in all discrepant cases. the extracellular proteoglycan versican was overrepresented in the myxoid matrix of mpe. employing digital pathology tools and vcan immunohistochemistry, we demonstrated that discrepant cases frequently contain myxoid foci. 67 cases that were not in the training cohort of hetairos, an ai-assisted morphology tool, were evaluated. 16 of the 17 discrepant cases (94 %) had a prediction for mpe. conclusion(s): the different layers of information demonstrated that discrepant cases show a pattern more similar to mpe. we therefore propose classifying tumors that resemble morphologically spe but exhibit a mcmpe profile or with nuclear expression of hoxb13 as mpe irrespective of their morphological appearance. p35 free neuropathol 6:17:53 addressing mgmt activity in glial cell lines helene blatt1, julia kettern1, walter j. schulz-schaeffer1 1 universität des saarlandes, medizinische fakultät, institut für neuropathologie, homburg, germany the silencing of the o6-methylguanine-dna methyltransferase (mgmt) by methylation of its promoter is the most important predictive marker for the current standard chemotherapy in malignant glioma. however, methylation detection is indirect and other parameters may contribute. therefore, it is our objective to establish a method for the direct detection of mgmt activity in different samples using synthetic methylated oligonucleotides. for our method validation we chose commercial glioblastoma cell lines (t98g, u-87 mg) and the mgmt knock-out cell line hap1 to test their suitability as general models for glioma before transitioning to patient samples. we characterized them using our molecular diagnostics standard panel for gliomas, which, among others, includes mgmt analysis by pyrosequencing and methylation-specific-pcr. additionally, we performed qpcr and western blot. simultaneously, we quantified the demethylation activity of recombinant mgmt and cell extracts using reverse-phase liquid chromatography (lc). our analysis revealed that both cell lines show genetic markers which classify according to who as a grade 4 glioblastoma. by pyrosequencing, mgmt-promotor-methylation in hap1 was ~10 %, t98g 31 % and u-87 mg 54 %. qpcr confirmed successful knock-out and no detectable mgmt-mrna in u-87 mg, but in t98g. western blot confirmed these results. the lc-method was developed and optimized with (un)methylated standards and validated using recombinant mgmt: methylated oligonucleotides (substrate) can be well separated from its demethylated counterparts (product) in submicromolar ranges. we currently test cellular extracts with differing mgmt amounts to mimic complex samples. a successful transfer to patient samples can support therapy decision using mgmt as a biomarker. p36 free neuropathol 6:17:54 loss of global dna methylation is prognostic in oligodendrogliomas felix hinz1,2, dennis friedel1,2,3, franziska maria ippen2,4,5, martin sill6,7, andrey korshunov1,2,6, leonille schweizer8,9,10,11, daniel schrimpf1,2, kirsten göbel1,2, fuat aras1,2, lukas s friedrich1,2, henri bogumil1,2, rouzbeh banan1,2, hildegard dohmen12, sebastian brandner13,14, simone schmid15,16, david capper15,16, niklas grassl17,18,19, henning b boldt20, pieter wesseling21,22, sybren l. n. maas23,24, juan pablo garces martinez25, christine stadelmann-nessler25, guido reifenberger26, thomas stehle27, alonso barrantes-freer28, tareq juratli29, stefan pusch1,2, daniel haag1, david reuss1,2, christel herold-mende30, sandro m. krieg30, wolfgang wick4,5,31, nima etminan32, michael platten17,18,19,33,34, stefan pfister4,6,7,35, david jones4,6,36, felix sahm1,2,6, andreas von deimling1,2, abigail k. suwala1,2 1 dept. of neuropathology, pathological institute, heidelberg university hospital, heidelberg, germany 2 clinical cooperation unit neuropathology, german consortium for translational cancer research (dktk), german cancer research center (dkfz), heidelberg, germany 3 faculty of bioscience, heidelberg university, heidelberg, germany 4 national center for tumor diseases (nct), nct heidelberg, a partnership between dkfz and university hospital heidelberg, heidelberg, germany 5 department of neurology, university hospital heidelberg, heidelberg, germany 6 hopp children's cancer center heidelberg (kitz), heidelberg, germany 7 division of pediatric neurooncology, german cancer consortium (dktk), german cancer research center (dkfz), heidelberg, germany 8 neurological institute (edinger institute), goethe university, frankfurt am main, germany 9 partner site frankfurt, and german cancer research center (dkfz), german cancer consortium (dktk), frankfurt am main, germany 10 university cancer center, goethe university frankfurt, frankfurt am main, germany 11 frankfurt cancer institute (fci), frankfurt am main, germany 12 institute of neuropathology, justus-liebig university giessen, giessen, germany 13 division of neuropathology, national hospital for neurology and neurosurgery, university college london hospitals nhs foundation trust, queen square, london, united kingdom 14 department of neurodegenerative disease, ucl queen square institute of neurology, queen square, london, united kingdom 15 department of neuropathology, charité-universitätsmedizin berlin, corporate member of freie universität berlin and humboldt-universität zu berlin, berlin, germany 16 german cancer consortium (dktk), partner site berlin, german cancer research center (dkfz), heidelberg, germany 17 dktk ccu neuroimmunology and brain tumor immunology, german cancer research center (dkfz), heidelberg, germany 18 department of neurology, medical faculty mannheim, mctn, heidelberg university, heidelberg, germany 19 dkfz-hector cancer institute at university medical center mannheim, mannheim, germany 20 department of pathology, odense university hospital, odense, denmark 21 department of pathology, amsterdam university medical center, amsterdam, netherlands 22 princess máxima center for pediatric oncology, utrecht, netherlands 23 department of pathology, erasmus mc cancer institute, erasmus medical center, rotterdam, netherlands 24 department of pathology, leiden university medical center, leiden, netherlands 25 department of neuropathology, university medical center göttingen, göttingen, germany 26 institute of neuropathology, medical faculty, and university hospital düsseldorf, heinrich heine university, and german cancer consortium (dktk), partner site essen/düsseldorf, düsseldorf, germany 27 institute for neuropathology, faculty of medicine, university hospital cologne, cologne, germany 28 university of leipzig medical center, paul-flechsig-institute of neuropathology, leipzig, germany 29 department for neurosurgery, tu dresden university of technology, dresden, germany 30 department of neurosurgery, heidelberg university hospital, heidelberg, germany 31 clinical cooperation unit neurooncology, german consortium for translational cancer research (dktk), german cancer research center (dkfz), heidelberg, germany 32 department of neurosurgery, heidelberg university, mannheim, germany 33 immune monitoring unit, german cancer research center (dkfz) and national center for tumor diseases (nct), heidelberg, germany 34 helmholtz institute for translational oncology (hi-tron) mainz, german cancer research center, mainz, germany 35 department of pediatric hematology and oncology, heidelberg university hospital, heidelberg, germany 36 division of pediatric glioma research, german cancer research center (dkfz), heidelberg, germany background: idh-mutant gliomas are categorized into idh-mutant astrocytomas (cns who grades 2, 3, 4) and idh-mutant, 1p/19q-codeleted oligodendrogliomas (cns who grades 2, 3). apart from specific subgroups, idh-mutant and 1p/19q-codeleted oligodendrogliomas are assigned to a single dna methylation class using the heidelberg brain tumor classifier, whereas idh-mutant astrocytomas are divided into two methylation classes: astrocytoma, idh-mutant lower grade and astrocytoma, idh-mutant high grade. occasionally, idh-mutant and 1p/19q-codeleted oligodendrogliomas are classified as astrocytomas by methylation profiling despite the clear presence of 1p/19q codeletion. this study aims to investigate the significance of this alternative classification. method(s): we collected 69 idh-mutant and 1p/19q-codeleted oligodendrogliomas classified by the heidelberg brain tumor classifier (v12.8) as either astrocytoma, idh-mutant, lower grade or high grade. methylation data were analysed for methylation class assignment and deconvoluted using methylcibersort to determine cell type proportions. clinical follow-up data were retrospectively obtained for 40 of the 69 patients. result(s): idh-mutant and 1p/19q-codeleted oligodendrogliomas assigned to the astrocytoma, idh-mutant high grade methylation class exhibited a loss of global dna hypermethylation and significantly worse overall survival compared to cns who grade 3 idh-mutant and 1p/19q-codeleted oligodendrogliomas assigned to the oligodendroglioma methylation class (p = 0.0019). deconvolution revealed a higher proportion of residual brain tissue in oligodendrogliomas classified as idh-mutant astrocytoma, lower grade compared to other methylation classes of idh-mutant gliomas. conclusion(s): our findings suggest that decreased global dna methylation in idh-mutant and 1p/19q-codeleted oligodendrogliomas, as indicated by methylation classification into the astrocytoma, idh-mutant, high grade methylation class, is associated with poorer overall survival. p37 free neuropathol 6:17:57 leveraging peptide-level proteomics to detect brain cancer specific proteoforms ian fichtner1,2, fuat kaan1,2, gianluca sigismondo1,2, christel herold-mende1, sandro krieg1, wolfgang wick1, andreas von deimling1,2, felix sahm1,2, moritz gerstung2, isabell bludau1,2 1 heidelberg university hospital, heidelberg, germany 2 german cancer research center (dkfz), heidelberg, germany background: conventional bottom-up proteomics approaches rely on protein-group inference. this strategy overlooks peptide-level information of the proteome — particularly the existence of proteoforms, which are distinct molecular species originating from a single gene locus via mechanisms such as alternative splicing, proteolytic cleavage, and post-translational modifications. the commonly overlooked proteoform level is expected to provide clinically valuable information, especially in the context of complex and molecularly heterogeneous diseases such as brain cancers. objective(s): this study leverages peptide-level data to identify and characterize differentially regulated proteoform-groups across brain cancer subtypes. question(s): do specific brain cancer subtypes exhibit distinct patterns of proteoform-group regulation? if so, which subtypes are affected, how do these patterns differ from those in other tumor classes, and what molecular mechanisms underlie these regulatory differences? method(s): to address these questions, we applied the copf algorithm (bludau et al., 2021), which infers proteoform-groups based on peptide correlation patterns, to a clinical mass-spectrometry-based proteomics dataset comprising 31 patient samples across 4 brain tumor subtypes, including astrocytoma, glioblastoma, meningioma and oligodendroglioma. result(s): our analysis revealed multiple proteins with differentially regulated proteoform groups across brain cancer types. notably, rtn4 (nogo) and lima1 showed differential proteoform patterns in meningioma compared to other subtypes, matching known rna splice isoforms, that would have been missed in conventional proteomics data analysis. conclusion(s): proteoform-group inference can reveal subtype-specific expression patterns, highlighting its utility for uncovering molecular diversity in brain cancer and the identification of novel biomarkers. p38 free neuropathol 6:17:58 glioblastoma-derived secreted proteins drive invasion and affect neuronal integrity tanja buhlmann1,2, youcef dahmani1,2, victor adrian perez1,2, karl heinz plate2,3,4,5, joachim p. steinbach1,3,4,5, ann-christin hau1,2,3,5,6 1 dr. senckenberg institute of neurooncology, university hospital frankfurt, frankfurt am main, germany 2 institute of neurology (edinger institute), university hospital frankfurt, frankfurt am main, germany 3 frankfurt cancer institute (fci), frankfurt am main, germany 4 german cancer consortium (dktk), partner site frankfurt am mainz, frankfurt am main, germany 5 university cancer centre frankfurt (uct), university hospital frankfurt, frankfurt am main, germany 6 mildred scheel nachwuchszentrum (msnz), frankfurt am main, germany background: glioblastoma (gb) is an aggressive brain tumor characterized by diffuse infiltration and reciprocal interactions with the surrounding brain tissue, including neurons. understanding how tumor-derived factors contribute to both invasion and neuronal disruption is critical to elucidating glioma-brain interactions. objective: to identify glioma-derived secreted proteins that promote tumor cell invasion and to investigate whether these factors also affect the structural and functional integrity of human neurons. methods: we combined two complementary screening approaches in patient-derived glioma stem-like cells (gscs): (1) serial selection of highly invasive subpopulations, and (2) a genome-wide crispr activation (crispra) screen. candidate factors were validated via overexpression and crispr/cas9-mediated knockout across multiple gsc lines. functional consequences were assessed using 2d assays and 3d cerebral organoids derived from human induced pluripotent stem cells (hipscs), including live-cell imaging in the glioma cerebral organoid (glico) model. tumor-neuron interactions were studied using a competition assay with fluorescently labelled wildtype and knockout cells in co-culture with hipsc-derived neurons, and via direct exposure of mature neurons to recombinant proteins. results: chi3l1 and igfbp5 were identified as secreted regulators of glioma invasion, enriched at the tumor invasive front. functional assays demonstrated consistent effects on cell motility and infiltration. exposure of neurons to recombinant chi3l1 or igfbp5 led to synaptic alterations and neurotoxic effects. conclusion: chi3l1 and igfbp5 are secreted drivers of glioma invasion and modulators of neuronal integrity. these findings underscore the importance of tumor-neuron interactions, particularly at the invasive front where single tumor cells interface with intact brain parenchyma. p39 free neuropathol 6:17:59 establishment of droplet digital™ pcr (ddpcr™)-based assays for the diagnostic detection of mgmt promoter methylation in malignant gliomas marietta wolter1, david pauck1, jörg felsberg1, guido reifenberger1,2 1 university hospital düsseldorf, medical faculty, heinrich-heine-university düsseldorf, institute of neuropathology, düsseldorf, germany 2 german cancer consortium (dktk), german cancer research center (dkfz) heidelberg, partner site essen/düsseldorf, germany, essen/düsseldorf, germany mgmt promoter methylation is the most relevant predictor of response to chemotherapy with temozolomide (tmz) in patients with newly diagnosed idh-wildtype glioblastoma. in routine diagnostics, the mgmt promoter methylation status is commonly assessed by either pyrosequencing (psq) of bisulfite-modified dna or global epicv2 bead array-based dna methylation profiling using the stp27 algorithm. we aimed to establish and validate ddpcr™ as a novel method to allow for rapid diagnostic detection of the methylation status at relevant cpg sites in the mgmt promoter-associated cpg98 island. results obtained by ddpcr™ were compared to those obtained by psq or stp27. in total, we established three different duplex-ddpcr™ assays to analyse the methylation status at selected cpg sites covering (1) four of the cpg sites commonly analysed by psq (cpgs 76–79, numbering of cpg sites in the mgmt-associated cpg98 island according to malley et al., acta neuropathol. 2011, 121:651–661), as well as (2) cpg30 and cpg31, and (3) cpg84, with cpg31 corresponding to cpg10, and cpg84 to cpg16 of the mgmt-stp27 algorithm. comparison of the newly established ddpcr™-based assays with results obtained by psq or epicv2-based stp27 analysis showed a high degree of consistency in the methylation levels detected at the respective cpg sites. moreover, differences in methylation status at the distinct cpg sites interrogated by either psq or stp27 were reproduced by the distinct ddpcr™ assays. our results thus implicate ddpcr™ as a novel, rapid, comprehensive and quantitative approach for the routine diagnostic determination of the mgmt promoter methylation status in malignant gliomas. p40 free neuropathol 6:17:60 exploring glioblastoma through cpg site-specific methylation and transcription factor occupancy kimia kafi cheraghi1,2, michel mittelbronn3,4, karl heinz plate2,5, joachim steinbach1,5, tanja buhlmann1,2, ann-christin hau1,2 1 dr. senckenberg institute of neurooncology, university hospital frankfurt, frankfurt am main, germany 2 edinger institute, institute of neurology, goethe university, frankfurt am main, germany 3 laboratoire national de santé, dudelange, luxembourg 4 luxembourg institute of health, luxembourg, luxembourg 5 frankfurt cancer institute, frankfurt am main, germany glioblastoma (gb) is a highly malignant primary brain tumor that shows extensive heterogeneity and invasiveness. while several studies highlight the role of epigenetic alterations in gb aggressiveness and heterogeneity, the functional consequences of individual cpg site methylation remains poorly understood. methylation at cpg sites can influence transcription factors (tf) binding, thereby affecting gene regulation and potentially impacting tumor cell behavior and interactions within the tumor microenvironment (tme), including invasion. the present study investigates the impact of differential cpg site-specific methylation on tf occupancy and its role in regulating the expression of chitinase 3-like-1 (chi3l1), a gene differentially expressed in highly invasive gb stem cells (gscs), aiming to examine the role of methylation in tf binding sites and its influence on gb invasiveness. this project employed methylation analysis utilizing illumina epic 850k array across various gb cell lines, revealing hypomethylation of chi3l1 promoter in more invasive gscs. to assess the functional relevance of this finding to promoter activity, site-directed mutagenesis of the specific cpg sites was performed, followed by luciferase reporter assay. in silico motif enrichment analyses identified tf binding motifs around the candidate cpg sites, suggesting that methylation status could modulate tf binding. finally, chromatin immunoprecipitation (chip)-qpcr experiments were established to confirm tf binding at differentially methylated cpg sites. future work will examine the role of these tfs in differential expression of other genes involved in gb invasiveness using chip sequencing. additionally, we seek to optimize the chip method for formalin-fixed paraffin-embedded tissue, as they are more physiologically relevant models. p41 free neuropathol 6:17:61 integrated analyses reveal four distinct molecular subgroups in corticotroph pituitary neuroendocrine tumors/adenomas matthias dottermusch1, alice ryba2, temor rafiq1, linus haberbosch3, leonille schweizer4, mateusz bujko5, wolfgang saeger1,6, ulrich schüller1,7,8, markus glatzel1, jörg flitsch2, franz ricklefs2, julia neumann1,9 1 institute of neuropathology, university medical center hamburg-eppendorf, hamburg, germany 2 department of neurosurgery, university medical center hamburg-eppendorf, hamburg, germany 3 department of endocrinology and metabolic diseases (including lipid metabolism), charité university medicine, berlin, germany 4 edinger institute (institute of neurology), university hospital frankfurt, goethe university, frankfurt am main, germany 5 department of molecular and translational oncology, maria sklodowska-curie national research institute of oncology, warsaw, poland 6 institute of pathology, university medical center hamburg-eppendorf, hamburg, germany 7 pediatric hematology and oncology, university medical center hamburg-eppendorf, hamburg, germany 8 children's cancer research center hamburg, hamburg, germany 9 center for molecular neurobiology (zmnh), university medical center hamburg-eppendorf, hamburg, germany background: corticotroph pituitary neuroendocrine tumors (pitnets/adenomas) are common sellar neoplasms with variable clinical presentations. according to the current who classification, tumor subtyping is based on histopathology and comprises subtypes with either sparse or dense granulation as well as crooke cell tumors. previous studies on confined case series have reported three distinct molecular profiles in corticotroph pitnets. these molecular profiles primarily related to usp8-mutation status and gata3-expression levels, while their alignment with the established who subtypes was limited. objective: in this ongoing work, we aim to further explore molecular subgroups of corticotroph pitnets and their clinical relevance. methods: we compiled previously published and publicly available global epigenomic and/or transcriptomic data of 171 corticotroph pitnets, derived from a total of 7 independent studies. gap statistics and consensus clustering were applied to delineate molecular subgroups within the compiled datasets. results: based on epigenomic as well as transcriptomic data, corticotroph pitnets separated into four distinct molecular subgroups, preliminarily named subgroups 1–4. usp8-wildtype and gata3-negative tumors split into subgroups 1 and 2. usp8-wildtype tumors with increased gata3 expression were mainly found in subgroup 3. usp8-mutated tumors with increased sstr5 expression were predominantly aggregated in subgroup 4. preliminary investigations indicated that these four molecular subgroups enable improved prognostic stratification beyond current concepts. conclusions: our findings demonstrate that corticotroph pitnets segregate into four molecular subgroups. ongoing investigations including detailed clinicopathological characterizations of each subgroup have the potential to improve the existing histopathological classification framework in corticotroph pitnets. p42 free neuropathol 6:17:63 harnessing stress adaptive response mechanisms for pharmacological targeting of cancer laura hruby1, david bickel2, laura drobe1, holger gohlke2, dieter willbold3, guido reifenberger1, gabriel leprivier1 1 universitätsklinikum düsseldorf, institut für neuropathologie, düsseldorf, germany 2 heinrich-heine-universität, institut für pharmazeutische und medizinische chemie, düsseldorf, germany 3 heinrich-heine-universität, institut für physikalische biologie, düsseldorf, germany brain cancer cells are subjected to metabolic stress, such as hypoxia and glucose restriction, due to defective tumor vasculature. this forces brain cancer cells to evolve adaptive mechanisms to reprogram their metabolism, which includes inhibition of mrna translation — a highly energetic process. the rate of mrna translation is controlled by the mechanistic target of rapamycin (mtor)/ eukaryotic initiation factor 4e binding protein (4ebp) pathway according to glucose concentrations. glucose starvation blocks mtor, which in turn leads to 4ebp1 activation. this results in the inhibition of mrna translation initiation as 4ebp1 binds to and blocks the translation initiation factor eif4e. we found that high 4ebp1 expression is a factor of poor prognosis in glioblastoma and medulloblastoma. furthermore, we demonstrated that 4ebp1 protects glioblastoma and medulloblastoma cells under glucose starvation and promotes tumorigenicity both in vitro and in vivo. these findings support that 4ebp1 may be a potential therapeutic target in glioblastoma and medulloblastoma. therefore, we are aiming to develop a targeting strategy against 4ebp1 to drive cancer cells to death under metabolic stress. using an in-silico screening approach we identified potential inhibitors of 4ebp1, predicted by molecular docking to disrupt the 4ebp1-eif4e interaction. using in vitro binding assays and cellular assays, these compound candidates are being validated for their ability to block the physical binding of 4ebp1 to eif4e, to induce cell death under glucose deprivation and to reduce tumorigenic potential. our strategy to target 4ebp1 may represent a novel therapeutic approach to treat, yet, incurable malignant brain tumors. p43 free neuropathol 6:17:64 mechanisms of transformation from subependymoma to ependymoma hannah jessner1,2, flavia watusi de faria1, clara inserte singla3, thomas albert1, ina lu1, claudia rossig1, ulrich schüller4, erik schüftan1, simon johann kaufmann1, angela brentrup5, ann-katrin bruns5, dorothee cäcilia spille5, christian thomas2, kornelius kerl1 1 department of pediatric hematology and oncology, university children’s hospital münster, münster, germany 2 institute of neuropathology, university hospital münster, münster, germany 3 institute of medical informatics, university of münster, münster, germany 4 institute of neuropathology, university medical center hamburg-eppendorf, hamburg, germany 5 department of neurosurgery, university hospital münster, münster, germany background: subependymomas (ses) are slow-growing central nervous system tumors, yet those in the posterior fossa (pf-ses) can behave more aggressively, often showing areas of higher-grade ependymal differentiation. recent studies suggest that chromosome 6 loss and tert promoter mutations are associated with this transition, supporting a model of progression from low-grade to more malignant phenotypes. this study investigates the molecular and spatial heterogeneity underlying this evolution. methods: we analyzed 27 pf-se samples from 25 patients, alongside 5 pf-a and 6 pf-b ependymomas. using single-nucleus rna sequencing, spatial transcriptomics, and bulk rna sequencing, we assessed transcriptional and spatial dynamics. hypoxia-inducible factor 1-alpha (hif1a) immunohistochemistry was also performed. results: a hypoxia-associated transcriptional program was enriched in ependymal-like regions of pf-ses. copy number analysis revealed increasing chromosome 6 loss correlating with ependymal differentiation and hypoxia-related gene expression. tumors with both subependymal and ependymal areas exhibited a reactive transcriptional state marked by stress and interferon signaling, suggesting an active transitional phase. immune profiling showed a shift from pro-inflammatory to immunosuppressive signatures along the histologic spectrum, indicating progressive immune evasion. conclusion: these findings highlight hypoxia and chromosome 6 loss as interconnected mechanisms driving malignant transformation in pf-ses. the parallels with aggressive pf-as suggest shared evolutionary pathways among posterior fossa ependymal tumors. p44 free neuropathol 6:17:65 leveraging off-target reads for genome-wide copy number profiling in ngs panels jan schnorrenberg1, yannis adrian1, werner paulus1, martin hasselblatt1, christian thomas1 1 institut für neuropathologie münster, münster, germany background: copy number variations (cnvs) are crucial for the diagnosis and prognosis of central nervous system (cns) tumors. although typically assessed using chromosomal or dna methylation microarrays, cnv data can also be extracted from next-generation sequencing (ngs) panels. off-target reads mapping outside the targeted regions are usually discarded in standard ngs workflows. objectives: our study evaluates whether these reads can be repurposed for genome-wide cnv profiling in cns tumors using a small, custom ngs panel not specifically designed for cnv analysis. methods: we analyzed 60 cns tumors, including idh-wildtype glioblastomas (n = 25), oligodendrogliomas (n = 15), ependymomas (n = 9), medulloblastomas (n = 6), and choroid plexus tumors (n = 5). each case underwent epic methylation profiling and hybrid-capture ngs panel (31 genes, 0.17 mb). cnv profiles were generated using cnvkit and custom python scripts. results: sequencing yielded an average of 26.5 million reads per sample (range: 5.1–72.2 million), with a mean off-target rate of 64.9 % (range: 45–92 %). epic data identified 260 chromosomal arm gains and 271 losses. ngs-derived cnvs showed strong concordance (average r = 0.915, p = 0.000014). hallmark alterations, including +7/−10 and 1p/19q codeletion, were reliably detected from off-target reads. nineteen focal amplifications were concordantly identified, including mdm4 and mycn, which were not covered by the panel. of 20 homozygous deletions identified by the array, 19 (95 %) were also observed in the ngs data. conclusion: taken together, our findings demonstrate the feasibility of using off-target reads for cnv profiling in cns tumors, enabling detection of clinically relevant events even outside targeted regions. p45 free neuropathol 6:17:66 establishing ultra low-input spatial proteomics for glioblastoma analysis rhaissa ribeiro da silva1, daniela valdes2, abigail suwala1, philipp sievers1, christel herold-mende1, sandro krieg1, wolfgang wick1, andreas von deimling1,3, gianluca sigismondo1, felix sahm1,3, fabian coscia2, isabell bludau1 1 university hospital, heidelberg, germany 2 max delbrück center for molecular medicine (mdc), berlin, germany 3 german cancer research center (dkfz), heidelberg, germany background: glioblastoma shows pronounced spatial heterogeneity that impacts its aggressive behavior and therapy resistance. high-resolution spatial proteomics is needed to characterize distinct tumor microenvironments at the protein level, which cannot be fully captured by transcriptomics alone. objective(s): to establish an ultra-low-input spatial proteomics workflow enabling the identification of thousands of proteins from minute, laser-microdissected glioblastoma tissue regions. this approach aims to facilitate detailed molecular profiling of heterogeneous tumor areas. question(s): can this workflow detect spatial protein gradients within glioblastoma microenvironments? how can such data support future investigations of biologically relevant tumor niches? method(s): glioblastoma sections are annotated after h&e staining, followed by precise laser microdissection (~30,000 μm² equivalent to ~250 cell bodies) of morphologically distinct tumor regions. proteins are analyzed via highly sensitive lc-ms/ms with an optimized ultra-low-input protocol. result(s): using this workflow, we are able to identify approximately 3,500 proteins per microdissected region. this high sensitivity and spatial resolution provide a powerful basis for future applications, such as analyzing perinecrotic zones to explore spatially resolved protein expression patterns relevant to tumor progression. conclusion(s): the established ultra-low-input spatial proteomics workflow offers a robust platform for investigating glioblastoma heterogeneity at the protein level. it enables detailed studies of tumor microenvironments and supports the discovery of spatially distinct biomarkers and therapeutic targets. p46 free neuropathol 6:17:67 comparison of diffuse glioma types with gliomatosis cerebri growth pattern in adult patients marco münzberg1, franziska maria ippen2,3,4, iris divé5, charlotte brandenburg1, philipp sievers4,6,7, mustafa mahmutoglu8, marianne schell8, katharina schregel8,9, katharina wenger-alakmeh10, leonille schweizer1,11 1 institute of neurology (edinger institute), university hospital frankfurt, goethe university, frankfurt am main, germany 2 national centre for tumour diseases (nct), nct heidelberg, a partnership between dkfz and university hospital heidelberg, heidelberg, germany 3 department of neurology, university hospital heidelberg, heidelberg, germany 4 clinical cooperation unit neuropathology, german cancer research centre (dkfz), german consortium for translational cancer research (dktk), heidelberg, germany 5 dr. senckenberg institute of neurooncology, university hospital frankfurt, goethe university, frankfurt am main, germany 6 department of neuropathology, institute of pathology, university hospital heidelberg, heidelberg, germany 7 hopp children's cancer centre heidelberg (kitz), heidelberg, germany 8 department of neuroradiology, heidelberg university hospital, heidelberg, germany 9 clinical cooperation unit neurooncology, german cancer research centre (dkfz), heidelberg, germany 10 institute of neuroradiology, university of frankfurt am main / brain imaging centre frankfurt am main, frankfurt am main, germany 11 german cancer consortium (dktk), partner site frankfurt am mainz, german cancer research centre (dkfz), heidelberg, germany gliomatosis cerebri (gc) type 1 is characterised by diffuse infiltration of glioma cells spanning three or more cerebral lobes without contrast enhancement. its prevalence and clinical relevance across glioma subtypes remain incompletely understood. in a cohort of 853 gliomas epigenetically profiled between 2017 to 2025, gc was found in 1 % of glioblastomas, idh wildtype (gbm; 5/587), 4.5 % of oligodendrogliomas, idh-mutant and 1p/19q-codeleted (4/88), 2.8 % of astrocytomas, idh-mutant (3/108), 4 % of diffuse paediatric-type high-grade gliomas, h3-wildtype and idh-wildtype (2/54), and 69 % of the novel glioma type "gliomatosis cerebri-like gliomas, idh-wildtype" (gclg, 11/16). gliomas exhibiting the gc phenotype showed significantly higher mgmt promoter methylation rates compared with non-gc tumours (e.g., 46 % vs. 28 % in gbm), whereas mgmt promoter methylation was uncommon in gclgs with gc growth pattern (7 % vs. 28 %, p < 0.001). idh-mutant gliomas with gc were more often treated with radiotherapy or chemotherapy alone. notably, gbms and gclgs with gc exhibited comparable radiochemotherapy rates (88 % vs. 86 %), while monotherapy and watchful waiting were infrequently employed (0 % vs. 7 % and 12 % vs. 7 %). outcome for gclg with gc was significantly better compared to gbm with gc (mpfs 25 vs 12 months, mos 49 vs 19 months, p < 0.05). in summary, while gc is uncommon in most glioma types, it represents the predominant growth pattern in gclg. despite infrequent mgmt promoter methylation, the prognosis of gclg with gc is considerably more favourable than gbm. we therefore recommend comprehensive epigenetic profiling in patients presenting radiologically with gc type 1. p47 free neuropathol 6:17:69 immunohistochemical expression and differential methylation of hoxb13 reliably distinguishes myxopapillary ependymoma from spinal ependymoma suvendu purkait1,2, sophia praeger3,4, jörg felsberg2, david pauck2, kerstin kaulich2,5, marietta wolter2, david koppstein3,4, guido reifenberger2,5 1 department of pathology and laboratory medicine, all india institute of medical sciences, bhubaneswar, odisha, india 2 institute of neuropathology, heinrich heine university medical faculty and university hospital düsseldorf, düsseldorf, germany 3 cancer bioinformatics and multiomics (ed08), german cancer research center heidelberg and german cancer consortium (dktk), partner site essen/düsseldorf, düsseldorf, germany 4 department of pediatric oncology, hematology and clinical immunology, heinrich heine university medical faculty and university hospital düsseldorf, düsseldorf, germany 5 german cancer consortium (dktk), partner site essen/düsseldorf, düsseldorf, germany background: histological distinction of spinal ependymoma from myxopapillary ependymoma may be difficult in individual cases, especially in tumors located in the lumbar region. according to the who classification of 2021 unresolved lesions require global dna methylation profiling for correct classification. recently, high expression of the homeobox gene hoxb13 at the mrna and protein levels has been reported in myxopapillary ependymoma. objective(s): we evaluated the diagnostic role of hoxb13 immunostaining in an institutional cohort of patients with spinal neoplasms (n = 143), including different types of spinal ependymal tumors from various locations and other relevant differential diagnoses. method(s): expression of hoxb13 protein was compared to molecular findings obtained by dna methylation profiling, targeted methylation analysis, and next generation sequencing. result(s): collectively, our findings indicate that strong nuclear hoxb13 immunopositivity is a specific diagnostic marker for myxopapillary ependymoma which enables reliable differentiation of spinal ependymoma, especially in lumbar spinal cord tumors whose precise classification otherwise would require dna methylation profiling. we additionally provide evidence for differential methylation of hoxb13-associated cpg sites and established a pyrosequencing-based assay to interrogate a hoxb13-associated cpg site that showed consistent differential methylation between spinal ependymoma and myxopapillary ependymoma. conclusion(s): thus, immunohistochemistry for hoxb13 and/or targeted dna methylation analysis may constitute fast, resource-friendly approaches to substitute for global dna methylation profiling in the precise classification of spinal ependymal tumors. p48 free neuropathol 6:17:71 spatial mapping of immunogenic niches in melanoma brain metastases kristin peters1, artür manukyan2, helena radbruch3, torben redmer4, josefine radke4 1 universitätsmedizin greifswald, institut für pathologie, greifswald, germany 2 berliner institut für medizinische systembiologie (mdc-bimsb), berlin, germany 3 charité universitätsmedizin berlin, institut für neuropathologie, berlin, germany 4 universitätsmedizin greifswald, institut für molecular genomics, greifswald, germany background: tumor development and progression are dynamic, time-dependent processes influenced by genetic alterations, environmental factors, and spatial mechanisms all shaped by interactions with the tumor microenvironment (tme). resistance to immune checkpoint inhibition (ici) significantly contributes to the emergence and metastatic progression of brain metastases (mbm) in approximately 40–60 % of melanoma patients. objective(s): we employed spatial transcriptomics at the single cell level to investigate spatially defined immunosuppressive programmes in mbm and their role in therapy resistance. question(s): what are the spatial characteristics and cellular compositions of immune-suppressive niches in mbm, and how do they relate to resistance against immune checkpoint inhibition? method(s): we applied xenium (10x genomics) spatial transcriptomic technology to 14 mbm specimens representing various stages of tumor progression and treatment history. single-cell resolution data were analyzed to deconvolute the immunogenic landscape and cellular phenotypes within the tumor microenvironment. result(s): we identified substantial cellular heterogeneity across mbm samples and highlighted tumor subsets expressing bzw2, sox4, or tap1. bzw2⁺ and sox4⁺ tumor cells were associated with immune-suppressive microenvironments characterized by poor infiltration of immune cells and tumor-associated macrophages/microglia (tams). bzw2 and tap1 expression showed an inverse correlation, with tap1 being enriched in "hot" immune cell–infiltrated niches, consistent across tumor stages. conclusion(s): spatially-defined immunosuppressive programs critically shape immune cell infiltration patterns in mbm and may influence the response to immune checkpoint therapies. understanding these spatial dynamics could inform targeted therapeutic strategies to overcome ici resistance. p49 free neuropathol 6:17:72 targeting phosphorylation events in glioblastoma: a semi-spatial (phospho)proteomic workflow for diffuse glioma ffpe samples ivan abdulrazak ahmed1,2, gianluca sigismondo1, fuat aras2, david reuss1, stefan pusch1 1 universitätsklinikum heidelberg, deutsches krebsforschungszentrum, heidelberg, germany 2 universitätsklinikum heidelberg, heidelberg, germany glioblastoma is the most frequent and aggressive among the glioma. despite dna methylation has improved tumor classification, and dissected different subtypes, glioblastoma is still characterized by traditional therapeutic options, resulting in poor patient response and median 18 months survival. egfr kinase amplification is observed in 60 % of glioblastoma, causing hyperactivation of key signaling pathways regulating proliferation, motility, and apoptosis, thereby contributing to cancer progression. in a fraction of patient, egfr activation is observed in the absence of egfr amplification, thus the dissection of egfr phosphorylation cascade is critical to characterize gbm biology. phosphorylation events are dynamic, and due to the very low abundance of phosphopeptides, phosphoproteomic analysis is particularly challenging, especially in formalin-fixed paraffin-embedded (ffpe) clinical tissues. overcoming these obstacles relies on robust phosphopeptide enrichment methods and highest sensitivity mass spectrometers coupled with in-depth computational data analysis pipelines. in this context, our team at the division of neuropathology in heidelberg has implemented a low-input phosphoproteomic workflow optimized for single punches of ffpe material. the robust and automatized phosphopeptide enrichment strategy highly improves the quantification of phosphorylated peptides in clinical samples, while the downstream bioinformatic analysis enables the systematic mapping of kinase-substrate relationships and elucidates on signaling networks deregulated in glioblastoma. our optimized phosphoproteomic pipeline dramatically increases our understanding of glioblastoma biology while identifying patients with active signaling in the absence of target amplification, and holds promise for a more personalized and effective intervention for glioblastoma patients. p50 free neuropathol 6:17:73 meningioma sanity check: increasing classifier credibility through prototypical feature statistics kai schmid1, hildegard dohmen1, jannik sehring1, carmen selignow1, marco stein2, eberhard uhl2, felix sahm3, till acker1, daniel amsel1 1 justus-liebig university giessen, institute of neuropathology, giessen, germany 2 university hospital giessen und marburg location giessen, department of neurosurgery, giessen, germany 3 institute of pathology, university hospital heidelberg, department of neuropathology, heidelberg, germany background: meningiomas are the most common primary brain tumors. dna methylation-based classification has significantly improved diagnostic accuracy and prognostication. however, current classifiers — such as the dkfz heidelberg classifier — operate largely as black boxes, providing limited insight into how representative or atypical an individual case is within its predicted class. enhancing classifier transparency is essential for clinical trust and interpretability. methods: we analyzed 2,300 meningioma samples profiled with the illumina epic array platform, including all cases classified into a meningioma subtype, regardless of confidence score. fingerprinting techniques were applied to exclude recurrent samples. for each subtype, we computed features statistics across multiple axes: cnv profiling (conumee2), tumor microenvironment (methylcibersort, methyresolver, epidish, mdbraint), age, differential methylation regions (dmrs) and oncotree-based cnv mapping. dimensionality reduction (t-sne) and gradient boosting machine (gbm) were used to assess subtype separability. a composite prototypicality score is being developed to quantify how typical a case is within its predicted class. results: subtype assignments were consistent across methods and reflected distinct biological profiles. cnv analyses uncovered subtype-specific alterations correlating with clinical features. deconvolution identified heterogeneous immune and stromal cell populations. preliminary gbm models demonstrated predictive performance for subtype assignment. conclusion: this large-scale methylation study enhances understanding of methylation-based meningioma classification and supports the development of trust-enhancing tools. ongoing work aims to implement a prototypicality score and provide a web interface for classifier inspection and visualization from uploaded .idat files and clinical metadata. copyright: © 2025 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. preservation of cellular structure via immersion fixation in brain banking feel free to add comments by clicking these icons on the sidebar free neuropathology 6:4 (2025) original paper preservation of cellular structure via immersion fixation in brain banking macy garrood1, emma l. thorn2,3, adam goldstein2,3, allison sowa4, william janssen4, alyssa wilson5, claudia s. lópez6,7, raakhee shankar6, erin s. stempinski6, kurt farrell2,3, john f. crary2,3, andrew t. mckenzie1† apex neuroscience, salem, oregon, usa friedman brain institute, departments of pathology, neuroscience, and artificial intelligence & human health, icahn school of medicine at mount sinai, new york, new york, usa neuropathology brain bank & research core and ronald m. loeb center for alzheimer's disease, icahn school of medicine at mount sinai, new york, new york, new york, usa microscopy and advanced bioimaging core, icahn school of medicine at mount sinai, new york, new york, usa departments of neurology and psychiatry, icahn school of medicine at mount sinai, new york, new york, usa multiscale microscopy core, oregon health and science university, portland, oregon, usa department of biomedical engineering, oregon health and science university, portland, oregon, usa corresponding author: andrew t. mckenzie · apex neuroscience · 3265 marietta st se · salem · or 97317 · usa amckenzie@apexneuro.org submitted: 18 november 2024 accepted: 27 december 2024 copyedited by: georg haase published: 04 february 2025 https://doi.org/10.17879/freeneuropathology-2025-6104 keywords: brain banking, immersion fixation, histology quality, postmortem interval, myelin, synapse, vacuolization, volume electron microscopy abstract immersing the brain in a solution containing formaldehyde is a commonly used method for preserving the structure of human brain tissue in brain banking. however, there are questions about the quality of preservation using this method, as formaldehyde takes a relatively long period of time to penetrate a large organ such as the human brain. as a result, there is a critical need to determine whether immersion fixation is an adequate initial preservation method. to address this, we present exploratory histologic findings from our brain bank following the immersion fixation of hemi-sectioned brain specimens under refrigeration. using light microscopy, we found that there was no significant change in the size of pericellular or perivascular rarefaction areas based on the postmortem interval (pmi) or on the progression from the outer (frontal cortex) to the inner (striatum) brain regions. additionally, we did not identify any significant number of ghost cells – a state of late-stage cellular necrosis – in the light micrographs analyzed. using transmission electron microscopy of tissue from the frontal cortex, we found that synapses could still be visualized, but there was vacuolization and variable degrees of myelin disbanding identified. using serial section transmission electron microscopy, we found that identified synapses could be traced from one section to the next. using serial block face scanning electron microscopy, we also found that myelinated axons on 2d images can be traced with high fidelity from one image to the next, even at pmis of up to 27 hours. collectively, our data corroborate previous findings that immersion fixation is effective for prevention of cellular necrosis and for visualizing many ultrastructural features in at least the surface areas of the brain. however, how structural preservation quality should best be assessed in brain banking is an open question that depends on the intended research applications. abbreviations em electron microscopy, h hours, hfw horizontal field width, kv kilovolts, lhe luxol fast blue counterstained with hematoxylin and eosin, pmi postmortem interval, roi region of interest, sem scanning electron microscopy, sbf-sem serial block-face scanning electron microscopy, sstem serial section transmission electron microscopy, tem transmission electron microscopy, wsi whole slide image. introduction high-quality preservation of large samples of brain tissue for subsequent study is critical for many areas, including clinical care, research, and education. historically, whole brains were often preserved via immersion in solutions containing ethanol (horsley, 1997; schweizer et al., 2014). with the first patent for the large-scale production of formaldehyde solutions in 1891, there was considerable interest around that time in the use of formaldehyde as an antiseptic and also as a fixative in pathology (musiał et al., 2016). by the mid 1890s, formalin – a version of formaldehyde with a small percentage of methanol added for chemical stabilization – had already started to be widely used around the world for brain preservation, because it was found to be so effective in preserving microscopic structure (fish, 1895; musiał et al., 2016). today, immersion of a whole human brain or a hemi-sectioned half brain in formalin is widely used as a first step for many downstream applications. however, the effectiveness of this technique has been a subject of some debate, particularly regarding its capacity to adequately preserve deep brain structures before the cells decompose (grinberg et al., 2008; mcfadden et al., 2019). this concern is heightened in the context of human brain disorders, where precise preservation is essential for accurate clinical diagnosis and research efforts (yang et al., 2022). as a result, better understanding immersion fixation methods is critical for ensuring the integrity of tissue samples used in neuropathological research, particularly for studies aimed at unraveling the underlying mechanisms of human brain disorders and paving the way for improved treatments. one key alternative to the immersion fixation of whole or hemi-sectioned brain specimens is the use of biopsy samples. surgical biopsies yield some of the best-preserved human brain tissue and have been used for the largest-scale volume electron microscopy (em) studies in humans to date (oost et al., 2023; shapson-coe et al., 2024). researchers have invested considerable effort in optimizing fixation and processing methods for these biopsies, particularly for em studies (karlupia et al., 2023; rollenhagen et al., 2024). the primary advantage of surgical biopsies is the minimal ischemic time, often just minutes between tissue removal and fixation initiation. however, the availability of brain biopsies is limited to specific clinical scenarios requiring surgical intervention. in the autopsy setting, it is also common practice to obtain small biopsy samples of the brain for em, and often these biopsies are fixed separately in glutaraldehyde (kay et al., 2013; sele et al., 2019). this approach also minimizes the ischemic time exposure for that sample of tissue. on the other hand, there are also advantages to using whole or hemi-sectioned brain specimens that have been immersion fixed for em studies, as this allows for the use of already archived tissue and for histological studies across the entire brain. another alternative to immersion fixation is perfusion fixation. this involves the pressure-driven delivery of fixative through the vascular system, which can lead to more rapid and uniform tissue preservation. perfusion fixation has the obvious potential advantage of reducing the time required for fixation, which can be useful for expediting the neuropathologic exam and for preventing overfixation in surface areas (beach et al., 1987; adickes et al., 1997; sharma and grieve, 2006). several studies have also reported that perfusion fixation can improve the preservation of histologic features compared to immersion fixation, especially in inner brain regions such as the thalamus and basal ganglia (grinberg et al., 2008; mcfadden et al., 2019). however, this latter finding has not been reported by all studies. one study found no significant difference in staining quality between perfusionand immersion-fixed brain tissue, provided that immersion fixation was allowed sufficient time to fix the entire brain (sharma and grieve, 2006). some of the reported relative advantages of perfusion fixation may have been accentuated because the control immersion fixation procedures did not use refrigeration during immersion, which has been found to be critical for preservation quality, because it dramatically slows down cellular decomposition (mckee, 1999). moreover, perfusion fixation is technically more complex, not always available, can introduce tissue edema, and can be ineffective in cases with long pmis or prolonged agonal states. taken together, perfusion fixation has potential advantages in certain circumstances, but additional research is also warranted to better characterize immersion fixation. one important question is how quickly formalin penetrates to preserve different parts of the immersed brain. there have been several neuroimaging studies on this topic (yong-hing et al., 2005; dawe et al., 2009; dadar et al., 2024). however, the time it takes for the fluid to alter imaging parameters is not necessarily the same as the time it takes for preservative chemicals to penetrate the brain tissue to sufficiently slow down cellular decomposition. moreover, there is an important potential distinction between two timeframes: the initial short time needed for the fixative to enter a region of tissue versus the subsequent longer time required for tissue fixation to be sufficiently crosslinked for downstream histological processing (helander, 1999). this potential difference is not well studied. formalin fixation has been found to occur in a manner where time is roughly proportional to the depth penetrated, unlike some other chemicals where time is more proportional to the square root of the depth (dempster, 1960). one study on rabbit liver tissue found that 10 % formalin fixed a tissue depth of approximately 0.5 cm in 9 h (dempster, 1960). another study found that after 24 h of immersion fixation of whole human brains in 20 % formalin at room temperature, formalin had fixed the tissue 1–2 cm from the surface of the brain and adjacent to the ventricles (scott and macdonald, 2013). formalin penetration rates have been reported to become faster with higher temperature, a higher concentration of formaldehyde, and more postmortem decomposition (dempster, 1960). while penetration speed is of academic interest, the main practical question for histological quality is the degree to which the tissue has degraded prior to the moment when reached by the chemical preservative, which also depends on the rate of postmortem decomposition (krassner et al., 2023). there have been some empirical studies on the microscopic preservation quality of immersion-fixed brain tissue in non-human animals. outcomes vary based on the intended research application and the level of structural detail required. among applications using light microscopy, several studies have reported favorable results. for example, one study found that staining in mouse brains was of acceptable quality whether the tissue was fixed by perfusion or immersion (wahlsten et al., 2003). even in extremely large brains such as those of minke whales, researchers reported that immersion fixation resulted in generally good preservation across the brain, with only occasional artifacts observed in inner brain regions (knudsen et al., 2002). however, for certain types of downstream applications, perfusion fixation has been found to have advantages. one review noted that while immersion fixation is generally sufficient for routine toxicology studies, perfusion fixation is preferred for detailed neurotoxicity investigations (bolon et al., 2013). some studies have highlighted specific limitations of immersion fixation for em. one investigation observed alterations in mitochondrial morphology in large tissue blocks from mouse brains that were fixed via immersion (hinton et al., 2023), while another reported potential issues with visualizing microvasculature in scanning electron microscopy (sem) studies due to vessel collapse (lossinsky and shivers, 2003). another study found adequate tissue preservation allowing synaptic quantification in the surface areas of mouse brains, but noted some qualitative artifacts (monfils et al., 2005). these findings suggest that while immersion fixation can provide adequate preservation for many applications, the choice of the fixation method should be carefully considered based on the specific requirements of each study, particularly when examining ultrastructural details. in the context of human brain banking, there have also been some empirical reports on the preservation quality achieved when using immersion fixation of large tissue specimens. a common finding is that while immersion fixation can preserve certain cellular structures, it may not be optimal for all types of histochemical analyses. for example, one study comparing immersion and perfusion fixation in human brains found that immersion fixation led to decreased parvalbumin staining for fine neural processes and reduced staining for cytochrome oxidase, although nissl and myelin staining remained unchanged (wallace et al., 2002). studies focusing on ultrastructural preservation have yielded mixed results. one group found that synapses could still be visualized in the human cerebral cortex using focused ion beam/scanning em, even in tissue obtained at autopsy with a pmi of up to 4 h (cano-astorga et al., 2021). a separate study reported that immersion fixation of brains in formalin from donors with pmis of 18 to 50 h led to acceptable ultrastructural quality in the anterior cingulate cortex, with the exception of myelin lamellae splitting (krause et al., 2016). however, it has also been reported that although vascular morphology is reasonably preserved, cellular organelles can be lost in immersion-fixed human brain tissue (yamamoto et al., 2013). despite the widespread use of immersion fixation in brain banking, there remains a critical need to further evaluate its effectiveness in practical brain banking settings. the present study aims to bridge this gap by examining the quality of tissue preservation achieved through immersion fixation of human hemi-sectioned brain specimens under refrigeration. immersion fixation of hemi-sectioned brain samples is a common practice in brain banking because the other of the two hemispheres is often used non-fixed for cryopreservation. in this study, we employ a multimodal approach, using light microscopy to assess large areas of brain tissue and em techniques to evaluate detailed histologic features. our focus is on quantifying the degree of cellular structure preservation in tissues that may not be rapidly fixed via immersion, with particular attention to pericellular and perivascular spaces, cellular necrosis, synapse identification, and myelin integrity. by providing a detailed characterization of preservation quality across different brain regions and various pmis, our goal is to inform best practices in brain banking and to contribute to better understanding the limitations and capabilities of immersion fixation for various research applications. methods brain banking procedures all specimens were obtained and de-identified at the icahn school of medicine at mount sinai in accordance with its policies, regulations, and institutional review board. the brain hemisphere specimens were preserved with the standard protocol of immersion fixation that has been established in the neuropathology brain bank & research core. after extraction, the brain is transported via a plastic bag to the neuropathology brain bank & research core. the brainstem and cerebellum are isolated via dissection. the rest of the brain is then sectioned into two hemispheres at the level of the corpus callosum. one hemisphere is immersed in 10 % neutral buffered formalin (fisher 245–685) under refrigeration at 4 °c for at least two weeks and up to a period of several years. the hemisphere is then dissected into 3–5 mm thick coronal slabs and specific brain regions are sampled for neuropathologic study, as previously described (mckenzie et al., 2022). eventually, the storage solution is switched to phosphate buffered saline containing 0.1 % sodium azide (sigma aldrich, cas 26628-22-8) for long-term fluid preservation at 4 °c. light microscopy brain tissue sampled for light microscopy was placed into cassettes for processing and embedded in paraffin. next, 5–7 μm thick sections were cut on a microtome, which were mounted on glass slides, deparaffinized, and stained with luxol fast blue counterstained with hematoxylin and eosin (lhe). slides were then imaged as whole slide images (wsis) using a digital slide scanner (hamamatsu photonics). using ndp.view2 software (hamamatsu photonics), three representative regions of interest (rois) from the gray matter were captured from each wsi for subsequent analysis, attempting to provide coverage of distinct parts of the slide. our assessment focused on two key indicators of tissue preservation: (a) pericellular/perivascular rarefaction and (b) cellular necrosis (krassner et al., 2023). rarefactions were quantified in each roi by (a) measuring the average of the non-staining pericellular space for at least 3 of the largest cells and (b) measuring the average of the non-staining perivascular space for up to 3 of the largest blood vessels. the maximum width of the pericellular spaces was measured using the measurement tool in ndp.view2. perivascular spaces were measured at the widest part of the shortest axis using the measurement tool in imagej. the presence of late-stage cellular necrosis was assessed in each roi using a binary (present/absent) grading system. this assessment was based on the identification of a substantial proportion of ghost cells in the roi by two independent reviewers, which is a characteristic finding of brain tissue in late-stage necrosis (finnie et al., 2016). ghost cells were identified by (a) nuclei that are either absent or barely visible, (b) pale staining cytoplasm, and (c) indistinct cell membrane morphology (finnie et al., 2016). any grading discrepancies were resolved through a consensus review process among the raters. electron microscopy for em, gray matter regions of the frontal cortex from fluid preserved specimens were dissected and further fixed in a solution of 2 % paraformaldehyde and 2 % glutaraldehyde in 0.1 m sodium cacodylate buffer, as previously described (krassner et al., 2023). a version of the national center for microscopy and imaging research (ncmir) protocol was adapted to provide enhanced contrast (deerinck et al., 2010). this approach uses multiple methods of chemical fixation standard for em. the brain sample was then dehydrated through a graded ethanol series, infiltrated with embed 812 epoxy resin (ems), and polymerized for 72 h at 60 ° c. semithin sections (0.5 μm) were cut using a leica uc7 ultramicrotome (leica, buffalo grove, il) and counterstained with 1 % toluidine blue to identify the regions of interest within layers. ultra-thin sections of 80 nm thickness were collected onto nickel slot grids (ems, fcf2010-ni) and the grids were imaged on an ht7700 transmission electron microscope (tem) (hitachi high-technologies, tokyo, japan) using an advantage ccd camera (advanced microscopy techniques, danvers, ma). some of the tem images presented were adjusted for contrast, image size, and with a sharpening filter if needed. both the raw and adjusted tem images were made publicly available. for two of the samples, we performed serial section transmission electron microscopy (sstem), using ultra-thin sections of 80 nm thickness. each series consisted of 3 to 10 consecutive sections. the same embedded tissue was also imaged using serial block-face scanning electron microscopy (sbf-sem). images were acquired with a pixel size of 10 x 10 x 50 nm3, using a thermo fisher scientific apreo volumescope ii serial block face sem. data from both samples was collected with a horizontal field width (hfw) of 61 μm and an accelerating voltage of 2 kv. imaging data from one sample had one region of interest and a depth of 25 μm, while imaging data from the other sample was collected with depths of 10 μm and 20 μm, in two regions of interest each. all datasets were aligned and cropped to correct for sample drift using amira software (thermo fisher™). the sbf-sem datasets were named based on the donor identifier and the area where the images were taken from. the first number was the donor number, and the second number was the depth of the image. l and r correspond to the hemisphere of the frontal lobe. donor 100 had multiple sets of images taken, from two different depths (10 μm and 20 μm), and from two different representative regions at each depth. em images were viewed with the fiji distribution of imagej as well as with webknossos (boergens et al., 2017). four distinct analyses were conducted on the different types of em images, measuring myelin disbanding, myelinated axon density, synapse traceability, and myelinated axon traceability. the first two analyses were performed on standard 2d images using imagej software. to measure myelin disbanding, we selected the myelinated axon in the image with the thickest myelin sheath and measured the length of this thickest part (adapted from (sele et al., 2019)). along this length, we identified segments that appeared distinctly lighter than the surrounding dark myelin and measured their lengths. the percentage of disbanding was then calculated using the formula: (sum of light segment lengths / total measured length) × 100. for myelinated axon density, we outlined each myelinated axon in the image and calculated the area of each outlined axon using imagej. we then summed the areas of all myelinated axons and calculated the density as (total area of myelinated axons / total image area) × 100. the next two analyses, on synapses and myelinated axons, were performed on the serial section tem and sbf-sem data sets, respectively. synapses within the serial sections were chosen for analysis based on having clear visibility of synaptic structures and the ability to identify both the beginning and end of the synapse within consecutive sections. synaptic cleft length and width measurements were performed using imagej software. these measurements were then multiplied by the known section thickness (80 nm) to calculate the synaptic volume per image in cubic nanometers and then converted to cubic micrometers (adapted from (bloss et al., 2013)). the total synaptic cleft volume was determined by summing the calculated volumes across all relevant sections. for the traceability analysis on myelinated axons, the sbf-sem data was uploaded to webknossos for analysis. myelinated axons were identified by their characteristic dark banding and then traced to the last image containing the axon. specifically, using the skeleton tool on webknossos, the axon was traced until the axon ended or there were no longer any images containing the axon. results light microscopy we studied a convenience sample of n = 40 brains from the mount sinai neuropathology brain bank & research core. the age range was from 33 to 87 years (mean = 65.4 years), the pmi ranged from 9.1 to 81.1 h (mean = 26.5 h). neuropathological workups revealed a range of diagnoses including alzheimer's disease, cerebrovascular disease, and cases with no diagnostic abnormality recognized. we studied lhe-stained wsis from three brain regions, which had variable coverage across the cohort: the frontal cortex (at least one wsi available in n = 32 or 80.0 % donors), anterior striatum (n = 35, 87.5 %), and posterior striatum (n = 28, 70.0 %). in n = 23 (57.5 %) donors, at least one wsi was available from all three brain regions. these three brain regions were chosen in order to include both a surface area (frontal cortex) as well as inner areas (anterior striatum, posterior striatum) of the hemi-sectioned brain specimen. our goal was to look for differences in preservation quality associated with the relatively slower fixation achieved in the inner areas. qualitatively, we found that the overall tissue preservation visible on light microscopy across all examined samples maintained cellular structures that were generally discernible and identifiable (figure 1). expected postmortem artifacts were present, especially pericellular rarefactions, perivascular rarefactions, and vacuolization of the parenchyma. otherwise, the cellular morphology appeared largely as expected across all regions, with neurons, glia, and blood vessels retaining their characteristic shapes. luxol fast blue staining of myelin was also present across samples. we could not identify clear qualitative differences in the preservation quality between the surface areas (frontal cortex) and the inner areas (anterior and posterior striatum) of the hemi-sectioned brain specimens, although there were inherent differences in cellular morphology between regions that prevented us from performing quantitative comparisons of cell shape or other cellular features as a proxy for preservation quality. in a few cells across all three brain regions, we detected what potential signs of early necrosis, such as partially disrupted membranes, but these were not widespread or common enough to distinguish them from potential artifacts, such as staining inconsistencies or image blurring. figure 1. representative light microscopy images from several brain region studied. lhe-stained tissue sections from the frontal cortex (a–c), anterior striatum (d–f), and posterior striatum (g–i) of an immersion-fixed hemi-sectioned brain specimen. panel (a) shows a low-magnification overview of the frontal cortex, with black rectangles indicating regions of interest examined at higher magnification in (b) of gray matter and in (c) of white matter. similar triads of representative images are shown for the anterior striatum (d–f) and the posterior striatum (g–i). all samples were obtained from a 63-year-old male brain donor who died of respiratory failure due to malignant mesothelioma, with a pmi of 23.6 h before tissue preservation. scale bars in (a, d, and g) are 5 mm; scale bars in higher magnification images are 100 μm. to evaluate for potential late-stage necrosis, two reviewers evaluated the presence of ghost cells in each of three gray matter regions of interest for all the wsis available. any given cell may have apparent characteristics of ghost cells, because of problems with staining or visualization, with a preponderance of these aspects at late-stage necrosis (finnie et al., 2016). out of the 321 regions of interest (rois) analyzed, we identified two rois that appeared to potentially have a substantially elevated number of ghost cells, although this may reflect differences in staining or imaging (figure 2). these two rois with elevated numbers of ghost cells were found in samples from the posterior striatum, with pmis of 53.5 h and 70.5 h. the tissue in these rois may represent areas that degraded especially rapidly for an unknown reason, or that may have been differentially affected by an artifact in the processing or imaging procedure. for the other two rois from these two samples, there was not a substantial number of ghost cells identified. taken together, these observations indicate that the immersion fixation procedure generally maintained the histologic architecture on light microscopy as might be expected on a biopsy sample from autopsy brain tissue, with very few or no areas exhibiting substantial cellular necrosis. figure 2. light microscopy of regions of interest showing a potentially significant number of ghost cells. the regions of interest were classified as having at least three potential ghost cells identified. ghost cells are characterized by having absent or barely visible nuclei, pale-staining cytoplasm, and indistinct cell membrane morphology. asterisks indicate cells potentially having these characteristics. it is unclear if staining or imaging factors could also contribute to this appearance. a: lhe-stained tissue section from the posterior striatum of a 62-year-old brain donor, with a pmi of 53.5 h. b: lhe-stained tissue section from the posterior striatum of a 76-year-old brain donor, with a pmi of 70.5 h. scale bars: 100 μm. to measure whether decomposition might vary quantitatively with pmi or across brain regions, we measured the size of pericellular and perivascular rarefaction areas (figure 3). these rarefaction areas are common postmortem artifacts that are visible within the early postmortem period and appear to be due to the swelling of tissue elements such as astrocyte processes (krassner et al., 2023). for each brain region sampled, we determined the arithmetic mean sizes of the pericellular and perivascular rarefaction areas measured across all three of the gray matter regions of interest selected. we found that there was no significant difference between these measures in the surface brain region studied (frontal cortex) and the two inner brain regions studied (anterior striatum and posterior striatum) (kruskal-wallis tests: for pericellular rarefaction, h(2) = 1.93, p = 0.381; for perivascular rarefaction, h(2) = 3.71, p = 0.156). we did not identify significant rank correlations between these measures and the pmi in any of the three brain regions studied (all |ρ| < 0.25, p > 0.05). we did find one nominally significant pearson correlation between the pericellular rarefaction area size and the pmi in the frontal cortex (r = 0.371, p = 0.033), but this became non-significant after adjusting for multiple comparisons using the benjamini-hochberg false discovery rate correction (fdr-adjusted p = 0.20). note that our pericellular and perivascular area measurements may have been influenced by 3d tissue sectioning orientation, independent of postmortem fluid accumulation. that is, it is possible that the visible space around cells and vessels varied based on the angle of the cut during sample preparation. additionally, it is possible that an effect would have been distinguished with a larger sample size. acknowledging these potential sources of variability, we did not identify a clear relationship between the measures of rarefaction and either the pmi or the brain region. this finding suggests that while these areas of rarefaction are consistently seen in postmortem brain tissue, they do not seem to be a linear proxy for the duration of ischemia prior to fixation, at least in this data set. these results are consistent with a model in which the rarefactions first occur due to the retrograde flow of cerebrospinal fluid occurring early in the pmi and then remain relatively stable for a period of time (du et al., 2022; krassner et al., 2023). it is possible that the rarefaction areas may break down in more advanced decomposition, as the cell membranes of astrocytes and other cell types begin to degrade and therefore allow water to equilibrate across the sample. this phenomenon could then be interpreted as a sign of advanced necrosis. however, we did not observe this phenomenon in the data we analyzed. figure 3. the sizes of pericellular and perivascular rarefaction areas are not significantly different across brain regions nor significantly correlated with the pmi. a: representative image showing measurements of the three largest pericellular rarefaction areas in a region of interest. scale bar: 100 μm. b: distributions of the average sizes of pericellular rarefaction areas in each brain region. horizontal lines represent arithmetic means. no significant differences were found across brain regions. c–e: scatter plots showing the pmis and average pericellular rarefaction size measured for each brain donor across the frontal cortex, anterior striatum, and posterior striatum, respectively. f: representative image showing sizes of the three largest perivascular rarefaction areas in a region of interest. scale bar: 100 μm. g: distributions of average sizes of perivascular rarefaction areas within each brain region. horizontal lines represent arithmetic means. no significant difference was found across brain regions. h–j: scatter plots showing the pmis and average sizes of perivascular rarefaction areas measured for each brain across the frontal cortex, anterior striatum, and posterior striatum, respectively. for all scatter plots, the black lines represent linear regression fits, with gray areas indicating 95 % confidence intervals. spearman's rho and associated p values are shown for each brain region. 2d electron microscopy using em, we studied ten brain donors from the same brain bank (table 1). we dissected fixed tissue from gray matter of the middle frontal gyrus, a part of the frontal cortex, and performed tem on the embedded samples. the pmis of the brains prior to immersion fixation ranged from 10.75 h to 27 h, with an arithmetic mean of 20.6 h. qualitatively, the tem images showed expected postmortem artifacts such as vacuolization and some areas with apparently decreased cytoplasmic contrast (figure 4). however, the images showed that cell membrane contours were largely discernible and maintained much of their expected morphology, despite some potential postmortem alterations. synapses and mitochondria could be identified in at least one image from all ten samples studied. notably, the duration of refrigerated fluid preservation prior to sample processing ranged from 0.1 to 3.4 years, with no apparent differences depending on this variable. table 1: characteristics of brain donors whose tissue was profiled with em. sample number age sex postmortem interval (hours) duration of fluid preservation (years) npbb 36* 50 m 27 3.4 npbb 40 52 m 20 3.3 npbb 57† 53 f 25 2.9 npbb 98 77 f 15 2.2 npbb 100† 85 f 10.75 2.1 npbb 113 89 f 21 1.8 npbb 198 71 m 21.1 0.8 npbb 233* 87 m 26.5 0.4 npbb 265 67 f 22.75 0.2 npbb 267 86 m 17.5 0.1 the duration of fluid preservation refers to the length of time that samples were stored in 10 % neutral buffered formalin and/or phosphate buffered saline with 0.1 % sodium azide prior to sample preparation for em. *: samples used for sstem, †: samples used for sbf-sem. figure 4. representative tem images show qualitative preservation of synapses and mitochondria. all tissues are from the frontal cortex. asterisks indicate two representative structures: synapses (red) and mitochondria (blue). donor numbers: 36 (a), 40 (b), 57 (c), 98 (d), 100 (e), 113 (f), 198 (g). 233 (h), 265 (i), 267 (j). scale bars: a-c, e, h, i: 1 μm; d, f, g, j: 2 μm. quantitative analysis of the tem images revealed several findings. varying degrees of myelin disbanding were seen across samples, a phenomenon known to occur early in the pmi (krassner et al., 2023). myelin disbanding was found to be present in 81 % (87/107) of the largest myelinated axons seen in each high-resolution image. the degree of myelin disbanding ranged from 0.0 % to 71.5 % across samples. note that some degree of myelin disbanding can be present even in em images from human surgical biopsy specimens that are preserved via immersion fixation (shapson-coe et al., 2024). the density of myelinated axons varied across images, ranging from 0 % to 21.5 %, with an average density of 4.2 %. the highest number of myelinated axons identified in one image was eleven. notably, in the ten brain donors, there was no significant rank correlation between the myelin disbanding percentage or the myelinated axon density and the pmi (myelin disbanding percentage: ρ = 0.21, p = 0.56; myelinated axon density: ρ = 0.006, p = 1.00; figure 5). these results suggest that while postmortem changes are evident, substantial structural integrity is maintained in many myelinated axons. figure 5. quantification of the degree of myelin disbanding and the density of myelinated axons in tem images. a, b: representative images of myelin disbanding in tem images, showing examples of highly disbanded (a) and non-disbanded (b) myelin. c: scatter plot showing the percentage of myelin disbanding and the pmi. blue points indicate the mean myelin disbanding for each sample; gray points indicate measurements from each individual image. d, e: a representative image with a particularly high density of myelinated axons (d) and the quantification of myelinated axon density in this image (e), i.e. the sum of areas containing myelinated axons (red) divided by the total area. f: scatter plot showing the myelinated axon density and the pmi. blue points indicate the mean myelinated axon density for each sample; gray points indicate measurements from each individual image. all points in scatter plots are partially transparent to account for overplotting. scale bars: 2 μm. volume electron microscopy to further characterize synaptic preservation, we conducted sstem on two samples with pmis of 26.5 and 27 h. we focused the imaging on cortical layers ii/iii, which have been found to have a relatively moderate to high synaptic density (anton-sanchez et al., 2014). qualitative analysis of the resulting image stacks revealed that all 18 synapses that met our criteria for tracing could be consistently traced across adjacent sections (figure 6). to quantify synaptic dimensions, we approximated synaptic cleft volumes by measuring cleft length and width in each section and multiplying this area by the section thickness. the mean synaptic cleft volume was 1.731 × 10-3 μm3, with a range of 4.01 × 10-3 μm3 to 3.609 × 10-3 μm3, and a standard error of the mean of 7.16 × 10-4 μm3. this substantial variability in synaptic size aligns with previous reports on the variability of synaptic size measurements (morales et al., 2013; santuy et al., 2018). it is important to note that our analysis excluded synapses extending beyond the image stack boundaries, with a potential bias of our sample towards smaller synapses. our ability to trace identified synapses across multiple sections suggests that the structure of the synaptic cleft remains largely intact in these samples, despite the pmi, at least within the subset of synapses we were able to measure. figure 6. representative images showing synapses that are traceable in sstem images. all synapses are all fully encapsulated in the set of four images. the samples are from donor numbers 36 (a, b) and 233 (c, d, e). scale bars: 800 nm. to measure the traceability of neural structures over longer distances, we next performed sbf-sem on two samples (samples 57 and 100, with pmis of 25 h and 10.75 h respectively). we tested the degree to which myelinated axons could be traced across the available volumes (figure 7). we found that all 25 myelinated axons that we attempted to trace were easily traceable (figure 8). overall, these findings suggest that the 3d structural integrity of myelinated axons is maintained in these immersion-fixed tissue samples, allowing for reliable tracing. figure 7. example of tracing a myelinated axon in the sbf-sem data set. the images were taken at a depth of 25 μm in donor sample number 57. scale bars: 2.0 μm. figure 8. skeleton tracings of myelinated axons from sbf-sem data sets. tissues were from brain donor numbers 57 (a) and 100 (b, c, d, e). volumes were imaged at three different depths: 25 μm (a), 20 μm (c, e), and 10 μm (b, d). discussion in this study, we employed both light and electron microscopy to characterize the histological quality of human hemi-sectioned brain specimens preserved via immersion fixation under refrigeration, which is a common method of brain banking. our findings demonstrate that immersion fixation can effectively maintain overall tissue architecture and cellular structure. expected postmortem changes were observed, including pericellular and perivascular rarefaction areas, but the extent of these rarefaction areas did not correlate significantly with the brain region or the pmi over the ranges we studied. we also found that neural structures, including myelinated axons and synapses, remained traceable across volume em data in samples with pmis of up to slightly more than 24 h. this observation may help to define an extended time window where em analyses of autopsy tissue can be effectively performed (cano-astorga et al., 2024). of note, several of our samples were preserved in fluid for more than one year, without any identified effect on cellular morphology, adding to our knowledge about the effects of fluid preservation on brain cell structure (liu and schumann, 2014; mckenzie et al., 2024). while perfusion fixation or immersion fixation of small biopsy brain samples may have advantages over immersion fixation, our results suggest that routine immersion fixation of brain tissue may provide sufficient preservation quality for many types of downstream applications. there are many limitations of our current data set and analysis methods, which warrant careful consideration. measuring histologic preservation quality is indeed a challenging and multifaceted problem. previous state-of-the-art approaches in this area have tended to be semi-quantitative (kiernan, 2009; lindblom et al., 2021). a fundamental issue of our approach is whether our metrics for measuring postmortem decomposition are valid. using light microscopy, we quantified the pericellular and perivascular rarefaction areas and the presence of ghost cells, which may not necessarily capture the most important postmortem changes. the selection of these metrics was based on our own observations and on previous literature, rather than on an unbiased, comprehensive understanding of the mechanistic alterations that occur in the postmortem brain. we may thus have missed more nuanced changes that may escape the attention of a trained histologist. this limitation underscores the need for more unbiased approaches to assessing tissue preservation. future research could benefit from employing machine learning algorithms to identify subtle structural and molecular changes that occur during the pmi. for example, with larger sample sizes, it might be possible to use deep learning to discover features that distinguish tissue that has experienced short versus long pmis in an unbiased manner. such a project would advance our ability to more objectively measure the loss of preservation quality due to tissue decomposition. em data presents particular complexities in assessing tissue preservation quality, due to the many potential artifacts or distortions that can be introduced during sample preparation and imaging (hayat, 1981). it is therefore challenging to distinguish between genuine loss of microstructural integrity in banked brain tissue and artifacts from the assay procedure itself. for example, image blurriness in tem can be a function of magnification rather than tissue quality. given these complexities, our em analysis focused on structures with characteristic appearances, primarily synapses and myelin. we found that myelin, which has been reported to degrade relatively rapidly during the pmi (krassner et al., 2023), can serve as a practical metric for preservation quality due to its easy identification in em images. however, this approach has downsides. while measuring myelinated axon density can help bound the degree of tissue degradation, it varies significantly across brain regions and disease states, limiting its standalone utility. also, while myelin decomposition could theoretically be a proxy for the decomposition of other cell membranes as well, it is difficult to establish this, because other cell membranes have less reliable contrast and a less characteristic form. ideally, a comprehensive assessment would involve annotating all visible structures, amounting to a type of panoptic segmentation (peters et al., 1991; nahirney and tremblay, 2021; liu et al., 2024). developing such tools for em data would require training on a large set of expertly annotated images from partially degraded brain tissue, a resource that is currently lacking. moreover, many membrane-bound structures in 2d em images are ambiguous (e.g., axons, dendrites, or glial processes) and require volume imaging data for identification. these challenges underscore the need for advanced analytical tools and standardized datasets to improve our ability to assess preservation quality in em studies of imperfectly preserved brain tissue. in volume em, traceability has emerged as a key metric for assessing the quality of connectome data sets. for example, one study found that finer neural structures are more challenging to trace across sections than larger structures, requiring thinner sections for reliable reconstruction (hayworth et al., 2015). in our volume em data sets, we found that both synapses and myelinated axons, which are relatively thicker compared to fine, unmyelinated cell processes, were easily traceable. this suggests that our immersion fixation protocol adequately preserves these thicker structures for potential volumetric analysis. however, it is important to note that our study did not attempt to trace fine, unmyelinated processes, which may be more susceptible to postmortem changes. the traceability of neural structures in volume em raises another practical question: to what extent can 3d traceability be predicted from 2d images? based on our qualitative experience analyzing this data, we suspect that the signal-to-noise ratio of cell membranes in individual 2d images may serve as a useful proxy for 3d traceability. however, this hypothesis requires formal testing. future studies could systematically evaluate the relationship between 2d image quality metrics – such as cell membrane contrast and continuity – and the success rate of 3d tracing in volume em datasets. such research could potentially lead to the development of ultrastructural quality assessment tools for brain bank samples, allowing researchers to predict the suitability of tissue for volume em studies. another key limitation of our study is the absence of em data from inner brain regions. our initial approach was to start with a surface brain region for em analysis, as we were uncertain about the overall quality of preservation achievable anywhere in the brain using our immersion fixation protocol at the given pmis. we reasoned that if the surface regions, which are exposed earliest to the fixative, showed poor preservation, it would be necessary to reassess our methods for exploring deeper structures. however, since our approach yielded acceptable preservation quality in the surface region analyzed, important questions about the quality of tissue preservation after immersion fixation can now be addressed in inner brain regions. while some previous data has suggested that ultrastructural quality can be at least acceptable in deeper brain regions (krause et al., 2016), the existing data on this topic are minimal, so substantial additional data will be required to corroborate these findings across various brain regions and under different fixation conditions. currently, the extent to which cellular features are altered in deep brain regions when visualized via electron microscopy is not well established. to address this limitation in a separate future publication, we plan to perform similar analyses of ultrastructural preservation quality in multiple inner brain regions. such data will be critical for understanding the full capabilities and limitations of immersion fixation in brain banking. over recent years there has been research on new technologies to “unlock” genomic data in specimens preserved in formalin in museum collections around the world (hahn et al., 2022). drawing parallels to this research, we propose that it may one day be possible to unlock connectome data from immersion-fixed brain tissue stored in brain banks. the feasibility of this approach likely depends on the degree of artifacts present in the samples, which can vary based on the specific fixation and fluid preservation protocols used (mckenzie et al., 2024). additionally, realizing this possibility would require substantial advances in embedding long-term fluid-preserved specimens. the duration of ischemia and prolonged storage almost certainly alter the optimal parameters for sample preparation and imaging. nevertheless, based on our current findings, we suggest that the fundamental structural elements required for connectome analysis may be present in at least a subset of immersion-fixed brains. this is particularly significant given that the vast majority of fixed brain bank specimens have been, and continue to be, preserved using immersion fixation. while technical challenges and scientific questions about feasibility certainly remain, we propose that more exploratory research should be performed on the possibility of leveraging existing brain bank resources for human connectomics studies. conclusions our study of immersion-fixed hemi-sectioned brain specimens provides evidence that this brain banking method results in adequate preservation of cellular and subcellular structures, even at pmis that are commonly achievable in brain banking. light microscopy revealed generally well-maintained tissue architecture, with no significant correlation between the measured features of decomposition and the pmi or the brain region. em demonstrated the preservation of characteristic ultrastructural features, including traceable synapses and myelinated axons in volume imaging. while expected postmortem changes such as vacuolization and myelin disbanding were observed, these did not preclude the identification and analysis of critical neural structures. our findings suggest that immersion-fixed brain bank specimens may be suitable for a wider range of research applications than commonly recognized, potentially including connectomics studies. however, we acknowledge the limitations of our current metrics for assessing preservation quality and the need for more sophisticated, unbiased approaches. we suggest that future research should focus on developing advanced analytical tools for measuring tissue integrity, expanding ultrastructural analyses to deeper brain regions, and exploring the potential for profiling volume em data from archival brain bank specimens. as our understanding of postmortem tissue preservation advances, we may unlock new ways to leverage brain bank resources to elucidate the mechanisms of neurobiological disorders. author contributions m.g., k.f., j.c., and a.t.m. conceptualized the article. a.s., w.j., j.c., and a.t.m. designed the electron microscopy experiments. a.t.m. performed article searching and screening. e.t. performed light microscopy experiments. a.s., w.j., c.s.l., r.s., and e.s.s. performed electron microscopy experiments. m.g., a.g., a.w., and a.t.m. performed data analysis. m.g. and a.t.m. wrote the initial draft of the manuscript. all authors reviewed the manuscript. all authors approved the final manuscript. acknowledgements we would like to thank kenneth hayworth for helpful personal communications on this topic. the icahn school of medicine at mount sinai provided access to library resources. electron microscopy tissue preparation and imaging were performed at the microscopy and advanced bioimaging core at the icahn school of medicine at mount sinai. serial block face electron microscopy was performed at the multiscale microscopy core, a member of the ohsu university shared resource cores rrid:scr_022652. funding this work was supported by the rainwater charitable foundation as well as nih grants p30 ag066514, k01 ag070326, rf1 ag062348, p30 ag066514, rf1 ns095252, u54 ns115266, and rf1 mh128969. the funders had no role in the design of the study or in the collection or interpretation of the data. conflict of interest macy garrood and andrew mckenzie are employees of oregon brain preservation, a non-profit brain preservation 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of progressive formalin fixation of the human brain. magn. reson. med. 54, 324–332. https://doi.org/10.1002/mrm.20578 copyright: © 2024 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. capillary basal lamina in human brain and spinal cord has fibrillar collagen type i and type iii: ignorance may not be bliss feel free to add comments by clicking these icons on the sidebar free neuropathology 6:6 (2025) original paper capillary basal lamina in human brain and spinal cord has fibrillar collagen type i and type iii: ignorance may not be bliss wen-lang lin1, dennis w. dickson1 1 department of neuroscience, mayo clinic, jacksonville, florida, usa corresponding author: dennis w. dickson · department of neuroscience · mayo clinic · 4500 san pablo road · jacksonville, fl 32224 · usa · dickson.dennis@mayo.edu additional resources and electronic supplementary material: supplementary material submitted: 18 december 2024 accepted: 25 january 2025 copyedited by: vanessa s. goodwill published: 18 february 2025 https://doi.org/10.17879/freeneuropathology-2025-6159 keywords: capillary basal lamina, fibrillar collagens, central nervous system, electron microscopy, immunohistochemistry, species difference abstract the capillary basal lamina (bl) located between the endothelial cell, pericyte and perivascular astrocyte plays important roles in normal and diseased central nervous system (cns). using immunohistochemistry (ihc), electron microscopy (em) and post-embedding immunogold em (iem), we studied capillary bl in biopsy and autopsy tissues of human cns from cases with and without significant brain pathology and aged from 4 days to 49 years. in all cases, ihc showed, in the bl of microvessels, immunoreactivity for collagen types i, iii, iv, vi and fibronectin. em revealed fusion of the bl of capillary endothelial cells or pericyte with perivascular astrocyte bl, which was focally split, resulting in expanded spaces bordered by bl and containing striated fibrils. there was no significant thickening of fused or split bl. iem showed localization of collagen i and iii to banded fibrils, and of collagen iv to split and fused bl. these characteristic ultrastructural findings in human capillary bl were not found in normal or transgenic mice. our observations of fibrillar collagen in young individuals complement previous observations of similar findings in older individuals. this raises the possibility that fibrillar collagen in human vascular bl plays a significant role in cns capillary physiology and pathophysiology. the species-specific differences in capillary morphology between humans and mice might have relevance to poor correlations between benefits of immunotherapy and drug treatment in mice compared with human. introduction capillaries are the smallest segment of the vascular network throughout the body and play crucial roles in exchange of oxygen, nutrients, pharmaceuticals, and waste between vascular and perivascular tissues [1]. electron microscopic (em) studies show capillary lumens are covered by a single layer of endothelial cells and the abluminal side is covered by a basal lamina (bl, basement membrane], which is formed by the fused bl of endothelial cells and pericapillary astrocytes [2–4]. pericytes and their processes are present focally in the fused bl. fig 1 illustrates the anatomical relationship between capillary lumen, endothelial cell, pericyte, pericapillary astrocyte, and basal lamina. fig 1. electron micrograph of a capillary in cross section from the cortex of a 5.5-month-old non-tg mouse. perfusion of fixative cleared the lumen (l) that is lined with a thin layer of endothelial cells (e) abutting a homogenous basal lamina (bl), pericapillary astrocytes (as) and their end-feet are tightly attached on the other side of the bl. pericyte (p) and its processes are embedded in the bl. the sketch illustrates the anatomical relationship between vascular cells and bl. in the central nervous system (cns), capillary endothelial cells have specific structures that control movements of molecules between the blood and brain parenchyma to maintain homeostasis of the cns [5], as well as regulation of blood flow [6]. these structures include interendothelial tight junctions, luminal and abluminal molecular transporters, and a paucity of vesicular transport, which constitutes the blood-brain-barrier (bbb). pericytes are also involved in bbb function and blood flow [7, 8]. em of well-preserved/fixed mouse and rat cns has made this ultrastructural definition of the capillary bbb universally accepted in other animal species. the non-cellular capillary structure, bl, sandwiched between the abluminal side of endothelial cells and pericapillary glial cells, also plays a key role in barrier functions and immune response in the cns [9–11]. early biochemical studies of bl from non-cns tissues of human and mouse have identified major and minor components and their molecular interactions [12]. non-fibrillar collagen type iv [col 4] is biochemically the most abundant component of bl. col 4 interconnects with non-collagenous components, including laminin, perlecan, nidogen and fibronectin, to form an amorphous meshwork with a homogeneous density on em. these biochemical data, like morphological data, are also accepted universally. the col 4-dominant biochemical view also prevails in cns capillary bl. compared to col 4, other bl components are understudied [13]. early studies in rats have shown that bl could be a physical barrier to certain intravenously injected molecules after they pass the bbb [14, 15]. it is hypothesized that electric charges of bl components contribute to barrier function. investigators have tried to translate data from mouse models of human disease to clinical trials in humans; however, such translational studies have often not succeeded. the difference between human and mouse capillaries may contribute to this finding. intravenously administered molecules that can bypass bbb have some success treating mouse models of human neurodegenerative diseases but have minimal effect in humans for currently unknown reasons, and the role of vascular cells and bl needs to be considered. recent studies have shown differences in brain cells and microvascular cells between humans and mice. significant differences in expression were found in specific brain regions [16–20]. similar studies have not been performed in human and mouse spinal cords. the differences in expression in cell types between species could produce different bl properties in human and mouse cns, which might interfere with the movement of molecules/drugs within the perivascular compartment. fifty years ago, cervós-navarro and ferszt [21, 22] described ultrastructural characteristics of capillaries in human spinal cords of individuals ranging in age from 7-months to 97 years. they found a consistent “dilated pericapillary space” bordered on one side by endothelial bl and on the other side by perivascular glial cells, with collagen fibers in about 70 % of the capillaries. in 1998, ono et al. [23] studied spinal cord capillaries in patients ranging in age from 37 to 74 years with sporadic amyotrophic lateral sclerosis (sals), other neurologic diseases and in neurologically normal controls. they found that about 40 % of capillaries in the three groups had a “focally split bl to encompass a large area containing accumulation of collagen fibrils.” in 2012, garbuzova-davis et al. [24] described expanded capillary bl with collagen fibrils in spinal cords of 18 controls [39–86 years] and in sporadic als [45–83 years]. in 2013, we showed similar findings in a preliminary report of 8 controls (28–81 years), 5 sals (47–75 years) and 7 familial als (15–69 years) patients [25]. in 2015, sasaki [26] examined 358 and 366 capillaries in 12 controls (50–80 years) and 12 sals (59–83 years) cases and found “slight or abundant accumulation of collagen fibers in the perivascular space within expansion of the capillary basement membrane” in controls and als cases. expanded spaces in bl of spinal cord capillaries have not been reported in normal mice or als mouse models. in contrast to spinal cord capillaries, early em studies of human cerebral capillaries only briefly comment on collagen fibrils in bl, with a few images: 61-year-old [27], age unknown [28], fetus [29, 30], 50–66 years [31], 56 years [32] and age unknown [33]. splitting of the bl has also been observed in capillaries of biopsy tissues from patients with cerebral edema (58–80 years) [34], multiple sclerosis (25–30 years) and alzheimer’s disease (ad) (age unknown) [35, 36]. later, farkas et al [37] examined human cerebral periventricular white matter and showed collagen fibrils within capillary bl in patients 40 to 90 years of age. they also reported increased bl pathology in ad and parkinson’s disease. szpak et al [38] showed cerebral capillaries with numerous collagen fibers in two ad patients (36–46 years) with a p.p117l psen1 mutation. they all showed banded collagen fibrils with em. in a preliminary em report of human brain capillary bl (11 cases, 7–49 years of age), we showed banded collagen in expanded spaces in the basal lamina [39]. some studies have reported collagen types other than col 4 in human brain capillaries. van duinen et al [40] showed immunohistochemical (ihc) staining of collagen type i (col 1) and collagen type iii (col 3) and fibronectin in capillaries of normal controls aged 36–78 years. dong et al [41] studied six patients (46–83 years old) with cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy with notch 3 mutations (cadasil) and six controls (54–82 years old). they found col 1 and col 3 in capillaries of both cadasil and control cases. staining was heterogeneous with much lower frequency and intensity in controls. in our preliminary em reports of human cns capillary bl, iem localized col 1 and col 3 to fibrillar collagens in the expanded spaces [25, 39]. ihc and proteomic analysis showed col 6α3 in controls, ad and cerebral amyloid angiopathy (caa) and col 6ɑ2 in individuals with caa ranging in age from 65–95 years [42], and col 1ɑ1 in ad with caa ranging in age from 58–97 years [43], and in all vessels of caa cases ranging from 61–83 years old [44]. the sparse em studies of cns capillary bl in young human cases prompted our studied of capillary bl with ihc, em and iem in cases ranging in age from 4 days to 49 years. the cases had no significant brain pathology or a range of neurological disorders. we limited the study to those 50 years of age or younger to minimize the possible effects of age-related neurodegenerative or cerebrovascular changes [45, 46]. additionally, considering the increased attention on species differences between brain vasculature of humans and mice [19, 20, 47], we included normal and transgenic (tg) mouse models of human neurodegenerative diseases. material and methods brains and spinal cords table 1 listed human brains and spinal cords from the brain bank in mayo clinic, jacksonville, fl. a total of 45 cases aged from 4 days to 49 years old that included 18 cases with no significant brain pathology and 27 cases with different neurological disorders. among them were two biopsies and 10 cases with postmortem interval (pmi) of less than 24 hours. neuropathologic assessments were based on light microscopic examination of paraffin sections. table 2 cns tissues were from non-tg and tg mice developed in mayo clinic, jacksonville, fl [48–50]. table 1: studies of human brain and spinal cord microvasculature age, sex pmi, hour brain pathology region em immunogold em immunohistochemistry 4 days, f na no hippocampus yes no no 1.5 months, m na leigh syndrome     no col 1, 4 7 yr, f na occipital lobe malformation frontal cortex yes fn col 1, 3, fn 9 yr, m na tauopathy, nifid, als cingulate yes no no 15 yr, m na als [fus] spinal cord yes col 1, 3, 4 no 18 yr, m 18 no frontal cortex yes col 1, 3 col 1, 3, fn 20 yr, m na no frontal cortex no no col 1, 3, 4 23 yr, f na no frontal cortex no no col 1, 3, 4 24 yr, m na no frontal cortex no no col 1, 3, 4 16 yr, m na no cerebellum no no col 1, 3, 4, 6 19 yr, f na no cerebellum no no col 1, 3, 4, 6 23 yr, m na no cerebellum no no col 1, 3, 4, 6 24 yr, f na no cerebellum no no col 1, 3, 4, 6 27 yr, m na no cerebellum no no col 1, 3, 4, 6 35 yr, m na no cerebellum no no col 1, 3, 4, 6 47 yr, m na no cerebellum no no col 1, 3, 4, 6 25 yr, m 0 tuberous sclerosis frontal cortex yes col 1, fn col 1, 3, fn 26 yr, f na no frontal cortex yes col 3 col 1, 3, fn 28 yr, m na no (dbhd) frontal cortex yes no col 1, 3, fn       spinal cord yes col 1, 3, 4, fn col 4 28 yr, m 2 no (polg) subthalamic nucleus yes no col 1, 3, fn 30 yr, m na no frontal cortex no no col 1, 3, 4 33 yr, m 21 acute contusion of frontal, temporal lobe substantia nigra yes col 3, fn no 35 yr, f 0 vascular mineralization cerebral cortex (wm>gm) yes no no 38 yr, m 13 als, cte frontal cortex yes no col 1, 3, 4, fn 40 yr, f na no frontal cortex yes fn col 1, 3, fn 41 yr, f na no frontal cortex yes fn col 1, 3, fn 42 y, f na ftld thalamus yes col 4 no 42 yr, m na ftld frontal cortex yes no no 45 yr, m 12 ftld-tauopathy frontal cortex yes fn col 1, 3, fn 44 yr, m 7 perry's syndrome substantia nigra yes no no 45 yr, m 25 als spinal cord no no col, 1, 3, 4, fn 47 yr, f 8 als spinal cord yes col 1, 3, 4 no 41 yr, f 3 als (c9orf72) spinal cord yes no no 49 yr, f 6 als (c9orf72) spinal cord yes no col 1, 3 47 yr, f na als (fus) spinal cord yes no col 1, 3 49 yr, f 3 als (sod1) spinal cord yes no col, 1, 3 42 yr, m na familial parkinsonism putamen no col 1, 3 no 48 yr, m na familial parkinsonism amygdala yes no no 49 yr, m na diffuse lewy body disease (dj-1) basal nucleus of meynert yes no col 1, 3, fn 46 yr, f na ftdp-17 frontal cortex yes col 1, 3 col 1, 3, fn 48 yr, f na ftdp-17 spinal cord no fn, tau no 48 yr, f na down's syndrome frontal cortex yes no col 1, 3, fn 48 yr, f na polyglucosan body disease optic tract yes no no 46 yr, f 45 hdls frontal cortex white matter yes col 1, 3, 4, fn no 49 yr, f na hdls frontal cortex gray matter yes no no als, amyotrophic lateral sclerosis; c9orf72, chromosome 9 open reading frame 72; cte, chronic traumatic encephalopathy; dbhd, dopamine β-hydroxylase deficiency; em, electron microscopy; f, female; fn, fibronectin; ftld, frontotemporal lobar degeneration; ftdp-17, frontotemporal degeneration with parkinsonism linked to chromosome 17; fus, fused sarcoma; hdls, hereditary diffuse leukoencephalopathy with spheroids; m, male; na, not available; nifid, neuronal intermediate filament inclusion disease; polg, mitochondrial dna polymerase gamma; sod1, copper/zinc superoxide dismutase 1 table 2: studies of mouse brain and spinal cord microvasculature age, months pmi, hour genotype region em iem ihc 1 0 tdp-43 cortex yes no no 2.5 0 normal. hepatic encephalopathy globus pallidus yes no no 5.5 0 non-transgenic cortex yes no no 5.5 0 non-transgenic spinal cord yes no no 7 0 c9orf72 motor cortex yes no no 9 0 tau p301, appsw cortex yes no no 10 0 tau p301l spinal cord yes no no tdp-43, tar dna-binding protein 43 kd; appsw, amyloid precursor protein swedish mutation antibodies affinity purified rabbit antibodies to col 1, col 3, col 4 (rockland, limerick, pa; chemicon, temecula, ca), col 6a1 (atlas antibodies, american research products, inc., waltham, ma), and to plasma fibronectin (f3648, sigma, st. louis, mo), monoclonal mouse antibody to col 4 (mp biomedicals, solon, oh; chemicon, temecula, ca). goat anti-rabbit igg conjugated with 10or 18-nm colloid gold particles (jackson immunoresearch laboratories, west grove, pa). immunohistochemistry formalin-fixed human and mouse tissues were processed for paraffin embedding by a tissuetek embedding console system, tec5 (sakura finetek usa, inc., torrance, ca). five μm-thick sections mounted on fisherbrand plus glass slides were pre-treated in a steamer with deionized water before immunostaining with an autostainer (dako, carpinteria, ca) using 3,3’-diaminobenzidine as the chromogen. sections are examined with an olympus bx 50 fitted with an olympus dp12 digital camera. electron microscopy small pieces (2 x 2 m2) of formalin-fixed human tissues were further fixed in 2.5 % glutaraldehyde-2 % paraformaldehyde in 0.1 m cacodylate buffer. biopsy tissues were directed fixed in the above fixative. mice were perfused transcardially with saline followed by 2.5 % glutaraldehyde-2 % paraformaldehyde in 0.1 m cacodylate buffer. all tissues were fixed overnight at 4 °c. the tissues were processed for embedding in epon as previously described [48, 51]. ultrathin sections were collected on formvar-coated copper grids, stained with uranyl acetate and lead citrate, and examined with a philips 208s electron microscope (fei, hillsboro, or) fitted with a gatan 831 orius camera (gatan, ca), and a jem-1400 flash microscope (jeol, peabody, ma). digital images were processed with adobe photoshop software. post-embedding immunogold em formalin-fixed tissue pieces (2 x 2 mm2) were processed for embedding in lr white resin, and subjected to iem as previously described [48, 51]. for fibronectin, ultrathin sections collected on formvar-coated nickel grids were pre-treated by floating with section-side down on 1 ml of 0.1 m sodium citrate, ph 6, for 10 min at 95 °c and cooled to room temperature for 15 minutes before incubation with antibody. results only selected figures were described here; more figures can be found in supplementary material. the morphological descriptions were similar, if not the same, in all cases, regardless of age, sex, disease, tissue region and pmi. in this report capillary refers to vessels less than 10 μm in diameter. fig 1 shows an em of a capillary from the cortex of a normal 5.5-month-old mouse illustrating the anatomical relationship between the vascular cells and bl. ihc of the same large vessels (arterioles) in serial sections established the specificity of antibodies. sections of cerebral cortex, cerebellar vermis, and spinal cord anterior horn in figs 2–4 show col 4 outlined endothelial bl and the outermost vascular bl, but not the media or adventitia, which were immunostained with col 1 and col 3. vascular media in large vessels normally contains smooth muscle cells surrounded by fibrillar col 1 and col 3. adventitia is the outermost area of large vessels that also contains fibrillar collagens. fig 2. 18-year-old male with no significant brain pathology. mid-frontal cortex. postmortem interval, 18 hours. immunohistochemistry of collagen type i, iii, and iv (col 1, col 3, col 4) in serial sections of the same field (cortex next to a deep sulcus, arrowheads) showing many microvessels stained with col 4. however, several microvessels (arrows) are also stained with col 1 and col 3 with stronger col 3 than col 1 staining. staining of col 3 and col 1 but not col 4 to the adventitia of small arterioles (curved arrows) and leptomeningeal vessels (arrowhead) indicate specificity of antibodies. vascular adventitia and leptomeningeal vessels normally contain fibrillar collagens, e.g., col 1 and col 3. scale bar, 50 μm. fig 3. 16-year-old male with no significant brain pathology. cerebellum, vermis, granular layer. immunohistochemistry of collagen type i, iii, and iv (col 1, col 3, col 4) in serial sections of the same field with arrowheads pointing to the same large vessel. many col 4-stained microvessels are also stained with col 3 and col 1, with col 3 being stronger (arrows). note in the large vessel, col 4 outlines the vessel and the endothelial basal lamina, while col 1 and col 3 stained the media that normally contains fibrillar collagens surrounding vascular smooth muscle cells. scale bar, 50 μm. fig 4. 38-year-old male with amyotrophic lateral sclerosis. spinal cord. immunohistochemistry of collagen type i, iii, and iv (col 1, col 3, col 4) in serial sections of the same field near central canal epithelium (arrowheads). note some of col 4-stained microvessels are also stained with col 3 and col 1 (arrows). in the large vessels, col 4 outlines the vessel and the endothelial basal lamina, while col 1 and col 3 stained the media that normally contains fibrillar collagens surrounding vascular smooth muscle cells. scale bar, 50 μm. as would be expected, ihc of col 4 showed immunostaining of many microvessels in various cerebral regions, cerebellar vermis and spinal cord as col 4 is the major bl component. importantly, in all cases, many of the same vessels in serial sections were also immunostained strongly for col 3 and weakly for col 1. em (figs 5–13) and iem (figs 14–22) confirmed the ihc findings of microvessels. cross sections of capillaries were selected to ensure their proper orientation for measurement of diameter and their ultrastructural organization. like capillaries in non-cns regions of the human body, cns capillaries have luminal endothelium and abluminal bl with pericytes embedded within the bl. the bl were composed of a homogeneous structure representing the fused bl of endothelial cell/pericyte and perivascular astrocyte. also observed were segments with apparent splitting of the bl (fig 5). some of the split bl were shown to be continuous with fused bl in the same section. the most striking finding was the small to large amounts of fibrils deposited in various focally expanded spaces bounded by split bl. these fibrils displayed characteristic banding patterns of fibrillar collagens, with a width ranging from 50–80 nm diameter. sometimes, collagens larger than 120 nm were found (fig 8, supplementary fig 32). all fibrils appeared randomly oriented in the expanded space. very sparse cytoplasmic elements were present in the expanded space. tissues from short pmi autopsies and biopsies showed pericapillary parenchyma tightly wrapping the capillaries, even in areas with massive, banded collagen deposits (fig 10, supplementary figs 26, 41, 57). no banded fibrils were observed outside of the bl. no phagocytotic cells or inflammatory cells were present. there was minimal astrogliosis, and despite autolytic changes, astroglial processes maintained hemi-desmosome attachments on fused or split bl (fig 20–22, supplementary figs 55) [52]. there was no evidence of extensive bl remodeling (e.g., duplication or thickening) of either fused or split bl. fig 5. 9-year-old male with tauopathy, neurofilament inclusion disease, motor neuron disease. cingulate. top. electron micrograph of a capillary at low magnification showing fused basal lamina (bl) of endothelial cell (e) and perivascular astrocyte (as) at the top and bottom, and split bl at the left and right side of the capillary. arrowheads point to split of fused bl. rbc, red blood cell in capillary lumen. bottom. enlargements of boxed areas showing expanded spaces [double head arrows] in the split bl contain bundles of banded fibrils in cross, oblique and longitudinal sections [arrows]. scale bars, top, 1μm; bottom, 0.2μm. fig 6. 18-year-old male with no significant brain pathology. superior frontal cortex. postmortem interval, 18 hours. same case with ihc depicted in figure 2. left. electron micrograph of two capillaries with homogenous fused basal lamina (bl) of endothelial cell (e) and perivascular astrocyte (as). note the right capillary has an expanded space [double head arrow] formed by split bl (arrowheads). right. enlargement of boxed area shows the expanded space containing many banded fibrils (arrows) in random orientation. arrowheads indicate splits of the two bls. scale bar, right, 0.2μm. fig 7. 28-year-old male with congenital dopamine β-hydroxylase deficiency, but no significant brain pathology. spinal cord. left. cross section of a capillary completely wrapped by expanded spaces [double head arrows] in split basal lamina (bl) of the endothelial cell (e), pericyte (p) and perivascular astrocyte (as). right. boxed area is enlarged to show the expanded space filled with cross and longitudinal sections of banded fibrils (arrows). rbc, red blood cell occupying the capillary lumen. gf, astrocytic glial fibrils. fig 8. 33-year-old male with acute contusion of frontal and temporal lobes, but no other significant brain pathology. substantia nigra. postmortem interval, 21 hours. left. a capillary with split basal lamina (bl) of endothelial cell (e), pericyte (p) and perivascular astrocyte (as) forming wide and narrow expanded spaces (double head arrows) filled with fibrils. l, capillary lumen. scale bar, 1μm. right. enlarged boxed area showing large and small banded fibrils in cross and longitudinal sections (arrow) in the expanded space. fig 9. 41-year-old female with no significant brain pathology. superior frontal cortex. left. cross section of a capillary with large and small expanded spaces (double head arrows) between split basal lamina (bl) of endothelial cell (e), pericyte (p) and perivascular astrocyte (as). rbc, red blood cell in the capillary lumen. right. enlarged box area shows many banded fibrils [arrows] in the expanded space. fig 10. 41-year-old female with amyotrophic lateral sclerosis with chromosome 9 open reading frame 72 (c9orf72) mutation. spinal cord. postmortem interval, 3 hours. left. cross section of a capillary with large and small expanded spaces (double head arrows) between split basal lamina (bl) of endothelial cell (e), pericyte (p) and perivascular astrocyte (as). rbc, red blood cell in the capillary lumen (l). ax, myelinated axon. right. enlarged box area shows the large, expanded space is filled with many fibrils in cross-sections (arrows), and a longitudinal banded fibril (long arrow). fig 11. 44-year-old male with perry’s syndrome. substantia nigra. postmortem interval, 7 hours. left. cross section of a capillary enwrapped with large and small expanded spaces (double head arrows) due to separation of basal lamina (bl) of the endothelial cell (e), pericyte (p) and perivascular astrocyte (as). rbc, red blood cell in capillary lumen. right. enlarged boxed area shows the expanded space contains longitudinal banded fibrils [long arrows] among many cross-sectioned fibrils (short arrows). note some large-diameter fibrils in cross sections (double arrows). the split bl are not thickened. fig 12. 48-year-old female with polyglucosan body disease. optic tract. cross section of a capillary with large and small expanded spaces (double head arrows) between split basal lamina (bl) of endothelial cell (e), pericyte (p) and perivascular astrocyte (as). the expanded space is filled with fibrils in crossand longitudinal sections (arrows). rbc, red blood cell in the capillary lumen. pb, polyglucosan body. fig 13. 49-year-old male with diffuse lewy body disease (dj-1 mutation). basal nucleus of meynert. left. cross section of a capillary with fused and split basal lamina (bl) of endothelial cell (e), pericyte (p) and perivascular astrocyte (as). rbc, red blood cell in the capillary lumen. right. enlarged box area shows the expanded space between split bl (double head arrow) contains a few longitudinal banded fibrils (long arrows), many cross-sectioned fibrils in various diameters [short arrows] and a cluster of small fibrils. fig 14. 28-year-old male with congenital dopamine β-hydroxylase deficiency but no significant brain pathology. spinal cord. immunogold electron micrographs of collagen type i (col 1). left. cross section of a capillary enclosed completely by wide (left and right side) and narrow (top and bottom) expanded spaces between the basal lamina (bl) of endothelial cell (e), pericyte (p) and perivascular astrocyte (as) (double head arrows). the space is filled with fibrils. rbc, red blood cells in the capillary lumen. right. enlargement of boxed area showing localization of col 1 to the fibrils. arrows point to gold particles. note the absence of gold particles from the split homogeneous bl of as and p. scale bar, right, 0.2μm. fig 15. 28-year-old male with congenital dopamine β-hydroxylase deficiency and no significant brain pathology. spinal cord. immunogold electron micrographs of collagen type iii (col 3). left. cross section of a capillary with a very wide expanded space [double head arrows] between the split basal lamina (bl) of endothelial cell (e), pericyte (p) and perivascular astrocyte (as). the space is filled with fibrils. the lower half of the capillary has narrow spaces between the split bl. rbc, red blood cell in the capillary lumen. right. enlargement of boxed area showing localization of col 3 to the fibrils. arrows point to gold particles. note the absence of gold particles from the split homogeneous bl of as and e. scale bar, left, 1μm. fig 16. 28-year-old male with congenital dopamine β-hydroxylase deficiency, no significant brain pathology. spinal cord. immunogold electron micrographs of collagen type iv (col 4). left. cross section of a capillary with wide and narrow expanded spaces (double head arrows) between the split basal lamina (bl) of endothelial cell (e) and perivascular astrocyte (as). the spaces are filled with fibrils. rbc, red blood cell in the capillary lumen. right. enlargement of boxed area showing localization of col 4 to the homogeneous split bl of e and as, but not the banded fibrils in the expanded space. arrows point to gold particles. fig 17. 28-year-old male with congenital dopamine β-hydroxylase deficiency and no significant brain pathology. spinal cord. immunogold electron micrographs of fibronectin. left. cross section of a capillary with wide and narrow expanded spaces [double head arrows] between the split basal lamina (bl) of endothelial cell (e), pericyte (p) and perivascular astrocyte (as). the spaces are filled with fibrils. rbc, red blood cell in the capillary lumen. right. enlargement of boxed area showing localization of fibronectin mostly to the fibrils in the space, but also to the homogeneous split bl. arrows point to gold particles. fig 18. 42-year-old male with familial parkinson’s disease. putamen. immunoelectron micrographs of collagen type i (col 1) and type iii (col 3). left. serial sections of a capillary with fused and split basal lamina (bl) of endothelial cell (e) and perivascular astrocyte [as]. double head arrows indicate expanded spaces between the split bl. rbc, ghost red blood cell in capillary lumen (l). middle, right. enlargements of respective boxed area. gold particles (arrows) are localized mainly to banded collagen fibrils in the expanded space and rarely to the split thin bl surrounding the space. scale bar, left, 1 μm; middle, right, 300 nm. fig 19. 46-year-old female with hereditary diffuse leukoencephalopathy with spheroids. frontal cortex white matter. immunogold electron micrographs of collagen type i (col 1). left. cross section of a capillary showing fused basal laminas (bl) of endothelial cell (e) and perivascular astrocyte (as) splits and the resulting expanded spaces [double head arrows] with fibrillar deposits. rbc, red blood cell in capillary lumen (l). right. enlargement of boxed area reveals localization of col 1 (arrows pointing to gold particles) to longitudinal banded fibrils and cross-sectioned fibrils in the expanded space, but not the split thin bl. scale bars, left, 1 μm; right, 300 nm. fig 20. 46-year-old female with hereditary diffuse leukoencephalopathy with spheroids. frontal cortex white matter. immunogold electron micrographs of collagen type iii (col 3). left. cross section of a capillary showing fused basal laminas (bl) of endothelial cell (e) and perivascular astrocyte (as) splits and the resulting large and small expanded spaces [double head arrows] with deposits. rbc, red blood cell in capillary lumen (l). right. enlargement of boxed area reveals localization of col 3 to longitudinal banded fibrils [arrows] in the expanded space, but not the split thin bl. scale bars, left, 1 μm; right, 300 nm. fig 21. 46-year-old female with hereditary diffuse leukoencephalopathy with spheroids. frontal cortex white matter. immunogold electron micrographs of collagen type iv (col 4). left. cross section of a capillary showing fused basal lamina (bl) of endothelial cell (e) and perivascular astrocyte (as) splits and the resulting large and small expanded spaces [double head arrows] with deposits. rbc, red blood cell in capillary lumen (l). right. enlargement of boxed area reveals localization of col 4 to the split thin bl (arrows point to gold particles), but not banded fibrils [long arrows] in the expanded space. fig 22. 46-year-old female with hereditary diffuse leukoencephalopathy with spheroids. frontal cortex white matter. immunogold electron micrographs of fibronectin (fn). left. cross section of a capillary showing fused basal lamina (bl) of endothelial cell (e), pericyte [p] and perivascular astrocyte (as) splits and the resulting large and small expanded spaces [double head arrows] with deposits. l, capillary lumen; rbc, red blood cell. right. enlargement of boxed area reveals many gold particles [arrows] over banded fibrils and split thin bl. immunoreactivity for col 1 and col 3 was localized to collagen fibrils, while col 4 was mainly localized to the split bl and the normal appearing fused bl. col 3 was more abundant than col 1 in some cases, as seen on ihc. the reason for this differential immunoreactivity is not clear. it could be differential antibody reactivity, differential amount of collagen in the expanded space, or an age-related finding. fibronectin was heavily localized to fibrils and bl where col 6 was weakly localized. in normal or transgenic mice younger than 10 months (equivalent to 50 years of human age), the fused bl of the endothelial cell, pericyte and perivascular astrocytes did not split to form expanded spaces (figs 23–28). fig 23. 1-month-old mouse (equivalent to 14-years-old human), expressing tar dna-binding protein 43 kd (tdp-43). cortex. left. a capillary is surrounded by fused basal lamina (bl) of endothelial cell (e) and perivascular astrocyte (as). note the homogeneity and uniform thickness of amorphous bl. l, lumen. right. enlarged boxed area. fig 24. 2.5-months-old mice (equivalent to 22-years-old human). globus pallidus. left. normal. a capillary with a fused basal lamina [bl] of endothelial cell (e), pericyte (p) and perivascular astrocyte (as). note the homogeneity and uniform thickness of amorphous bl. l, lumen. right. drug-induced hepatic encephalopathy. despite swelling of perivascular astrocyte (as), bl remains normal. fig 25. 5.5-months-old non-transgenic mouse (equivalent to 30-years-old human). cortex. two capillaries both have a fused basal lamina (bl) of endothelial cell (e), pericyte (p) and perivascular astrocyte (as). note the homogeneity and uniform thickness of amorphous bl. rbc, red blood cell in the lumen (l); ax, myelinated axon. fig 26. 5.5-month-old non-transgenic mouse (equivalent to 30-years-old human). spinal cord. top. longitudinal section of a capillary showing fused basal lamina (bl) of endothelial cell (e), pericyte (p) and perivascular astrocyte (as). bottom. enlarged box areas. note the homogeneity and uniform thickness of amorphous bl. l, capillary lumen; ax, myelinated axon. fig 27. 7-months-old, transgenic mouse (equivalent to 38-years-old human) expressing c9orf72 mutation. motor cortex. a capillary (cap) with perivascular edema [*], but the fused basal lamina (bl) of endothelial cell (e), pericyte (p) and perivascular astrocyte (as) maintains amorphous homogeneity and uniform thickness. scale bar, 1 μm. fig 28. 9-months-old, transgenic mouse (equivalent to 46-years-old human) expressing tau/amyloid precursor protein mutations. cortex. left. a red blood cell (rbc) fills the lumen of a capillary adjacent to a neuron [n] with heavy deposits of tau filaments [*]. right. enlargement of the capillary shows the fused basal lamina (bl) of endothelial cell (e), pericyte (p) and perivascular astrocyte (as) maintains its amorphous homogeneity and uniform thickness, despite edema around the vessel [*]. scale bars, left, 3 μm; right, 1 μm. discussion summary of major findings this study includes two aspects of capillary bl in human cns that have long been ignored. the first being em evidence of collagen fibrils in capillary bl in human cns. we confirmed early findings and expanded upon our previous reports [25, 39] with ihc, em and post-embedding iem in a cohort of 45 patients ranging in age from 4 days to 49 years. the case number is relatively modest, but it includes a spectrum of cns disorders, as well as those without significant brain pathology. in all cases, a similar morphologic observation was made, regardless of age, sex, disease, tissue region or pmi. the common findings included cns microvessels that were ihc-positive for col 1, col 3, col 4 and col 6. em studies revealed fused bl of endothelial cells, pericytes and perivascular astrocytes with focal splitting of the bl forming small to large spaces bordered by split bl and filled with fibrillar collagens, but no cellular components. no pericapillary space or astrogliosis was detected on the external bl. iem showed localization of col 1 and col 3 in fibrils in the expanded spaces, but not the split bl. non-fibrillar col 4 was absent from expanded spaces, but was localized to the bl, including the split bl, which was not thickened or duplicated. importantly, ultrastructural findings similar to those observed in humans were rarely detected in mouse brain and spinal cord. evidence that the findings are genuine and not artifactual it is highly unlikely that the observed ultrastructural change in capillaries were due to postmortem changes. previous investigators who have found similar results have argued that short pmi, uniform tissue handling/processing, as well as the inherent biochemical stability of collagen fibrils argues against these observations being artifactual [21–23, 26]. although vascular cells produce fibrillar collagens, it is unlikely that the collagen fibrils that we observed are produced and deposited as a terminal event given that the pmi of most of the cases studied was short. additionally, biopsy tissues in our cases, as well as in previous reports [34–36] argue against these being postmortem changes. underrecognized features of human cns capillaries it is not clear why capillary bl expansions with fibrillar collagen deposits have not been previously reported in humans. several factors could explain the oversight. for one, there is a prevailing assumption that brain capillaries are the same in humans and mice, and comparative studies are rare. in addition, detailed studies on the ultrastructure of human microvasculature are few. another factor to consider is that there may be variability in microvascular structures in different regions of the human brain. the cortical microvascular network is organized like a grid structure, while non-anastomotic vessels and capillary beds are common in the striatum [56, 57]. neocortical regions are not equivalent and there are also differences related to aging and disease [58]. a recent study showed that human capillary endothelial cells from isolated microvessels from fresh brain tissue of fetuses,adult controls, and diseased brains had high transcriptional heterogeneity [59]. since bl is produced by endothelial cells, it is possible that bl morphology is also heterogeneous. in this report we studied capillary bl of normal fetal and adult humans in cortical and subcortical gray matter and in cerebral white matter. in addition, we studied microvessels in a range of neurologic disorders in people younger than 50 years of age. other investigators may not have such advantages. the main findings of this study are based on qualitative analysis by em, which is a labor intensive procedure. access to human samples, as well as limited em resources and the time and effort involved in ultrastructural analysis may in part explain the paucity of similar studies in the scientific literature [9, 11]. advances in microscopic techniques at the light microscopic level [60] could make comparative studies of microvessels in humans and animals more approachable. additional comprehensive structural studies are needed to validate our findings and address their potential contribution to normal microvascular physiology and pathology. evidence that the findings are not due to aging our findings of extensive collagen fibril deposits in expanded bl spaces of young individuals argue against the observations being due to aging or specific disorders associated with bl pathology in human cns capillaries [37, 53–55]. while detailed cardiovascular histories were not routinely recorded, chronic cardiovascular disease is uncommon at young ages. more studies of capillary bl from young people are needed to address this novel finding. our ultrastructural observations are unlikely specific to a particular disease because they were found in a range of cns disorders. comparable findings in cases without significant brain pathologies further support the notion that capillary bl morphologies are characteristic of human cns capillaries. physiological implications of collagen deposition in blood vessels collagens in extracellular matrix are now recognized as being more than an inert scaffold for mechanical support, instead they are thought to play an active role in homeostasis between vascular and perivascular elements [61]. the adult human brain has an extensive microvasculature for physiologic metabolic and energy demands, while these requirements are less in the mouse. consequently, it is reasonable to expect more regulatory controls of blood flow in human cns, even under normal physiological conditions, than in mice. studies in both human and mouse brain show that the capillary bed has the highest resistance to blood flow within the cerebral vasculature [6, 62]. blood flow regulation in capillaries has been studied in endothelial cells [63], pericytes [8] and astrocytes [64]. these cells are closely adherent to the capillary bl. therefore, bl components may also have a role in blood flow control by their molecular interaction with vascular cells. previous studies on brain vascular bl components frequently highlight col 4 [9, 11, 65]; however, our recent study questioned the involvement of col 4 in small vessel disease and in the hypertensive rat [66]. the ihc findings in this report show col 4 in bl of capillary endothelial cells and the split pericapillary bl, but not in the space produced by the bl splitting. iem further confirmed the absence of col 4 in expanded bl spaces. this, plus the absence of remodeling in the split bl in capillaries of normal and pathological brain samples, suggests that col 4 has a minimal role in producing this unique capillary structure, which is found even in young individuals less than 50 years of age. an in vitro study of contractile cells grown on experimental matrix composed of col 1 responded to strain/deformation faster than on matrigel (mouse sarcoma derived matrix containing 30 % col 4, 60 % laminin, and 8 % entactin) suggesting cells grown on col 1-matrix could communicate mechanical information to other cells over considerable distances (67). we propose that in addition to other bl components, fibrillar col 1 and col 3 might provide an extra control mechanism to regulate capillary blood flow related to their inherent tensile and elastic properties. functions of collagens in human capillary basal lamina in pathological conditions structural defects of col 1 and col 3 are thought to contribute to vascular wall fragility in disorders such as ehlers-danlos syndrome [68]. on the other hand, increased col 1 and col 3 in media of arterioles and pericapillary spaces of subcortical white matter is characteristic of binswanger’s disease [69, 70]. it has been suggested that fibrillar collagens strengthen the vascular wall in response to degeneration in the media in familial and sporadic arteriosclerotic disorders. in human cystic fibrosis, damage to the lung epithelial barrier will allow protease such as matrix metalloproteases (mmp) and prolyl endopeptidase to reach and digest the underlying matrix containing col 1. the ensuing collagen fragments then act as a neutrophile chemoattractant in chronic inflammation [71]. it has been shown that mmp-9 can degrade native col 1 and col 3 [72], and increased expression or activity of mmp is known in neurodegenerative diseases [73]. our data showed abundant col 1 and col 3 fibrils in the expanded spaces in human brain capillary bl. in a similar scenario to lung epithelial barrier breakdown, these collagens could be targeted by proteases that breach the bbb, leading to fragment peptides that are involved in inflammation during neurodegeneration. col 1 binds within residues 448–465 of amyloid protein precursor protein (app-695) [74]. app was previously shown to trigger activation of monocytes upon adhesion to col 1, which typically occurs during adhesion of monocytes to vascular endothelium or during tissue infiltration by monocyte/macrophages [74]. interestingly, a recent report of amyloid immunotherapy in the pdapp mouse model of ad showed upregulated protein levels of mmt 9 by activated perivascular macrophages [76]. this may be related to the pathogenesis of amyloid-related imaging abnormalities (aria) in certain ad patients after immunotherapies [77]. also, macrophages could phagocytize drugs that pass the bbb and prevent them from reaching their target cell. species specify of observed findings our data is the first to show ultrastructural difference between human and mouse csf capillary bl, with prevailing presence of fibrillar col 1 and col 3 and minimal involvement of col 4. the expanded spaces in bl containing fibrillar collagens were observed in human cns even at lower magnifications, but similar structures are not observed in even old mice. the large, expanded spaces with fibrillar collagen deposits are not found in adult mice (up to 10 months of age in this study and 2 years of age in an unpublished study). rarely, a few fibrillar collagens fibrils are found in bl at higher magnifications. if these expanded bl spaces were a common feature in capillaries of mouse cns, it is unlikely previous em studies would have missed them. two recent studies of brain capillary bl in young and aged mice, as well as early studies of spinal cord capillaries in als mouse models, showed thickened bl, but no fibrillar collagens [78–81]. mouse models of human diseases are valuable tools for new avenues of research and therapy development; however, species differences between humans and mice have been noted [82]. furthermore, translational data from mouse models to humans does not always succeed, including in neurodegenerative disorders/diseases [83–85]. the implication of species differences, if any, remains to be elucidated. recent genomic studies showed species differences between human and mouse cortical neurons and glial cells [86], as well as differences in recent transcriptomic analyses of human and mouse brain vascular cells [16–18, 58, 59, 87]. future studies need to address the role of specific collagens, given the recent report showing difference in expression of col3a1 gene in mouse and human lung [88]. in conclusion, our data showed that, in the human cns, capillary bl contains fibrillar col 1 and col 3 in expanded spaces formed by splitting of the bl that are not found in mouse cns. we suggest that in human cns capillary bl, a “col 4-centric” dogma needs to be modified to include fibrillar collagens, in particular col 1 and col 3. aspects of cns vascular biology in humans has been inferred from mouse studies, but these results may need to be revised. additional studies that focus on unique capillary bl structure and the functional significance of fibrillary collagens in human cns in physiology and pathology are needed. acknowledgements we are grateful to the tissue donors and their families. we thank virginia phillips, monica castanedes-casey, nathan perez, and whitney davis for their assistance in histology and immunohistochemistry. conflict of interest statement the authors declare no conflict of interests. funding statement funded by nih grants and mayo foundation. supplementary material 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are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. increased frontocortical microvascular raspberry density in frontotemporal lobar degeneration compared to lewy body disease and control cases: a neuropathological study free neuropathology 6:7 (2025) ek olofsson and englund doi: https://doi.org/10.17879/freeneuropathology-2025-6178 page 1 of 14 copyright: © 2025 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. increased frontocortical microvascular raspberry density in frontotemporal lobar degeneration compared to lewy body disease and control cases: a neuropathological study henric ek olofsson¹, elisabet englund¹ 1 division of pathology, department of clinical sciences lund, lund university, lund, sweden corresponding author: henric ek olofsson · department of clinical sciences lund · lund university · sölvegatan 25b · lund, 22362 · sweden henric.ek_olofsson@med.lu.se submitted: 01 january 2025 · accepted: 20 february 2025 · copyedited by: lauren walker · published: 04 march 2025 abstract background: brain raspberries are histologically defined microvascular entities that are highly prevalent in the neocortex. increased cortical raspberry density occurs in vascular dementia, but also with advancing age. here, we examined the raspberry density in two neurodegenerative diseases, wherein vascular alterations distinct from conventional vascular risk factors have been indicated: frontotemporal lobar degeneration (ftld) and lewy body disease (lbd). methods: this retrospective study included 283 clinically autopsied individuals: 105 control cases without neurodegenerative disease, 98 ftld cases (mainly ftld-tau and ftld-tdp), and 80 lbd cases (mainly neocortical). the raspberry density was quantified on haematoxylin-eosin-stained tissue sections from the frontal cortex, and the frontocortical atrophy was ranked 0–3. results: there was a higher raspberry density in the ftld group compared to both other groups (p ≤ 0.001; games-howell post hoc test). the difference between the ftld and lbd groups remained significant in multiple linear regression models that included age, sex, and either brain weight (p = 0.034) or cortical atrophy (p = 0.012). the difference between the ftld and control groups remained significant when including age, sex, and brain weight in the model (p = 0.004), while a trend towards significance was demonstrated when including age, sex, and cortical atrophy (p = 0.054). further analyses of the ftld group revealed a trend towards a positive correlation between raspberry density and cortical atrophy (p = 0.062; spearman rank correlation). comparisons of ftld subgroups were inconclusive. conclusion: the frontocortical raspberry density is increased in ftld. an examination of the raspberry density in relation to a quantitative measure of cortical atrophy is motivated to validate the results. future studies are needed to determine whether increased raspberry density in ftld could function as a marker for more widespread vascular alterations, and to elucidate the relation between microvascular alterations and neurodegenerative disease. keywords: angiogenesis, cerebrovascular disorders, frontotemporal lobar degeneration, lewy body disease, neurodegenerative diseases original paper https://doi.org/10.17879/freeneuropathology-2025-6178 https://creativecommons.org/licenses/by/4.0/ https://creativecommons.org/publicdomain/zero/1.0/ mailto:henric.ek_olofsson@med.lu.se free neuropathology 6:7 (2025) ek olofsson and englund doi: https://doi.org/10.17879/freeneuropathology-2025-6178 page 2 of 14 abbreviations c9orf72 chromosome 9 open reading frame 72 gene, cbd corticobasal degeneration, ftd frontotemporal dementia, ftld frontotemporal lobar degeneration, fus fused in sarcoma, grn progranulin gene, lbd lewy body disease, mapt microtubule-associated protein tau gene, nft neurofibrillary tangle, nos not otherwise specified, pid pick’s disease, psp progressive supranuclear palsy, tdp-43 transactivation response dna-binding protein 43, ups ubiquitin proteasome system. introduction frontotemporal lobar degeneration (ftld) and lewy body disease (lbd) are two heterogeneous neuropathological entities that manifest with cellular inclusions and neuronal loss. ftld cases are classified based on the abnormal protein contents of the cellular inclusions: microtubule-associated protein tau, transactivation response dna-binding protein (tdp) 43, fused in sarcoma (fus), or proteins of the ubiquitin proteasome system (ups) [1, 2]. ftld is not known to be associated with conventional vascular risk factors [3], but recent studies on human tissue indicate structural and functional cerebrovascular alterations that may be intrinsic to the disease [4–10]. lewy body disease (lbd) is characterised neuropathologically by alpha-synuclein containing inclusions [11, 12]. like ftld, lbd has not been associated with conventional vascular risk factors [13, 14], but potential mechanisms of cortical hypoperfusion have been described, including cardiovascular autonomic dysfunction [15–17], intrinsic cholinergic denervation of blood vessels [18–20], impaired cortical angiogenesis [21], and reduced cortical capillary density [22, 23]. information on histopathological signs of cerebrovascular alterations in ftld and lbd is limited. we have previously applied the term “raspberry” to refer to a histological finding of ≥ 3 transversally sectioned vascular lumen within a common perivascular space (fig. 1) [24–27]. a more recently introduced and similarly defined term is “multi-lumen vascular profiles” [28]. to illustrate the link to our previous studies, the term “raspberry” will be used in the present work. there are older terms (including glomerular loop formations, vascular bundles, vascular wickerworks, and vascular convolutes) that refer to several distinct vascular formations which are best visualised three-dimensionally but lack definitions adapted for a two-dimensional histological context [29–32]. raspberries may constitute cross-sectional counterparts to some of these vascular formations, but the extent of this overlap has not been established. fig. 1 a cortical microvascular raspberry, measuring 60 µm in diameter. stain: haematoxylin-eosin. https://doi.org/10.17879/freeneuropathology-2025-6178 free neuropathology 6:7 (2025) ek olofsson and englund doi: https://doi.org/10.17879/freeneuropathology-2025-6178 page 3 of 14 in archival brain tissue from postmortem neuropathological examinations, raspberries are highly prevalent in the neocortex, basal ganglia, hippocampus, and brainstem, while occurring only rarely in white matter and in the cerebellum [26]. in our experience, raspberries are typically composed of arterioles and may focally constitute up to 3 % of the cortical blood vessels. we have previously demonstrated increased cortical raspberry density in association with vascular dementia [24], cerebral atherosclerosis [25, 26], and hypertensive disease (exploratory finding [27]), potentially indicating that the mechanisms of raspberry formation overlap with those that lead to cerebrovascular disease. however, an independent, positive association between raspberry density [27], or multi-lumen vascular profiles [28], and advancing age has also been observed, indicating that additional mechanisms may be involved, as ageing is associated with distinct effects on the vasculature [33, 34]. knowledge on the distribution of raspberries among neurodegenerative diseases would be valuable to further explore the extent to which increased raspberry density may occur separately from conventional vascular risk factors and cerebrovascular disease. we have previously observed an increased cortical raspberry density in a small group of ftld patients (n = 10) compared to control cases, but this finding did not achieve statistical significance [24]. the same study indicated a similar but weaker trend for patients with alzheimer’s disease [24], while a later study provided no indication of an increased raspberry density in patients with alzheimer pathology [27]. no previous study has been designed to examine the raspberry density in lbd. an investigation including lbd would also enable stratification according to co-occurring alzheimer pathology, which frequently accompanies this disease [35]. as such, the aim of the current study was to examine the frontocortical raspberry density in frontotemporal lobar degeneration (ftld) and lewy body disease (lbd) in an adequately dimensioned study sample, and to progress with exploratory subgroup analyses if supported by the primary statistical analysis. materials and methods study population the study population was drawn retrospectively from patients who had undergone a diagnostic postmortem neuropathological examination at clinical genetics, pathology, and molecular diagnostics, region skane, lund. the autopsy reports were accessed via the digital systems sympathy (cases prior to april 2019) and lims rs. the medical records were accessed via the digital system melior, in which public specialist healthcare performed in region skane is documented. all histotechnical procedures and diagnostic examinations had been performed prior to the start of the study; the details of the macroand microscopic neuropathological examination have been described previously [36]. cases with ftld and lbd were retrieved from january 2001 to december 2022. the ftld cases had been diagnosed according to published consensus recommendations, including, when applicable, assignment of a specific diagnosis [1, 2, 37–40]. likewise, the lbd cases had been diagnosed according to consensus recommendations; for inclusion, the cases needed to be of neocortical or limbic category [11, 12]. the control cases were drawn from april 2019 to december 2022 and consisted of individuals who were over 49 years old at death and who were not diagnosed with dementia according to the medical records. in all cases, the autopsy reports were as sessed regarding co-occurring neuropathology. brain weight and findings of atherosclerotic plaques in the basilar artery and circle of willis were noted (referred to as cerebral atherosclerosis), as were findings of cerebral arteriolosclerosis [41]. mixed pathology defined as more than one cerebral infarct resulted in exclusion. likewise, co-occurring neurodegenerative disease resulted in exclusion, with the exception of primary age-related tauopathy or alzheimer’s disease neuropathologic change with neurofibrillary tangle (nft) pathology corresponding to braak stage ii or lower [42–44]. in cases with lbd, nft pathology up to braak stage iv was accepted due to the known co-occurrence of lbd https://doi.org/10.17879/freeneuropathology-2025-6178 free neuropathology 6:7 (2025) ek olofsson and englund doi: https://doi.org/10.17879/freeneuropathology-2025-6178 page 4 of 14 and alzheimer-type pathology [35]. other forms of neuropathological comorbidity were noted, but did not result in exclusion. patients included in our previous study on raspberries in ftld were deemed eligible for inclusion, since they were considered not to differ from the rest of the study sample in any systematic way [24]. after assessment, nine of the ten ftld cases from this previous study were included (in one case, an adequately dimensioned glass slide was not available; see next section). because the raspberry density of these cases had been measured using an older method, they were re-quantified along with the other cases, as described in the next section. raspberry quantification and assessment of cortical atrophy the raspberry quantification was performed on whole slide images of scanned tissue sections in the same manner as in our latest study on this topic [27]. for each included individual, the cortical area of one 6-µm thick haematoxylin-eosin-stained tissue section from the frontal lobe (prepared from a formalin-fixed paraffin-embedded tissue block and mounted on a 76 × 26 mm glass slide) was viewed in either aperio imagescope (cases prior to april 2019) or sectra ids7. the tissue section had been sampled from brodmann area 10 in most cases, but tissue sections from other parts of the frontal lobe were also considered acceptable. for each tissue section, the number of cortical raspberries was noted, correlated with the size of the examined area (median 1.89 cm²), and reported as raspberries/cm² (fig. 2). all quantification was performed blinded to patient characteristics. the degree of frontocortical atrophy was assessed in well-orientated parts of the tissue samples and ranked on a scale from 0 to 3 (none, mild, moderate, or severe) based on the overall impression of cortical thinning, neuropil rarefaction, and neuronal loss. to aid in the assessment, each slide was compared to a template of representative micrographs (fig. 3). all assessments were performed by a single examiner without access to data on raspberry density or other patient characteristics. fig. 2 overview of a tissue section from the frontal lobe. the cortex has been delineated, and the raspberries within the delineated area have been marked. stain: haematoxylin-eosin. https://doi.org/10.17879/freeneuropathology-2025-6178 free neuropathology 6:7 (2025) ek olofsson and englund doi: https://doi.org/10.17879/freeneuropathology-2025-6178 page 5 of 14 fig. 3 a–d template with representative micrographs ranked as a no (0), b mild (1), c moderate (2) and d severe (3) frontocortical atrophy. stain: haematoxylin-eosin. scalebar: 200 µm. statistics a sample size calculation performed in epitools [45] indicated that 88 individuals per group would enable the detection of a mean difference in raspberry density of 3 raspberries/cm² with a 95 % level of confidence and 80 % statistical power. when increasing the level of confidence to 98 % to provide an approximate adjustment for multiple tests (three tests), a group size of 112 individuals was indicated. as such, we aimed to include at least 88 individuals per group, and optimally 112 individuals per group. ibm spss statistics version 28 was used to perform the statistical analyses. the difference in mean raspberry density between the three groups was analysed using one-way welch’s anova, followed by the games-howell post hoc test due to the unbalanced design. when the post hoc test indicated a difference between any two of the groups, they were further analysed with two multiple linear regression models wherein age, sex, and either brain weight or microscopic cortical atrophy were included as potential confounding factors. exploratory subgroup analyses addressed the raspberry density in relation to neurodegenerative disease subtype (tested with the non-parametric kruskal-wallis test), and in relation to brain weight and cortical atrophy (tested with spearman rank correlation). a p value of ≤ 0.05 was considered statistically significant. results the control group consisted of 105 individuals, the lbd group of 80 individuals, and the ftld group of 98 individuals (the distribution of lbd and ftld subtypes is shown in fig. 4). demographics, markers of brain atrophy, and neuropathological findings of cerebrovascular disease are reported in table 1; other neuropathological findings did not occur in the ftld and lbd groups, with the exception of two cases in the lbd group, who had been diagnosed with cerebral metastases. in the control group, the neuropathological findings were more diverse, but the raspberry density of control cases with neuropathological comorbidity (which constituted 31 % of the control group) was similar to that of control cases with no comorbidity (table 2). https://doi.org/10.17879/freeneuropathology-2025-6178 free neuropathology 6:7 (2025) ek olofsson and englund doi: https://doi.org/10.17879/freeneuropathology-2025-6178 page 6 of 14 fig. 4 neuropathological classification of the lbd and ftld groups. numbers in brackets indicate n. c9orf72 chromosome 9 open reading frame 72 gene; cbd corticobasal degeneration; ftld frontotemporal lobar degeneration; fus fused in sarcoma; lbd lewy body disease; mapt microtubule-associated protein tau gene; nft neurofibrillary tangle; nos not otherwise specified; pid pick’s disease; psp progressive supranuclear palsy; tdp transactivation response dna-binding protein; ups ubiquitin proteasome system. table 1: individual variables including neuropathology details variable control lbd ftld total n 105 80 98 283 female sex 42 (40 %) 24 (30 %) 48 (49 %) 114 (40 %) median age at death (yr) 72 (50–92) 77 (51–99) 73 (44–92) 74 (44–99) median brain weight (g) 1367 (875–1900) 1367 (986–1698) 1185 (610–1570) 1301 (610–1900) no cortical atrophy 87 (83 %) 24 (30 %) 12 (12 %) 123 (43 %) mild cortical atrophy 18 (17 %) 50 (63 %) 43 (44 %) 111 (39 %) moderate cortical atrophy 0 6 (8 %) 27 (28 %) 33 (12 %) severe cortical atrophy 0 0 16 (16 %) 16 (6 %) single cerebral infarct 24 (23 %) 13 (16 %) 15 (15 %) 52 (18 %) cerebral arteriolosclerosis 29 (28 %) 18 (23 %) 32 (33 %) 79 (28 %) cerebral atherosclerosis 25/87 (29 %) 21/69 (30 %) 27/87 (31 %) 73/243 (30 %) table 1 characteristics of the study sample. numbers represent frequency unless otherwise indicated in the variable column. intervals represent min–max. for variables with incomplete data, full quotients are provided. other neurodegenerative diseases and cases with multiple infarcts had been excluded from the sample as described under study population (materials and methods). lbd lewy body disease; ftld frontotemporal lobar degeneration. https://doi.org/10.17879/freeneuropathology-2025-6178 free neuropathology 6:7 (2025) ek olofsson and englund doi: https://doi.org/10.17879/freeneuropathology-2025-6178 page 7 of 14 table 2: neuropathological comorbidity in the control group variable frequency median raspberry density (min–max) demyelinating disease 2 (2 %) 2.0 (0.8–3.2) infection 17 (16 %) 3.1 (0.5–19.2) malignancy 8 (4 %) 2.8 (1.2–7.8) malformation 2 (2 %) 1.7 (0.8–2.6) toxic disease 4 (4 %) 5.0 (3.2–9.2) traumatic brain injury 1 (1 %) 16.4 any finding* 33 (31 %) 3.1 (0.5–19.2) no finding* 72 (69 %) 3.9 (0–19.2) total 105 (100 %) 3.3 (0–19.2) table 2 frontocortical raspberry density (raspberries/cm²) among control cases in relation to neuropathological comorbidity. * neuropathologically verified demyelinating disease, infection, malignancy, malformation, toxic disease, and traumatic brain injury. there was a higher raspberry density in the ftld group compared to both the lbd and control groups, and a slightly higher raspberry density in the lbd group compared to the control group (fig. 5). the one-way welch’s anova indicated statistically significant differences between the three groups (p < 0.001), and the games-howell post hoc test demonstrated significant differences between the ftld and control groups (p < 0.001), as well as between the ftld and lbd groups (p = 0.001). in contrast, the lbd and control groups did not differ at a statistically significant level (p = 0.69). when comparing the ftld group to the lbd group using multiple linear regression, the difference in raspberry density remained statistically significant when including age, sex, and either brain weight or microscopic cortical atrophy in the analysis (table 3). when comparing the ftld group to the control group, the results remained statistically significant when including age, sex, and brain weight in the analysis, while a trend towards statistical significance was observed when instead including age, sex, and cortical atrophy (table 4). fig. 5 raspberry density (raspberries/cm²) of the frontal cortex in relation to ftld, lbd, and control cases. lines and dots indicate medians and interquartile ranges, respectively. * p = 0.001 (games-howell post hoc test). ** p < 0.001 (games-howell post hoc test). ftld frontotemporal lobar degeneration; lbd lewy body disease; ns not significant. table 3: statistical comparison of raspberry density between the ftld and lbd groups games-howell post hoc test* independent variable p value (95 % ci) ftld 0.001 (1.0;5.0) multiple linear regression, model 1 independent variable p value (95 % ci) ftld 0.034 (0.2;4.6) age at death (y) 0.34 (-0.5;0.2) female sex 0.85 (-2.2;1.9) brain weight (g) 0.02 (-0.1;-0.001) multiple linear regression, model 2 independent variable p value (95 % ci) ftld 0.012 (0.6;4.5) age at death (y) 0.069 (-0.007;0.2) female sex 0.68 (-1.4;2.1) cortical atrophy (0–3) 0.02 (0.2;2.4) table 3 p values with 95 % ci from the games-howell post hoc test and multiple linear regression models aimed at comparing the ftld group (n = 98) to the lbd group (n = 80). statistically significant results have been marked in bold. outcome variable: frontocortical raspberry density (raspberries/cm²). *the post hoc test was preceded by one-way welch anova which included all three groups (n = 283) and achieved statistical significance (p < 0.001). ci confidence interval; lbd lewy body disease; ftld frontotemporal lobar degeneration. https://doi.org/10.17879/freeneuropathology-2025-6178 free neuropathology 6:7 (2025) ek olofsson and englund doi: https://doi.org/10.17879/freeneuropathology-2025-6178 page 8 of 14 table 4: statistical comparison of raspberry density between the ftld and control groups games-howell post hoc test* independent variable p value (95 % ci) ftld < 0.001 (1.7;5.5) multiple linear regression, model 1 independent variable p value (95 % ci) ftld 0.004 (1.0;5.0) age at death (y) 0.072 (-0.007;0.2) female sex 0.93 (-1.8;2.0) brain weight (g) 0.38 (-0.008;0.003) multiple linear regression, model 2 independent variable p value (95 % ci) ftld 0.054 (-0.04;4.1) age at death (y) 0.014 (0.02;0.2) female sex 0.88 (-1.4;1.7) cortical atrophy (0–3) 0.061 (-0.05;2.2) table 4 p values with 95 % ci from the games-howell post hoc test and multiple linear regression models aimed at comparing the ftld group (n = 98) to the control group (n = 105). statistically significant results have been marked in bold. outcome variable: frontocortical raspberry density (raspberries/cm²). *the post hoc test was preceded by one-way welch anova which included all three groups (n = 283) and achieved statistical significance (p < 0.001). ci confidence interval; ftld frontotemporal lobar degeneration. given the results of the primary statistical analysis, further analyses were undertaken to explore the variation in raspberry density within the fltd group. these demonstrated a trend towards a positive correlation between raspberry density and microscopic cortical atrophy (p = 0.062; rho = 0.19; fig. 6, a), and a corresponding trend towards a negative correlation between raspberry density and brain weight (p = 0.078; rho = -0.19; fig. 7, a). the most frequent diagnosis among ftld cases with no cortical atrophy was progressive supranuclear palsy, whereas most cases with severe cortical atrophy were diagnosed with ftld-tdp (various types). as expected, cortical atrophy correlated negatively with brain weight (p < 0.001; rho = -0.55). non-parametric testing (kruskal-wallis) was inconclusive when analysing the raspberry density in ftld in relation to proteinopathy (p = 0.46) and specific diagnosis; the latter comparisons were restricted to ftld-tau (p = 0.28) and -tdp (p = 0.52; fig. 7, b–d). these analyses remained inconclusive also after excluding small groups (n < 5; not shown). for descriptive data on the raspberry density in lbd in relation to limbic / neocortical category and presence / absence of braak stage iii–iv nft pathology, see fig. 8, a–b. fig. 6 a–c raspberry density (raspberries/cm²) of the frontal cortex in relation to microscopic cortical atrophy in a ftld (p = 0.062; rho = 0.19; spearman rank correlation), b lbd (not statistically analysed) and c control cases (not statistically analysed). lines indicate medians; numbers in brackets indicate n. ftld frontotemporal lobar degeneration; lbd lewy body disease. https://doi.org/10.17879/freeneuropathology-2025-6178 free neuropathology 6:7 (2025) ek olofsson and englund doi: https://doi.org/10.17879/freeneuropathology-2025-6178 page 9 of 14 fig. 7 a–d raspberry density (raspberries/cm²) of the frontal cortex in ftld in relation to a brain weight (p = 0.078; rho = -0.19; spearman rank correlation), b proteinopathy (p = 0.46; kruskal-wallis test), c ftld-tau (p = 0.28; kruskal-wallis test), and d ftld-tdp (p = 0.52; kruskal-wallis test). lines indicate medians; numbers in brackets indicate n. the statistical analyses of raspberry density in relation to b–d remained inconclusive also after excluding small groups (n < 5; not shown). cbd corticobasal degeneration; ftld frontotemporal lobar degeneration; fus fused in sarcoma; nos not otherwise specified; pid pick’s disease; psp progressive supranuclear palsy; tdp-43 transactivation response dna-binding protein 43, ups ubiquitin proteasome system. https://doi.org/10.17879/freeneuropathology-2025-6178 free neuropathology 6:7 (2025) ek olofsson and englund doi: https://doi.org/10.17879/freeneuropathology-2025-6178 page 10 of 14 fig. 8 a–b raspberry density (raspberries/cm²) of the frontal cortex in lbd in relation to a lbd category (not statistically analysed), and b co-occurring nft pathology (not statistically analysed). lines indicate medians; numbers in brackets indicate n. lbd lewy body disease; nft neurofibrillary tangle. discussion the current study examined the raspberry density of the frontal cortex in ftld, lbd and control cases, and found it to be increased in ftld compared to both of the other groups. the results did not indicate any difference between the lbd and control groups. based on our sample size calculation, the ftld (n = 98) and control groups (n = 105) were considered sufficiently large, but the comparisons involving the lbd group (n = 80) were at risk of being underpowered. low power could reduce the chances of detecting true differences, while also increasing the risk of overestimating effect sizes. with this limitation in mind, our results provide no indication of a relevant difference in frontocortical raspberry density between lbd and control cases. according to our multiple linear regression analysis, the increased raspberry density in ftld relative to the lbd group was independent of variations in age, sex, brain weight, and microscopic cortical atrophy. the difference in raspberry density between the ftld and control groups was statistically significant when including age, sex, and brain weight in the analysis, but not when exchanging brain weight for cortical atrophy (p = 0.054). it should be noted that most ftld and lbd cases exhibited some degree of cortical atrophy, whereas most control cases did not. as such, it is possible that a larger study sample – wherein more ftld and control cases would exhibit the same degree of cortical atrophy – could have achieved statistical significance. however, we also observed a trend towards a positive correlation between raspberry density and cortical atrophy in the ftld group (p = 0.062), which is similar to the finding of an increasing endothelial cell proportion with increasing cortical atrophy described in a recent study on ftld-tdp [9]. in one respect, our finding could indicate that the increased raspberry density in ftld is at least partially secondary to cortical atrophy. our previous studies on raspberries have not indicated an association between raspberry density and brain atrophy https://doi.org/10.17879/freeneuropathology-2025-6178 free neuropathology 6:7 (2025) ek olofsson and englund doi: https://doi.org/10.17879/freeneuropathology-2025-6178 page 11 of 14 [26, 27], but such an association could still exist within the ftld population (or in a broader context of neurodegeneration, as potentially indicated by the multiple linear regression models). cortical atrophy could appear to increase the raspberry density by reducing the distance between blood vessels while the absolute number of vessels remains constant. alternatively, neuropil rarefaction could reduce the stability around cortical arterioles and lower the threshold for arteriolar buckling and stretching with ensuing elongation [46], possibly resulting in crowding of multiple arteriolar branches within the same perivascular space. however, the trend towards a positive correlation between raspberry density and cortical atrophy could also be due to the ftld disease process causing both cortical atrophy and raspberry formation. notably, vascular alterations have been described in experimental studies on the pathomechanisms of tau and tdp-43 [47–52], in autopsy studies on ftld [4–10], and in amyotrophic lateral sclerosis [53], an entity that overlaps clinically, pathologically, and genetically with ftld-tdp [54]. these alterations include abnormal remodelling of the extracellular matrix [8–10, 48, 51, 52], increased angiogenesis [8–10, 48, 51], altered microvascular density and morphology [6, 8, 48], compromised blood brain barrier integrity [5, 8, 47, 50, 52], and abnormal blood flow [48, 49]. among these mechanisms, remodelling of the vascular wall extracellular matrix could alter the stiffness of cortical arterioles and lower the threshold for arteriolar buckling [46], in addition to the possible effects of neuropil rarefaction discussed above. alternatively, the increased raspberry density in ftld could be a manifestation of abnormal angiogenesis and arterialisation, as previously hypothesised due to the increased raspberry density in vascular dementia [24]. our exploratory analyses of the raspberry density in ftld in relation to proteinopathy and specific diagnosis were inconclusive. looking at the descriptive data, the raspberry density in ftld-tdp was slightly higher than in ftld-tau, but the ftld-tdp group also exhibited more severe cortical atrophy. among the ftld-tau and -tdp subgroups, the highest raspberry density was found in ftld-tdp type b. among the eight patients with a documented c9orf72 mutation, six manifested as ftld-tdp type b (one of these cases also had tau pathology), and the other two as type a. as a group, these eight patients exhibited a notably high raspberry density (median 11.6 raspberries/cm², compared to 7.1 raspberries/cm² for the entire ftld group). due to the low number of known c9orf72 mutation carriers and large proportion of individuals with unknown genetic status in our study population, the relevance of this observation is unclear. interestingly, individuals with a c9orf72 mutation exhibit a presymptomatic decline in cerebral blood flow that may be less dependent on brain atrophy compared to grn and mapt mutation carriers [55], and a recent experimental study indicated that endothelial cells derived from c9orf72 mutation carriers have an abnormal phenotype that potentially affects the substrate exchange with neurons (other dysfunctional features were also reported [56]). our results do not indicate an increased frontocortical raspberry density in lbd compared to control cases. it is possible that the pathomechanisms of lbd simply do not induce raspberry formation. alternatively, raspberry formation could be partially inhibited by anti-angiogenic effects of alpha-synuclein accumulation [21]. it should be noted that hypoperfusion of the occipital lobes is considered characteristic of lbd [57]; however, the hypoperfusion in lbd is often more widespread and involve also the frontal lobes [58, 59]. as such, we would have expected to detect an increased frontocortical raspberry density in the lbd group if an association between increased raspberry density and lbd were to exist, but an examination of the occipital raspberry density in lbd could be valuable to further explore this relationship. the main limitations of this study are the retrospective design and the semiquantitative assessment of cortical atrophy. because all autopsies were initiated based on clinical indications, relatively lenient exclusion criteria were deemed necessary to acquire a sufficiently large study sample. consequently, a proportion of the control cases exhibited neuropathological comorbidity that could affect the external validity, although importantly, the raspberry density of cases with neuropathological comorbidity was similar to that of cases with no such findings (with the exception of the single case with traumatic brain injury; table 2). ordinal ranking of https://doi.org/10.17879/freeneuropathology-2025-6178 free neuropathology 6:7 (2025) ek olofsson and englund doi: https://doi.org/10.17879/freeneuropathology-2025-6178 page 12 of 14 cortical atrophy was considered less dependent on sample orientation than measuring cortical thickness. with this semiquantitative approach, a difference in raspberry density beyond what may be explained by cortical atrophy was indicated, but the comparison between ftld and control cases was statistically inconclusive, and our results also indicated a potential correlation between raspberry density and cortical atrophy. as such, an assessment of the raspberry density in ftld in relation to a quantitative measure of cortical atrophy is motivated to validate the results. with these limitations in mind, the study adds to previous findings of increased raspberry density in relation to advancing age and vascular dementia, potentially indicating that various underlying causes may converge in increased raspberry formation. the study does not enable conclusions on whether the raspberry density is higher within a certain part of the ftld spectrum. however, given the lack of association between raspberry density and lbd, altered raspberry density may not be a general feature of neurodegenerative disease. future studies are needed to determine whether increased raspberry density in ftld could function as a marker for more widespread vascular alterations, by examining the raspberry density in ftld in relation to overall vascular density, protein aggregates in the vascular wall, signs of compromised blood brain barrier, or altered extracellular matrix deposition. an examination of the raspberry density in other conditions that manifest with tau or tdp43 pathology may also be motivated. declarations ethics statement the study was approved by the swedish ethical review authority, application number 2019-00080 and amendments number 2021-05753-02 and 202303097-02. conflicts of interest statement the authors declare no conflicts of interest. funding statement the study was supported by lund university, region skane, and the trolle-wachtmeister foundation for medical research. acknowledgements the authors extend their gratitude to the staff at clinical genetics, pathology, and molecular diagnostics, region skane, lund, for invaluable technical assistance throughout the project. references 1. mackenzie ira, neumann m, bigio eh, cairns nj, alafuzoff i, kril j, et al. nomenclature for neuropathologic subtypes of frontotemporal lobar degeneration: consensus recommendations. acta neuropathologica. 2009;117(1):15-8. https://doi.org/10.1007/s00401-008-0460-5 2. mackenzie ira, neumann m, bigio eh, cairns 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statistics results discussion declarations ethics statement conflicts of interest statement funding statement acknowledgements references free neuropathology 6:13 (2025) garrood et al doi: https://doi.org/10.17879/freeneuropathology-2025-6763 page 1 of 27 copyright: © 2025 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. evaluating ultrastructural preservation quality in banked brain tissue macy garrood1*, alicia keberle1*, allison sowa2, william janssen2, emma l. thorn3,4, claudia de sanctis3,4, kurt farrell3,4, john f. crary3,4, andrew t. mckenzie1 1 apex neuroscience, salem, oregon, usa 2 microscopy and advanced bioimaging core, icahn school of medicine at mount sinai, new york, new york, usa 3 friedman brain institute, departments of pathology, neuroscience, and artificial intelligence & human health, icahn school of medicine at mount sinai, new york, new york, usa 4 neuropathology brain bank & research core and ronald m. loeb center for alzheimer's disease, icahn school of medicine at mount sinai, new york, new york, usa  the authors contributed equally to this work. corresponding author: andrew t. mckenzie · apex neuroscience · 3265 marietta st se · salem · or 97317 · usa amckenzie@apexneuro.org submitted: 06 may 2025 · accepted: 09 june 2025 · copyedited by: alessia sciortino · published: 25 june 2025 abstract the ultrastructural analysis of postmortem brain tissue can provide important insights into cellular architecture and disease-related changes. for example, connectomics studies offer a powerful emerging approach for understanding neural circuit organization. however, electron microscopy (em) data is difficult to interpret when the preservation quality is imperfect, which is common in brain banking and may render it unsuitable for certain research applications. one common issue is that em images of postmortem brain tissue can have an expansion of regions that appear to be made up of extracellular space and / or degraded cellular material, which we call ambiguous interstitial zones. in this study, we report a method to assess whether em images have ambiguous interstitial zone artifacts in a cohort of 10 postmortem brains with samples from each of the cortex and thalamus. next, in matched samples from the contralateral hemisphere of the same brains, we evaluate the structural preservation quality of light microscopy images, including immunostaining for cytoskeletal proteins. through this analysis, we show that on light microscopy, cell membrane morphology can be largely maintained, and neurite trajectory visualized over micrometer distances, even in specimens for which there are ambiguous interstitial zone artifacts on em. additionally, we demonstrate that synaptic structures can be successfully traced across serial em sections in some postmortem samples, indicating the potential for connectivity studies in banked human brain tissue when appropriate preservation and visualization protocols are employed. taken together, our analysis may assist in maximizing the usefulness of donated brain tissue by informing tissue selection and preparation protocols for various research goals. keywords: brain banking, postmortem changes, perfusion fixation, neurofilaments, ultrastructural quality, connectomics original paper https://doi.org/10.17879/freeneuropathology-2025-6763 https://creativecommons.org/licenses/by/4.0/ https://creativecommons.org/publicdomain/zero/1.0/ mailto:amckenzie@apexneuro.org free neuropathology 6:13 (2025) garrood et al doi: https://doi.org/10.17879/freeneuropathology-2025-6763 page 2 of 27 abbreviations aiz ambiguous interstitial zones, em electron microscopy, gfap glial fibrillary acidic protein, h hour, h&e hematoxylin and eosin, irr inter-rater reliability, lm light microscopy, nbf neutral buffered formalin, pmi postmortem interval, wsi whole slide image. introduction since the 1950s, when the synapse was first visualized with an electron microscope, the ultrastructural analysis of brain tissue has enabled many critical discoveries about the structure of the brain (sotelo, 2020; nahirney and tremblay, 2021). in recent years, em-based connectomics, in particular, has enabled key insights into larger-scale neural circuitry across diverse species (lichtman et al., 2014; shiu et al., 2024). expanding connectomics to a larger set of human brains may allow us to answer fundamental questions about our brains in health and disease. however, data collection for connectomics relies on high-quality tissue preservation that enables the reliable tracing of neuronal processes and identification of synaptic connections. these requirements are challenging to achieve in human brain tissue samples, which generally have quality limitations due to agonal factors, the postmortem interval (pmi), and impediments in preservation methods (mckee, 1999; mcfadden et al., 2019; mckenzie et al., 2022). to date, most volume electron microscopy studies have been performed on non-human brain tissue (chiappini et al., 2025). the successful volume electron microscopy and connectomics studies on human brain tissue have been limited to tissue that is selected for having ideal preservation quality, such as surgical biopsy tissue or deceased donor tissue with very low pmi (shapson-coe et al., 2024; plaza-alonso et al., 2025). while mapping an entire human brain with electron microscopy far exceeds our current technological capabilities, the ability to image nanoscale neural connectivity in tissue from a wider set of banked brains would still be valuable for several reasons. first, brain banks serve diverse research communities, and investigators require access to tissue from different brain regions and with different donor characteristics, which may not always be possible to acquire from surgical biopsies. second, larger sample sizes are critical for statistically robust inferences regarding the structural correlates of disease, as the history of genomics has shown (uffelmann et al., 2021). third, advances in imaging and computing technologies are rapidly improving our ability to analyze neural circuits at scale. as these tools continue to develop, having access to more banked brain tissue whose ultrastructure can be profiled may enable increasingly larger-scale connectomics studies of the human brain. given these considerations, there is a critical need to investigate whether current techniques used in brain banking are sufficient to maintain the structural integrity needed for future connectome analyses, despite the fact that our present connectome imaging and reconstruction capabilities are still limited. there is a lack of standardized methods to assess brain preservation quality, which means that it may be useful to consider first principles (mcfadden et al., 2019; wahyudi et al., 2025). structural changes can occur prior to fixation, during fixation, and during subsequent tissue processing, and may include membrane blebbing, vacuoles, cytoplasmic washout, and chromatin alterations (garman, 2011; krassner et al., 2023; mckenzie et al., 2024). some of these artifacts, such as blebbing or vacuoles, may not prevent us from tracing the connectome, instead only leading to the expansion or compression of certain cellular structures, which can still be adequately visualized. a more significant challenge arises from the observed phenomenon in some samples of enlarged, electron-lucent, nonmembrane-bound regions between cells, which we call ambiguous interstitial zones (aizs). crucially, these aizs are usually also associated with an apparent decrease in the density of visualized cellular structures, such as thin neurites. instead, the aizs often contain what appears to be poorly defined structures, possibly cellular debris. these poorly defined structures are not usually visible in tissue that has been preserved with a negligible duration of ischemia prior to preservation. these aizs and associated poorly defined structures have been described in previous literature, for example, as causing a “lacey” appearance in postmortem https://doi.org/10.17879/freeneuropathology-2025-6763 free neuropathology 6:13 (2025) garrood et al doi: https://doi.org/10.17879/freeneuropathology-2025-6763 page 3 of 27 brain tissue (palay et al., 1962; liewald et al., 2014; lewis et al., 2019). mechanistically, very early in the pmi, the extracellular space is actually expected to shrink due to ischemic cell swelling (nicholson and syková, 1998). as the pmi becomes prolonged, biomolecular structures break down and cell membrane integrity is eventually lost, leading to passive fluid redistribution and an expected expansion of the apparent extracellular space (krassner et al., 2023). this excess fluid may affect fine neural structures in various ways, from complete dissolution in severe cases to preservation of some elements but with insufficient compactness for visualization in others. the difficulty in visualization may be in part because microscopy depends on biomolecules being densely aggregated enough to maintain their structural integrity during tissue processing and bind adequately with staining chemicals (wang and minassian, 1987). however, during prolonged pmi, these biomolecular networks progressively dissociate, reducing their density. previous studies have found that fixation and sample preparation methods can influence the visualized ultrastructure of brain tissue (schiff and gennaro jr., 1979; small, 1981; sele et al., 2019; eberhardt et al., 2022; shafiei et al., 2024). for example, one study reported that there can be significant extraction of proteins and lipids during em preparation, creating large empty spaces, which is an issue that worsens with longer pmi (lewis et al., 2019). they found that adding 0.1 % glutaraldehyde to their formalin fixative partially rescued this. another study found that the use of embedding techniques for em at room temperature, which is standard for the field, introduced ultrastructural artifacts such as swollen mitochondria, disrupted membranes, and extraction of cellular components (sosinsky et al., 2008). however, these artifacts were avoided when fixation was instead followed by a high-pressure freezing protocol, which led to images with smoother cell membranes and more dense cytoplasm. finally, one study tested more than one hundred protocol variations on human brain tissue and found that even subtle differences in fixation, washing, dehydration, and embedding can affect the ultrastructural appearance, including the preservation of extra cellular space (karlupia et al., 2023). our working hypothesis is that the observed artifacts on ultrastructural imaging may result solely from structural degradation due to decomposition prior to fixation, and / or may be partially accounted for by our current methods of sample preparation, staining, and imaging, which are not optimized for decomposed tissue. if true structural loss is the dominant factor, then strict constraints on pmi and preservation procedures may be necessary for selecting samples that are suitable for connectome imaging. on the other hand, if enough morpho molecular markers are still theoretically present to reconstruct the cellular skeleton but are not visualized with current methods due to insufficient optimization for this purpose, then improved postfixation, embedding, staining, imaging, and analysis methods could potentially recover them. in this study, we attempt to address the challenge of distinguishing true structural degradation from visualization artifacts in postmortem brain em images. we develop a metric to assess the extent of aizs in the em images as an index for evaluating preservation quality. we also analyze matched samples from the contralateral hemisphere stained with hematoxylin and eosin (h&e) and via immunohistochemistry, to attempt to identify cases where cellular structures remain intact on light microscopy. our goal is to help improve our methods for assessing ultrastructural preservation quality and explore the differences between the measured preservation quality at the light and electron microscopy levels. methods brain banking procedures anatomical whole-body donations were performed by a partner whole-body donation organization operating under oregon health authority regulations. additionally, through our canine brain bank program, we were donated the body of one deceased canine following euthanasia by a licensed veterinarian, with signed owner consent for research use (sándor et al., 2021). the apex neuroscience brain and tissue bank operates https://doi.org/10.17879/freeneuropathology-2025-6763 free neuropathology 6:13 (2025) garrood et al doi: https://doi.org/10.17879/freeneuropathology-2025-6763 page 4 of 27 under an exemption determination issued by the pearl institutional review board (irb) after the submission of our protocols for review. the pmi was calculated as the time elapsed between death and the initiation of the preservation procedure. when the day of death but not the hour was known, the time of death was estimated at the middle of the day (12 pm) to provide a standardized approach for pmi calculation in cases with incomplete time data. during the pmi, the donor bodies were generally stored in refrigeration, for example, in a hospital morgue. however, we do not have the exact durations of this refrigerated storage period for each donor, which is a potential confound, as higher temperatures during the pmi are associated with more structural breakdown. one of the brain specimens (donor #57) was fixed by immersion in 10 % neutral buffered formalin (nbf, azer scientific nbf55g) alone. note that 10 % nbf contains 3.7 % formaldehyde (snyder et al., 2022). the rest of the human brains were perfused in situ with the use of a peristaltic pump, following cannulation of the bilateral carotid arteries with 10 % nbf. for several of the donors (donors #7, 30, 34, and 37), the carotid arteries were accessed via dissection in the anterior cervical region. for the remaining human donors, the cephalon was isolated via dissection at the approximate spinal level of c4-c5 (turkoglu et al., 2014). this allowed for cannulation of the bilateral internal carotid arteries and clamping of the vertebral arteries to prevent outflow through them. the one canine brain was from a 2.3 kg miniature pinscher whose brain was perfused in situ with 10 % nbf via cannulation of the left ventricle and the use of a peristaltic pump. the brains were removed from the skull following standard procedures (adams and murray, 1982). they were then immersed in 10 % nbf at 4 °c for at least one month prior to further processing (mckee, 1999). small samples were taken from grey matter of the sensorimotor cortex and the grey matter of the thalamus, from one hemisphere for light microscopy and the contralateral for em. electron microscopy tissue for em was post-fixed in a solution of 2 % paraformaldehyde and 2.5 % glutaraldehyde in 0.1m sodium cacodylate buffer. a version of the national center for microscopy and imaging research (ncmir) protocol was adapted to provide enhanced contrast (deerinck et al., 2010). specifically, after fixation, the tissue underwent a multi-step enhanced contrast protocol at room temperature including sequential treatments with tannic acid, reduced osmium, thiocarbohydrazide, osmium, and uranyl acetate. this was followed by lead aspartate staining at 60 °c. the brain sample was then dehydrated through a graded ethanol series, infiltrated with embed 812 epoxy resin (ems), and polymerized for 72 h at 60 °c. semithin sections (0.5 μm) were cut using a leica uc7 ultramicrotome (leica, buffalo grove, il) and counterstained with 1 % toluidine blue to identify the regions of interest within layers. images were taken on a ht7500 transmission electron microscope (hitachi high-technologies, tokyo, japan) using an amt nanosprint12 12-megapixel cmos tem camera system, software version 7.0.1.485 (advanced microscopy techniques, danvers, ma). images were only adjusted for contrast on the amt software. for three of the samples, we performed serial section transmission electron microscopy (sstem), using ultra-thin sections of 80 nm thickness, collected onto nickel slot grids. there were 6 image series from the identified region of interest across 10 serial sections. we first performed fine-scale manual annotation of a subset of em images, including one high-magnification image from all 20 unique brain samples (from different donors and brain regions). we annotated the boundaries of four mutually exclusive classes of structures across the image: (a) cell bodies, (b) membrane-bound structures with electron-dense interior, (c) membrane-bound structures with electron-lucent interior, and (d) myelinated axons. category (c) is expected to include fluid-filled, swollen astrocyte processes, which is a common artifact seen in postmortem https://doi.org/10.17879/freeneuropathology-2025-6763 free neuropathology 6:13 (2025) garrood et al doi: https://doi.org/10.17879/freeneuropathology-2025-6763 page 5 of 27 brain tissue, but is not necessarily expected to make it more difficult to trace neural processes (krassner et al., 2023). notably, category (b) could include neurites as well as astrocyte, oligodendrocyte, or microglia processes. because we only have 2d images for most of the em data, we cannot more definitively annotate these on the basis of their 3d structure. the remainder of the image is designated as aizs. these extracellular spaces may or may not contain partially degraded cellular membranes or other structural elements that cannot be reliably visualized due to postmortem changes or preparation artifacts. to assess inter-rater reliability for the fine-scale manual annotation, two independent raters annotated the same image. the spatial agreement between their annotations was quantified using the dice similarity coefficient, with the median pairwise dice score calculated across all polygons that were identified as having the most similar coordinates between the annotations. we next graded two-dimensional em images on whether or not they had aiz artifacts, rating each image as either having or not having both (a) extensive aizs and (b) a low degree of cell membrane intactness. we added the additional criterion of (b) to ensure that the images did not merely have expanded extracellular space, which could theoretically occur in conjunction with clearly delineated, well-preserved lipid membranes, for example depending upon the osmolarity of the preservative solutions (pallotto et al., 2015). two raters worked together to determine the grade for each image and resolved any discrepancies via consensus review. we calculated the percentage of images graded as having aiz artifacts across each of the 5 medium-magnification em images available for all 20 samples. light microscopy brain tissue sampled for light microscopy was placed into cassettes for processing and embedded in paraffin. paraffin-embedded brain sections 6 µm thick were baked, deparaffinized, and stained for h&e. chromogenic immunohistochemistry was performed on ventana discovery ultra according to the manufacturer’s directions (neuropathology brain bank and research core, at mount sinai). the slides 6 µm thick were baked, deparaffinized, and pretreated using cell conditioning (cc1) antigen retrieval buffer (tris / borate/ edta buffer, ph 8.0–8.5, 950-224, roche diagnostics). primary antibodies (table 1) were diluted in antibody dilution buffer (adb250, ventana medical system inc., roche diagnostics). smi-311 and smi-312 are antibody cocktails that target neurofilament heavy (nf-h) and medium (nf-m) chain proteins (ulfig et al., 1998). smi-311 recognizes non-phosphorylated epitopes and therefore primarily stains dendrites and perikarya, while smi-312 targets the highly phosphorylated epitopes predominantly found in axons. the detection was performed using the ultraview universal dab detection kit (760-500, roche diagnostics). hematoxylin and bluing reagent (760-2021, 760-2037, roche diagnostics) were used as a nuclear counterstain. digital images of the stained sections were captured at 40x as whole slide images (wsis) using the aperio gt450 high-resolution scanner (leica biosystems). wsis were viewed using the default display settings in qupath (v. 0.4.3). table 1: list of antibodies used in this study. antibody company catalog # dilution vimentin (v9) roche, cell marque 05278139001 2.5 ug/ml gfap (ep672y) roche, cell marque 5269784001 1 ug/ml smi-311 biolegend 837904 1/1000 smi-312 biolegend 837801 1/1000 https://doi.org/10.17879/freeneuropathology-2025-6763 free neuropathology 6:13 (2025) garrood et al doi: https://doi.org/10.17879/freeneuropathology-2025-6763 page 6 of 27 for the h&eand immunohistochemically stained wsis, we first performed a qualitative assessment of preservation quality, examining the images for whether cell membrane morphology appeared intact across samples and also for common postmortem artifacts. for semi-quantitative grading of the samples, two raters collaboratively evaluated each sample and decided upon consensus grades. perfusion quality on h&e staining was graded on a 0-3 scale by examining for the presence of intravascular material (including blood cells) across the wsi, with 0 indicating minimal intravascular material and 3 indicating extensive intravascular material. this analysis was done with the raters blinded to the preservation method used. we attempted to perform a grading scale for glial fibrillary acidic protein (gfap), smi-311, and smi-312 immunostaining. however, for gfap immunostaining, we determined that variability in staining could reflect multiple factors, including pre-mortem influence, the region sampled, and the fixation time, making it unsuitable as a reliable metric for preservation quality assessment. for smi-311 and smi-312, we were unable to identify enough variability between the samples, as they all appeared to have robust and consistent immunoreactivity, with the exception of smi-311 for the two canine samples, which had negligible immunostaining. results preservation quality in electron microscopy images our cohort consisted of nine human brains and one canine brain, which we selected following their preservation to have a wide range of pmis, from 1.5 hours to 4 days (table 2). for each brain, small samples were obtained from the grey matter of the sensorimotor cortex and thalamus. qualitatively, we observed varying degrees of ultrastructural preservation across the specimens. across all samples, we detected several artifacts commonly found in postmortem brain tissue, including cellular swelling and shrinkage, vacuolization, myelin disbanding, and partially disrupted cell membranes whose potential shapes could still be at least partially distinguished (krassner et al., 2023). the canine brain, which had the shortest pmi of 1.5 hours, was found to have the lowest burden of these postmortem artifacts. table 2: characteristics of brain donors included in this study. donor id age sex pmi reported cause of death preservation method fixation time 7 78 male 4.25 hours cancer* perfusion fixation 5 months 30 77 male 17 hours kidney failure, failure to thrive perfusion fixation 4 months 34 88 male 20 hours cancer* perfusion fixation 4 months 37 70 male 72 hours myocardial infarction perfusion fixation 4 months 55 70 male 36 hours cardiac causes perfusion fixation 3 months 57 75 male 96 hours leukemia immersion fixation 3 months 59 41 female 91 hours leukemia perfusion fixation 2.5 months 65 17** female 1.5 hours euthanasia (donated canine) perfusion fixation 2 months 66 89 female 61 hours sepsis and acute renal failure perfusion fixation 2 months 78 89 male 2.5 hours failure to thrive perfusion fixation 1 month fixation time refers to the amount of time in 10 % neutral buffered formalin at 4 °c. *: this donor utilized medical aid in dying (maid) for end-of-life care. **: canine brain. pmi: postmortem interval https://doi.org/10.17879/freeneuropathology-2025-6763 free neuropathology 6:13 (2025) garrood et al doi: https://doi.org/10.17879/freeneuropathology-2025-6763 page 7 of 27 we next performed fine-scale manual annotation of ultrastructural components in the images (figure 1). to assess the reproducibility of our annotation approach, two independent raters annotated the same image, which was from the cortex sample of donor #66. inter-rater reliability was quantified by calculating the dice similarity coefficient for each pair of matching polygons, yielding a median dice score of 0.79 (figure 2). in one high-magnification image from each unique sample, we also quantified the percentage of area occupied by aizs (figure 3, table 3). figure 1. fine-scale annotation of ultrastructural components in one representative em image from the cortex of donor #7. color coding identifies distinct cellular structures: green: membrane-bound structures with electron-dense interior (e.g., organelle-rich neurites); purple: membrane-bound structures with electron-lucent interior (e.g., swollen astrocytic processes); blue: myelinated axons; yellow: cell bodies. non-colored regions represent ambiguous interstitial zones (aizs) that lack clearly defined membrane boundaries. upper scale bar: 2 µm. lower scale bar: 1 µm. https://doi.org/10.17879/freeneuropathology-2025-6763 free neuropathology 6:13 (2025) garrood et al doi: https://doi.org/10.17879/freeneuropathology-2025-6763 page 8 of 27 figure 2. overlap of annotations of a sample from the cortex of donor #66 by independent annotators. the top and middle image were done by two independent annotators. the bottom image is a combination of the overlays to compare how alike the two annotations are. color coding identifies distinct cellular structures: green: membrane-bound structures with electron-dense interior (e.g., organelle-rich neurites); purple: membrane-bound structures with electron-lucent interior (e.g., swollen astrocytic processes); blue: myelinated axons; yellow: cell bodies. non-colored regions represent ambiguous interstitial zones (aizs) that lack clearly defined membrane boundaries. scale bars: 1 µm. https://doi.org/10.17879/freeneuropathology-2025-6763 free neuropathology 6:13 (2025) garrood et al doi: https://doi.org/10.17879/freeneuropathology-2025-6763 page 9 of 27 figure 3. fine-scale manual annotation of ambiguous interstitial zones (aizs) from one em image across all samples. samples are sorted by the percentage of ambiguous interstitial zones (aizs), from the lowest to the highest: 7-c (a), 37-t (b), 34-c (c), 65-c (d), 59-c (e), 55-c (f), 37-c (g), 78-c (h), 57-c (i), 30-c (j), 65-t (k), 57-t (l), 55-t (m), 78-t (n), 30-t (o), 34-t (p), 66-c (q), 7-t (r), 66-t (s), 59-t (t), where the number is the donor id number, and then “-t” indicates that it is from the thalamus and “-c” from the cortex. color coding identifies distinct cellular structures: green: membrane-bound structures with electron-dense interior (e.g., organelle-rich neurites); purple: membrane-bound structures with electron-lucent interior (e.g., swollen astrocytic processes); blue: myelinated axons; yellow: cell bodies. non-colored regions aizs that lack clearly defined membrane boundaries. scale bars: 500 nm. https://doi.org/10.17879/freeneuropathology-2025-6763 free neuropathology 6:13 (2025) garrood et al doi: https://doi.org/10.17879/freeneuropathology-2025-6763 page 10 of 27 table 3: summary of tissue preservation quality metrics by donor and brain region. donor id pmi fixation time region aiz percentage (in one annotated image) percentage of images with aiz artifacts (count/total) 7 4.25 hours 5 months thalamus 37.18 % 100 % (5/5) cortex 7.97 % 0 % (0/5) 30 17 hours 4 months thalamus 33.14 % 100 % (5/5) cortex 17.76 % 100 % (5/5) 34 20 hours 4 months thalamus 34.79 % 80 % (4/5) cortex 8.91 % 60 % (3/5) 37 72 hours 4 months thalamus 8.46 % 100 % (5/5) cortex 10.61 % 0 % (0/5) 55 36 hours 3 months thalamus 24.51 % 100 % (5/5) cortex 17.52 % 100 % (5/5) 57 96 hours 3 months thalamus 21.51 % 20 % (1/5) cortex 17.52 % 40 % (2/5) 59 91 hours 2.5 months thalamus 40.44 % 100 % (5/5) cortex 11.29 % 100 % (5/5) 65 1.5 hours 2 months thalamus 20.95 % 0 % (0/5) cortex 9.57 % 0 % (0/5) 66 61 hours 2 months thalamus 39.37 % 100 % (5/5) cortex 35.31 % 20 % (1/5) 78 2.5 hours 1 month thalamus 27.49 % 100 % (5/5) cortex 15.65 % 40 % (2/5) the “aiz percentage” column shows the proportion of area occupied by aizs in one high magnification, annotated em image, representing electron-lucent, non-membrane-bound regions that may contain degraded cellular structures. the “percentage of images with aiz artifacts” column indicates how many em images were graded as having aiz artifacts, with the raw number of affected images shown in parentheses (out of 5 total images evaluated per sample). pmi: postmortem interval; aiz: ambiguous interstitial zone. we next developed a grading method to rate em images for aizs that could both be performed more rapidly by trained annotators and could also take into account the degree of cell membrane intactness. specifically, the images were graded as having aiz artifacts if they displayed both (a) extensive aizs and (b) poor delineation of cellular membrane boundaries (figure 4). this combined assessment is meant to distinguish artifactual aizs from potential scenarios with non-artifactual extracellular space expansion. the frequency of images meeting these criteria for having aiz artifacts was calculated for each sample (table 3). we found that samples from a donated canine preserved after a pmi of 1.5 hours yielded em images with no aiz artifacts in either brain region. the brains from two of the human donors, one with a pmi of 4.25 hours (donor #7) and one with a pmi of 72 hours (donor #37), also yielded em images with no aiz artifacts in the cortical samples, but both of these brains had aiz artifacts detected in images from the thalamus samples. the cortex and thalamus samples from the other 7 brains produced em images graded as having aiz artifacts in at least one of the five available em images. https://doi.org/10.17879/freeneuropathology-2025-6763 free neuropathology 6:13 (2025) garrood et al doi: https://doi.org/10.17879/freeneuropathology-2025-6763 page 11 of 27 figure 4. representative em images graded as having or not having ambiguous interstitial zone (aiz) artifacts. five examples images are shown that were graded as having aiz artifacts: 30-t (b), 39-c (d), 59-c (f), 59-t (h), 66-t (j), as well as five examples that were not: 7-c (a), 57-c (c), 65-c (e), 65-t (g), 66-c (i), where the number is the donor id number, and then “-t” indicates that it is from the thalamus and “-c” from the cortex. scale bars: 1 µm. https://doi.org/10.17879/freeneuropathology-2025-6763 free neuropathology 6:13 (2025) garrood et al doi: https://doi.org/10.17879/freeneuropathology-2025-6763 page 12 of 27 we found that there was no significant rank correlation between the pmi and the annotated aiz percentage in the images from either the cortex (rho = 0.35, p = 0.327) or the thalamus (rho = 0.09, p = 0.811). similarly, we found no significant rank correlation between the pmi and the percentage of images graded as having aiz artifacts in the samples from either the cortex (rho = 0.27, p = 0.454) or the thalamus (rho = 0.10, p = 0.790). we also found that there was no significant rank correlation between the fixation time and either of these metrics related to the percentage of aizs in either brain region (all p-values > 0.1). next, we found that there was a significantly higher annotated aiz percentage in images from the thalamus than the cortex (mean in thalamus = 28.8 %, mean in cortex = 15.2 %, t-test p-value = 0.004). this result may reflect differences in preservation quality, baseline differences between the regions, or other factors. there was also a non-significant regional trend in the percentage of images graded as having aiz artifacts, which was higher in the thalamus than the cortex (mean in thalamus = 80 %, mean in cortex = 46 %, t-test p-value = 0.074). we next performed a qualitative analysis of the sstem data, which was derived from cortical samples. first, synapses were identified in the available image stacks, all of which could be traced through the image stack until synapse termination or the end of the image stack, without apparent loss of structure (figure 5). next, we attempted to trace figure 5. representative images in the serial section tem data sets showing that synapses can be traced. samples from the cortex of donor ids #7 (a), #35 (b), #65 (c). synapses are highlighted in color. scale bars: 1 µm. https://doi.org/10.17879/freeneuropathology-2025-6763 free neuropathology 6:13 (2025) garrood et al doi: https://doi.org/10.17879/freeneuropathology-2025-6763 page 13 of 27 figure 6. representative serial section image stack with annotations for afferent neurites from one synapse. all images are from a serial section from the cortex sample of donor #65. scale bars: 1 µm. the neural connections arising from synapses to their associated dendrites and axons across the image stacks. qualitatively, we found that for the sample from donor #35, which had a more extensive burden of aiz artifacts on the 2d em images, it was more difficult to manually trace the neurite structures. for the other two samples imaged with sstem (donor ids #7 and #65), we successfully identified traceable neurites across the image stack in at least some instances (for example, figure 6). however, we were unable to rigorously quantify the traceability due to two key limitations: (a) an insufficient sample size to establish reliable metrics, and (b) technical constraints related to our section thickness. specifically, the resolution between sequential images made it challenging to https://doi.org/10.17879/freeneuropathology-2025-6763 free neuropathology 6:13 (2025) garrood et al doi: https://doi.org/10.17879/freeneuropathology-2025-6763 page 14 of 27 differentiate between potential structural damage and normal morphological variations where processes might naturally terminate or significantly change direction. preservation quality in light microscopy images we next set out to compare preservation quality between modalities. we started by performing an analysis of h&e-stained light microscopy sections. these samples were obtained from the contralateral hemisphere of the same brain regions that were examined by electron microscopy. we detected expected postmortem changes in all images, including pericellular rarefactions, perivascular rarefactions, and neuropil vacuolization (krassner et al., 2023). qualitatively, the extent of pericellular rarefaction and neuropil vacuolization appeared to potentially be more pronounced in some cases with longer pmis. however, no obvious differences in the preservation quality of cell membrane morphology were observed that corresponded with the pmi on these h&e-stained images (figure 7). additionally, the postmortem artifacts visualized did not have obvious qualitative differences based on the fixation time. taken together, these findings suggest that basic cellular morphology remains largely intact at the light microscopy level in the samples from these brains, despite the range of pmis. notably, in cases where perfusion fixation was performed, the extent to which perfusate was successfully delivered to the brain regions examined varied across cases, as evidenced by differences in brain stiffness and the extent of blood clearance from surface vessels. this variability is consistent with previous findings on postmortem brain perfusion (mcfadden et al., 2019). as one additional way to measure this, we assessed h&e-stained images to measure the degree to which blood vessels were cleared of material such as red blood cells (figure 8, table 4). these data demonstrate that blood vessels were not fully cleared, and therefore, perfusion was not complete, in the majority of the brains. this is further supported by the observation that the immersion-fixed brain (donor #57) had a similar grade for intravascular material clearance as several of the perfusion-fixed samples. to address a possible confounding variable, we also found that there was no significant rank correlation between the fixation time and the intravascular material grade on the h&e images, in the data from either the cortex (rho = 0.04, p = 0.92) or the thalamus (rho = 0.24, p = 0.51). taken together, these results suggest that in cases where a perfusion procedure was performed, the fixative may not have fully penetrated the vasculature throughout the brain, perhaps instead only traversing through a subset of blood vessels. we next performed immunohistochemical staining to evaluate the preservation of specific neural components across our samples. staining for smi-312, a pan-axonal stain targeting highly phosphorylated neurofilaments, revealed consistent preservation of axonal architecture across samples with varying pmis (figure 9) (ulfig et al., 1998). even in samples with pmis exceeding 60 hours, we observed well-defined axonal processes with clear continuity and distinct morphology. in the cortical samples, neurofilament-positive axons were readily identifiable traversing throughout the neuropil, with minimal disruption of their trajectory detected on qualitative analysis. staining for smi-311, an antibody targeting non-phosphorylated neurofilaments that are primarily located in the dendrites and perikarya, also revealed consistent staining quality across samples with varying pmis (figure 10) (ulfig et al., 1998). dendritic morphology appeared to be largely intact, without the diffuse staining that would be expected with widespread breakdown of the dendritic cytoskeleton. a key exception was the canine brain, which did not stain adequately for this antibody in either brain region, possibly reflecting molecular differences in neurofilaments between human and canine brains (dimakopoulos and mayer, 2002). https://doi.org/10.17879/freeneuropathology-2025-6763 free neuropathology 6:13 (2025) garrood et al doi: https://doi.org/10.17879/freeneuropathology-2025-6763 page 15 of 27 figure 7. representative h&e-stained images demonstrate that cell membrane morphology is generally intact across a wide range of pmis. samples from the thalamus of donor ids #7 (a, b), #65 (c, d), #57 (e, f), and #59 (g, h) with pmis of 4.25 h, 1.5 h, 96 h, and 91 h, respectively. insets on the lower magnification images correspond to the regions shown in the higher magnification images. scale bars: (a, c, and g) 1 mm; (e) 2 mm; (b, d, f, and h) 50 μm. https://doi.org/10.17879/freeneuropathology-2025-6763 free neuropathology 6:13 (2025) garrood et al doi: https://doi.org/10.17879/freeneuropathology-2025-6763 page 16 of 27 figure 8. representative h&e-stained images from the thalamus showing variable vascular clearance. a, b: images from donor #30 show extensive intravascular material. c, d: images from donor #59 show minimal intravascular material. scale bars: (a, c) 2 mm; (b, d) 200 μm. https://doi.org/10.17879/freeneuropathology-2025-6763 free neuropathology 6:13 (2025) garrood et al doi: https://doi.org/10.17879/freeneuropathology-2025-6763 page 17 of 27 figure 9. representative smi-312-stained images demonstrate that axonal morphology is generally intact across a wide range of pmis. samples from the thalamus of donor ids #65 (a), #78 (b), #7 (c), #37 (d), #59 (e), and #57 (f) with pmis of 1.5 h, 2.5 h, 4.25 h, 72 h, 91 h, and 96 h, respectively. scale bars: 50 μm. https://doi.org/10.17879/freeneuropathology-2025-6763 free neuropathology 6:13 (2025) garrood et al doi: https://doi.org/10.17879/freeneuropathology-2025-6763 page 18 of 27 figure 10. representative smi-311-stained images demonstrate that dendritosomatic morphology on light microscopy is generally intact across a wide range of pmis in human brains. samples from the thalamus of donor ids #78 (a), #7 (b), #30 (c), #37 (d), #59 (e), and #57 (f) with pmis of 2.5 h, 4.25 h, 17 h, 72 h, 91 h, and 96 h, respectively. scale bars: 50 μm. https://doi.org/10.17879/freeneuropathology-2025-6763 free neuropathology 6:13 (2025) garrood et al doi: https://doi.org/10.17879/freeneuropathology-2025-6763 page 19 of 27 table 4: semi-quantitative grades to evaluate preservation quality on light microscopy. donor id pmi fixation time region intravascular material grade on h&e images 7 4.25 hours 5 months thalamus 1 cortex 1 30 17 hours 4 months thalamus 3 cortex 1 34 20 hours 4 months thalamus 2 cortex 2 37 72 hours 4 months thalamus 2 cortex 2 55 36 hours 3 months thalamus 2 cortex 1 57 96 hours 3 months thalamus 2 cortex 2 59 91 hours 2.5 months thalamus 0 cortex 2 65 1.5 hours 2 months thalamus 2 cortex 1 66 61 hours 2 months thalamus 2 cortex 2 78 2.5 hours 1 month thalamus 1 cortex 1 the presence of intravascular material was graded on a scale of 0–3, where 0 indicates minimal intravascular material and 3 indicates extensive intravascular material. pmi: postmortem interval. we next assessed immunostaining for the astrocyte marker gfap (figure 11). in samples from the thalamus, we observed that gfap staining was generally lower compared to the cortical samples, with the exception of the subependymal area, which consistently showed higher immunoreactivity. in the cortical samples, the subpial area consistently had the highest density of staining, which is expected given previous findings about gfap staining patterns (halliday et al., 1996). the density of gfap staining in the cortex appeared to be lower in some cases with longer pmis, which had very few astrocytes stained by anti-gfap (for example, see figure 11e). however, there was no robust trend for lower gfap staining with longer pmis, as evidenced by strong staining in some samples that had relatively longer pmis, such as one brain with a pmi of 96 hours (figure 11f). moreover, when gfap-positive astrocytes were visible in the longer pmi cases, the individual cells that were stained appeared to still have intact morphology without obvious signs of partial decomposition, arguing against cellular decomposition playing the major role in mediating staining differences. however, given our relatively small sample size, we cannot clearly distinguish the reasons for variability in gfap staining across samples. in a subset (n = 6) of the samples, we also performed immunostaining with vimentin (see data https://doi.org/10.17879/freeneuropathology-2025-6763 free neuropathology 6:13 (2025) garrood et al doi: https://doi.org/10.17879/freeneuropathology-2025-6763 page 20 of 27 availability for access to these wsis). while this marker did stain around blood vessels, its staining for astrocytes appeared highly sensitive to the duration of fixation, more so than gfap, showing only weak and inconsistent staining in samples fixed for longer than one month. due to this limitation of vimentin staining, we did not extend the vimentin staining to the full cohort. figure 11. representative gfap-stained images of the cortex show intact subpial astrocyte morphology in images from samples with a wide range of pmis. samples from donor ids #65 (a), #78 (b), #7 (c), #37 (d), #59 (e), and #57 (f) with pmis of 1.5 h, 2.5 h, 4.25 h, 72 h, 91 h, and 96 h, respectively. scale bars: 50 μm. https://doi.org/10.17879/freeneuropathology-2025-6763 free neuropathology 6:13 (2025) garrood et al doi: https://doi.org/10.17879/freeneuropathology-2025-6763 page 21 of 27 discussion in this study, we first present a framework for evaluating preservation quality in postmortem brain tissue through the assessment of ultrastructural integrity in em images. we found that aiz artifacts occurred in at least one of the available em images from our tested samples, with the exception of a canine sample with a relatively shorter pmi of 1.5 hours and two cortical samples from human donors. notably, in the cortical sample from the canine and one of these two human cortical samples, we were able to trace the neurites arising from synapses across sstem image stacks in some instances, though technical limitations and insufficient sample volumes prevented rigorous quantification. our observations suggest that samples without aiz artifacts on 2d images may be a proxy for the degree of ultrastructural integrity that could potentially support connectomics studies, though this requires more imaging and larger sample sizes for corroboration. we also detected a trend towards a higher percentage of aizs in thalamic samples compared to cortical ones. given that our perfusion quality was far from perfect, these observed regional differences could be due to the deeper thalamic structures being preserved more slowly by the post-perfusion immersion fixation of the entire brain, potentially leading to a longer period of autolysis prior to preservation. alternatively, the result may also be due to statistical noise, baseline differences between the regions, or other factors. next, we performed light microscopy on samples from the same brains, using both non-specific morphological staining as well as immunostaining for neurofilaments and gfap. the subjective quality of the morphological staining and neurofilament staining appeared similar across samples from all 10 brains, despite the varying pmis and differences observed on em images. our data suggests a complex relationship between the imaging findings and the underlying tissue preservation depending on the imaging modality used, which warrants further investigation. although our study identified several challenges in this field, it is important to emphasize the significant potential value that em-based connectomics could provide. while a complete human brain connectome remains technologically unfeasible, volume em imaging of targeted brain regions or circuits could complement emerging whole-brain volumetric methods (hillman et al., 2019). such focused volume em studies could be particularly valuable for investigating diseasespecific alterations in synaptic connectivity patterns. for example, alterations in dendritic spine density and synaptic connectivity have been reported in schizophrenia, which has potentially significant implications for the development of treatments for this disorder (glausier and lewis, 2013). however, pharmaceutical companies may not be confident enough in existing ultrastructural biomarkers of disease to pursue them as therapeutic targets, as these findings have often been primarily reported in relatively small tissue samples and in studies with modest sample sizes. studies with larger volumes and larger sample sizes may be useful to corroborate existing ultrastructural correlates of disease and discover new ones, which could potentially be achieved through the improved use of banked brain samples. an important variable in brain banking is the agonal state of the donor, which includes factors such as fever, hypoxia, acidosis, and alterations in metabolism. variability in the agonal state is often found to have an even larger effect on brain tissue quality than the pmi, and can affect cellular morphology (williams et al., 1978; hardy et al., 1985; ohm and diekmann, 1994; paasila et al., 2019). in addition to the direct effect on brain structure, a prolonged or severe agonal phase is likely to lead to the accumulation of thrombi and other factors promoting impairment of postmortem perfusion of the brain (mcfadden et al., 2019). we note that the canine brain sample was donated after the canine was euthanized by a licensed veterinarian, which entails effectively a negligible agonal duration. this is likely another reason that this brain had the highest ultrastructural quality, in addition to the shorter pmi. although the agonal state is more difficult to quantify than the pmi, a better understanding of how the agonal state affects the quality of the tissue for downstream research applications is a critical goal for brain banking, in order to make the field more rigorous. there is an extensive literature on the visualization of extracellular space in brain tissue, with https://doi.org/10.17879/freeneuropathology-2025-6763 free neuropathology 6:13 (2025) garrood et al doi: https://doi.org/10.17879/freeneuropathology-2025-6763 page 22 of 27 many aspects of the preservation procedure, including the delivery technique, fixative, buffer, and osmolarity, all significantly affecting it (van harreveld and malhotra, 1967; cragg, 1980; fix and garman, 2000; korogod et al., 2015; fulton and briggman, 2021). living brain tissue generally contains around 20 % extracellular space (nicholson and syková, 1998). standard methods of perfusion fixation in laboratory animals, which involve a minimal duration of ischemia prior to preservation, can decrease the extracellular space (ecs) in an artifactual manner from its typical in vivo volume (pallotto et al., 2015). our findings suggest that although there may be an initial decrease in the ecs from in vivo levels with minimal pmi, which may involve ischemic cell swelling, as the pmi progresses, the ecs may once again grow to in vivo levels and beyond as fluid redistributes. indeed, one would expect that given a sufficiently long pmi, the brain would be entirely “extracellular,” as all of the cellular structures would eventually break down and the brain liquefy. although we expect that molecular decomposition and fluid redistribution are the most likely explanation for aizs, there are several non-mutually exclusive explanations for the aizs we observed in our samples with longer pmis. some degree of extracellular space is expected even in ideally preserved tissue, and may in fact be desirable as this better reflects in vivo conditions and can aid in neurite tracing (korogod et al., 2015; pallotto et al., 2015). however, the appearance of potential debris alongside the expansion of the extracellular space suggests a significant change from normal tissue architecture. this is why our grading method for aiz artifacts takes into account both the presence of aizs and a qualitative determination of poor cell membrane intactness. one notable pattern we observed in our data is that the occurrence of aiz artifacts qualitatively appears to coincide with other postmortem changes involving fluid shifts. for example, images with aiz artifacts also tend to exhibit increased astrocyte process swelling, vacuolization, and cytoplasmic washout. this pattern is evident in both our images and in other publicly available electron microscopy data sets of postmortem human brain tissue (oost et al., 2023). these observations suggest that there may be a generalized “excess fluid” phenomenon in some postmortem brain tissue, with aiz artifacts being just one manifestation of this. theoretically, one possibility is that it may be possible to develop sample preparation, staining, or analysis methods that can partially adjust for the consequences of excessive fluid accumulation, including aiz artifacts, potentially allowing for inference of neural morphology despite these artifacts. a previous approach to address extracellular space shrinkage in em images classified the space into sheet-like and tunnel-like regions, then used computational models to redistribute volume between these compartments (kinney et al., 2013). however, it is highly questionable that approaches designed for controlled shrinkage artifacts could be sufficient for adjustment of postmortem brain em images, as the aiz artifacts present are anisotropic, haphazard, and enigmatic. instead, more complex computational approaches, developed in consideration of the physiology of agonal and postmortem degradation, would likely be required, to the extent that it is feasible at all. our light microscopy findings provide complementary data to help interpret the em results. h&e and immunohistochemical staining revealed relatively well-preserved cellular morphology in samples from the same brains that had substantial changes on em images. particularly notable was the integrity of neurofilament staining, which allowed for visualization of axonal and dendritic trajectories across substantial distances despite varying pmis. this is consistent with previous data showing the robustness of neurofilament antigen staining to extended pmi (ulfig et al., 1998; blair et al., 2016; bouvier et al., 2016). as a result, our findings corroborate previous observations that electron microscopy is more sensitive to postmortem changes than light microscopy (krassner et al., 2023). one obvious potential explanation for this difference is simply that em has better resolution and is therefore better able to detect early, minute aspects of tissue decomposition. it is also possible that relative differences in the sensitivity of light microscopy and em staining procedures to postmortem changes could help to explain the inconsistency between the modalities. for example, osmium tetroxide, a key staining agent for em, has been found to bind primarily to unsaturated fatty acids arranged in lipid membranes (wigglesworth, 1957, 1981; belazi et al., https://doi.org/10.17879/freeneuropathology-2025-6763 free neuropathology 6:13 (2025) garrood et al doi: https://doi.org/10.17879/freeneuropathology-2025-6763 page 23 of 27 2009; li et al., 2024). autolysis causes a rapid release and redistribution of membrane lipids, including unsaturated fatty acids, via the activity of phospholipase a2 (van der vusse et al., 1989; pueyo et al., 2000). if unsaturated fatty acids in lipid membranes degrade relatively faster than proteins in the pmi, then this would disproportionately affect visualization following standard em protocols, compared to other methods that target protein antigens. notably, previous data has found that the more lipid-dense myelinated axons are more recognizable than unmyelinated axons via em after postmortem disintegration prior to fixation (liewald et al., 2014). as another possibility, cellular structures might remain partially present during the postmortem period, but their constituent biomolecules could become insufficiently compact for adequate visualization by non-specific morphological stains alone. immunostaining, in particular, may be better able to amplify partially intact structures and therefore not be affected as rapidly by the postmortem breakdown of gel-like biomolecular networks as the typical non-specific em staining methods (krassner et al., 2023). this possibility would parallel light microscopy studies on postmortem brain tissue, showing that immunostaining can sometimes reveal cellular structures not visible with non-specific morphological stains (monroy-gómez et al., 2020). further research using complementary methods, such as immunoelectron microscopy, correlative light and electron microscopy, or expansion microscopy, may help to clarify the reasons for the differences in results between light and electron microscopy. several limitations should be considered when interpreting our findings. first, our sample size of ten brains obviously does not capture the full spectrum of preservation quality variation seen in brain banking. information about the donors, such as the pmi, may have inaccuracies, which becomes a bigger problem with smaller sample sizes. as a result, our findings will require larger subsequent studies for corroboration. second, our analysis of the contralateral hemispheres for light and electron microscopy introduces obvious potential confounding factors. preservation quality, including the quality of perfusion, can clearly vary between the hemispheres, which might affect our results. third, although formaldehyde alone has been used successfully for fixation in some studies, others recommend the addition of glutaraldehyde (gonzalez aguilar and de robertis, 1963; westrum and lund, 1966; lewis et al., 2019). on initial fixation we used formaldehyde alone without the addition of glutaraldehyde, which may have reduced the ultrastructural quality of our resulting em images. a future study comparing post-perfusion immersion fixation of small samples from the same brain with either paraformaldehyde-glutaraldehyde or neutral buffered formalin could directly test the relative contribution of fixative composition to ultrastructural preservation quality. fourth, our semi-quantitative grading systems for the em and immunohistochemically stained images represent a simplified assessment of complex data. further, our analysis of the light microscopy images was largely qualitative, which renders this more vulnerable to bias. we encourage any interested readers to analyze the raw image data, all of which we have made available, to come to their own conclusions. lastly, our study focused primarily on 2d ultrastructural features. the implications for 3d connectivity reconstruction remain speculative without more direct volumetric data and analysis. these limitations highlight the need for future studies with larger sample sizes and more direct comparisons via multiple imaging techniques. conclusions our study provides insights into the relationship between preservation quality, pmi, and imaging modality in banked brain tissue. we found that the level of ultrastructural preservation required for performing connectomics studies appears to be achievable in some postmortem samples. these findings suggest that under close to optimal brain banking conditions, postmortem human brain tissue could potentially be suitable for connectomics studies using current em methods. however, in at least a subset of em images from the other samples, our analysis revealed the presence of aiz artifacts, which may indicate significant challenges for the reliable tracing of neural processes. these aiz artifacts may result from true structural degradation prior to fixation, inadequate https://doi.org/10.17879/freeneuropathology-2025-6763 free neuropathology 6:13 (2025) garrood et al doi: https://doi.org/10.17879/freeneuropathology-2025-6763 page 24 of 27 visualization of thin axons and other cellular components, or both. notably, our light microscopy data from these same brains indicates that many cellular structures remain intact and potentially traceable at this level of resolution via neurofilament staining. this suggests a difference between preservation quality as observed via different imaging modalities. however, this is not necessarily surprising, because while both light and electron microscopy can provide windows into cell membrane morphology, they examine structures at different resolutions and thus can be considered complementary. to advance connectomics research using banked brain tissue, further methodological development may be valuable. current approaches can only reliably visualize key ultrastructural features in a small subset of banked brain samples, constraining the possible sample sizes. it is not yet clear if these are the true biological limits to ultrastructural visualization. future research could focus on optimizing fixation protocols, staining techniques, and computational methods specifically for postmortem brain tissue. such research may be critical for realizing the full potential of human brain connectomics as a tool for understanding neural circuit organization in both health and disease. author contributions a.k., m.g., k.f., j.c., and a.t.m. conceptualized the article. a.s. and w.j. performed electron microscopy experiments. e.t. and c.d.s. performed light microscopy experiments. a.k., m.g., and a.t.m. performed data analysis. a.t.m. wrote the initial draft of the manuscript. all authors reviewed the manuscript. all authors approved the final manuscript. acknowledgements we would like to acknowledge the technical assistance of laura paredes and alexander parra, and we would like to thank kenneth hayworth for helpful personal communications on this topic. we acknowledge the neuropathology brain bank & research core at the icahn school of medicine at mount sinai for their histology and tissue processing services. electron microscopy tissue preparation and imaging were performed at the microscopy and advanced bioimaging core at the icahn school of medicine at mount sinai. the icahn school of medicine at mount sinai provided access to library resources. funding statement this work was supported by the rainwater charitable foundation as well as nih grants p30 ag066514, k01 ag070326, rf1 ag062348, p30 ag066514, rf1 ns095252, u54 ns115266, and rf1 mh128969. the funders had no role in the design of the study or in the collection or interpretation of the data. conflict of interest statement alicia keberle, macy garrood, and andrew mckenzie are employees of oregon brain preservation, a non-profit brain preservation organization. andrew mckenzie is a director of apex neuroscience, a non-profit research organization. data availability code used for data analysis is available here: https://github.com/andymckenzie/ultrastructure_ quality_manuscript. all raw image data can be accessed in a public repository 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https://doi.org/10.1098/rspb.1957.0043 williams, r.s., ferrante, r.j., caviness, v.s., 1978. the golgi rapid method in clinical neuropathology: the morphologic consequences of suboptimal fixation. journal of neuropathology and experimental neurology 37, 13–33. https://doi.org/10.1097/00005072-197801000-00002 https://doi.org/10.17879/freeneuropathology-2025-6763 https://doi.org/10.1016/s0046-8177(87)80243-5 https://doi.org/10.1242/jcs.1.2.229 https://doi.org/10.1016/0040-8166(81)90035-5 https://doi.org/10.1098/rspb.1957.0043 https://doi.org/10.1097/00005072-197801000-00002 original paper abbreviations introduction methods brain banking procedures electron microscopy light microscopy results preservation quality in electron microscopy images preservation quality in light microscopy images discussion conclusions author contributions acknowledgements funding statement conflict of interest statement data availability references 68th annual meeting of the german society of neuropathology and neuroanatomy (dgnn) meeting abstracts free neuropathology 5:19 (2024) meeting abstracts 68th annual meeting of the german society of neuropathology and neuroanatomy (dgnn) meeting abstracts september 12–14, 2024, regensburg, germany german society of neuropathology and neuroanatomy (dgnn) submitted: 29 august 2024 accepted: 02 september 2024 published: 03 september 2024 https://doi.org/10.17879/freeneuropathology-2024-5842 keywords: german society of neuropathology and neuroanatomy, dgnn, meeting abstracts dear colleagues, it is my pleasure and honor to host the 68th annual meeting of the german society of neuropathology and neuroanatomy (dgnn) in regensburg. since the magdeburg meeting in 2019 this is the first pure national meeting of our society that will be held in presence after five long years. while the meeting in 2020 was cancelled due to the corona pandemic, the 2021 meeting (organized by the colleagues in gießen) took place as a mere online meeting. in 2022 and 2023 our national society meetings were embedded in the “neurowoche” and the international congress of neuropathology in berlin. we are enthusiastic about this years’ reunion of our society in regensburg. in our regensburg meeting, we aim to provide a comprehensive update on the major and hot topics in neuropathology. neuropathologists address some of the currently most relevant and discussed health care issues, such as for example cancer, neuroimmunological diseases like multiple sclerosis, neurodegenerative diseases including alzheimer’s and parkinson’s, and muscle/nerve diseases. as tissue specialists, neuropathologists directly study diseases in human materials. neuropathologists use state of-the-art methods to uncover disease processes on the molecular level. during our congress, we will hear a lot on the methodical progress made in this regard. neuropathology is also becoming increasingly clinical as many of our scientific and diagnostic findings influence and directly guide treatment decisions. we were able to attract renowned national and international speakers and our meeting will allow for an intensive interchange both within our society and with our neighboring disciplines. program highlights include a pre-congress hands-on workshop on next generation sequencing, a session on molecular tumorboards and a mini-symposium on quality assurance in neuropathology. we are delighted about the submission of 31 abstracts covering the research fields neurooncology, neuroimmunology, muscle/nerve, neurodegeneration, and methods/free topics. the abstracts are published below in this edition of free neuropathology. i want to thank the scientific committee of our congress for helping in evaluating the submissions and selecting the poster talks and poster spotlight presentations. many of the abstracts were submitted by our young researchers. they deserve our special attention! posters will be exhibited throughout the entire congress and we will have plenty of time for poster viewing and discussions on thursday evening at the welcome reception and at the main poster session on friday at noon. so let me again welcome you all to our beautiful city of regensburg. i am looking forward to inspiring talks, vivid discussions and enriching encounters with like-minded people. yours, prof. dr. markus j. riemenschneider regensburg university hospital, department of neuropathology congress president dgnn annual meeting 2024 contents i. neurooncology p01 identification and description of a novel type of medulloblastoma p02 glutamatergic synaptic input to brain metastases drives metastatic colonization p03 brain invasion in otherwise benign meningiomas: molecular characteristics and prognostic relevance p04 electrophysiological characterization of slow inward currents in glioblastoma p05 nanopore sequencing from formalin-fixed paraffin-embedded specimens for copy number profiling and methylation-based cns tumor classification p06 epigenetic profiling can improve diagnostics of mpnst with intratumoral histological heterogeneity p07 anaplastic histology and distinct molecular features in a small series of spinal cord ependymomas p08 integrated analyses reveal two molecularly and clinically distinct subtypes of h3 k27m-mutant diffuse midline gliomas with prognostic significance p09 spatial multiomics profiling reveals heterogeneity of b:t cell interactions and plasma cell formation in tertiary lymphoid structures in human gliomas p10 digital biomarkers for an improved clinical stratification of meningioma patients p11 cellular senescence as a shared contributor to disease progression in glioblastoma and alzheimer's disease p12 vopp1::egfr gene fusion as an oncogenic driver via nfκb pathway activation in a case of ganglioglioma p13 insights from the additional immunohistochemical work-up of molecular tumor board patients from a regional brain tumor center p14 cytokine response in the cerebrospinal fluid after intraoperative radiation of primary and metastatic brain tumors ii. neuroimmunology p15 in-depth analysis of viral distribution and immunological profiling in human bornavirus encephalitis p16 histopathological analysis of bodv-1 distribution in a non-human primate model: comparing intranasal and subcutaneous inoculation p17 systematic analysis of virus spread to the peripheral nervous system in fatal borna virus encephalitis p18 global cerebral hypoxia-ischemia: ex vivo ultrahigh field mri signals correlate with differential cortical localization of microglia and gemistocytes characterized by chit1 and chi3l1 (ykl 40) expression iii. muscle / nerv p19 adipo-glial signaling mediates metabolic adaptation in peripheral nerve regeneration iv. neurodegeneration p20 alpha-synuclein co-pathology in alzheimer’s disease drives tau accumulation p21 frontotemporal dementia patient neurons with the mapt-n279k mutation are responsive to tau filaments and contribute to neuroinflammation in vivo p22 alterations of er-co-chaperone sil1 in amyotrophic lateral sclerosis (als) p23 characterizing granular tau aggregates in astrocytes in multiple system atrophy v. methods and free topics p24 ultra-rapid bacterial detection from neuropathology specimens using next-generation pcr and nanopore sequencing p25 ccnv: r package for enhanced cumulative copy number variation analysis p26 rapid slice-free intraoperative histology in neurooncology using multiphoton microscopy – first study results p27 predicting epigenetic ependymoma types from histological whole-slide images using neural networks p28 established in 1902: a brief history of the institute of neurology (edinger institute) in frankfurt, germany p29 paving the path: the powerful effect of substrate topography on axon-repulsive schwann cell-astrocyte barrier formation for spinal cord injury repair p30 using eye-tracking to find differences in the analysis of whole-slide images between physicians and machine learning models – a study design p31 why we don’t save whole-slide images as lego mosaics – putting the scale of whole-slide images into perspective i. neurooncology p01 free neuropathol 5:19:5 identification and description of a novel type of medulloblastoma alicia eckhardt1,2,*, chris meulenbroeks3,*, neal geisemeyer4,*, michael bockmayr1,2, helena bode1,2, karoline hack1,2, marthe sönksen2, denise obrecht-sturm2, christian thomas5, melanie schoof1,2, arend koch6, sage green7,8, sidharth mahapatra9, dave doss10, ekin guney11, arie perry11, kenneth aldape12, anna darabi13, stephan frank14, peter kuzman15, miriam ratliff16,17, abigail suwala18,19, andrey korshunov18,19, olivier ayrault20, christine haberler21, ales vicha22, markus glatzel23, martin mynarek2, stefan rutkowski2, martin hasselblatt5, michael d taylor24, david jones25,26, marcel kool3,4,*, ulrich schüller1,2,23,* 1 research institute children’s cancer center hamburg, hamburg, germany 2 pediatric hematology and oncology, university medical center hamburg-eppendorf, hamburg, germany 3 princess máxima center for pediatric oncology, utrecht, netherlands 4 hopp children’s cancer center heidelberg (kitz) / german cancer research center (dkfz), heidelberg, germany 5 institute of neuropathology, university hospital münster, münster, germany 6 department of neuropathology, charité universitätsmedizin berlin, corporate member of freie universität berlin, and berlin institute of health, charité university medicine, berlin, germany 7 dell children's medical center, austin, tx, usa 8 nemours children's health, wilmington, de, usa 9 university of nebraska medical center, omaha, ne, usa 10 creighton university school of medicine, omaha, ne, usa 11 department of pathology, university of california, san francisco, san francisco, ca, usa 12 laboratory of pathology, center for cancer research, national cancer institute, bethesda, md, usa 13 glioma immunotherapy group, neurosurgery, department of clinical sciences, lund university, lund, sweden 14 division of neuropathology, institute of medical genetics and pathology, university hospital basel, university of basel, basel, switzerland 15 institute of neuropathology, university hospital leipzig, leipzig, germany 16 clinical cooperation unit neurooncology, german cancer consortium (dktk), german cancer research center (dkfz), heidelberg, germany 17 neurosurgery clinic, university hospital mannheim, mannheim, germany 18 department of neuropathology, institute of pathology, university of heidelberg, heidelberg, germany 19 clinical cooperation unit neuropathology, german cancer research center (dkfz), german consortium for translational cancer research (dktk), heidelberg, germany 20 institut curie, cnrs umr, inserm, psl research university, orsay, france; cnrs umr 3347, inserm u1021, universite paris sud, universite paris-saclay, orsay, france 21 institute of neurology, medical university of vienna, vienna, austria 22 prague brain tumor research group, second faculty of medicine, charles university and university hospital motol, v uvalu 84, 15006, prague 5, czech republic 23 institute of neuropathology, university medical center hamburg-eppendorf, hamburg, germany 24 division of neurosurgery, the hospital for sick children, toronto, ontario, canada 25 hopp children's cancer center heidelberg (kitz) and nct heidelberg, heidelberg, germany 26 division of pediatric glioma research, german cancer research center (dkfz), heidelberg, germany * shared authorship medulloblastoma (mb) is the most frequent malignant brain tumor of childhood and adolescence. based on biology, histology, and its clinical course, mb is a heterogeneous disease with four different types (wnt, shh, group 3, and group 4), which are most reliably distinguishable by their global dna methylation pattern. as a result of integrating dna methylation data of > 2,600 mbs and screening of > 140,000 data sets uploaded to the dkfz brain tumor classifier (www.molecularneuropathology.org), we identified a small group of mb (n = 49) that displayed a homogeneous dna methylation pattern, which was clearly distinct from previously known mb types. tumors within this group have also been recognized as a separate methylation class by the latest version of the classifier (provisionally named as mb_myo). transcriptomic data (n = 14) were similarly distinct from other mb types. comparison of these data to various cell types of the developing hindbrain revealed similarities to precursor cells of the rhombic lip with signatures of wnt signaling and myogenic differentiation. in line with the latter findings, 3/13 analyzed cases harbored hotspot ctnnb1 mutations and 7/10 cases showed a myogenic differentiation based on histology. myc amplifications were present in 12/49 (24.5 %). median age at diagnosis was 16 years, and five-year overall survival was ~ 70 %. in summary, we describe a novel type of mb identified by dna methylation profiling that likely needs to be addressed separately during the retrospective analyses of mb patient cohorts, for the design of future clinical trials, and when evaluating targeted therapies. p02 free neuropathol 5:19:7 glutamatergic synaptic input to brain metastases drives metastatic colonization linh-cathrin nguyen1, matthia karremann2,3, thomas kuner1, frank winkler2,3, varun venkataramani1,2,3 1 department of functional neuroanatomy, institute for anatomy and cell biology, heidelberg university, heidelberg, germany 2 clinical cooperation unit neurooncology, german cancer consortium (dktk), german cancer research center (dkfz), heidelberg, germany 3 neurology clinic and national center for tumor diseases, university hospital heidelberg, heidelberg, germany brain metastases frequently occur in patients with cancer, primarily originating from lung carcinoma, breast carcinoma, and melanoma, resulting in high morbidity and mortality. the brain microenvironment significantly influences the progression of brain metastases. recent studies have demonstrated that direct, glutamatergic synapses on glioma cells drive brain tumor progression. however, it is unclear if direct synaptic communication occurs between neurons and cancer cells from non-neural tumors, and if so, whether this can promote metastasis and tumor progression. this study aims to identify and characterize direct glutamatergic synapses between neurons and brain metastatic cells in breast cancer and melanoma models. we hypothesize that direct glutamatergic synapses are formed on brain metastases, mediating signals via ampa receptors (ampars). we employed ex-vivo and in-vitro electrophysiology. patch-clamp recordings from melanoma and breast cancer cells revealed spontaneous excitatory postsynaptic currents (sepscs). the application of the ampar antagonist cnqx inhibited sepscs, confirming the involvement of ampars. in addition, genetic modification and pharmacological blockade of ampars using the approved antiepileptic drug perampanel in breast and melanoma cancer models led to a reduction in the number of brain metastases and overall brain metastatic burden. this study demonstrates that brain metastases can integrate into the neuronal network by forming direct chemical synapses with neurons. ampars on cancer cells play a functional role for brain metastatic progression that can be pharmacologically targeted. this is the first evidence of direct synapses on brain metastases, highlighting the need for further characterization of these synapses in brain tumor biology to find novel therapeutic opportunities. p03 free neuropathol 5:19:8 brain invasion in otherwise benign meningiomas: molecular characteristics and prognostic relevance rouzbeh banan1, thomas hielscher2, yiheng tang1, mark youngblood3, daniel schrimpf1, kirsten göbel1, sybren l.n. maas3,4, christian thomas5, mara popovic6, jernej mlakar6, katharina j. weber7, karl h. plate7, werner paulus5, david reuss1, miriam ratliff8, nima etminan8, patrick n. harter9, christian hartmann10, ayça e. danyeli11, yanghao hou12, christel herold-mende13, stephen magill, sandro krieg13, craig m. horbinski14, sonika dahiya15, andreas von deimling1, bhuvic patel16, felix sahm1 1 heidelberg university hospital, department of neuropathology and dktk, dkfz, heidelberg, germany 2 biostatistics, dkfz, heidelberg, germany (th) 3 northwestern university, dept. of neurological surgery chicago, il, usa 4 department of pathology, leiden university medical center, leiden, netherlands and department of pathology, erasmus mc cancer institute, university medical center rotterdam, rotterdam, the netherlands 5 university hospital münster, institute of neuropathology, münster, germany 6 university of ljubljana, faculty of medicine, institute of pathology, ljubljana, slovenia 7 goethe university, university hospital, institute of neurology (edinger institute), institute of neurology, frankfurt, germany 8 university hospital mannheim, dept. of neurosurgery, mannheim, germany 9 center for neuropathology and prion research, faculty of medicine, ludwig-maximilians-university munich, munich, germany 10 hannover medical school, dept. of neuropathology, hannover, germany 11 acibadem mehmet ali aydinlar university, dept. of pathology, istanbul, turkey 12 chongqing medical university, dept. of pathology, chongqing, p. r. china 13 university hospital heidelberg, dept. of neurosurgery, heidelberg, germany 14 northwestern university, department of pathology, chicago, il, usa 15 washington university school of medicine, div. of neuropathology, dept. of pathology and immunology, st. louis, mo, usa 16 washington university school of medicine, dept. of neurological surgery, st. louis, mo, usa introduction: the prognostic value of brain invasion (bi) in meningiomas and thus the grading of these tumors has controversially been discussed for decades. the 2016 revised 4th edition of who classification of cns tumors defined bi as a stand-alone criterion for grade 2. still, the data published since then have been inconsistent. in the 2021 classification cns5, brain-invasive otherwise benign meningiomas (biob) are still graded as cns who grade 2, yet with emphasis on the lingering controversy. materials and methods: a multicentric series of 304 brain-invasive cases was analyzed. 673 meningiomas from previous studies were used for comparison. dna-methylation classification, cnv-profiling (both epic, v12) and sequencing data (panel/whole-exome) were generated. kaplan-mayer analysis and multivariate cox regression were applied to analyze clinical outcomes. results: biob cases were more frequently of male sex and showed chr14q loss compared to non-invasive cns who grade 1 tumors, (p = 0.027 and p = 0.006 respectively). biob had shorter pfs than cns who grade 1 (p = 0.003), yet more favorable outcome than non-invasive cns who grade 2 cases (p = 0.006). in multivariate analyses considering sex, who grade, methylation families (intermediate, malignant) and prognostically relevant cnvs, bi was an independent risk factor for shorter pfs (hr: 1.73; 95 %-ci: 1.34–2.22; p < 0.001). conclusion: in this study, bi confers an independently higher risk of reduced pfs even among otherwise cns who grade 1 cases. however, the risk and outcome align neither with cns who grade 1 nor 2 in our reference cohort. further analyses are warranted for alternative risk stratification and data validation. p04 free neuropathol 5:19:10 electrophysiological characterization of slow inward currents in glioblastoma tamara chopurian1, frank winkler2,3, thomas kuner1, varun venkataramani1,2,3 1 department of functional neuroanatomy, institute for anatomy and cell biology, heidelberg university, germany 2 neurology clinic and national center for tumor diseases, university hospital heidelberg, germany 3 clinical cooperation unit neurooncology, german cancer consortium (dktk), german cancer research center (dkfz), heidelberg, germany introduction: glioblastomas are incurable primary brain tumors colonizing the entire brain. long membrane protrusions called tumor microtubes (tms) as well synapses on glioblastoma cells (gbcs) contribute to glioma progression. in a subset of gbcs neuronal input leads to excitatory postsynaptic currents (epscs) and slow inward currents (sics). the exact molecular mechanisms of sics and their biological relevance are incompletely understood. objective: this project addresses the question of the biological relevance of sics for the growth of glioblastomas. materials & methods: patient derived xenograft models and in-vitro co-culture models of neurons and glioma cells are used. whole cell patch-clamp recordings of glioma cells are performed to characterize sics. simultaneous patch-clamp recordings and calcium imaging are performed to understand the downstream effects. results: acute stimulation with glutamate evokes sics in gbcs from acute brain slices as well as from cocultures. a percentage of the evoked sics are followed by intracellular calcium signals. the calcium signals are heterogeneous in size and subcellular localization. sic-positive gbcs exhibit a total higher growth of tms than sic-negative gbcs. this is a new connection between electrical activity and the functional relevance for glioblastoma. conclusion: neuronal input of glutamatergic synapses leads to sics and heterogeneous, intracellular calcium events in a subset of gbcs. it expands upon these by showing a positive correlation between gbcs with sics and the growth of tms. targeting sics and subsequent calcium events in gbcs may be a novel therapeutic approach in this intractable disease warranting further investigation. p05 free neuropathol 5:19:11 nanopore sequencing from formalin-fixed paraffin-embedded specimens for copy number profiling and methylation-based cns tumor classification ann-kristin afflerbach1,2,3, anne albers4, anton appelt1,2, leonille schweizer5,6,7, werner paulus4, michael bockmayr1,2,8, ulrich schüller1,2,9#, christian thomas4# 1 department of pediatric hematology and oncology, university medical center hamburg-eppendorf, hamburg, germany 2 research institute children’s cancer center hamburg, hamburg, germany 3 institute for tumor biology, university medical center hamburg-eppendorf, hamburg, germany 4 institute of neuropathology, university hospital münster, münster, germany 5 institute of neurology (edinger institute), university hospital frankfurt, goethe university, frankfurt am main, germany 6 german cancer consortium (dktk), partner site frankfurt/mainz, german cancer research center (dkfz), heidelberg, germany 7 frankfurt cancer institute (fci), frankfurt am main, germany 8 baiome center for biomedical ai, university medical center hamburg-eppendorf, hamburg, germany 9 institute of neuropathology, university medical center hamburg-eppendorf, hamburg, germany # equal contribution microarray-based dna methylation profiling has emerged as a powerful tool for central nervous system tumor classification and investigation of diagnostically relevant copy-number alterations such as 1p/19q-codeletion and the +7/-10 signature. methylation arrays are well compatible with formalin-fixed and paraffin-embedded (ffpe) derived dna but are time-consuming and requires batch processing. nanopore sequencing has emerged as a rapid and scalable method, enabling measurement of dna methylation and generation of copy-number profiles, but has been limited to high-quality dna from native or cryopreserved samples so far. here, we demonstrate the feasibility of nanopore sequencing from ffpe-derived dna for methylation-based classification and generation of genome-wide copy-number profiles. ffpe-derived dna was isolated from 40 cns tumors (average storage time: 19 months) including idh-wildtype glioblastomas (n = 8), oligodendrogliomas (n = 6), posterior fossa ependymomas (pfa: n = 6, pfb: n = 6), medulloblastomas (wnt: n = 4, shh: n = 5), pilocytic astrocytomas (n = 4), and one meningioma. all samples were analyzed with the illumina epic methylation array and nanopore sequencing on minion devices. on average, sequencing runs produced 205,000 reads and 201 mb per sample. methylation-based analysis using the random forest classifier nanodx resulted in correct classification in 25/40 samples (63 %), whereas sturgeon classified the vast majority of samples correctly (37/40, 93 %), including 14/16 samples with poor dna quality (din < 5). all idh-wildtype glioblastomas showed the +7/-10 signature, and all oligodendrogliomas harbored a 1p/19q-codeletion. taken together, our study demonstrates the feasibility of rapid methylation profiling and copy-number analysis of ffpe specimens using nanopore sequencing. sturgeon, a neural network-based classifier, performed considerably better than the random forest-based classifier nanodx. p06 free neuropathol 5:19:13 epigenetic profiling can improve diagnostics of mpnst with intratumoral histological heterogeneity tomas phan1, karoline hack1,2, lennart well3,4, inka ristow3,4, lan kluwe5, said farschtschi5, markus glatzel6, victor-felix mautner5, ulrich schüller1,7, catena kresbach1,2,6,7 1 research institute children's cancer center, hamburg, germany 2 department of pediatric hematology and oncology, university medical center hamburg-eppendorf, hamburg, germany 3 lab of radiobiology & experimental radiation oncology, hubertus wald tumorzentrum, university cancer center hamburg, university medical center hamburg-eppendorf, hamburg, germany 4 department of diagnostic and interventional radiology and nuclear medicine, university medical center hamburg-eppendorf, hamburg, germany 5 department of neurology, university medical center hamburg-eppendorf, hamburg, germany 6 department of diagnostics, institute of neuropathology, university medical center hamburg-eppendorf, germany 7 mildred scheel cancer career center hatrics4, university medical center hamburgeppendorf, hamburg, germany introduction: approximately 10 % of nf1 patients develop malignant peripheral nerve sheath tumors (mpnst) with a poor prognosis, making early detection crucial. mpnst exhibit pronounced intratumoral heterogeneity, often containing areas resembling plexiform neurofibroma (pnf), atypical neurofibroma (annubp), and mpnst. consequently, ct-guided biopsies may miss critical tumor features. this study aims to delineate mpnst heterogeneity histologically and molecularly to improve diagnostic precision and to better understand the mechanisms of malignant progression. methods: evaluation of tissue morphology on h&e-stained sections, immunohistochemistry, global methylation profiling (850k illumina epic arrays), and gene sequencing panels. results: we chose one area with premalignant and one area with mpnst morphology per tumor for molecular analysis. clustering analysis showed similar epigenetic characteristics in both areas in 5/10 cases. the remaining 5 cases displayed distinct epigenetic profiles, with premalignant areas clustering with annubp and malignant areas clustering with mpnst. copy number profiles showed marked alterations not only in the high-grade areas but also in the histologically benign areas in 8/10 cases. gene sequencing identified identical mutations in suz12 and tp53 in premalignant and malignant areas in 2/10 cases, and mpnst-typical mutations in tp53 and eed observed exclusively in high-grade areas in 3/10 cases. conclusions: our findings highlight that the histology of mpnst biopsies may often not fully represent the underlying tumor biology. we show that genetic and epigenetic changes occur before histological features of malignancy become apparent. this study underscores the diagnostic relevance of mpnst intratumoral heterogeneity and the need for comprehensive diagnostic approaches especially in needle biopsies. p07 free neuropathol 5:19:15 anaplastic histology and distinct molecular features in a small series of spinal cord ependymomas ulrich schüller1,2,3,*, antonia gocke4,5,*, karoline hack1,2, shweta godbole4, claire delbridge6, christian thomas7, julia e. neumann3,4 1 department of pediatric hematology and oncology, university medical center hamburg-eppendorf, hamburg, germany 2 research institute children's cancer center hamburg, hamburg, germany 3 institute of neuropathology, university medical center hamburg-eppendorf, hamburg, germany 4 center for molecular neurobiology hamburg (zmnh), university medical center hamburg-eppendorf, hamburg, germany 5 section of mass spectrometric proteomics, university medical center hamburg-eppendorf, hamburg, germany 6 institute of pathology, department of neuropathology, tum school of medicine and health, technical university munich, munich, germany 7 institute of neuropathology, university hospital münster, münster, germany * these authors contributed equally to this work ependymomas (epn) of the spinal cord encompass multiple types and subtypes with distinct histological, molecular and clinical features. apart from subependymomas (cns who 1), spinal epn, and myxopapillary epn (both cns who 2), spinal epn with mycn amplification have been identified as the most aggressive type in the spinal cord. the latter include most of the spinal tumors that had been diagnosed as ‘anaplastic ependymoma (cns who 3)’ in the pre-molecular era. while amplifications of mycn therefore need to be investigated specifically, global dna methylation profiling has emerged as a valuable tool to classify epn. here, in depth analysis of the methylome and the proteome of spinal cord epn revealed a novel distinct molecular epn subtype. unsupervised clustering of integrated methylation data of epn revealed a distinct cluster of cases, that the established brain tumor classifier (capper et al. (2018)) mainly defined as spinal subependymomas. further liquid chromatography mass spectrometry-based proteomics measurements were conducted and an unsupervised analyses of matched proteome data revealed a similarity of the corresponding samples with spinal epn, mycn amplified. reevaluation of histological features showed an anaplastic histology with high ki67 indices and positive nuclear staining for mycn and olig2, which has not been described for subependymomas before. although our cases had anaplastic features and showed expression of mycn protein by immunohistochemistry, mycn amplifications were neither detectable by fish nor by whole genome copy number profiles. limited follow up data indicated that respective cases may not relapse as commonly as mycn amplified epn. we report a series of spinal epn displaying distinct histomorphology, epigenetic and proteomic patterns and propose the provisional designation as mycn-like spinal ependymoma (sp-epn-mycn-like). more in-depth investigations are warranted to uncover e.g. genetic drivers of the tumors and clinical outcomes of respective patients. p08 free neuropathol 5:19:17 integrated analyses reveal two molecularly and clinically distinct subtypes of h3 k27m-mutant diffuse midline gliomas with prognostic significance lotte stegat1, alicia eckhardt1,2, antonia gocke3,4, sina neyazi2,5, lara pohl2,5, simone schmid6,7, matthias dottermusch1,3, stephan frank8, hans pinnschmidt9, jochen herms10, markus glatzel1, matija snuderl11, leonille schweizer12,13,14, christian thomas15, julia neumann1,3, mario m. dorostkar10,16, ulrich schüller1,2,5, annika k. wefers1,17,18 1 institute of neuropathology, university medical center hamburg-eppendorf, hamburg, germany 2 research institute children’s cancer center hamburg, martinistrasse 52, n63 (hpi), 20251 hamburg, germany 3 center for molecular neurobiology hamburg, university medical center hamburg-eppendorf, hamburg, germany 4 section of mass spectrometric proteomics, university medical center hamburg-eppendorf, hamburg, germany 5 department of pediatric hematology and oncology, university medical center hamburg-eppendorf, hamburg, germany 6 charité universitätsmedizin berlin, corporate member of freie universität berlin, humboldt-universität zu berlin, and berlin institute of health, department of neuropathology, berlin, germany 7 german cancer consortium (dktk), partner site berlin, and german cancer research center (dkfz), heidelberg, germany 8 department of neuropathology, institute of pathology, basel university hospital, basel, switzerland 9 institute of medical biometry and epidemiology, center for experimental medicine, university medical center hamburg-eppendorf, hamburg, germany 10 center for neuropathology and prion research, ludwig-maximilians-university, munich, germany 11 department of pathology, nyu langone medical center, new york, usa 12 edinger institute (institute of neurology), university hospital frankfurt, goethe university, frankfurt am main, germany 13 german cancer consortium (dktk), partner site frankfurt-mainz, german cancer research center (dkfz), heidelberg, germany 14 frankfurt cancer institute (fci), frankfurt am main, germany 15 institute of neuropathology, university hospital münster, münster, germany 16 karl landsteiner privatuniversität für gesundheitswissenschaften, st. pölten, austria 17 mildred scheel cancer career center hatrics4, university medical center hamburg-eppendorf, hamburg, germany 18 present address: regensburg university hospital, department of neuropathology, regensburg, germany histone h3 k27m-mutant diffuse midline gliomas (dmgs) are highly malignant tumours arising in the midline structures of the cns. recent studies suggested that epigenetic subgroups of dmgs can be distinguished based on alterations in the mapk-signalling pathway, tumour localisation, mutant h3-gene, or overall survival. however, it is unclear how these parameters collectively influence survival. hence, we analysed dependencies between different parameters, to define novel epigenetic, clinically meaningful subgroups of dmgs. we collected a cohort of 149 h3 k27m-mutant dmgs, that could clearly be allocated to the spinal cord (n = 31; one patient with an additional sellar tumour), medulla (n = 20), pons (n = 64) or thalamus (n = 33), including published data. we then performed dna methylation profiling and, for a subset, dna sequencing and survival analyses. unsupervised hierarchical clustering of dna methylation data indicated two clusters of dmgs, i.e. subtypes dmg-a and dmg-b. these subtypes differed in mutational spectrum, localisation, age at diagnosis and overall survival. dmg-a was enriched for mapk-associated mutations, medullary localisation and adult age. 13 % had a methylated mgmt promoter. contrarily, dmg-b was enriched for tp53-mutations, pdgfra-amplifications, pontine localisation and paediatric patients. in univariate analyses, the features enriched in dmg-b were associated with a poorer survival. however, these parameters were dependent on the cluster attribution, which had the largest effect on survival: dmg-a had a significantly better survival compared to dmg-b. hence, the subtype attribution based on two methylation clusters is best suited to predict survival as it integrates different molecular and clinical parameters. p09 free neuropathol 5:19:19 spatial multiomics profiling reveals heterogeneity of b:t cell interactions and plasma cell formation in tertiary lymphoid structures in human gliomas pinar cakmak1,2,*, jennifer h. lun1,2,*, aakanksha singh1,2,3,*, jadranka macas1,2,*, michael c. burger2,3,4, lucie marie hasse1,2,3, elke hattingen2,3,5,6, miriam köhler1, jonathan schupp1,2, tatjana starzetz1, eike steidl5, karl h. plate1,2,3,6,ƚ, yvonne reiss1,2,6,ƚ, katharina imkeller1,2,3,ƚ 1 institute of neurology (edinger institute), university hospital, goethe university, frankfurt, germany 2 frankfurt cancer institute (fci), frankfurt, germany 3 university cancer center (uct), frankfurt, germany 4 dr. senckenberg institute of neurooncology, university hospital, goethe university, frankfurt, germany 5 institute of neuroradiology, university hospital, goethe university, frankfurt, germany 6 german cancer consortium (dktk), partner site frankfurt/mainz and german cancer research center (dkfz), heidelberg, germany * these authors contributed equally ƚ these authors contributed equally adult-type diffuse gliomas, the most common primary brain tumors, remain a clinical challenge in oncology with limited treatment options, restricted anti-tumor immune response and dismal patient prognosis. despite their immunosuppressive microenvironment, the formation of lymphoid aggregates containing adaptive immune cells has been reported in gliomas. however, the cellular composition, immunological function and relevance of lymphoid aggregates for adaptive anti-glioma immunity is not well understood. therefore, we performed a comprehensive, unbiased analysis of lymphoid aggregation in 642 adult-type diffuse gliomas using a multi-modal approach; combining dna methylation, rna sequencing with spatial transcriptome and proteome profiling. overall, b cell aggregates and tertiary lymphoid structures (tls) were observed in 15 % of tumors and associated with an improved overall survival. gliomas containing tls displayed a remodeled perivascular space, characterized by transcriptional upregulation and spatial redistribution of collagens associated with barrier functions. spatial transcriptome and proteome profiling revealed heterogeneous b:t cell interactions that were associated with elevated cd8 t-cell numbers and differences in iga and igg plasma-cell forming capacity, suggestive of dynamic adaptive immune responses. p10 free neuropathol 5:19:20 digital biomarkers for an improved clinical stratification of meningioma patients moritz armbrust1, eike steidl2,3,4, nadine flinner2,5,6,7, lina-elisabeth qasem2,8, ali al-hilou8, florian buettner4,7,9,10, karl h. plate1,2,4,7, marcus czabanka2,4,7,8, katharina j. weber1,2,4,7 1 neurological institute (edinger institute), university hospital, goethe university, frankfurt am main, germany 2 university cancer center (uct) frankfurt-marburg, university hospital, goethe university, frankfurt am main, germany 3 institute of neuroradiology, university hospital, goethe university, frankfurt am main, germany 4 german cancer consortium (dktk), partner site frankfurt / mainz and german cancer research center (dkfz), heidelberg, germany 5 dr. senckenberg institute of pathology, university hospital, goethe university, frankfurt am main, germany 6 frankfurt institute for advanced studies (fias), frankfurt am main, germany 7 frankfurt cancer institute (fci), goethe university, frankfurt am main, germany 8 department of neurosurgery, university hospital, goethe university, frankfurt am main, germany 9 department of medicine, goethe university, frankfurt am main, germany 10 department of informatics, goethe university, frankfurt am main, germany introduction: postsurgical therapy concepts in meningiomas are influenced by the world health organisation (who) three-tiered histomorphological grading, which is subjected to a high interobserver variability and low reproducibility. risk-of-recurrence models, integrating histology and molecular tumor data, have been developed to improve patient stratification. however, the distinguishment between meningioma patients with a low risk of recurrence from those with an intermediate risk remains particularly challenging. here, we propose imageand methylome-based biomarkers for a refined outcome prognostication. material and methods: whole slide images (wsi) of ki67-stained meningioma samples were generated and dna methylomes were collected (n = 173). a random-forest based pixel classifier for a machine-learning based tumor segmentation was generated on training images (n = 110). in a scripted batch processing, the ki67 marker was spatially quantified within randomly distributed, segmented image tiles (10 tiles, tumor area 10 mm2). further, samples were epigenetically subcharacterized by reference-free tumor deconvolution. results: the wsi data is assignable to methylation subclasses within the heidelberg v12.8 brain tumor classifier (median ki67 grouped subclasses benign 1-3 and intermediate-a = 5.0 %; median ki67 grouped subclasses intermediate-b and malignant = 7.0 %; p = 0.0096; mann-whitney u test; n = 135). integrated risk-of-recurrence scores (ints, hielscher et al., 2022) are distinguishable based on proliferation (median ki67 ints low = 4.1 %; median ki67 ints intermediate/high = 6.5 %; p < 0.0001; mann-whitney u test; n = 118). based on methylation-based tumor deconvolution, a meningioma clustering according to histological who grades is identifiable (n = 107). conclusion: ki67-focused wsi and tumor deconvolution data translate into different patient outcome groups, representing promising novel biomarkers for future integrated models in disease course prognostication of meningioma patients. p11 free neuropathol 5:19:22 cellular senescence as a shared contributor to disease progression in glioblastoma and alzheimer's disease elena krause1,2, lisa greutter1, daria romanovskaia3, peter stepper3, felix sahm4, adelheid wöhrer1 1 medical university of vienna, department of neurology, division of neuropathology and neurochemistry, vienna, austria 2 university of heidelberg, heidelberg, germany 3 cemm research center for molecular medicine of the austrian academy of sciences, vienna, austria 4 department of neuropathology, university hospital heidelberg, heidelberg, germany introduction: neurodegenerative disorders and cancer are increasingly prevalent in aging populations. over 50 % of glioblastoma (gb) patients show alzheimer's disease neuropathological changes (adnc) in tumor-adjacent cortex, but the biological link remains unclear. here, we investigate cellular senescence, one hallmark of aging, as a shared mechanism of disease progression. methods: we analyzed single cell/nucleus rna sequencing datasets from 110 gb patients (gbmap core atlas) and 89 ad individuals (sea-ad mtg atlas). using established senescence gene lists, gene set enrichment analysis and machine learning we identified senescent cells. this allowed us to compare senescence in cell types, subclusters, and disease stages. weighted gene co-expression network analysis (wgcna) identified senescence-associated co-expression modules. quantitative neuropathological analysis of published measurements supported the findings. results: leveraging published datasets, we identified senescent cells in gb and ad. gb showed higher variance in senescence scores, indicating greater inter-patient variability. among glial cells, microglia were enriched for senescence gene sets, with the highest scores in proinflammatory subtypes. using wgcna we found senescence genes co-expressed with known disease-associated genes like pten or spp1 in ad or gb, respectively. in both diseases, microglia senescence correlated with disease progression. accordingly, we identified higher adnc and altered microglia morphology with signs of functional exhaustion in ad specimens with high senescence scores. conclusion: our atlas-level transcriptomic analysis identifies cellular senescence as a shared mechanism in gb and ad pathology and disease progression, particularly enriched in microglia. co-expression of senescence-associated and disease-specific genes in microglial modules highlights the complex role of senescence in these diseases. p12 free neuropathol 5:19:23 vopp1::egfr gene fusion as an oncogenic driver via nfκb pathway activation in a case of ganglioglioma max braune1, mathias stiller2, cordula scherlach3, katja jähne4, alonso barrantes-freer1 1 paul-flechsig-institute of neuropathology, university hospital leipzig, leipzig, germany. 2 institute of pathology, university hospital leipzig, leipzig, germany 3 institute of neuroradiology, university hospital leipzig, leipzig, germany 4 department of neurosurgery, university hospital leipzig, germany ganglioglioma is a well-differentiated glioneuronal tumor characterized by a combination of neoplastic ganglion and glial cells. oncogenic driver mutations and gene fusions have been shown to be of prognostic significance in gangliogliomas and can offer potential therapeutic targets. typical molecular alterations are mapk pathway activations with braf p.v600e being the most frequent one. here, we report for the first time a vopp1::egfr gene fusion as the single oncogenic driver in a case of ganglioglioma leading to activation of nfκb signaling. we show the respective histological and molecular evidence including gene fusion and mutational analysis, methylation profiling and clinical outcome. the case expands the known molecular spectrum of oncogenic drivers in ganglioglioma linking it with prognostic and potentially therapeutically relevant data. p13 free neuropathol 5:19:24 insights from the additional immunohistochemical work-up of molecular tumor board patients from a regional brain tumor center elena geitner1, tanja rothhammer-hampl1, christina mikolajek1, atik baborie1, martin proescholdt3, nils-ole schmidt3, elisabeth bumes2, peter hau2, markus j. riemenschneider1 1 department of neuropathology, regensburg university hospital, regensburg, germany 2 department of neurology and wilhelm sander-neurooncology unit, regensburg university hospital, regensburg, germany 3 department of neurosurgery, regensburg university hospital, regensburg, germany patients with cns/pns tumors are included into the molecular tumor board of our local brain tumor center to defined criteria and on a regular basis. patients discussed in the period from 22.06.2021 to 30.06.2023 were selected for accompanying immunohistochemical (ihc) work-up in addition to comprehensive molecular analysis. main objectives were to substantiate signaling pathway activation and to identify biomarkers harboring predictive value. the project was approved by the local ethics committee, and informed consent was obtained from all patients. ffpe tumor tissues (same material as used for molecular analyses) from 84 patients were included on tissue-microarrays comprising the following entities: adult-/pediatric-type diffuse glioma, circumscribed glioma, ependymoma, meningioma, medulloblastoma, peripheral nerve sheath and pineal tumor. tissue-microarrays were analyzed immunohistochemically using 14 different antibodies: p-akt, p-mtor, p-tsc1, p-tsc2, p-s6-rp, p-stat3 (ser727 and tyr705), p-p42/44 mapk(erk1/2), p-mek, p-rb, mlh1, pms2, msh2 and msh6. the h-score was used to classify results semi-quantitatively. immunohistochemical results were correlated to molecular alterations and revealed two significant associations: (1) patients with at least one activating egfr alteration exhibit significantly lower phosphorylation and activation of tsc1 than those without egfr alterations. (2) expression of the mismatch-repair proteins msh2, msh6, mlh1 and pms2 was reduced in patients with a nf1 mutation. in summary, additional ihc work-up of molecular tumor board patients generated two interesting scientific findings: (1) p-tsc1 might serve as a surrogate marker for egfr alterations and (2) a potential interrelation between mmr-deficiency and mutational inactivation of nf1 exists. the exact mechanisms of this finding, however, need further investigation. p14 free neuropathol 5:19:25 cytokine response in the cerebrospinal fluid after intraoperative radiation of primary and metastatic brain tumors zuzanna mielewczyk1,2, philipp krauss3, björn sommer3, klaus-henning kahl4, tatiana mögele2, bruno märkl2, ehab shiban3, friederike liesche-starnecker1,2 1 department of neuropathology, pathology, medical faculty, university of augsburg, germany 2 pathology, medical faculty, university of augsburg, germany 3 department of neurosurgery, medical faculty, university of augsburg, germany 4 department of radiooncology, medical faculty, university of augsburg, germany aim of our study is to characterize the immunological signature represented by the cytokine profile in the cerebrospinal fluid (csf) after iort of primary and secondary brain tumors. for this, the cytokine patterns in 67 csf samples obtained from 19 patients with brain tumor resection were analyzed. samples were collected at four time points: 1. intraoperatively, before tumor resection and iort, 2. intraoperatively, after tumor resection, before iort, 3. intraoperatively, after tumor resection, after iort and 4. approx. 24 hours postoperatively from drainage. multiplex immunoassay codeplex secretome for the isospark technology was used for level determination of 19 cytokines involved in the innate immunoresponse. an increase of signal intensity was observed for 14 of the 19 analyzed cytokines (73 %) in the iort group, including egf, granzyme b, il-10, il-1β, il-6, il-7, il-8, ip-10, mcp-1, mip-1α, mip-1β, pdgf-bb, scd137 and vegf. seven of those cytokines (37 %) displayed a significant increase. when comparing the time points 1 and 4 between iort and non-iort group, the increase in signal intensity for il-1β, il-8, ip-10, mip-1β, il-10, and tnf-α in the iort group is noticeably greater than in the non-iort group, although not significantly. our results allow first conclusions about changes in the cytokine profile of csf after iort of primary and secondary brain tumors. the results indicate that il-1β, il-8, ip-10, mip-1β, il-10 and tnf-α are involved in the inflammatory response induced by exposure to ionizing radiation of the tumor bed during surgery. ii. neuroimmunology p15 free neuropathol 5:19:26 in-depth analysis of viral distribution and immunological profiling in human bornavirus encephalitis nicola jungbäck1,2, tatiana mögele2, przemyslaw grochowski2, patrick adam3,4, thomas pfefferkorn5, frank lippek6, antonios bayas7, thomas richter8, georg rieder9, bruno märkl2, jürgen schlegel1,2,4, dennis tappe10, friederike liesche-starnecker1,2 1 department of neuropathology, pathology, medical faculty, university of augsburg, augsburg, germany 2 pathology, medical faculty, university of augsburg, augsburg, germany 3 pathology, ingolstadt, germany 4 institute of pathology, school of medicine and health, technical university of munich, munich, germany 5 department of neurology, ingolstadt hospital, ingolstadt, germany 6 institute of pathology and cytology, university hospital ruppin-brandenburg, neuruppin, germany 7 clinic for neurology and clinical neurophysiology, university hospital augsburg, augsburg, germany 8 pathology rosenheim, rosenheim, germany 9 department of neurology, innklinikum, altötting, germany 10 bernhard nocht institute for tropical medicine, national reference centre for tropical pathogens, hamburg, germany the borna disease virus 1 (bodv-1) has recently been shown to cause a usually fatal encephalitis in humans. to date, little is known about the pathogenesis and distribution pattern of the virus in human diseases. aim of the present study is to characterize the virus distribution in the cns and profile the immunological processes in human bornavirus encephalitis (bve). complete sagittal and coronary sections of the brains from four individuals who died of bve were embedded and stained immunohistochemically for the bodv-1 nucleoprotein (antibody bo18). the cross-sections were then digitally reconstructed, and the amount of bodv-1-positive cells quantified using the software cellquant. furthermore, the viral loads were estimated for each block using qpcr. immunological profiling was done using the ncounter technology (nanostring). while most cases show relatively low virus intensities in the cerebellum, the basal ganglia, brainstem and thalamus are frequently heavily affected as estimated by both, immunohistochemistry and qpcr. the immunological profile was examined for one bodv-1 case based on low, medium and high viral loads. cd56dim natural killer cells have the highest scores in areas with high viral loads. interestingly, regions with medium viral loads show the highest signatures for immunological cells, like cytotoxic cells and macrophages. bve can be considered a model disease for neurotropic virus infections. understanding the pathogenesis and viral spread mechanisms is crucial despite the rarity of the disease. a standardized, comprehensive analysis of autopsy cases, including the peripheral nervous system, is essential. this will significantly enhance our understanding and management of such infections. p16 free neuropathol 5:19:28 histopathological analysis of bodv-1 distribution in a non-human primate model: comparing intranasal and subcutaneous inoculation nicola jungbäck1,2, przemyslaw grochowski2, tatiana mögele2, friederike feldmann3, heinz feldmann4, greg saturday3, bruno märkl2, martin beer5, kore schlottau5, friederike liesche-starnecker1,2 1 department of neuropathology, pathology, medical faculty, university of augsburg, augsburg, germany 2 pathology, medical faculty, university of augsburg, augsburg, germany 3 rocky mountain veterinary branch, division of intramural research, national institute of allergy and infectious diseases, national institutes of health, hamilton, usa 4 laboratory of virology, division of intramural research, national institute of allergy and infectious diseases, national institutes of health, hamilton, usa 5 institute of diagnostic virology, friedrich-loeffler-institut, greifswald-insel riems, germany borna disease virus 1 (bodv-1) is known to cause fatal encephalitis in humans, yet the mechanisms and distribution of the virus within the brain remain poorly understood. this study investigates the histopathological impact of bodv-1 in a non-human primate model. twelve macaques were infected with bodv-1, with six receiving intranasal inoculation which reflects the currently hypothesized portal of entry, and six receiving subcutaneous inoculation as setting for infection via the peripheral nervous system. the primary focus of this project is the histopathological examination of the brains to determine the differences of the viral distribution in the two settings of infection. complete sagittal sections of the brains were embedded and immunohistochemically stained targeting the bodv-1 nucleoprotein. the stained sections were digitally reconstructed, allowing for a detailed analysis of the virus' distribution. quantitative assessment was performed using the software cellquant. preliminary results indicate differential patterns of viral spread and intensity between the two inoculation methods. intranasally infected macaques showed a higher concentration of bodv-1-positive cells in the olfactory bulb, spreading to the frontal cortex and deeper brain structures. subcutaneously infected macaques demonstrated a more dispersed viral distribution with generally lower virus detection. this study provides valuable insights into the pathogenesis of bodv-1, highlighting the importance of the route of infection in determining viral spread within the cns. the findings emphasize the need for comprehensive histopathological analysis to enhance our understanding of bodv-1 and its implications for neurotropic virus infections. p17 free neuropathol 5:19:29 systematic analysis of virus spread to the peripheral nervous system in fatal borna virus encephalitis przemyslaw grochowski1, nicola jungbäck1,2, tina schaller1, frank lippek3, patrick adam4,5, thomas pfefferkorn6, jürgen schlegel1,2,4, bruno märkl1, friederike liesche-starnecker1,2 1 pathology, medical faculty, university of augsburg, augsburg, germany 2 department of neuropathology, pathology, medical faculty, university of augsburg, augsburg, germany 3 university hospital ruppin-brandenburg, institute of pathology and cytology, neuruppin, germany 4 pathology ingolstadt, ingolstadt, germany 5 technical university of munich, school of medicine and health institute of pathology, munich, germany 6 klinikum ingolstadt, department of neurology, ingolstadt, germany borna disease virus 1 (bodv-1) causes fatal encephalitis in endemic regions. even if there are reports on spread of this highly neurotropic virus to the peripheral nervous system, there is no systematic analysis of the virus distribution aside from the central nervous system yet. we analyzed autopsy material from four deceased with bodv-1 infection regarding the presence of bodv-1 nucleoprotein in visceral organs (including heart, lung, liver, thyroid gland, pancreas, stomach) with immunohistochemistry. additionally, we evaluated the tissue morphology considering local inflammatory reaction, which is commonly observed in central nervous system and contributes to the fatal outcome. bodv-1 could be shown in small peripheral nerves in the thyroid gland, pancreas, adrenal glands, left side heart and in the stomach, without spread outside the nerve structures. one patient was a transplant recipient from earliest reported infection cluster and showed additional infiltration in peripheral skeletal nerves, esophagus, mediastinal and retroperitoneal adipose tissue, lungs, liver as well as in the implanted but not their own kidney. neither inflammatory nor any other tissue reaction was observed. in one case, no peripheral spread could be shown (with also atypical brain distribution due to high-dose immunosuppression). no patient showed bodv-1 positivity aside of the peripheral nerve tissue. we confirm that bodv-1 spreads to small peripheral nerves, pronouncing in organs innervated by the vagus nerve. although neither specific reaction nor local damage was observed, consecutive autonomic nerve system dysregulation may occur in some patients. investigations regarding alternative clinical presentations apart from the typical encephalitis should be considered. p18 free neuropathol 5:19:30 global cerebral hypoxia-ischemia: ex vivo ultrahigh field mri signals correlate with differential cortical localization of microglia and gemistocytes characterized by chit1 and chi3l1 (ykl 40) expression deniz yilmazer-hanke1, elisa tuzzi1, najwa ouali alami1, lubin fang1, sigrid klotz2, ellen gelpi2, kelly del tredici1 1 clinical neuroanatomy, neurology, university ulm, university hospital ulm, ulm, germany 2 division of neuropathology and neurochemistry, department of neurology, medical university of vienna, vienna, austria previously, we have shown in survivors of global hypoxic-ischemic brain injury (hibi) that the neuroinflammationand ischemia-related glial markers chitotriosidase 1 (chit1) and chitinase-3-like protein 1 (chi3l1, alias ykl-40) are expressed by microglia and astrocytic gemistocytes, respectively (yilmazer-hanke et al., 2022, neuroscience 506:91–11). in the current study, we investigated the distribution of chit1-positive microglia and chi3l1-positive gemistocytes in the cerebral cortex after hibi. tissue from hibi cases was scanned using high-resolution ultrahigh field mri (uhf-mri) with ex vivo t1w, t2w and t2*w sequences. chit1 and chi3l1 expression was compared to changes in microglial (e.g., iba1, cd68) and astrocytic (e.g., gfap) markers using immunohistochemistry and multiple-label immunofluorescence. results indicated altered mri signals in different cortical layers. these altered mri signals correlated with severe neuronal cell loss, enhanced densities of rounded chit1-positive microglia in deep cortical layers, and the emergence of chi3l1-positive gemistocytes, which populated superficial cortical layers and the juxtacortical boundary. further analyses revealed that chit1-positive microglia aggregated around microvessels of deep cortical layers. in conclusion, altered cortical mri signals that reflect the cortical damage as well as chit1and chi3l1-positive glial cell pathology with a differential cortical distribution could be a valuable in vivo biomarker for monitoring the outcome of global hibi and for determining the prognosis of long-term hibi survivors. iii. muscle / nerv p19 free neuropathol 5:19:31 adipo-glial signaling mediates metabolic adaptation in peripheral nerve regeneration v.k. sundaram1, v. schütza2, n. schröter2, e. ernst sanchez2, a. backhaus2, a. bilsing2, l. joneck2, a. seelbach1, jose a gomez-sanchez 3, d. akkermann1, t. kungl2, m. ost2, peter arthur-farraj4, r. stassart1, r. fledrich2 1 paul flechsig institute center of neuropathology and brain research, leipzig university, leipzig, germany 2 institute of anatomy, leipzig university, leipzig, germany 3 instituto de investigación sanitaria y biomédica de alicante (isabial), alicante, spain; instituto de neurociencias csic-umh, san juan de alicante, spain 4 john van geest centre for brain repair, department of clinical neurosciences, university of cambridge, cambridge cb2 0py, uk the peripheral nervous system exhibits an impressive potential to regenerate following acute nerve injury. however, complete functional recovery is uncommon and heavily relies on peripheral nerve schwann cells, which orchestrate both the degradation and re-synthesis of myelin while also supporting axonal regrowth. these cellular processes elicit a substantial metabolic demand and the exact mechanisms by which schwann cells cater to the high metabolic demands of nerve repair are still not well understood. in this study, we demonstrate that nerve injury triggers signaling from adipocytes to glial cells and we identify the adipokine, leptin, as a key regulator of glial metabolic adaptation during regeneration. using conditional mutagenesis in mice, we demonstrate that leptin receptor ablation in schwann cells or leptin ablation in adipocytes renders a congruent phenotype characterized by abrogated injury-specific catabolic processes such as myelin autophagy and mitochondrial respiration in schwann cells during nerve repair. our research thus proposes a model wherein acute nerve injury initiates an adipo-glial communication that can be therapeutically targeted to modulate glial metabolism, ensuring sufficient energy for effective nerve repair. iv. neurodegeneration p20 free neuropathol 5:19:32 alpha-synuclein co-pathology in alzheimer’s disease drives tau accumulation felix l. struebing1,2, teodoro de vecchi1,2, jeannine widmann1,2, xiaoxuan song1,2, federico fierli1,2, viktoria ruf1,2, qilin tang2,4, thomas arzberger1, sigrun roeber1, thomas koeglsperger2,4, otto windl1, christian haass2,3, juliane winkelmann3, jochen herms1,2,3 1 center for neuropathology and prion research, ludwig maximilian university of munich, munich, germany 2 german center for neurodegenerative diseases (dzne), munich, germany 3 munich cluster of systems neurology (synergy), munich, germany 4 department of neurology, ludwig maximilian university, munich, germany background: the molecular basis for accelerated cognitive decline seen in alzheimer’s disease (ad) cases presenting with cortical alpha-synuclein co-pathology is not well understood. mouse experiments have shown adverse interactions between tau and alpha-synuclein, but how this finding translates to humans from a genome-centered point of view remains unknown. materials and methods: whole genome sequencing was performed on 137 neuropathologically defined ad cases, 36 of which presented with neocortical alpha-synuclein co-pathology (braak stage 6). polygenic risk scores were calculated. single-nucleus rna sequencing and western blot data were collected from post-mortem tissue. bioinformatic analysis of a large external dataset (n > 300) served as external validation. transcriptomic and proteomic experiments were carried out in cell lines and cortical organoids derived from ipscs. results: ad brains with alpha-synuclein co-pathology had significantly higher polygenic risk scores for parkinson’s disease, which could be partially explained by variants associated with higher expression of snca (the gene symbol for alpha-synuclein). single-nucleus rna sequencing revealed a higher expression of mapt, the gene encoding microtubule-associated protein tau, in co-pathology cases. protein and mrna expression of mapt and snca were positively correlated in an external cohort. ultimately, cell culture experiments demonstrated that overexpression of snca was sufficient to drive accumulation of soluble tau. conclusion: we show that alpha-synuclein co-pathology brains are characterized by higher tau levels and that increasing alpha-synuclein expression is sufficient to drive tau accumulation. our results reveal that tau and alpha-synuclein can synergistically drive dementia-related pathology. p21 free neuropathol 5:19:33 frontotemporal dementia patient neurons with the mapt-n279k mutation are responsive to tau filaments and contribute to neuroinflammation in vivo julie j. mcinvale1, matti lam2, river kim1, markus d. siegelin1, james e. goldman1,3, dennis w. dickson4, vilas menon2,3, peter canoll1, gunnar hargus1,3 1 department of pathology & cell biology, columbia university, new york, ny, 10032, usa 2 center for translational & computational neuroimmunology, department of neurology, columbia university irving medical center, new york, ny, 10032, usa 3 taub institute for research on alzheimer's disease & the aging brain, columbia university, new york, ny, 10032, usa 4 department of neuroscience, the mayo clinic florida, jacksonville, fl, 32224, usa frontotemporal dementia (ftd) is a heterogeneous group of early-onset dementias leading to impairment of cognition, language and behavior. ftd can be caused by deposition of hyperphosphorylated tau (p-tau) in neurons and glial cells in various brain regions throughout the patient brains. however, the mechanisms leading to neurodegeneration are still largely unknown and a curative therapy does not exist. here, we applied a stem cell-based approach combined with single-cell analyses of ftd patient brains and differentiated ftd patient-derived induced pluripotent stem cells (ipscs) carrying the mapt-n279k mutation and healthy control ipscs into neurons. we found disease-relevant changes in ftd neurons associated with mis-splicing of tau, p-tau pathology, neurite outgrowth deficits, and increased oxidative stress and neuroinflammation with an upregulation of pro-inflammatory genes, several of which were also upregulated in neurons in ftd patient brains carrying the same mapt-n279k mutation. tau filaments isolated from ftd patient brains with mapt-n279k further upregulated expression of the pro-inflammatory marker osteopontin in ftd neurons and altered pathways related to the unfolded protein response and proteasomal function. when injected into the mouse brains, ftd neurons showed decreased survival and induced an increased microglial response. decreased survival of ftd neurons was also noted when cells were co-injected with ftd tau filaments into mouse brains. interestingly, alterations of inflammatory gene expression in engrafted neurons resulted in altered engraftment and microglial infiltration. these findings point towards an immune-modulatory role of neurons in ftd and indicate that its alteration may represent a potential therapeutic target in ftd. p22 free neuropathol 5:19:34 alterations of er-co-chaperone sil1 in amyotrophic lateral sclerosis (als) haihong guo1, alfred yamoah1,2,3, priyanka tripathi1,2,3, antonio sechi4, istvan katona1,5, eleonora aronica6, marcel naumann7, andreas hermann7,8, harry steinbusch2,3, andreas roos9,10, joachim weis1,*, anand goswami1, 11, 12,* 1 institute of neuropathology, rwth aachen university hospital, 52074 aachen, germany 2 department of psychiatry and neuropsychology, school for mental health and neuroscience, maastricht university, 6229 er maastricht, the netherlands 3 euron european graduate school of neuroscience, maastricht university, 6229 er maastricht, the netherlands 4 institute of cell and tumorbiology, rwth aachen university hospital, 52074 aachen, germany 5 department of neurology, houston methodist research institute, tx 77030 houston, united states 6 division of neuropathology, department of pathology, academic medical centre, 1105 az, amsterdam, the netherlands 7 translational neurodegeneration section “albrecht-kossel’’, department of neurology and centre for transdisciplinary neurosciences rostock (ctnr), university medical centre rostock, 18147 rostock, germany 8 german centre for neurodegenerative diseases (dzne) rostock/greifswald, 18147 rostock, germany 9 department of neurology, heimer institute for muscle research, university hospital bergmannsheil, ruhr university bochum, 44789 bochum, germany 10 department of neuropediatrics and neuromuscular centre for children and adolescents, centre for translational neuroand behavioural sciences, university duisburg-essen, 45147 essen, germany 11 department of neurology, centre for motor neuron biology and disease, columbia university, 10032 new york, united states 12 department of neurology, eleanor and lou gehrig als centre, columbia university, 10032 new york, united states * equal contribution amyotrophic lateral sclerosis (als) is a devastating disease characterized by progressive loss of upper and lower motor neurons (mns). recent studies showed that selectively viable neurons are often equipped with protective factors, while vulnerable neurons lack them. among others, the er co-chaperone sil1, mutated in marinesco-sjögren syndrome (mss), supports such selective mn viability not only in mss pathology but also in als pathology. however, the precise molecular mechanism(s) of such neuroprotection as well as its role in maintaining neuronal proteostasis is largely unclear. we aimed to validate the correlation between sil1 and als-associated protein aggregates in human als together with investigating the neuroprotective role of sil1 associated with the protein quality control (pqc) mechanism. we used als postmortem autopsy materials and compared them with als cell models expressing als-causing mutant proteins. our results confirmed that in the als mns, sil1 protein levels were specifically elevated in slow fatigue-resistant mns (smns), and reduced sil1 levels were linked to als-associated protein aggregates. besides, sil1 was found to be sequestered with various forms of ptdp-43 and other aggregates in als spinal mns as well as als cell culture models. overexpression of sil1 facilitated the clearance of the above-mentioned pathogenic aggregates in these models. furthermore, sil1 could activate autophagy pathways by promoting fusion of autophagosomes with lysosomes. in summary, our results suggest that sil1 is promoting neuronal pqc and survival. conversely, disturbed sil1 function could be detrimental to neurons. p23 free neuropathol 5:19:36 characterizing granular tau aggregates in astrocytes in multiple system atrophy viktoria c. ruf1, doris weissenbrunner1,2, paul feyen1,2, jochen herms1,2,3 1 center for neuropathology and prion research, faculty of medicine, lmu munich, munich, germany 2 german center for neurodegenerative diseases (dzne) e. v., site munich, munich, germany 3 ludwig-maximilians-university, munich cluster of systems neurology (synergy), munich, germany introduction: multiple system atrophy (msa) is a rare adult-onset fatal neurodegenerative disorder characterized by alpha-synuclein aggregation in oligodendrocytes. moreover, small granular tau inclusions in the white matter were occasionally, but not extensively described in the literature and have been controversially discussed. based on the finding from our snrna-seq dataset that mapt is significantly upregulated in astrocytes in the frontal white matter of msa patients, we sought to better and more comprehensively characterize the observation of granular tau inclusions in msa. materials and methods: immunohistochemistry and immunofluorescence as well as rnascope were applied. results: at8-positive granules were a common finding although to a highly variable extent in the white matter of msa patients, whereas no such staining was observed in a series of non-neurodegenerative controls or patients with lbd. the granules partially colocalized with ptau-thr231 and ptau-ser396 as well as with rd3 or rd4. co-staining of at8 with gfap revealed an association with astrocytes without any obvious association with oligodendrocytes or microglia. while there was no noticeable colocalization of ptau and alpha-synuclein inclusions, we find a positive relationship between the load of alpha-synuclein pathology and granular tau inclusions. conclusion: granular tau accumulations are a common feature in msa. the inclusions, however, seem to be different from other known tau aggregates. while it is generally assumed that glial cells take up misfolded tau from neurons, the upregulation of mapt in astrocytes may indicate that in this case tau accumulation may be a consequence of an increased expression of endogenous tau. v. methods and free topics p24 free neuropathol 5:19:37 ultra-rapid bacterial detection from neuropathology specimens using next-generation pcr and nanopore sequencing daan brackel1, sander boden1, anne albers2, robin struijk3, christian thomas2, eefje schrauwen1 1 centre of expertise perspectives in health, research group analytical techniques in the life sciences, avans university of applied sciences, breda, netherlands 2 institute of neuropathology, university hospital münster, münster, germany 3 department of research & development, molecular biology systems b.v., goes, the netherlands bacterial infections of the central nervous system (cns) are severe and potentially fatal diseases. early detection of causative pathogens is crucial for the rapid administration of a tailored antibiotic regime. routine neuropathology diagnostics is typically limited to histological staining and microbiological culture, which are time consuming and result in false-negative results in one-third of cases. metagenomic sequencing of the bacterial 16s rrna gene offers a rapid and unbiased alternative. in this study, we present a method for detecting bacterial pathogens in neuropathology specimens using ultra-rapid pcr combined with nanopore sequencing of 16s rdna amplicons. for protocol optimization, a microbial community standard and varying concentrations (5 pg to 5 ng) of gdna from e. coli and s. aureus were used. ten ffpe samples with known bacterial cns infections were retrieved from the institute of neuropathology münster. after dna isolation, library preparation was performed using classic pcr and nextgenpcr with four primer pairs covering the variable regions v3–v7 of the 16s rrna gene. amplicons were sequenced on a minion device and data was analysed with epi2me, maba16s and emu. nextgenpcr followed by nanopore sequencing identified bacterial taxa more accurately (64 % vs. 26 % correct hits, p < 0.001, t-test). compared to classic pcr, the optimized nextgenpcr protocol significantly reduced the amplification time from 165 to 35 minutes. in conclusion, nextgenpcr of 16s rdna followed by nanopore sequencing offers an ultra-rapid method for detecting bacteria in neuropathology specimens. further investigation of a larger cohort including cerebrospinal fluid samples is warranted. p25 free neuropathol 5:19:38 ccnv: r package for enhanced cumulative copy number variation analysis antonia gocke1,2, yannis schumann3, jelena navolic1, melanie schoof4,5, matthias dottermusch1,6, julia e. neumann1,6 1 center for molecular neurobiology hamburg (zmnh), university medical center hamburg-eppendorf, hamburg, germany 2 section of mass spectrometric proteomics, university medical center hamburg-eppendorf, hamburg, germany 3 chair for high performance computing, helmut-schmidt university, hamburg, germany 4 research institute children’s cancer center hamburg, hamburg, germany 5 department of pediatric hematology and oncology, university medical center hamburg-eppendorf, hamburg, germany 6 institute of neuropathology, university medical center hamburg-eppendorf, hamburg, germany copy number variation (cnv) analysis is the study of genomic alterations resulting in an abnormal copy number of genes, chromosomal segments or whole chromosomes. the identification of chromosomal aberrations can help identify targetable proteins or markers for specific tumour entities (e.g. the mycn amplification in spinal ependymomas, mycn-amplified or the loss of smarcb1 in atypical teratoid/rhabdoid tumours). cnv information can be derived from global dna methylation data, which has become an integral part of tumor diagnostics. cnvs can help in or even guide tumor classification and the number of available data constantly increases. in order to characterize cnvs in large datasets comprising tumor subgroups or types we developed ccnv, a tool for fast, accurate and efficient cumulative cnv analyses. ccnv is an r package that integrates two main algorithms for the segmentation of inferred copy number data from methylation data; the circular binary segmentation integrated in conumee/conumee2.0 (hovestadt, zapatka (2015), daekanas (2024)) and the piecewise curve fit algorithm (nilsen (2012)). the package is compatible with all major available dna methylation chips (450k, epic; epicv2, mouse chip) and can automatically read array data from .idat files. each segmentation algorithm produces one intensity plot, representing how strong aberrations are across the cohort, and one frequency plot, representing how often an aberration occurs. while the circular binary segmentation is applied consecutively to each sample, the piecewise curve fit algorithm can be applied to all samples simultaneously, achieving a significant decrease in runtime. the latter further extracts the main aberrations of the investigated samples, making it particularly suitable for large datasets. with different selectable segmentation algorithms and parameters the ccnv package is a flexible and efficient tool to generate cumulative cnv plots from big datasets. p26 free neuropathol 5:19:39 rapid slice-free intraoperative histology in neurooncology using multiphoton microscopy – first study results jessica kren1, jan philip kolb2, matthias strauch2, niloofar khosravi2, alvaro vega perez2, matteo m. bonsanto1, julia e. neumann3,4 1 university hospital schleswig-holstein, lubeck clinic for neurosurgery, lübeck, germany 2 histolution gmbh, lübeck, germany 3 institute for neuropathology, university hospital hamburg-eppendorf, hamburg, germany 4 center for molecular neurobiology hamburg (zmnh), university hospital hamburg-eppendorf, hamburg, germany multiphoton microscopy (mpm) enables histological examination of tissue samples without the prior production of thin sections using paraffin or frozen preparation. it can be applied in the operating room (or) with less effort and in just a few minutes. in a proof of concept study, we evaluated mpm on neurosurgical tumor samples and compared the diagnostic results to the routine histology. mpm uses a laser to create non-linear optical effects that allow tissue blocks to be scanned at adjustable depths. after a 2:30 mins long staining process the mpm scanned the tissue at a speed of 4 min/cm², with the potential for further acceleration to 30 s/cm². the results are digital images with high similarity to standard h&e-stained slides. 12 tissue samples were collected at the clinic for neurosurgery at uksh lübeck. the digital images were evaluated in a blinded manner by a board-certified neuropathologist with no prior training in mpm image diagnosis. only minimal clinical input data was provided (patient's age, sex, and sample localization). in 11 of the 12 cases the diagnosis was consistent with the result of the routine analysis. tissue histomorphology assessment revealed a good assessability of the cell morphology and tissue texture compared to standard slides. the initial results show that the mpm is a valuable tool for accelerating and simplifying intraoperative histological examinations in neurosurgery without the need for device-specific training of the neuropathologist. in ongoing research, we will further optimize the technical settings and increase the cohort size to validate a broad applicability in neurooncology. p27 free neuropathol 5:19:40 predicting epigenetic ependymoma types from histological whole-slide images using neural networks maximillian middelkamp1,*, yannis schumann2,*, matthias dottermusch3,4, leonille schweizer5,6,7, maja krech8, tasja lempertz2,3, jens gempt1, ulrich schüller3,9,10, philipp neumann2, julia e. neumann3,4 1 department of neurosurgery, university medical center hamburg-eppendorf, 20246 hamburg, germany 2 chair for high performance computing, helmut-schmidt-university/university of the federal armed forces hamburg, holstenhofweg 85, 22043 hamburg, germany 3 institute for neuropathology, university medical center hamburg-eppendorf, martinistrasse 52 20246 hamburg, germany 4 center for molecular neurobiology hamburg, university medical center hamburg-eppendorf, falkenried 94, 20251 hamburg, germany 5 institute of neurology (edinger institute), university hospital frankfurt, theodor-stern-kai 7, 60590 frankfurt am main, germany 6 german cancer consortium (dktk), partner site frankfurt/mainz, german cancer research center (dkfz), heidelberg, germany 7 frankfurt cancer institute (fci), paul-ehrlich-str. 42–44, 60596 frankfurt am main, germany 8 institute for neuropathology, charité berlin, charitéplatz 1, virchowweg 15, 10117 berlin, germany 9 research institute children’s cancer center hamburg, uke, martinistrasse 52, 20246 hamburg, germany 10 department of pediatric hematology and oncology, uke, martinistrasse 52, 20246 hamburg, germany * authors contributed equally keywords: neuropathology, cancer, multiple instance learning, self-supervised learning, attention ependymomas are neuroepithelial neoplasms of the central nervous system and comprise (at least) 10 main clinically distinct types based on epigenetic (dna methylation) profiles. for their diagnosis, the current standard practice is to integrate time consuming epigenetic analyses with histological assessment. we asked whether neural networks can predict the dna methylation class of ependymoma types from hematoxylin and eosin stained whole-slide images. using explainable ai, we further aimed to prospectively improve the consistency of histology-based diagnoses with dna methylation profiling by identifying and quantifying distinct morphological patterns of these molecular ependymoma types. we collected sample-matched epigenetic profiles and whole-slide images (hematoxylinand eosin stain) of > 500 ependymomas from different anatomical compartments. attention-based classification models (clam) or multi-grid vision transformers (vits) were trained to predict the epigenetic ependymoma types from the slide images and the models were compared the results from histological annotations by neuropathologists for a fraction of cases. our approach yielded reliable predictions of the epigenetic types based on histomorphological data. self-supervised encoder training was crucial for classification performance. the classifiers improved over board-certified neuropathologists and its attentions scores were leveraged to correlate epigenetic and morphological ependymoma characteristics. image normalization and augmentation facilitated domain adaptation towards whole-slide images from other medical facilities and brightfield microscopy images. we established an interpretable method to reliably predict epigenetic ependymoma types from histological whole-slide images. our approach provides a fast and inexpensive way for first assessment of molecular ependymoma classification, provides morphological interpretability and may prospectively enable rapid decisions on patient-specific treatment in the upcoming era of digital pathology. p28 free neuropathol 5:19:42 established in 1902: a brief history of the institute of neurology (edinger institute) in frankfurt, germany karl h. plate1 1 goethe university, university hospital, institute of neurology (edinger institute), frankfurt, germany the institute of neurology was established by the jewish neuroscientist ludwig edinger on the premises of the dr. senckenberg institute of pathology in frankfurt, headed by carl weigert. edinger was considered to be the leader in comparative neuroscience of its time by the spanish nobel prize winner ramon y cajal. edinger was among the founders of the “königliche universität zu frankfurt” (later renamed as goethe university) in 1914 and was appointed as the first professor of neurology in germany. during the “third reich”, jewish scientists, including edinger's successor kurt goldstein, were forced to leave the institute. as a consequence, scientific activities in the institute of neurology ceased almost completely. after wwii, wilhelm krücke, an expert on peripheral neuropathies, became the director of the institute and founder of the german society of neuropathology in 1950. in 1960, the edinger institute served as founder for the max-planck institute for brain research, the legal successor of the kaiser-wilhelm institute for brain research (kwih, established 1914). as a consequence of the merger of edinger's institute with the department of histopathology of the former kwih, previously unrecognized specimens of victims of the nazi-regime were detected. wolfgang schlote became the director of the institute in 1984 until his retirement in 1999. this position was carried on by karl h. plate in 2001. the year 2002 marked the 100th anniversary of the edinger institute. several members of the edinger family returned to germany for the first time after wwii on this occasion. 122 years after its foundation, the institute of neurology continues to serve as the department of neuropathology of frankfurt university hospital, hosting three professors and six research groups, consisting of neuropathologists, molecular biologists, developmental biologists, bioinformaticians and research staff to provide a unique, lively environment for translational neuropathology, coming close to how it was initially intended by its founder ludwig edinger. p29 free neuropathol 5:19:43 paving the path: the powerful effect of substrate topography on axon-repulsive schwann cell-astrocyte barrier formation for spinal cord injury repair pascal achenbach1, laura hillerbrand2, josé l. gerardo-nava3,4, axel dievernich5,6, dorothee hodde1,7, antonio sechi8, paul d. dalton9, andrij pich3,10, joachim weis1, haktan altinova1,11,12, gary a. brook1 1 institute of neuropathology, rwth aachen university hospital, aachen, germany 2 department of functional materials in medicine and dentistry, university hospital würzburg, würzburg, germany 3 dwi leibniz institute for interactive materials, aachen, germany 4 advanced materials for biomedicine (amb), institute of applied medical engineering (ame), rwth aachen university hospital, aachen, germany 5 feg textiltechnik forschungsund entwicklungsgesellschaft mbh, aachen, germany 6 institute of laboratory animal science, rwth aachen university hospital, aachen, germany 7 hospital of the ludwig maximilians university munich, munich, germany 8 department of cell and tumor biology, rwth aachen university hospital, aachen, germany 9 phil and penny knight campus for accelerating scientific impact, university of oregon, eugene, oregon, united states 10 institute of technical and macromolecular chemistry, rwth aachen university, aachen, germany 11 department of neurosurgery, rwth aachen university hospital, aachen, germany 12 berlin state office for health and social affairs (lageso), berlin, germany spinal cord injury is a devastating condition that leads to permanent impairment of sensory, motor and autonomous functions. the severely limited regenerative capacity of the lesioned central nervous system (cns) is mainly attributed to the formation of a glial scar, a process that is mediated by reactive astrocytes (acs). transplantation of schwann cells (scs) into the severed spinal cord represents a promising therapeutic approach to support tissue regeneration but is limited by mutually repulsive interactions with resident acs, resulting in the formation of well-defined interfaces. such interfaces act as effective barriers for regenerating axons, allowing the transition from the ac-dominated cns environment into the sc compartment but not vice versa, leaving regenerated axons trapped within the sc graft. for this investigation, the well-established confrontation assay model was adapted to study the effect of topographical cues derived from substrate-bound, highly oriented poly(ε-caprolactone) nanofibers on such devastating sc-ac interactions as well as axon regeneration in vitro. immunocytochemistry was performed to quantify the extent of sc-ac intermingling and the success of axon regeneration across the modified sc-ac interface. our study demonstrates the powerful effect of oriented substrate topography on sc-ac interactions, preventing the formation of barrier-forming sc-ac interfaces and enabling substantial directed and long-distance axon outgrowth across the sc-ac interface and into the ac compartment. this is the first demonstration of non-functionalized nanofibers providing a substrate that can override such mutually repulsive cell-cell interactions. it is anticipated that these findings will profoundly influence the design of biomaterial-based scaffolds for spinal cord injury repair. p30 free neuropathol 5:19:45 using eye-tracking to find differences in the analysis of whole-slide images between physicians and machine learning models – a study design daniel hieber1,2,3,4, nicola jungbäck1,3, felix holl2, bruno märkl3, rüdiger pryss4, johannes schobel2, friederike liesche starnecker1,3 1 department of neuropathology, pathology, medical faculty, university of augsburg, augsburg, germany 2 digihealth institute, neu-ulm university of applied sciences, neu-ulm, germany 3 pathology, medical faculty, university of augsburg, augsburg, germany 4 institute of medical data science, university hospital würzburg, würzburg, germany neuropathologists are experts in their field, quickly detecting and classifying tumors in whole-slide images (wsis). however, it is often unclear what features they focus on and which aspects they rely on for their decision. machine learning (ml) is also increasingly used in pathology, but its decision-making process is similarly opaque. to better understand expert analysis and evaluate ml reasoning, an eye-tracking study will compare detection accuracy and region of interest (roi) selection among neuropathologists, pathologists in general, medical students, and ml models. the study involves experts (neuropathologists and pathologists with high experience in brain tumor diagnostics), intermediates (pathologists without any particular expertise in brain tumor diagnosis), and beginners (students, physicians from other domains) analyzing hematoxylin-and-eosin (he) stained sections of 50 glioblastoma (gbm) wsis. participants must determine whether neoplastic tissue is present in the section and, if so, mark the most relevant regions for their analysis. the eyelogic logicone eye-tracking device records their gaze during this process. post-study, gaze data from different participant groups will be compared with ml probability masks to identify differences and similarities between human and artificial analysts. the study will also correlate selected rois with ml probabilities and gaze duration to see if the most critical areas are those most viewed by humans and deemed most important by ml. an eye-tracking study is planned for october 2024, comparing the analysis of he-stained wsis of gbms between human analysts and ml models. the study is currently in the recruitment phase, with results expected by end of the year. p31 free neuropathol 5:19:46 why we don’t save whole-slide images as lego mosaics – putting the scale of whole-slide images into perspective daniel hieber1,2,3, nicola jungbäck1, felix holl2, bruno märkl4, rüdiger pryss3, johannes schobel2, friederike liesche starnecker1 1 department of neuropathology, pathology, medical faculty, university of augsburg, augsburg, germany 2 digihealth institute, neu-ulm university of applied sciences, neu-ulm, germany 3 institute of medical data science, university hospital würzburg, würzburg, germany 4 pathology, medical faculty, university of augsburg, augsburg, germany why do we save whole-slide images (wsis) as dicom-wsi, tiff, svs, ... and not as lego mosaics? while the answer to this question might be pretty obvious (working with digital data is much easier, and the staining is closer to the original), we want to use the latter to put the scale of wsis into perspective. wsis typically scale up to 80,000 x 60,000 pixels (larger sizes are possible). when transforming such images into lego, we need 4.8 billion 1 x 1 lego plates and 4,687,500 32 x 32 lego base plates. this would accumulate to a total cost of 330 million euros for a single wsi using original lego blocks. the final mosaic would result in a size of 625 m by 468.75 m, a total area of 292,968.75 m2 (41.03 soccer fields or approx. 2.09 times legoland germany). since lego currently offers only 40 different colors for its plates – of which only about 10 to 20 are relevant for pathology (depending on the staining method) – the conversion to a lego mosaic would result in a significant loss of information. while these numbers may seem absurdly large, processing a digital wsi with all its information at once using a machine learning model would result in 14.4 billion input parameters. while converting a wsi to lego does not make sense from a practical view point, it illustrates the enormous amount of data in such images. these numbers further demonstrate the immense value of digital pathology, allowing computers to process such data quantities for diagnostic uses. copyright: © 2024 the author(s). this is an open access article distributed under the terms of the creative commons attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the creative commons license is provided, and any changes are indicated. the creative commons public domain dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. postmortem changes in brain cell structure: a review feel free to add comments by clicking these icons on the sidebar free neuropathology 4:10 (2023) review postmortem changes in brain cell structure: a review margaret m. krassner1,2,3, justin kauffman1,2,3, allison sowa4, katarzyna cialowicz4, samantha walsh5, kurt farrell1,2,3, john f. crary1,2,3, andrew t. mckenzie1,2,3,6 department of neuroscience, icahn school of medicine at mount sinai, new york, new york, usa friedman brain institute, departments of pathology, neuroscience and artificial intelligence & human health, icahn school of medicine at mount sinai, new york, new york, usa neuropathology brain bank & research core and ronald m. loeb center for alzheimer's disease, icahn school of medicine at mount sinai, new york, new york, usa microscopy and advanced bioimaging core, icahn school of medicine at mount sinai, new york, new york, usa hunter college libraries, cuny hunter college, new york, ny department of psychiatry, icahn school of medicine at mount sinai, new york, new york, usa corresponding author: andrew t. mckenzie · icahn school of medicine at mount sinai · icahn building 9th floor, room 20 · 1425 madison avenue · new york, ny 10029 · usa andrew.mckenzie@icahn.mssm.edu additional resources and electronic supplementary material: supplementary material submitted: 14 april 2023 accepted: 15 may 2023 copyedited by: georg haase published: 31 may 2023 https://doi.org/10.17879/freeneuropathology-2023-4790 keywords: postmortem changes, oncotic necrosis, autolysis, staining methods, species differences, brain mapping abstract brain cell structure is a key determinant of neural function that is frequently altered in neurobiological disorders. following the global loss of blood flow to the brain that initiates the postmortem interval (pmi), cells rapidly become depleted of energy and begin to decompose. to ensure that our methods for studying the brain using autopsy tissue are robust and reproducible, there is a critical need to delineate the expected changes in brain cell morphometry during the pmi. we searched multiple databases to identify studies measuring the effects of pmi on the morphometry (i.e. external dimensions) of brain cells. we screened 2119 abstracts, 361 full texts, and included 172 studies. mechanistically, fluid shifts causing cell volume alterations and vacuolization are an early event in the pmi, while the loss of the ability to visualize cell membranes altogether is a later event. decomposition rates are highly heterogenous and depend on the methods for visualization, the structural feature of interest, and modifying variables such as the storage temperature or the species. geometrically, deformations of cell membranes are common early events that initiate within minutes. on the other hand, topological relationships between cellular features appear to remain intact for more extended periods. taken together, there is an uncertain period of time, usually ranging from several hours to several days, over which cell membrane structure is progressively lost. this review may be helpful for investigators studying human postmortem brain tissue, wherein the pmi is an unavoidable aspect of the research. table of contents introduction review methods review framework eligibility criteria qualitative data analysis characteristics of included studies mechanisms and associated microscopic outcomes cell death by oncotic necrosis intracellular structures temporary versus permanent ischemia compacted or “dark” neurons apoptosis postmortem fluid shifts perivascular rarefaction pericellular rarefaction intracellular vacuolization neuropil and white matter vacuolization biomolecule degradation alterations of biomolecule distribution summary rates of postmortem decomposition time series correlational studies case reports summary differences in decomposition rates across assessment methods immunohistochemistry morphological staining electron microscopy summary selective vulnerability to decomposition models of selective vulnerability brain region and cell type heterogeneity alterations in cellular and subcellular volumes dendrites axons synapses myelin summary variables modifying decomposition rates temperature acidity and agonal damage hydration oxygen content putrefaction in situ versus ex situ brain storage variation across species in metabolic rate premortem metabolic state age premortem pathology summary interactions between postmortem changes and preservation methods morphological staging of decomposition implications for human brain mapping further studies of postmortem changes comparison to other reviews strengths and limitations of this review conclusions abbreviations author contributions acknowledgements funding data availability supplementary files references introduction in studying the structure, function, and etiopathology of neurobiological disorders in the human brain, direct examination of postmortem autopsy human brain tissue has an unparalleled role (buja et al., 2019). compared to postmortem autopsy tissue, biopsy tissue is limited in size and obtainable only in a narrow set of conditions justifying neurosurgical intervention, neuroimaging studies have limited spatial and biomolecular resolution, and animal models have lower fidelity to human neurobiology. however, studying autopsy tissue presents significant confounds, crucial among them alterations occurring during the postmortem interval (pmi). the pmi is defined as the time elapsed between the subject's death and the autopsy and preservation of the brain tissue. in practical brain banking settings, the pmi generally lasts from a period of hours to several days until the autopsy is completed and the tissue is processed (beach et al., 2015; henstridge et al., 2015; samarasekera et al., 2013; vonsattel et al., 2008). left unprocessed, postmortem brain tissue will eventually disintegrate and liquefy, which has the obvious potential to significantly confound neuropathologic investigation (gonzalez-riano et al., 2017; hayman and oxenham, 2017). because of the value of studying donated autopsy human brain tissue, there is a critical need to understand and account for the changes that occur during the pmi. a diverse set of assessments can be performed on autopsy brain tissue, which we can loosely bracket into three categories: functional, biomolecular, and morphological properties. functional properties such as electrophysiological activity or cellular viability tend to be lost relatively rapidly during the pmi but can also be maintained for a surprising amount of time, on a timescale of minutes to hours (bailey, 2019; charpak and audinat, 1998; madea, 1994). biomolecular properties vary widely regarding their maintenance during the pmi, depending on the biomolecule's class (e.g. rna, protein, or lipid), individual type (e.g. particular rna transcripts), and property (e.g. enzyme activity, conformation state, or subcellular location). several studies and reviews have discussed the rate of postmortem decomposition of biomolecules (beach et al., 2015; kretzschmar, 2009; nagy et al., 2015; samarasekera et al., 2013; stan et al., 2006). in contrast, morphological properties have received less attention. morphological properties of the brain can be macroscopic (i.e., gross) or microscopic. macroscopically, brains with the least decomposition have clear internal anatomy, no softening, and are able to be extracted from the calvaria without fragmentation (hayman and oxenham, 2017). as the brain begins to decompose, which generally occurs over a time course of days, it liquefies and eventually reaches a pasteor fluid-like consistency (hayman and oxenham, 2017). microscopically, a great deal of morphologic information can be measured in the postmortem brain. here, we focus on the decomposition of cell membrane morphology, also known as cell “morphometry,” which refers to the external shape of a cell. specifically, we focus on general morphometric properties such as the presence of visible dendritic spines, rather than on detailed properties such as cell membrane width. this focus is because numerous neurobiological disorders have been associated with morphometric alterations, including those in dendrites, synapses, or myelin (kulkarni and firestein, 2012; stadelmann et al., 2019). for example, in alzheimer’s disease, evidence suggests that disruptions in the actin cytoskeleton of dendritic spines is a key mediator of disease pathogenesis (pelucchi et al., 2020). as another example, dendritic spine density has been found to be lower in the cortical tissue of brain donors with a diagnosis of schizophrenia (berdenis van berlekom et al., 2020). thus, understanding how cell morphometry degrades in the pmi is a crucial consideration. cell membranes are made up of a multiple classes of biomolecules; by mass, about equal amounts of proteins and lipids with much more diversity in the types of proteins (lodish and rothman, 1979). because cell morphometry is dependent on a multitude of biomolecules, morphometric alterations may proceed at either a slower or faster rate than the decay of any one type of the constituent biomolecules. to keep the review tractable, we do not focus on intracellular morphologic features, such as nuclear shape, the presence of rough endoplasmic reticulum, or other aspects of organelles, except insofar as they affect cellular morphometry. to integrate diverse empirical findings of postmortem changes into a unified understanding, it is essential to have a model of how cell membranes decompose after death. brain cell membrane shape is largely maintained through the cytoskeleton, a gel-like network of proteins that exhibits both passive elastic and active viscous behavior (ananthakrishnan et al., 2006; eberhardt et al., 2022; mogilner and manhart, 2018). in particular, cell membrane biomolecules are tethered to the underlying actin cortex through interacting proteins (chugh and paluch, 2018; svitkina, 2020). during the postmortem interval, the cytoskeleton and other gel-like networks in the cell break down. this is initially due to fragmentation and diffusion associated with autolysis, and later putrefaction if microorganisms are present (hau et al., 2014). after an extended postmortem period, the cytoskeleton and other gel-like structures will ultimately liquefy. it is essential to distinguish the chronological pmi from the amount of biological decomposition that occurs during that time. several modifying factors may affect the rate of postmortem decomposition, such as the storage temperature, which need to be accounted for as well. there are three complementary approaches to address the confound of the pmi in the study of neurobiological disorders. the first is to match cohorts by the duration of the pmi; for example, to ensure that the cases and controls each have the same average pmi (swaab and bao, 2021). the second is to adjust any quantitative traits under investigation by the measured pmi prior to or alongside statistical inference. the third is to restrict the pmi to a relatively short amount of time. for example, one recent study on the size of the synaptic surface in alzheimer's disease limited the sample to autopsy brains with pmis of less than 4.5 hours, yielding very high quality ultrastructure of the samples (montero-crespo et al., 2021). another study also reported that restricting the pmi to four hours or less provided higher quality ultrastructural data (roberts et al., 1996). all these approaches may benefit from an improved understanding of changes in the pmi, which otherwise has the potential to confound inference about group differences due to disease (schwab et al., 1994). an accurate estimate of how long it is expected for a particular morphologic feature to degrade in the postmortem period can help in designing a study with the highest possible statistical power. a better understanding of postmortem changes may also help in addressing the possibility that postmortem changes interact with disease states or agonal factors, i.e. the terminal state before death or the manner of death. indeed, agonal factors are often thought to contribute more to donated brain tissue quality than relatively short pmis (vonsattel et al., 2008; williams et al., 1978). while there have been many empirical studies measuring the degree of histologic degradation after different pmis, to the best of our knowledge there has been no recent, large-scale review that attempts to summarize these studies and to construct a model of how cell membranes decompose during the postmortem period. here, we perform a comprehensive literature search to build a database of studies addressing this topic. we enumerate the mechanisms by which brain cells have been proposed to decompose and we build a database of the timescales over which cell morphometry has been found to degrade in different contexts. we discuss how variation in decomposition outcomes can be explained by different visualization methods, the aspect of cell membrane morphology under study, and modifying variables affecting the state of the brain tissue. our overarching goal is to review progress towards building a coherent model of how brain cell morphometry decomposes during the pmi, which investigators who are banking or studying autopsy brain tissue can use to guide their approaches. review methods review framework we adopted a “realist synthesis” approach which incorporates aspects of a systematic review but focuses on theoretical understanding and pragmatism (wong et al., 2013). we chose this review style because of the wide-ranging and variably defined nature of the topic. we report on our adherence to the associated rameses criteria (supplementary file 1) (wong et al., 2013). prior to the formal search method development as described below, scoping of the literature was performed primarily via searches on pubmed and google scholar, alongside discussions among the authors. additional methods, including the search query, can be found in supplementary file 2. eligibility criteria any scholarly publication such as a journal article that describes the effect of the pmi on cell membrane morphology in the brain was included. the pmi was defined as the amount of time that elapses between when (a) death is declared, which generally means that blood flow to the brain ceases, and (b) the brain tissue is preserved or otherwise processed. a wide range of durations of pmi, from minutes to weeks or months, were considered. cell morphometry could be evaluated with any form of histology. to be included, studies had to contain a measurement of shape rather than solely a quantification of biomolecules. additionally, the study needed to measure cell membranes, not solely intracellular features such as nuclear or other organellar morphology. studies on humans or non-human animals of any age were included. to exclude the archaeological literature, pmi lengths of a year or more were not considered (morton-hayward et al., 2020). review articles, studies on the retina, and non-english studies were also excluded. qualitative data analysis we performed an assessment of the degree to which cell membrane structural features tend to degrade. for qualitative synthesis, decomposition timelines were considered both as a whole and, where possible, grouped by structural features (e.g. dendrites, somata, and axons), visualization methods (e.g. morphological staining, immunohistochemistry, or electron microscopy), or modifying variables (e.g. storage temperature during the pmi). we also reviewed the decomposition mechanisms posited by the different included studies. building upon these, we attempted to describe a model of how cells in the brain degrade after death and how this affects the ability to visualize cell morphometry in autopsy brain tissue. characteristics of included studies screening identified 172 studies that met our inclusion criteria, including 22 outside of the formal search (figure 1; supplementary file 3; supplementary file 4). these studies were classified as correlational studies (n = 90), time series studies (n = 84), and case reports (n = 6). of the 172 included studies, 133 (77%) used only light microscopy, 33 (19%) used only electron microscopy, and 6 (3%) used a combination of the two. there was substantial heterogeneity in the methods. among the 84 time series studies, there was a diversity of species studied, with 31 (37%) studying rat brains, 11 (13%) human brains, 9 (11%) mouse brains, and the rest studying brains from other or multiple species. all but one of the correlational and case report studies were on human brains. figure 1. study selection flow diagram. studies were screened and selected using the web-based software covidence (available at https://www.covidence.org/). an export of the covidence database for this review containing the individual study screening decisions is available (supplementary file 3). mechanisms and associated microscopic outcomes cell death by oncotic necrosis a cell death pathway describes a stereotyped sequence of events by which the functions and structure of a cell are lost. delineating cell death is complex, as there are usually not exact boundaries for when a cell has undergone an irreversible cessation of its functions and is therefore considered dead (galluzzi et al., 2018). it is a widely replicated finding that brain cells do not necessarily “die” immediately after somatic death. neurons can retain electrophysiological functions for hours after somatic death (abbas et al., 2022; charpak and audinat, 1998). according to a consensus definition, cell death is mediated by the loss of cell membrane integrity (galluzzi et al., 2018). however, cell morphometry can potentially be visualized – or partially visualized – for a window of time even after the loss of cell membrane integrity. therefore, even if a cell is dead, it may still be possible to extract useful data from visualizing it. the major cell death pathway associated with global cerebral ischemia, a sine qua non of the postmortem brain, is oncotic cell death, also known as oncosis (fricker et al., 2018; loh et al., 2019; majno and joris, 1995; weerasinghe and buja, 2012). oncosis was coined by von recklinghausen in 1910 to describe cell death with swelling, from the greek root onkos, which refers to “mass” or “bulk” (majno and joris, 1995). subsequently oncosis fell out of favor as a concept, but in recent years has become more commonly used as cell death pathways are precisely dissected (fricker et al., 2018; majno and joris, 1995). the driver of oncotic cell death, which can also be triggered by causes other than ischemia, is the loss of cellular atp (fricker et al., 2018). global cerebral ischemia causes the depletion of atp because oxygen is no longer delivered to cells through the blood, thus halting oxidative phosphorylation, after which energy stores such as glycogen are rapidly consumed (pélissier-alicot et al., 2003). global cerebral ischemia also stops the process of metabolic waste product removal that is normally ensured by the blood flow (jenkins et al., 1979). oncosis is a non-regulated form of cell death, thus distinguishing it from the many different types of regulated cell death (galluzzi et al., 2018; lossi, 2022). in causes of death that directly affect the brain, such as death due to a toxin or traumatic brain injury, the postmortem cell death pathway may be much different. there are three stages of oncotic cell death (d’arcy, 2019; majno and joris, 1995; pélissier-alicot et al., 2003; weerasinghe and buja, 2012). in the first stage, loss of atp causes inactivation of the sodium–potassium atpase, resulting in increased intracellular sodium and chloride concentrations, a net gain in solute, and usually an associated osmotic influx of water leading to cell swelling (kramer and myers, 2013). there is a concomitant increase in intracellular calcium, leading to activation of catabolic enzymes (majno and joris, 1995; trump et al., 1997). in the second stage, there is a non-selective increase in membrane permeabilization, leading to vacuolization and cell membrane blebbing. in the third stage, there is physical disruption of the cell membrane, leading to a loss of membrane integrity. at this point, the cell is generally considered dead, initiating the necrosis phase. during the process of necrosis, cellular contents fragment, condense, convert from a gel-like phase to a liquid phase, and ultimately progress towards equilibrium with the environment (majno and joris, 1995; weerasinghe and buja, 2012). alternatively, some investigators use the term necrosis to describe the morphological alterations at this stage of cell death (fink and cookson, 2005; fricker et al., 2018). we describe this entire sequence of decomposition as cell death by oncotic necrosis. this cell death pathway has also been described as coagulative or ischemic necrosis (levin et al., 1999). among the articles included in our review, several mention cell death pathways. shepherd and colleagues, in their study of postmortem rat brains, provide the only article that specifically describes oncotic cell death (shepherd et al., 2009). they found a substantial increase in calpain-specific spectrin hydrolysis products at 1 and 4 h postmortem, which they attributed to catabolic molecular changes consistent with oncosis (shepherd et al., 2009). another frequently used term to describe postmortem cell death is autolysis, which is used to describe the self-destruction of a cell due to its own enzymes (nakabayashi et al., 2021; tafrali, 2019). for example, wenzlow and colleagues describe the postmortem cell death pathway observed in horse brains as cell autolysis (wenzlow et al., 2021). they note that the observed morphological changes are similar to in vivo necrosis, with the exception of inflammatory cell infiltrates in the latter. most of the early decomposition in the postmortem brain is due to autolysis as opposed to putrefaction, which is decomposition driven by microbial agents (ith et al., 2011). however, we prefer the term oncotic necrosis to describe the postmortem cell death pathway, in part because the term autolysis assumes an aseptic mechanism that is usually not directly tested. moreover, autolytic cell death can also occur in vivo and is not always associated with atp depletion, making it a less specific term than oncotic necrosis (fricker et al., 2018). intracellular structures several studies we identified note that cell membranes are generally more resistant to postmortem decomposition than intracellular organelles (karlsson and schultz, 1966; van nimwegen and sheldon, 1966). for example, schulz 1980 reported that after 22 h of pmi, there were significant intracellular changes including cytoplasm lysis, but there were no associated significant changes in the size and form of cells at this time point (schulz et al., 1980). the loss of cytoplasmic components can lead to cells becoming hypereosinophilic on h&e, which are sometimes then referred to as “red neurons” (garman, 2011; finnie et al., 2022). most authors refer to red neurons as distinct from postmortem changes, because the preferential breakdown of cell content leading to hypereosinophilia may be an active, atp-requiring process. because intracellular content can theoretically be lost without significant alterations of cell shape, we do not focus on red neurons or this distinction here. however, red neurons can also be associated with cell shrinkage, which would be a change in cell morphometry (finnie et al., 2022). temporary versus permanent ischemia a common area of confusion, which raises concern about the value of studying brain tissue after extended pmis, is the finding that just a few minutes of temporary cerebral ischemia can cause severe structural and functional damage to the brain. however, this brain damage is a delayed phenomenon that occurs hours to days following reperfusion, due to the triggering of atp-dependent cell death pathways (lipton, 1999; lee et al., 2019). this process diverges from the cell death mechanisms in the postmortem brain, in part because it is an active process requiring atp. on the other hand, after permanent ischemia of one or more (but not all) cerebral blood vessels, the resulting histologic changes are similar to those observed in the postmortem period (tao-cheng et al., 2007). for example, solenski and colleagues found that in the ischemic core of the cortex exposed to permanent ischemia due to occlusion of the middle cerebral artery, neuronal swelling was present by 3 h, became more severe by 5 h, and by 24 h neurons had shrunken with edema of the neuropil, broadly consistent with the expectations of the pathway of cell death by oncotic necrosis (solenski et al., 2002). this study also corroborated that cell death was more advanced in tissue that had been reperfused (solenski et al., 2002). as another example of permanent focal ischemia, garcia and colleagues permanently occluded the right middle cerebral artery in rats and found that acute shrinkage and swelling were prominent within 6 h, followed by delayed necrotic changes occurring onwards from 6 to 12 h (garcia et al., 1995). using a similar methodology, this same group reported that leukocyte invasion into the ischemic parenchyma was present by 12 h and peaked at 24 h after occlusion (garcia et al., 1994b). leukocyte invasion is one mechanism through which the rate of tissue decomposition after occlusion of an isolated cerebral blood vessel can be faster than during the global cerebral ischemia that occurs postmortem (lipton, 1999). compacted or “dark” neurons another form of postmortem histological damage that has been investigated is the formation of compacted neurons. also known as “dark neurons” or basophilic neurons, these are a common artifact in preserved brain tissue described by several studies (bywater et al., 1962; cammermeyer, 1978). compacted neurons have a shrunken cell body, shrunken dendrites, intact membranes, and a hyper-basophilic staining pattern on h&e (cammermeyer, 1978; kovács et al., 2007). they occur following of a diverse set of stressors including mechanical trauma of unfixed tissue. the compaction phenomenon is considered haphazard, as it does not affect all neurons but tends to occur in clusters. biophysically, there is strong evidence that compaction involves rapid loss of water and gel-gel phase transition (kovács et al., 2007). following this striking change in cell morphology, compacted neurons are not expected to follow the typical changes in oncotic necrosis, although they still will eventually undergo necrosis (cammermeyer, 1979). the preponderance of studies report that compacted neurons do not become more common during the pmi, and in fact, that they may become less frequent (garcia et al., 1995; kherani and auer, 2008). this may be because intracellular gel-like networks are weakened during the pmi, making a gel-gel phase transition less likely. however, one study noted that solitary compacted neurons could be stimulated due to a perfusion fixation delay of five to ten min (cammermeyer, 1978). additionally, another study postulated that one of two types of compacted neurons they studied was associated with 3 h of pmi, because these cells also had other morphological signs of postmortem decomposition, such as vacuoles and nuclear damage (badonic et al., 1992). thus, shorter but not longer periods of postmortem decomposition may be associated with the formation of compacted neurons following fixation (garman, 2011). apoptosis apoptosis, a form of programmed cell death, is a tightly regulated and controlled process by which cells undergo self-destruction, often in response to cellular damage (galluzzi et al., 2018). this cell death pathway is characterized by distinct morphological findings, such as chromatin condensation, cell shrinkage, and the formation of apoptotic bodies. apoptosis is not prominent in postmortem tissue, because it is an active, atp-dependent process, and cellular atp stores are rapidly depleted postmortem. caspase inhibitors, which prevent apoptosis, have been found to protect against cell death in focal but not global cerebral ischemia (fricker et al., 2018). the preponderance of studies report that apoptosis markers are not associated with the pmi (brück et al., 1996; del bigio et al., 2000; geiger et al., 2006; hausmann et al., 2007; lucassen et al., 1995; müller et al., 2001). for example, one study found that apoptotic morphology was not present in any of the postmortem human brains studied (lucassen et al., 1997). these authors also noted that necrotic cell death takes longer to complete than apoptotic cell death, the latter of which would be expected to take only a few hours to be completed. as another example, one study found that that positive neuronal ssdna immunostaining, a marker of apoptosis, was not correlated with the pmi in a large (n = 335) study of postmortem brains with a pmi range of 2.8 to 48 h (michiue et al., 2009). however, apoptotic markers were associated with certain causes of death such as drowning and drug intoxication. this finding is consistent with the notion that markers of apoptotic cell death beginning prior to death can be identified in postmortem brains but these markers are not expected to progress postmortem. in contrast to most studies, a few studies have reported increases in certain markers of apoptosis in the pmi. for example, in postmortem rat brains, one study noted an increase in immunostaining for the apoptosis marker caspase-3 that peaked at 9 h of pmi (sheleg et al., 2008). at that time, 2.5% of cortical neurons were found to stain strongly positive for caspase-3. as another example, schallock and colleagues reported that one marker of apoptosis, i.e. clusters of cells labeled with the in situ end-labeling technique, appeared after 48 h of pmi in mouse brains stored at room temperature (schallock et al., 1997). labeled cells were generally found in isolation, not in clusters, and moreover the cells did not consistently show morphological changes of apoptosis. as a result, the authors reasoned that this marker was more likely due to dna fragmentation associated with postmortem degeneration. because of the potential for certain antigens to be unmasked during the pmi, some markers of apoptosis could also be seen due to decomposition during oncotic necrosis. postmortem fluid shifts at the time of death, the brain is largely composed of gel-like networks of different strengths, organized at a range of scales from the extracellular matrix to sub-cellular structures as well as localized compartments of liquids. after death, oncotic necrosis begins wherein catabolic enzymes break down tissue macromolecules. as gel-like networks break down, their components solubilize, increasing the fluid content of the brain. at the same time there is a disruption of the blood brain barrier and other tissue barriers allowing fluid that is typically cordoned off to diffuse into the parenchyma, further increasing the free fluid content of the brain. this fluid is made up of water, lipids, and other soluble molecules. the end result is that the brain is transformed to the liquid state (miller and zachary, 2017). consistent with these theoretical expectations, the fluid content of the brain has been found to increase after death. one study of rat brains found that the fluid content increased from an average of 77.8% at the time of death to 79.4% at 3 h pmi, 79.6% at 6 h pmi, and 80% at 24 h pmi (leonard et al., 2016). notably, in this study, a head-down position was not associated with increased brain fluid content, suggesting that postmortem fluid increases are primarily due to processes within the brain rather than migration from other areas of the body. another study reported an increase in the water content of the brain by around 10% in the pmi (ansari et al., 1976b). macroscopically, in neuroimaging studies, a decline in grey-white matter differentiation occurs at pmis longer than 24 h, which also occurs in cerebral edema and has been attributed to autolysis and fluid shifts (wagensveld et al., 2017; martin et al., 2022). large-scale fluid shifts can also manifest across the postmortem brain. for example, as a result of gravity, blood has been reported to migrate to dependent parts of the calvarium during the pmi in a phenomenon known as hypostasis (takahashi et al., 2010). microscopically, postmortem fluid shifts also lead to many of the defining cellular changes that occur in the pmi, including circumscribed rarefaction and vacuolization, discussed below. perivascular rarefaction several studies using light microscopy have reported that perivascular rarefactions appear and increase in frequency and size during the pmi (bywater et al., 1962; garcia et al., 1978; liu and windle, 1950; schwarzmaier et al., 2022). morphologically, these non-staining areas generally appear as ellipsoid shapes around blood vessels (figure 2a). they have been described as “a rose-branch beset with thorns” because intertwining glial fibers frequently remain between the non-staining areas (bruce and dawson, 1911). figure 2. representative micrographs demonstrating rarefaction and vacuolization in the postmortem brain. tissue from the frontal cortex tissue of an 87-year-old man with a pmi of 28 hours. a: h&e/lfb (luxol fast blue)-stained image demonstrating a perivascular rarefaction in the grey matter. arrowheads denote examples of intertwining fibers. scale bar = 10 μm. b: h&e/lfb-stained image showing variable degrees of asymmetric pericellular rarefactions (asterisks). scale bar = 20 μm. c: h&e/lfb-stained image of white matter vacuoles (asterisks) and pericellular rarefactions. white matter vacuoles are found haphazard in distribution, but appear more common near blood vessels, likely because of postmortem fluid extravasation. scale bar = 20 μm. d: electron photomicrograph in grey matter demonstrates prominent electron lucent areas that appear to be swollen cellular processes (asterisks). scale bar = 500 nm. the underlying nature of perivascular rarefactions seen on light microscopy has caused controversy for generations of neuroanatomists (bruce and dawson, 1911; maynard et al., 1957; weller et al., 2018). frequently described as a “space” or “cleft”, we do not favor these terms because they imply an underlying mechanism. similarly, the term “retraction artifact” implies a physical separation of the blood vessel from the parenchyma occurring during the tissue processing procedure. instead, we prefer the more agnostic term “rarefaction” to describe the non-staining areas. another potential point of confusion is that the perivascular rarefactions that develop in the pmi can be distinct from the dilated perivascular spaces seen on neuroimaging in vivo (kwee and kwee, 2007; weller et al., 2018). for example, in one correlative postmortem study, these were found to stain positive for collagen (haider et al., 2022). sometimes, these perivascular rarefactions are described as resulting from a fixation artifact, although this is considered less likely, as they also occur in postmortem brain tissue preserved via freezing in liquid nitrogen (de groot et al., 1995). instead, electron microscopy data from ischemic or postmortem states and detailed analysis of light microscopy data suggest that perivascular rarefactions are actually due to swollen astrocyte processes (maynard et al., 1957; arsénio-nunes et al., 1973; garcia et al., 1994a; garman, 2011; weller et al., 2018; dehghani et al., 2018). other than postulating that perivascular rarefactions on light microscopy are swollen astrocyte processes, or other swollen tissue elements, perhaps the only other explanation for the divergence between light and electron microscopy data would be differences in tissue processing between the two techniques, which is less consistent with the available data. these swollen areas of astrocytes have been described as vacuoles and contain predominantly fluid, so it makes sense that they are electron lucent and do not stain positive on light microscopy for astrocyte markers such as gfap (schultz et al., 1957; gibson and tomlinson, 1979; lafrenaye and simard, 2019). consistent with this, gfap staining decreases in brain tissue following a hypoxic/ischemic period (sullivan et al., 2010). protoplasmic astrocyte processes can have irregular geometries, allowing them to fit into narrow spaces (schultz et al., 1957). pericellular rarefaction as with perivascular rarefactions, pericellular rarefactions have also been found to develop in the pmi (figure 2b) (bywater et al., 1962; de groot et al., 1995; dehghani et al., 2018; liu and windle, 1950; shepherd et al., 2009). similarly to perivascular areas, electron microscopy data in grey matter shows that astrocyte processes, including end feet, swell in the perineuronal area in postmortem and ischemic conditions, thereby accounting for the change observed on light microscopy (figure 2d) (kuroiwa et al., 1998; suzuki, 1987). these non-staining areas are heterogenous, as they do not occur in all cells to the same degree and are more pronounced in some brain regions than others (snyder et al., 2021). in white matter, oligodendrocyte cell bodies have also been found to swell (kuroiwa et al., 1998). this accounts for the frequently described pericellular “halos” of oligodendrocytes, leading to an overall “fried egg” appearance on light microscopy (snyder et al., 2021). it is generally supported that the major mechanism of perivascular and pericellular swelling of tissue elements in the postmortem period is fluid shifts. for example, pericellular and perivascular swelling is a microscopic finding seen in brain edema (dreier et al., 2013). it is well-described that astrocyte processes become dramatically enlarged in ischemia, mediated via passive aquaporin-4 channels, which is thought to be a protective response to minimize brain damage (nahirney and tremblay, 2021). the observed astrocyte process swelling at 30 min of complete global cerebral ischemia has been found to be reversible with recirculation, consistent with the idea that this is a homeostatic process (arsénio-nunes et al., 1973). it is still undetermined why astrocyte processes are more liable to swell around cells and around blood vessels than in other areas of brain tissue. in breast cancer, where the presence of pericellular areas on histology correlates with an unfavorable prognosis, similar areas have been attributed to functional pre-lymphatic spaces (acs et al., 2012). the predilection for swelling in these areas in the postmortem brain may reflect aspects of the in vivo functioning of the glymphatic system, which operates by shuttling water via aquaporin-4 channels on astrocytes (silva et al., 2021). regardless, the extent to which fluid shifts into astrocytes or other cells will depend on the local anatomic and biochemical context. intracellular vacuolization the formation of vacuoles is a common finding in oncotic cell death and is also associated with fluid shifts in the postmortem brain (shubin et al., 2016; weerasinghe and buja, 2012). vacuoles are homogenous, spherical areas visualized under the microscope. vacuolization is a common endpoint of a heterogeneous set of physiologic or pathologic processes, such as water accumulation, lipid droplet formation, dilatation of organelles, fusion of small vesicles, invagination of the plasma membrane, degradation of cytoplasmic components, or artifact formation during slide preparation (henics and wheatley, 1999; ilse et al., 1979; wohlsein et al., 2013). as with perivascular and pericellular rarefactions, the precise underlying nature of vacuolization is often unknown. many studies describe vacuoles as a characteristic morphologic finding in postmortem brain cells (haines and jenkins, 1968; hilbig et al., 2004; koenig and koenig, 1952). for example, lindenberg 1956 found that in cat brains stored at 37° c, small vacuole-like transparencies were first seen at 30 min pmi, while at 6 h pmi larger vacuoles could be seen in some cells (lindenberg, 1956). in this study, the rate of development of the vacuoles was slower at lower temperatures: when the brains were stored at 18 °c, it took until 12 h pmi for vacuoles to appear. albrechtsen 1977 noted that small vacuoles were commonly seen in cerebellar granule cells, preceding necrotic changes as one of the first signs of decomposition, and that only minimal vacuole formation was seen in cerebella without necrosis of the granule layer (albrechtsen, 1977a). in an early paper about postmortem degeneration, koenig 1952 reported that cytoplasmic vacuoles appeared at 3 h of pmi, increase in size and frequency throughout the pmi, and stated that their nature was not known (koenig and koenig, 1952). subsequent electron microscopy studies have attributed the vacuoles in brain cells that accumulate postmortem or in ischemia to swollen mitochondria and dilatations of the endoplasmic reticulum or golgi apparatus (badonic et al., 1992; shibayama and kitoh, 1976; suzuki, 1987). it is not clear if all the vacuoles are due to the swelling of these organelles, or if some vacuoles could be due to other factors. also using electron microscopy, sele and colleagues found that the number of vacuoles increased during the pmi and speculated that they resulted from degraded cellular compartments (sele et al., 2019). intracellular vacuoles have the clear potential to affect cell morphometry by causing asymmetric cell membrane distortions. neuronal somata have also been found to have lateral expansions, or blisters, during the pmi (williams et al., 1978). the presence of organelle-free membrane blisters or blebs is also a commonly described finding in oncotic cell death that may be associated with vacuolization (weerasinghe and buja, 2012). gibson and colleagues report that there is an increase in electron translucent vacuoles in human cortical tissue during the pmi (gibson and tomlinson, 1979). they associate vacuolization with the swelling of cell processes, especially astrocytic processes, and found a highly significant correlation between the pmi and the degree of vacuolization up to 33 h, followed by no significant change up to 69 h pmi. while they did not detect any obvious structural degeneration of membranes during the pmi range studied, this vacuolization led to a severe compression of the tissue. for example, there was a significant decrease in the number of recognizable synapses during the pmi, even though there was no structural disintegration of synapses observed, indicating that the presence of large vacuoles could prevent the visualization of other cellular structures. neuropil and white matter vacuolization vacuolization can also occur during the pmi in parts of brain tissue that are not as clearly associated with a single cell, including the white matter (figure 2c) (hilbig et al., 2004). white matter vacuoles often look like randomly distributed holes and have been reported to occur in densely myelinated areas such as the corpus callosum or the internal capsule (snyder et al., 2021). in ischemia, electron microscopy shows that white matter vacuoles can result from swollen astrocyte processes, swollen axons, or the separation of the myelin sheath from the axon, with fluid filling the resulting potential space (pantoni et al., 1996). vacuoles can also be seen in the neuropil during the pmi. in their study of horse brains stored at 22 °c, wenzlow and colleagues found a non-linear trend for neuropil vacuolization: it increased up to 24 h pmi and then decreased until 72 h pmi (wenzlow et al., 2021). however, they did not find a significant change in cytoplasmic vacuolization over the pmi range studied. garcia 1978 reported that the sponginess seen in the neuropil on light microscopy in regional ischemia was due to extensive swelling of astrocytic processes and presynaptic terminals seen on electron microscopy (garcia et al., 1978). consistent with the idea that neuropil vacuoles result from fluid shifts, incubating brains in saline prior to fixation has also been found to lead to vacuolization of the neuropil (garman, 1990). biomolecule degradation the primary macromolecules making up brain cells are proteins and lipids while nucleic acids and carbohydrates account for only a small percentage (susaki et al., 2020). therefore, understanding how proteins and lipids disintegrate in the pmi is critical for charting how cell structure changes postmortem. in the postmortem brain, proteins are thought to be primarily broken down by enzyme-mediated proteolysis. this is consistent with findings that the uncatalyzed hydrolysis of peptide bonds at neutral ph is extremely slow, with reaction half-lives on the order of hundreds of years (mahesh et al., 2018; radzicka and wolfenden, 1996). calpains and cathepsins are two of the major enzyme families that contribute to autolytic proteolysis. calpain activation is not dependent upon atp, but thought to be triggered by the postmortem increase in intracellular calcium concentration (sorimachi et al., 1996; zissler et al., 2020). empirical results have indicated that calpain enzymes are indeed active in the postmortem brain (geddes et al., 1995; harada et al., 1997; sorimachi et al., 1996). cathepsin family enzymes, which are activated by acidic ph, also have activity in the postmortem brain (compaine et al., 1995). cathepsin family enzymes are usually cordoned in lysosomes, but lysosomal membrane integrity is lost during the pmi, which is thought to allow cathepsin-catalyzed proteolysis to catabolize cytoplasmic proteins (compaine et al., 1995). if lysosomes rupture or are permeabilized during the pmi, the release of catabolic enzymes would accelerate the breakdown of cell structure. indeed, one study defines lysosomal cell death, i.e. cell death resulting from lysosomal membrane permeabilization and the resulting activity of cathepsins and other proteases, as a synonym for autolysis (fricker et al., 2018). multiple studies have reported that lysosomal enzymes contribute substantially to decomposition in the postmortem brain (albrechtsen, 1977a; shibayama and kitoh, 1976). while some studies report that the number or size of lysosomes increase during the pmi, the evidence for this is mixed, the effect size is not strong, and lysosomal expansion is not required for lysosomal enzymes to contribute to decomposition (sheleg et al., 2008; tafrali, 2019; van nimwegen and sheldon, 1966). different protein substrates can have differential sensitivity to enzyme-mediated proteolysis. for example, geddes and colleagues report that map2, nf-m, and nf-l are relatively more sensitive to calpain-mediated proteolysis, while tau and nf-h are more resistant (geddes et al., 1995). as another example, sarnat and colleagues report that neun immunoreactivity tends to degrade within 6 or 12 h of pmi, while synaptophysin is much more resistant to postmortem autolysis and can be detected even in brains with pmis of more than 96 h (sarnat et al., 2010). different regions of the same protein can also have different rates of postmortem proteolysis. for example, li and colleagues studied the postmortem degradation of different epitopes of glutamate transporters in rat brains after 0-72 h pmi (li et al., 2012). they found that the termini of glt-1 degrade much faster than the central parts of the protein. they also found that the proportions of immunolabeling signal observed for different epitopes varies by the pmi. if the region of a protein recognized by an antibody is proteolyzed first, then the protein could appear to be totally absent when immunolabeled with that antibody, even though other parts of the protein could still be present. even if a biomolecule does not disintegrate into component parts during the early pmi, it could still undergo a change in conformation, which could also lead to a false negative result when attempting to measure the protein’s distribution with a particular label. postmortem proteolysis does not always decrease antigenicity, but instead is often found to increase the immunolabeling signal for certain proteins. for example, multiple studies reported that postmortem proteolysis leads to more accessible epitopes of the astrocyte marker gfap (de groot et al., 1995; hilbig et al., 2004). similarly, immunoreactivity for the vascular basement membrane marker laminin increased during the pmi, which has been attributed to unmasking by proteolytic processes (mori et al., 1992; szöllősi et al., 2018). hayes and colleagues found that during the pmi, staining for somatostatin 28 decreased, while staining for its breakdown products somatostatin 14 and somatostatin 281-12 increased, suggesting post-mortem proteolytic processing of somatostatin 28 (hayes et al., 1991). monroy-gomez and colleagues found that immunostaining for rabies antigens increased substantially in the postmortem period, which they attributed to autolytic disintegration of the intracytoplasmic viral inclusions and subsequent dispersion of the viral antigen (monroy-gómez et al., 2020). as with protein processing, lipid breakdown is also thought to be enzyme-mediated, via the activity of lipases such as phospholipase a2 (jernerén et al., 2015). lipids can be divided into metabolic and structural classes. metabolic lipids such as endocannabinoids are often rapidly degraded in the pmi (palkovits et al., 2008). however, the levels of structural lipids are liable to be stable for longer periods. for example, one study in rat brains found that the composition of glycerophospholipid, a major structural lipid component of cell membranes, did not change significantly up to a pmi of 18 h (pearce and komoroski, 2000). many studies have been performed on the biomolecular composition of the brain in the early pmi. while a full analysis is outside of the scope of this review, a general trend is that the levels of proteins in the brain, including synaptic proteins, tend to be surprisingly well maintained for many hours and often up to one or two days of pmi (fountoulakis et al., 2001; halim et al., 2003; knudsen and pallesen, 1986; siew et al., 2004; stan et al., 2006). it is worth noting, however, that the effect of the pmi on protein breakdown is highly variable depending upon the protein considered (ferrer et al., 2007). another source of variability in postmortem proteolysis is across cell types. li and colleagues reported that there was a significant heterogeneity of postmortem proteolysis rates across cells, resulting in patchy immunolabeling patterns (li et al., 2012). in the cerebellum, postmortem proteolysis has been suggested to occur earlier in the granule cells than in the purkinje cells (albrechtsen, 1977a). alterations of biomolecule distribution for this review, we are interested in the extent to which the postmortem spatial distribution of populations of biomolecules matches their in vivo distribution. we are not focused on the precise location of individual biomolecules, which are thought to be exchangeable in their functions and are often diffusing within a given cellular region in vivo regardless. we define two spatial scales when discussing altered location of biomolecules: the structural feature level, which considers alterations in the location of objects made of many biomolecules seen under the microscope, and the biomolecule level, which considers alteration of locations of individual types of biomolecules. cells themselves can be thought of as structural features and can also move in the pmi, although many non-blood cells are tethered to one another and to the extracellular matrix by connections that are relatively resistant to postmortem decomposition. although the structure of individual biomolecules is often stable in the early pmi, if the biomolecules that comprise cell membranes move sufficiently far from their original locations, then cell morphometry can still be lost. indeed, cell membrane morphology is often reported to break down prior to the breakdown of its biomolecular constituents. for example, hukkanen 1987 reported that myelin ultrastructure in surgical specimens had degenerated after 24 h of pmi at 25 °c, even though the levels of the major myelin glycoprotein were unaltered (hukkanen and röyttä, 1987). subcellular structural features such as organelles are frequently reported to move in the pmi. this can occur due to mechanical forces that arise by swelling, shrinkage, or other fluid shifts, leading to an increase in local pressure and thereby causing the intermolecular bonds stabilizing structural features to break. it can also occur due to catabolism of the biomolecules that typically maintain the structure in place. for example, several studies have found that myelin lamellae tend to split during the pmi, which is likely due to a combination of mechanical forces and the breakdown of the biomolecules typically connecting the lamellae (ansari et al., 1976a; hukkanen and röyttä, 1987; rees, 1976; shibayama and kitoh, 1976). as another example, one study found that cilia on ependymal cells had fused together and sunk down onto the ependymal surface after 1 h of pmi (hetzel, 1980). this may be due to a postmortem fluid shift within ventricular spaces. at the level of individual biomolecules, the situation can be more complex, requiring us first to understand how biomolecules are organized during life before we can understand how this organization is lost postmortem. there are three types of biomolecular organization patterns that we will consider, which are not mutually exclusive: confinement in compartments, inclusion in gel-like networks, and maintenance by active transport mechanisms. the most obvious form of biomolecular organization results from containment within cell compartments, such as within a cell or organelle membrane. in living cells, diffusion is often constrained to a local area, such as by an organelle membrane or cytoskeletal structure. the nature of the confinement depends on the size and properties of the molecule as well as the properties of the confining material. after death, membranes will eventually become permeable and therefore there will be a loss of confinement. the next form of biomolecular organization is the gel-like network. structural features seen under the microscope are often composed of densely aggregated, gel-like networks (douglas, 2018). for example, the nucleolus is made up in part by a concentrated gel of enmeshed rrna (lafontaine et al., 2021; riback et al., 2022). gel-like networks can be formed by covalent and non-covalent interactions that lead to the aggregation of biomolecules. during the pmi, intraand inter-molecular bonds will degrade due to hydrolysis and loss of active maintenance, causing these gel-like networks to break down. however, many of the constituent biomolecules may still be present in the local area, just no longer densely aggregated enough to be seen under the microscope. as a result, changes in the pmi can cause gel-like structures such as the nucleolus to no longer be visualized under the microscope, even though the local levels of their constituent biomolecules, such as rrna, may be relatively stable. notably, the ability to visualize gel-like networks under the microscope can also depend on the strength of crosslinking fixation, which stabilizes them (wang and minassian, 1987). another major form of in vivo biomolecular organization is active transport. in active transport, atp is expended to move biomolecules and ions against a concentration gradient. after death, atp will be depleted, so active transport will cease, causing a potential loss of the spatial distribution of biomolecules maintained in vivo. an example of this is the loss of sodium–potassium atpase activity very early in the pmi. we can next consider what happens to individual biomolecules once these organizing structures and functions are lost in the pmi. molecular diffusion is the primary driving force of individual biomolecule movement in the pmi, due to thermal fluctuations of molecules in the liquid, which causes constituent biomolecules to be displaced in random directions (schavemaker et al., 2018). each biomolecule has its own diffusion coefficient, resulting from its size, lipophilicity, the temperature, how it interacts with the solvent, and the extent to which it is bound to other molecules (schavemaker et al., 2018). if a population of biomolecules is localized to a particular location or “point” in vivo and the organizing factors maintaining the localization are lost postmortem, then its distribution is expected to increasingly spread out during the pmi as a result of diffusion (schavemaker et al., 2018; ślęzak and burov, 2021). in the absence of barriers, postmortem diffusion would be expected to cause the spread of biomolecules to follow a gaussian displacement distribution, with the degree of displacement depending on the pmi (manzo and garcia-parajo, 2015). however, in biological systems, barriers to diffusion, such as the actin cytoskeleton and extracellular matrix, are ubiquitous, meaning that non-gaussian spread of biomolecules is commonplace (manzo and garcia-parajo, 2015; ślęzak and burov, 2021). diffusion speeds are substantially decreased in the crowded cytoplasm compared to pure solution, often to an order of magnitude less (kekenes-huskey et al., 2016). as the cytoplasm becomes less crowded and structures break down during the pmi, effective diffusion coefficients will accelerate. because of the complex factors that affect the rate and spread of postmortem diffusion, the outcome of biomolecular diffusion after a given pmi is largely an empirical question about which it is difficult to make precise predictions a priori. many studies describe the phenomenon in which biomolecules redistribute from a localized to diffuse distribution in the pmi. sex steroid receptors in brain cells have been found to diffuse from a nuclear to a perinuclear distribution after 24 h of pmi (fodor et al., 2002). oehmichen reported that enzymes redistribute from localized to diffuse distributions at different postmortem time courses, beginning for several enzymes at 32 h pmi (oehmichen, 1980). hilbig and colleagues found that during the pmi, localized synaptophysin immunoreactivity was lost after 4 h when brain tissue was stored at 22 °c and after 12 h when it was stored at 4 °c (hilbig et al., 2004). it is also a well-known phenomenon that biomolecules eventually leak into the extracellular space as autolysis progresses (pélissier-alicot et al., 2003). as a result, cell membrane visualization will become less distinct when the tissue is labeled by a biomolecule that has undergone postmortem diffusion. another complicating factor for predicting the outcome of biomolecular diffusion is that population level movement patterns can be non-random due to sinks that capture biomolecules. as a possible example of this, mori 1991 studied changes in the distribution of immunostained immunoglobulin during the pmi (mori et al., 1991). they noted that immunoglobulin leaked out of blood vessels, evolving from a focal to diffuse pattern as the pmi increased, as expected based on random diffusion. additionally, they found that immunoglobulin underwent a selective neuronal uptake phenomenon where it was incorporated into shrunken, hyperchromatic neurons that had been damaged by trauma. because such compacted neurons result from a gel-gel phase transition, the uptake may occur as a result of the gel matrix in these neurons capturing diffusing immunoglobulins (kovács et al., 2007). while this is a plausible mechanism for the postmortem localization patterns of immunoglobulin, it requires further study. it is also critical to distinguish between the population level biomolecule distribution that is observed under the microscope and the changes in the tissue that led to that observation. as discussed in the previous section, postmortem metabolism can also cause dramatic changes to the composition and/or conformation of individual biomolecules. if postmortem metabolism is not uniform during the pmi, this can affect the observed localization of populations of biomolecules in the pmi. we can call this phenomenon differential metabolism. for example, there could be different concentrations of proteases, phospholipases, or endonucleases in different parts of the cell or across cells. alternatively, there could be a differential change in epitope accessibility between compartments during the pmi, for example due to a decrease in cytoplasmic crowding. as a term agnostic to mechanisms, we will use the term redistribution to describe an observed change in the spatial distribution of a type of biomolecule measured at a particular postmortem timepoint. several articles described redistribution of certain labeled biomolecules from neurites to cell bodies (i.e. the perikarya) (d’andrea et al., 2017; gärtner et al., 1998; geddes et al., 1995; irving et al., 1997; kitamura et al., 2005; schwab et al., 1994). among these, geddes and colleagues reported the redistribution of several different neurofilament proteins, including nf-h, nh-m, map2, and tau, from neurites to cell bodies in the pmi, which they call perikaryal accumulation (geddes et al., 1995). in their discussion, they posed the question of whether this redistribution was due to (a) a shift in the cellular localization of the proteins themselves or (b) a change in the antigenicity of neurofilament epitopes in different compartments, for example due to calpain activation, which we would consider a type of differential metabolism. gärtner and colleagues studied the spatial distribution of tau at baseline and after 30 min of pmi in rat brains using seven different tau-specific antibodies, which label for different epitopes of the protein (gärtner et al., 1998). they found that labeling with two of the antibodies, tau-1 and 12e8, led to a perikaryal accumulation of staining intensity following the pmi. on the other hand, labeling with three of the antibodies for tau led to no change in the distribution of tau immunostaining in the pmi. they reasoned that their results could be explained by differential dephosphorylation of tau in different compartments of neurons, as opposed to movement of the tau protein itself. irving and colleagues also reported that the postmortem redistribution of tau was dependent upon the antibody used (irving et al., 1997). taking these studies together, the observed perikaryal redistribution of many neurofilament proteins is more likely due to differential metabolism in different neuronal compartments, rather than a shift in the location of the individual protein molecules. a special type of postmortem differential metabolism is postmortem synthesis. during the pmi, new biomolecules can be synthesized, leading to population-level shifts in the distribution of the synthesized biomolecule. for example, certain rna transcripts have been suggested to be transcribed postmortem in the mouse brain (pozhitkov et al., 2017). while some of these findings could also be attributed to complex differential metabolism, the possibility of postmortem synthesis remains valid and has the potential to alter the spatial distribution of biomolecules (schwab et al., 1994). as an insight from an adjacent field, the postmortem redistribution of small molecule drugs across body compartments is a commonly studied phenomenon in forensic pathology (pélissier-alicot et al., 2003). mechanistically, drug redistribution can result from diffusion away from drug reservoirs, cell death, putrefaction, and postmortem metabolism (yarema and becker, 2005). more basic and more lipophilic drugs are more likely to move in the postmortem interval. one source notes that the brain is not clearly affected by the postmortem redistribution of drugs (pélissier-alicot et al., 2003). while many biomolecules are observed to have postmortem redistribution, these are not omnipresent findings. many of the included studies that measured the spatial distribution of biomolecules postmortem did not find a significant degree of postmortem redistribution (blair et al., 2016; quartu et al., 2005; serra et al., 2005). for example, blair and colleagues found that the expected neuronal localization of neun immunostaining localization in isolated human brain tissue did not change during the pmi time points analyzed, up to 53 h of pmi (blair et al., 2016). as another example, serra and colleagues found that immunostaining patterns for gfralpha-1, gfralpha-2, gfralpha-3 and ret receptor molecules were not substantially altered up to 72 h of pmi in rat brains (serra et al., 2005). summary there are numerous distinct mechanisms that lead to changes in observed cell morphometry during the pmi (table 1). these include the initiation of cell death by oncotic necrosis, fluid shifts causing swelling of tissue elements and vacuolization, biomolecular breakdown by catabolic enzymes, and biomolecular redistribution. by characterizing the mechanisms of postmortem decomposition, our goal is to allow predictions to be made about how cell membrane morphometry will degrade during the pmi. these mechanisms have the potential to differ substantially from biological processes occurring in vivo. they also have implications for which methodologies are likely to be most robust for use in postmortem brains. for example, individual biomolecules may have artifactual disintegration or redistribution during the pmi. as a result, using individual biomolecular labeling as a proxy for cell membrane morphology is not expected to be as robust of a method as staining nonspecifically for classes of biomolecules, unless the labeled biomolecule is highly stable in the pmi. mechanism or associated outcome role in postmortem brain decomposition cell death by oncotic necrosis the main cell death pathway in the postmortem brain, wherein the loss of atp leads to a sequence of morphological changes and eventual necrosis autolysis primary role in the early postmortem decomposition of brain cells apoptosis not a substantial contributor to postmortem decomposition, because this cell death pathway requires atp compacted neurons (a.k.a. dark neurons) may become more common after short periods of pmi, but less common with longer periods of pmi; is not thought to contribute to postmortem decomposition temporary ischemia temporary cerebral ischemia followed by reperfusion has much different mechanisms of decomposition (e.g. requiring atp) than the permanent global cerebral ischemia that occurs postmortem postmortem fluid shifts biomolecular breakdown and blood extravasation leads to dramatic fluid shifts in the early postmortem brain; eventually, the brain is transformed to a pasteor fluid-like consistency perivascular and pericellular rarefactions these areas expand and become more frequent in the pmi, which appears to be due to the swelling of astrocyte processes and other cells, causing those areas to not stain on light microscopy vacuolization common finding in postmortem brain cells and tissue spaces such as the neuropil that evolves throughout the pmi and can cause alteration of cell morphometry or compression of other structures biomolecule degradation largely driven by enzymes, biomolecule breakdown is a key driving force of postmortem decomposition, varying in rate based on many factors, such as the substrate and the temperature biomolecule redistribution the breakdown of the factors stabilizing biomolecules in place in vivo leads them to eventually diffuse away from their original locations, with degrees of both gaussian and non-gaussian spread table 1. summary of the role of postmortem brain cell decomposition mechanisms. rates of postmortem decomposition time series describing the mechanisms of postmortem decomposition does not tell us about the kinetics over which the decomposition occurs. there are three types of empirical studies that we will review next to address this question: time series studies, correlational studies, and case reports. in a time series study, the pmi is experimentally controlled, thus allowing an estimate of the effect of a given pmi on cell membrane morphology. because the time series study design is less susceptible to confounding biases, it allows for the most reliable estimates of the three study types. as an example of a time series study, we highlight the study by haines and jenkins (haines and jenkins, 1968). in this study, the authors measured the effect of pmi on cell structures in the habenulopeduncular tract and nucleus of adult dogs. after death, the brains were kept inside of the head (i.e., in situ) and stored at around 25°c. at multiple pmi time points – 0, 6, 12, 18, 24, 38, and 48 h – they extracted the brain, isolated the habenula, and fixed it via immersion in 10% buffered formalin. the tissue was morphologically stained with a method designed to distinguish myelin, axons, and general cell architecture. they reported that general cell architecture began to become indistinct by 18 h of pmi, while myelin and axons were relatively well preserved over the course of the study up until 48 h of pmi. this is an example of how one study can make multiple descriptions about the degree of decomposition of cell morphometry at different time points or for different structural features, which we call “observations”. to build a database and draw comparisons across studies, we extracted the observations from the text of each included time series study (supplementary file 5). for each observation, multiple raters independently graded the severity of decomposition on a subjective 0-3 scale. note that we are using the term “grade” in the sense of severity rating, as opposed to the use of the term in neoplastic grading. we next calculated inter-rater reliability scores based on these decomposition grades, using the intraclass correlation (icc) statistic. the icc value was calculated as 0.721, with a 95% confidence interval of 0.657 to 0.775 (f-test p-value = 7.9 * 10-43). this score is considered to be of moderate reliability, which likely reflects limitations in both the clarity of the categories we defined as well as the precision by which the observations were described in the included studies (koo and li, 2016). to elucidate the variability in outcomes between studies, we plotted these decomposition grades at different pmis (figure 3). we found a wide range of pmis after which histology reaches different decomposition severities across studies, experimental designs, and structural features of focus. because the studies were very heterogeneous, it was not possible to perform a detailed quantitative meta-analysis of decomposition kinetics across studies. however, as expected, we did identify a significant positive rank correlation between the pmi and the decomposition severity grade when pooling all observations (ρ = 0.29, p-value = 4.0 x 10-7). figure 3. temporal progression of structural decomposition in time series studies. correlational studies rather than making observations at defined time points, correlational studies make observations about how the appearance of a structural feature varies over a range of pmis. because these studies are correlative, before discussing them further, it is important to consider the limitations when using correlational studies to estimate the effect of pmi on cell membrane degradation (lewis, 2002; palmer et al., 1988). these are not limitations of the original studies, which often do not have this task as the primary goal, but rather limitations of using this data for our purposes in this review. the first limitation is the potential for confounding variables, such as agonal state and tissue ph (glausier et al., 2019). a second limitation is that many studies correlate multiple structural features with the pmi but do not perform an adjustment for multiple hypothesis testing, raising the probability of a spurious correlation. a third limitation are statistical floor or ceiling effects, based on the range of pmis. if the shortest pmi is several hours after death, or the longest pmi is relatively short, this restricts our ability to infer what occurs in brain tissue outside of the pmi range sampled. a fourth limitation in the many included studies that treat the pmi as a nuisance variable that must be considered is the possibility of publication bias. finally, a fifth limitation is selection bias if tissue requires a quality standard for inclusion in a study. this would clearly affect any naïve correlations measured with the pmi. as an example of quality selection bias, one study included 16 brains with pmi < 24 h (jacobs and scheibel, 1993). this study also included 4 brains with pmi of longer than 24 h because those brains had been refrigerated and the tissue appeared to be in good condition. while the study found no correlation between pmi and total dendritic length as measured by golgi-cox staining, this result is more difficult to interpret in terms of pmi effects given that brains with longer pmis were included because of relatively better markers of tissue quality. despite the potential limitations of correlational studies, they are still helpful, especially for exploring large effect sizes over the pmi range studied. correlational studies can aid in bounding the range of plausible postmortem effects over the time ranges studied. evaluating this literature is helpful for investigators studying tissue stored in brain banks, because it is most representative of the brain tissue that is available for study. we found as an overall trend from correlational studies that the pmi often does not have a substantial effect on cell morphometry (figure 4; supplementary file 6). some studies even find this with pmis of several days. for example, garey and colleagues used rapid golgi impregnation to study cell morphometry in 24 brains with a pmi range of 4 to 120 h, finding that there was no significant effect of this time range on the observed dendritic spine density of pyramidal neurons (garey et al., 1998). however, other studies have found that even relatively shorter pmis can have a substantial effect. for example, booze and colleagues found that there was a loss of staining for fine varicose axons over a pmi range from 1 to 6 h (booze et al., 1993). the effect of pmi likely depends on characteristics of the cohort studied, such as whether the bodies were refrigerated, as well as the visualization methods and structural feature investigated. figure 4. cell morphometry changes associated with the postmortem interval in the included correlational studies. in this plot, each correlational study is a data point. if the study describes an association between the postmortem interval (pmi) and a change in any type of cell morphometry, based either on statistical significance or the qualitative impression of the authors, then that data point is considered positive or “yes”, and vice versa for “no”. if it is unclear whether there was an association reported in the study, then the data for that study is not included in the plot. if there are multiple structural changes tested and at least one of them has an association with the pmi, then that is considered positive for the purposes of this visualization. both the sample size of the study (i.e., the number of brains considered, y-axis) and the highest recorded pmi (x-axis) are plotted on log scales. case reports case report studies, of which six were identified, describe the degree of preservation of cell morphometry in relation to the pmi of a single brain (supplementary file 7). for example, mackenzie reported a case of a body that was immersed in water in late autumn and winter for 10 weeks prior to autopsy (mackenzie, 2014). the histology quality of this brain was reportedly “good”, with morphological changes of anoxic neuronal injury and axonal spheroids still able to be identified. as another example, gelpi and colleagues described a case of where a body was stored in a mortuary cooling chamber for 2 months after death prior to autopsy (gelpi et al., 2007). histomorphology was described as “well preserved”, with all cell types, neuropil, and axons appropriately visualized, although they did note moderate vacuolization of brain parenchyma. on the other hand, suárez-pinilla and fernández-vega described a case with severe autolysis of granule and purkinje cells in the cerebellum after only 6 h of pmi, which was associated with a metabolic insult (suárez-pinilla and fernández-vega, 2015). notably, the rest of the brain was found to have essentially normal microscopic findings. case reports demonstrate exceptions from the typical way that practitioners in the field are perceived to think about the problem, thereby making these findings especially interesting for publication. in this sense, the case reports collectively show that it is considered surprising when cell membrane structure is maintained after a pmi of several weeks, or when it is lost in one brain region after a pmi of only 6 h. summary the studies we identified had a wide range of pmis, over which cell morphometry was altered to different degrees. time series studies offer experimental control but create artificial contexts divergent from naturalistic studies relevant in brain banking. correlational studies have several confounds limiting their interpretation, but they represent the practical contexts that investigators encounter. finally, the few case reports identified may illustrate the limits of our understanding and they may challenge paradigms. as an attempt to explain the high heterogeneity between studies, we next turn our attention to potentially modifying variables. differences in decomposition rates across assessment methods changes identified due to the pmi depend in complex ways on the methods used to visualize them. for example, there might be selective degradation or inaccessibility of the label measured in the visualization procedure, rather than the structure itself. intracellular regions could also become more difficult or easier to stain if the cell membranes are partially degraded, for example due to changes in membrane permeability. immunohistochemistry immunohistochemistry (ihc) is a method commonly used as a proxy of cell membrane morphology. ihc involves the use of specific antibodies that can bind to target antigens of biomolecules, which are then labeled with a detectable marker, allowing for the visualization of the antigen under the microscope. in ihc, an abnormal staining pattern could result if the labeled biomolecule (a) is totally lost from the tissue, (b) has been fragmented, (c) has undergone a conformation change, (d) has bound to another biomolecule that blocks the epitope, or (e) the antibody is not able to diffuse closely enough to the biomolecule due to accessibility issues. for example, gärtner and colleagues used different anti-tau antibodies to study how tau immunostaining is affected by 30 min of ex situ postmortem storage at 37° c (gärtner et al., 1998). they found that three antibodies were not affected by the pmi, two others revealed alterations in the labeling patterns identified, and two others showed complete loss of immunoreactivity. this is an example of how different epitopes of the same protein can be variably affected by the pmi. while caution is warranted in the interpretation of ihc signals for antigens that are unstable in the pmi used as a proxy for the structure of cell membranes more broadly, ihc staining for certain biomolecules that do not undergo changes postmortem can be useful. for example, one study found that immunostaining for calbindin-d28k was more sensitive in postmortem brain tissue than nissl staining for detecting purkinje cells, although neither of these methods were found to vary with the pmi range studied of 3 to 48 h (whitney et al., 2008). another study found that neuronal morphology was lost with toluidine blue staining at 30 h pmi, but that immunostaining for rabies antigen was still able to delimit the profile of the soma of infected cells at this time point (monroy-gómez et al., 2020). morphological staining the most common morphological stain is h&e. hematoxylin stains basophilic regions of tissue such as the nucleus and rough endoplasmic reticulum, while eosin stains acidophilic regions such as the cell membrane, extracellular matrix, proteins, and most organelles (chan, 2014). nissl staining is also common, wherein basic dyes such as cresyl violet or thionine bind to nucleic acids in the cell including rna in the rough endoplasmic reticulum or in free ribosomes (kádár et al., 2009). because certain neurons have high concentrations of rough endoplasmic reticulum, nissl staining can highlight neuronal cytoplasm, but morphology is poorly visualized, although basic structural aspects of somata may be discerned. while h&e and nissl stains are not effective at visualizing neuronal processes, other specialized chromogenic dyes can be helpful. for example, das and colleagues used staining with the lipophilic dye dii and found that dendritic spines can be visualized for up to 28 h of pmi in cases of sudden death (das et al., 2019). they conclude that this is a longer period of dendritic spine stability during the pmi than previously thought, and that they were able to detect dendritic spines at extended time points because of their novel method. the golgi impregnation is a different technique that completely stains a subset of cells including their entire dendritic tree (kang et al., 2017). there are differences in the sensitivity of different golgi stains to the pmi. specifically, rapid golgi stains are more sensitive to changes in the pmi than golgi-cox. one study found that a pmi of more than 4 h leads to a reduction in visualized dendrite length in rapid golgi stained tissue (de ruiter and uylings, 1987). another study suggested that golgi-cox impregnation had robust visualization of dendritic spines with longer pmis (up to 28 h tested), while rapid golgi staining only had similarly good visualization for one case with a shorter pmi of 6 h (buell, 1982). in contrast to golgi methods, the bielschowsky method uses silver to stain essentially all types of cells and neural processes in the brain (switzer, 2000). it is most often used to visualize axons and has been reported to be robust to changes in the pmi. for example, one study found that there was no change with bielschowsky staining after 24 h of pmi (as cited in (haines and jenkins, 1968)). in a case report of a brain with a pmi of 2 months, another study reported that bielschowsky silver impregnation was more robust for the visualization of axons than staining with anti-neurofilament antibodies (gelpi et al., 2007). however, as the likelihood of putrefaction increases with extended pmis, bielschowsky stained tissue needs to be examined with more caution, because colonies of microorganisms can also stain positive and mimic neuritic plaques (mackenzie, 2014). taken together, as opposed to ihc for potentially unreliable antigens, morphological stains are reported to be more robust to changes in the pmi, because they label classes of biomolecules rather than specific ones. electron microscopy the electron microscopy (em) studies included in this review generally stain tissue with osmium tetroxide, which primarily labels unsaturated lipids in membranes, and uranyl acetate, which has binding affinity towards proteins and provides contrast (hua et al., 2015; moscardini et al., 2020). however, staining protocols for em are fastidious and require precise optimization (tapia et al., 2012). because the properties of brain tissue might be substantially altered during the pmi, additional optimization of the staining protocol for postmortem conditions might be required, which is not necessarily always performed. as a result, as with all stains, it is possible that with increasing pmi, what is lost when visualizing tissue with em is not the structural components of cell membranes themselves, but rather the ability to identify them with the particular methods employed. for example, as previously discussed, gibson and tomlinson claim that the decrease in visualized synapses on em over the first day or two of the pmi is largely due to cell process swelling which compresses synapses and makes them more difficult to visualize in 2d sections (gibson and tomlinson, 1979). if this is the case, then the compressed synaptic membranes could still potentially be visualized by an alternative approach such as a high-resolution volumetric em method or an immuno-em method that labeled for a particular synaptic protein that is relatively resistant to postmortem decomposition. consistent with the sensitivity of em to changes in the pmi, sarnat and colleagues claim that em is more sensitive to pmi artifacts than their light microscopy method of visualizing synapses by immunostaining with synaptophysin (sarnat et al., 2010). other authors have also claimed that light microscopy is more robust than em to changes in the pmi in the visualization of synapses (peroski et al., 2016). summary taken together, various methods of visualizing brain cell membranes differ in their robustness to changes in the pmi (table 2). as a result, investigators need to be aware of the potential for bias when using methods that are not as robust to postmortem changes. method reported robustness to postmortem decomposition immunohistochemistry often sensitive to postmortem changes, but highly dependent on the antigen labeled h&e and nissl relatively robust to postmortem changes, but unable to visualize cell processes specialized chromogenic dye some methods are relatively more robust to postmortem decomposition(e.g., dii staining) golgi golgi-cox is more reliable than rapid golgi bielschowsky reported to be generally robust to changes in the pmi, but can show false positive staining for microorganisms electron microscopy may be more sensitive than light microscopy to postmortem changes. structures may be not visualized on two-dimensional images due to compression table 2. summary of the relative robustness of different visualization methods to postmortem decomposition in the brain. selective vulnerability to decomposition during the pmi, some aspects of cell membranes decompose faster than others. these structural features include various aspects of cell membranes, such as dendrites or synapses. each instance of a feature is generally composed of millions or billions of individual biomolecules, which make up the underlying biochemical material. our attempt to summarize these collections of biomolecules by describing them with a single name is meant to be a useful abstraction, although there is a significant heterogeneity of biomolecular content within features of the same type. models of selective vulnerability as an initial way to think about how quickly different features might degrade, we can consider a naïve model in which: (a) the density or composition of biomolecules does not vary significantly between features and (b) the decomposition rate of each biomolecule is independent of its local context. under this model, features will degrade at a rate proportional to their size. of course, both assumptions of this model are false. different features are clearly composed of different types of biomolecules; for example, the cytoskeleton of axons tends to be made of more longitudinal neurofilaments, whereas the cytoskeleton of dendrites tends to be made of microtubules, which may degrade at different rates postmortem (schwab et al., 1994). similarly, the decomposition rate of each biomolecule will clearly depend on its local context, such as the density of catabolic enzymes. a size-proportional decomposition model, under which structural features decompose at a rate proportional to their size, may still be a helpful first approximation. smaller features will have fewer biomolecules, a lower probability of a label binding to that biomolecule, and therefore a lower probability of being visualized as its constituent biomolecules begin to degrade and it eventually disintegrates during the pmi. brain region and cell type heterogeneity before discussing sub-cellular features, it is important to consider how rates of decomposition vary across brain regions and cell types. certain brain regions, such as the hippocampus, are well-known to be more susceptible to degeneration due to temporary cerebral ischemia followed by reperfusion injury. however, damage following temporary ischemia is an active process and therefore caused by a different decomposition mechanism than what occurs postmortem. multiple studies reported that postmortem decomposition generally progresses at equal rates in the different brain regions studied, instead of progressing faster in brain regions more susceptible to temporary ischemia (garcia et al., 1978; spector, 1963). the main brain region that has been associated with a faster rate of postmortem decomposition is the cerebellum (averback, 1980; furukawa et al., 2015; finnie et al., 2016). bywater and colleagues reported that the cerebellum is the most sensitive region to postmortem autolysis, with significant changes occurring within 20 min, which they attribute to the high enzyme content of the granular layer (bywater et al., 1962). ikuta and colleagues also reported that there is a selective disintegration of the granular layer of the cerebellum in the pmi, without a change in glia or adjacent purkinje cells, due to similar enzyme-mediated autolysis in granular cells (ikuta et al., 1963). albrechtsen noted that there is a significant correlation between the ph of the brain tissue and the degree of decomposition of the granule cell layer of the cerebellum, consistent with a role of enzyme-mediated autolysis (albrechtsen, 1977a). aside from the cerebellum, some sources described the relative vulnerability of other brain regions, although not as consistently. multiple sources reported that white matter tends to be better preserved than other regions of the brain (furukawa et al., 2015; mackenzie, 2014). one source noted that the cortex tends to be better preserved than the basal ganglia and thalamus (mackenzie, 2014). oehmichen noted that the pons and certain thalamic nuclei are the first to show damage in the pmi (oehmichen, 1980). yeung and colleagues, studying an extended pmi of 30 d, found that outer layers of the cortex (i-iii) were less well preserved than the inner layers of the cortex (iv-vi) (yeung et al., 2010). notably, there can also be a patchy heterogeneity of decomposition within a local area of the brain (lesnikova et al., 2018). some regional differences may also be confounded by immersion-based preservation methods, which is discussed below. in terms of cell types, one source noted that ventral thalamic nucleus neurons and small cells of the striatum are the most vulnerable to postmortem decomposition, while large pyramidal cells in the parietal cortex are the least vulnerable (as cited in (choe et al., 1995)). several other sources also report that larger neurons are better preserved and/or that smaller neurons have more rapid postmortem changes (irving et al., 1997; lindenberg, 1956; williams et al., 1978). it has been reported that there is a large heterogeneity in the decomposition rate within a cell class (as cited in (choe et al., 1995)). in the anterior pituitary, significant variability of decomposition rate has also been reported within cell type classes, attributed to the functional state of the cell at the time of death (ilse et al., 1979). overall, decompensation kinetics are highly variable even within a cell type class, but certain types of smaller cells may be relatively more vulnerable. alterations in cellular and subcellular volumes in cell death by oncotic necrosis, cells are expected to initially swell in the first stage, and eventually shrink as necrosis progresses. however, the presence or absence of cell swelling depends on the local environment, including any fluid take-up by other nearby cells. indeed, cells may even shrink initially if nearby cells take up relatively more fluid. moreover, swelling may also be transient and not captured by the histological techniques at any given timepoint (majno and joris, 1995). therefore, the potential effects of pmi on cell volume are challenging to predict. cell swelling is one of the most common morphometric findings early in the pmi. schulz and colleagues reported cell swelling after 30 seconds of pmi that disappeared with continued postmortem time (schulz et al., 1980). they explain the mechanism as due to an increase in intracellular cations, leading to a rise in intracellular oncotic pressure. shibayama and kitoh noted swelling of pyramidal cells at 1 h pmi, which they reported was more pronounced near the white matter (shibayama and kitoh, 1976). in their study of isolated biopsy brain tissue, dachet and colleagues found that neuronal swelling first appeared by 2 h of pmi and that by 4-8 h, a majority of neurons were swollen (dachet et al., 2021). cell volume changes can have complex evolution patterns over the pmi. hayes and colleagues found that average neuron size in the white matter of monkey brains increased only slightly following a 2 h pmi, then increased substantially at a 12 h pmi, and then there was no additional increase at 24 or 48 h (hayes et al., 1991). they note that the inclusion of brains with relatively longer pmis in human cohorts may be helpful to factor out the effect of pmi on neuron size. in their study of horse brains, wenzlow and colleagues noted a linear decrease in the size of the cytoplasm up to 3 d of pmi when brains were stored at stored at 8 °c, although no volume changes were detected when brains were stored at 22 °c (wenzlow et al., 2021). tafrali found that neurons shrank at 12 h pmi, while astrocytes and to a lesser extent oligodendrocytes increased in size (tafrali, 2019). as opposed to swelling of many cell types, capillaries have been found to have decreased volume at 22 h pmi (hunziker and schweizer, 1977). consistent with the previously described mechanism of fluid shifts, many studies describe swelling of astrocytes and astrocyte processes (dachet et al., 2021; gibson and tomlinson, 1979; shibayama and kitoh, 1976; tafrali, 2019; williams et al., 1978). jenkins 1979 noted that astrocytic cell bodies and processes were more dramatically swollen than neurons or oligodendrocytes, starting at 5 min of pmi and continuing until 25 min of pmi (jenkins et al., 1979). arsénio-nunes and colleagues described pronounced astrocyte process swelling in the molecular layer and at the level of vascular end feet at 30 min pmi (arsénio-nunes et al., 1973). finnie and colleagues reported that bergmann glia had cytoplasmic swelling much earlier than purkinje cells in the pmi (finnie et al., 2016). del bigio and colleagues described pronounced swelling of astrocytes and oligodendrocytes during the pmi that could be spatially associated with a hemorrhagic lesion prior to death (del bigio et al., 2000). they attributed the postmortem etiology of this swelling as due to the uptake of leaked plasma proteins from the extracellular space by these cell types during the pmi. rees found that astrocyte processes were “markedly swollen” at 2 h of pmi and that this was especially evident in astrocytes around blood vessels (rees, 1976). in summary, postmortem cell volume changes very frequently occur in the pmi. swelling can occur very early in the pmi and may either persist, revert, or convert to shrinkage. generally, the volume of a given cell or cell sub-region assessed after a given period of pmi cannot be assumed to be the same as it was in vivo. cell volume changes are also heterogenous across cell type, between brain regions, and dependent on local events such as hemorrhage. dendrites dendrites play a critical role in information processing in the brain and have a wide variety of shapes and configurations. they can be very narrow, with diameters of less than 500 nm in certain areas (harris and spacek, 2016). dendritic spines have especially narrow necks connecting them to dendritic shafts, with diameters of 50-400 nm (adrian et al., 2014). based on the size-proportional decomposition model, dendrites are expected to be among the most vulnerable features to postmortem decomposition. several studies using immunohistochemical approaches have noted that immunoreactivity is lost from dendrites relatively early in the pmi. boekhoorn and colleagues found that doublecortin immunoreactivity was greatly diminished in dendrites after a pmi of only 1 h in rodent brains and was effectively lost by 8 h pmi, while soma immunoreactivity was retained for a longer time (boekhoorn et al., 2006). using calretinin staining in the monkey hippocampus, lavenex and colleagues found that dendrite staining became coarser over a pmi of 2 to 48 h (lavenex et al., 2009). specifically, while dendrites could still be visualized in the later pmi brains, they appeared as a string of discontinuous agglomerates as opposed to a series of ovals connected by a continuous fiber. multiple studies used the antibody smi-32, which binds to neurofilament h and can be used to visualize neuronal processes including dendrites. gonzalez-riano and colleagues did not find any changes in the distribution of staining for smi-32 in mouse brains at a pmi of 5 h (gonzalez-riano et al., 2017). hilbig 2004 noted that details of the dendritic tree in large pyramidal cells were visible up to a pmi of 12 h with immunostaining for smi-32. with increasing pmi after 12 h, a granular-like immunostaining of disintegrated dendritic neurofilaments was found instead. there is a substantial literature on postmortem map2 immunostaining, which has also been generally found to degrade relatively rapidly during the pmi, thus reducing visualization of dendrites in map2-stained sections (irving et al., 1997; kitamura et al., 2005; lingwood et al., 2008; schwab et al., 1994). for example, nakabayashi 2021 performed immunostaining for map2 in rat brains, reporting a fragmentation of secondary dendrites at 6 h pmi and a disappearance of primary dendrites at 1 d pmi (nakabayashi et al., 2021). map2 postmortem redistribution has been speculated to be due to the sensitivity of the map2 protein to postmortem proteolysis, although the precise mechanism is unclear (d’andrea et al., 2017). some studies using morphological staining have also found that dendrite structure is altered relatively early in the pmi. for example, in a study using the golgi rapid method to visualize pyramidal neurons in mouse brains, williams and colleagues noted that dendrites degenerated in a “moniliform” fashion, i.e. with alternating varicosities and constrictions, which was first noticed at 6 h pmi (williams et al., 1978). at 6 h of pmi, there were also patchy areas of dendrites that had a decreased density of spines. they also reported a centripetal loss of dendrite visualization with increasing pmi, wherein distal segments were lost first followed by more proximal areas. using an alternative visualization method of differential interference contrast optics, they found that this loss was at least partially attributable to a failure of silver impregnation of distal dendritic segments with increasing pmi. as another example of early dendritic changes in the pmi, bywater and colleagues used a silver impregnation method and reported that dendrites began to “curl up in a corkscrew manner” by 4.5 h of pmi in monkey brains (bywater et al., 1962). in their em study, rees 1976 found that dendrites were swollen after 30 min of pmi (rees, 1976). roberts and colleagues, also using em, noted that dendrites, along with axon terminals, spines, and mitochondria, became “bloated and/or irregular in contour” at pmis of greater than several hours, and therefore they restricted their pmi range to less than 4 h (roberts et al., 1996). other studies using morphological staining methodologies found that dendrite morphology is stable for longer pmis. for example, as previously discussed in the visualization section, das and colleagues found that dendritic spines can be visualized via staining with the dye dil for up to a pmi of 28 h (das et al., 2019). within their sample, they found that the pmi was not associated with spine density, spine head diameter, or spine length. jacobs and scheibel reported that dendrite morphology was well-preserved in their cohort of human brains, with pmis up to 32 h (jacobs and scheibel, 1993). they used the golgi-cox staining method because it had been reported to be less sensitive to postmortem autolysis. they did not find the markers of autolysis (e.g., irregular varicose enlargements and loss of spines) that had been previously reported with the rapid golgi method by williams and colleagues (williams et al., 1978). yeung 2010 found that dendritic branching patterns could be visualized with bielschowsky silver staining in a subset of pyramidal cells from human brains after even 30 d of pmi with storage at 4 °c (yeung et al., 2010). much of the correlational literature in human brain cohorts suggests a relative stability of dendritic structures in the pmi, including studies with a pmi range up to 9 h using em (kolomeets et al., 2005), up to 28 h or 78 h using golgi-cox staining (boros et al., 2017; buell, 1982), up to 74 h using immunostaining for glutamate receptor subunits (benes et al., 2001), or up to 120 h using the rapid golgi method (garey et al., 1998). an exception is the study of tóth and colleagues that used ihc and found that dendrites on calretinin-immunoreactive cells appeared to be shorter, varicose, and degenerating with increasing pmi over a range of 2 to 10 h (tóth et al., 2010). it is challenging to reconcile differences in the decomposition rate of dendrites observed across these studies. however, problems with dendrite visualization in studies finding that dendrites degrade relatively faster could help to explain this divergence, whereas it is more difficult to imagine how the literature suggesting dendrites are stable for longer periods of time could be systematically flawed. in summary, dendritic swelling can occur early in the pmi (rees, 1976; roberts et al., 1996). additionally, there is evidence for clumping or beading of dendritic components (lavenex et al., 2009; williams et al., 1978). however, total loss of dendritic cell membranes is not a commonly reported early phenomenon in the pmi. as a result, dendrite tracing in volumetric microscopy data may be possible even in brains with relatively longer pmis if robust visualization methods are used. certain morphological staining methods, such as the golgi-cox impregnation method, are more resistant to pmi artifacts than others. experiments labeling for particular biomolecules, such as map2 or smi32, should not be considered dispositive of the state of dendrite morphology more generally. axons like dendrites, axons are narrow, and have been found to have similar sized or lower average diameters compared to dendrites (faitg et al., 2021). this makes axons similarly vulnerable to postmortem changes under a size-dependent model. in studies using morphological stains, axons tend to be relatively well preserved, albeit with volume changes at early pmis. using rapid golgi staining, williams and colleagues found that axons tend to exhibit bead-like changes and become more lightly impregnated after a pmi of more than 6 h, which was a slower change than they reported for dendrites (williams et al., 1978). in the em studies by rees and roberts and colleagues, it was reported that axons were swollen at the same early time periods as dendrites (rees, 1976; roberts et al., 1996). in their study using light microscopy to visualize the habenular nuclei of dog brains, haines and jenkins found that a few large axons started to show areas of slight dilation and constriction at 12 h pmi (haines and jenkins, 1968). at 36 h pmi, these areas of constriction and dilation were found in most axons, and almost every axon started to have areas with short loops. generally, they found that axons retained their integrity up to 48 h pmi and were relatively stable postmortem, which corroborated the previous reports they cited. segmental axon swelling has also been reported as a consequence of focal ischemia in ultrastructural studies (pantoni et al., 1996). notably, this postmortem artifact is distinct from axonal spheroids, which are marked by accumulation of amyloid precursor protein (app) and can occur in the context of blockage of fast axonal transport (coleman, 2005). in immunostaining studies, axon definition can be lost after an extended pmi (sarnat et al., 2010). for example, blair and colleagues found that immunostaining for α-tubulin was lost at 48 h pmi in 3/6 of the human brains profiled (blair et al., 2016). on the other hand, in this study, neurofilament proteins were still detectable at these extended pmis and axons were still able to be thereby visualized. eggan and lewis studied the immunoreactivity of cannabinoid receptor 1 in axons of monkey brain tissue sections with light microscopy (eggan and lewis, 2007). they found that after a 24 h pmi, axons became less distinct, and axon terminals became swollen. booze and colleagues found that immunostaining for fine varicose axons with tyrosine hydrolase was lost between 1 and 6 h of pmi (booze et al., 1993). taken together, axons have similar postmortem decomposition patterns as dendrites, with early swelling and patches of alternating constrictions and varicosities that become more common in the pmi. some evidence suggests that axons may degrade slightly slower than dendrites postmortem, although this is highly uncertain and may relate to visualization methodology. synapses time series studies using em and short pmis have found that aspects of synaptic morphometry can be altered very rapidly in the pmi, within minutes. routtenberg and tarrant found that after 1 min of pmi, there was a substantially diminished concavity of the synapse and a thickening of the postsynaptic membrane (routtenberg and tarrant, 1974). tao-cheng and colleagues also found that after 6.5 min of pmi, there was a change in curvature and an increased thickness of the postsynaptic density (tao-cheng et al., 2007). karlsson and schultz found that after 60 min of pmi there were fewer and flattened synaptic vesicles, as well as occasional plasma membrane separation and an increase in extracellular space (karlsson and schultz, 1966). roberts and colleagues noted that as the pmi extends beyond several hours in human brains, the postsynaptic density becomes thicker, thus obscuring whether the synapse is asymmetric or symmetric (roberts et al., 1996). huttenlocher noted that in human postmortem brains, presynaptic and postsynaptic markers are less sharply demarcated, and intracleft areas cannot always be seen (huttenlocher, 1979). while morphometric properties of synapses are rapidly altered, they are usually found to be demonstrable with em following a more extended pmi. rees found that synapses had not disintegrated by 4 h of pmi in cats or 5.5 h of pmi in monkeys (rees, 1976). vrselja and colleagues found that post-synaptic densities were preserved at 10 h of pmi, although the presynaptic vesicle pool was not maintained (vrselja et al., 2019). tang and colleagues found that there was no significant decrease in visualized synaptic density at 2 d of pmi in mammalian brains (tang et al., 2001). shibayama and kitoh found that synaptic contacts were preserved for pmi of up to 10 h although vesicles decreased in number (shibayama and kitoh, 1976). in isolated rat brains stored at 37 °c, de wolf and colleagues found that synapses were able to be visualized at a pmi of 13.5 h, but the ability to visualize them was lost by a pmi of 24 h (de wolf et al., 2020). when stored at 0 °c, this study found that synapses could be visualized at 48 h of pmi but were lost at the next time point studied of 168 h of pmi. one clear exception to the trend of postmortem synapse maintenance is the study of petit and leboutillier which analyzed rat brain tissue with two em staining methods, measuring both synaptic density and morphometry (petit and leboutillier, 1990). the study required the presence of synaptic vesicles to identify a synapse and only counted synapses had a clear preand postsynaptic density. with an osmium-based staining method, it was found that synapse density dropped markedly from 0 to 1 h of pmi, and then markedly once again from 10 to 15 h of pmi. with an ethanol phosphotungstic acid method of staining, synapse density dropped more slowly from 1 h to 15 h of pmi, with a total of 60-70% of synapses lost by the latest pmi time point tested. notably, synaptic structure within identified synapses was found to be “remarkably stable” up to the longest pmi they studied of 15 h, suggestive that synapses in an intermediate disintegrating state were not frequently found. in the osmium-stained tissue, the decline in the number of synapses from 1 h to 15 h of pmi mirrored an increase in the number of contacts without synaptic vesicles. the authors suggest that the observed decline in synaptic density in the pmi may be due to a loss of synaptic vesicles and therefore the ability to identify a connection as a synapse, although they noted that other explanations were also possible. correlational studies using em in human brain cohorts have had mixed results regarding synapse preservation. glausier and colleagues found that there was a significant decrease in the number of postsynaptic densities identified as the pmi increased up to 24 h (glausier et al., 2019). however, glausier and colleagues did not find a significant correlation between the pmi and the postsynaptic density length or with the total number of neuronal profiles identified. roberts and colleagues found that preand postsynaptic membrane structures were lost or distorted with pmi of greater than 7 h (roberts et al., 1996). on the other hand, roberts and colleagues reported that synapse classification and morphology was equivalent in cases with pmi of up to 7 h (roberts et al., 2005). scheff and colleagues found that there was no association between pmi up to 13 h and the synaptic features they studied (scheff et al., 1990; scheff and price, 1993). kolomeets and colleagues found that there was no correlation between the pmis of their cohort, which had an average of 6-7 h, and the number of synaptic contacts identified per mossy fiber terminal (kolomeets et al., 2007). gibson and tomlinson found that the numbers of recognizable synapses decreased by 77% at 33 h of pmi, at which point it stabilized until up to 69 h of pmi (gibson and tomlinson, 1979). as previously discussed, these authors suggested that the loss of recognizable synapses could be accounted for by the compression of synapses due to astrocyte vacuolization. it is not clear how to reconcile the differences between studies regarding the postmortem decomposition of synapses. several studies suggest that synapse visualization can be lost within the first hours of the pmi, while other studies suggest that the delay is longer and that the apparent loss of synapses is due to technical problems rather than synaptic disintegration. the latter prospect is consistent with the finding that partially disintegrated synapses are rarely reported, whereas one would expect to see these if synapses were disintegrating in the early pmi as opposed to becoming not recognizable with the techniques used. in summary, certain synaptic morphometric changes, such as thickening of the postsynaptic membrane and changes in curvature, can begin in the earliest minutes of the pmi. however, the preponderance of evidence suggests that synapses can remain generally intact for an extended period, up to many hours or even multiple days of pmi. certain methods of visualizing synapses are more robust in the postmortem period, suggesting that technical approaches are crucial to consider carefully when designing these experiments. myelin one of the major myelin artifacts that is consistently noted in the pmi is myelin lamellae separation. shibayama and kitoh noted that myelin lamellae are partially distorted at 30 mins of pmi (shibayama and kitoh, 1976). choe and colleagues described concentric unraveling of myelin at 1 h of pmi (choe et al., 1995). karlsson and schultz noted that there are infrequent myelin membrane separation defects after 1 h of pmi (karlsson and schultz, 1966). rees noted marked separation of lamellae in some myelin sheaths in monkey brains at 4 h of pmi (rees, 1976). hukkanen noted the formation of network-like structures in myelin lamellae at 24 h (hukkanen and röyttä, 1987). de wolf and colleagues noted unravelling of myelin sheaths at 9 h pmi with storage at 37 °c (de wolf et al., 2020). ansari and colleagues noted that myelin lamellae splitting can also occur in cerebral edema and that its occurrence may be associated with the postmortem increase in the water content of the brain (ansari et al., 1976b). another phenomenon, which may be related to lamellae splitting, is volume changes in myelin sheaths. haines and jenkins noted a slight swelling of myelin in some areas, first noticed at 18 h of pmi (haines and jenkins, 1968). on the other hand, de wolf and colleagues described myelin thinning, first noticed at 3.86 h of pmi with storage at 37 °c (de wolf et al., 2020). aside from these artifacts, myelin is generally reported to be stable in the pmi. myelin lamellae splitting is not associated with a break in the continuity of the separated lamellae (ansari et al., 1976a). several studies note that myelin is a relatively robust structure to postmortem decomposition (haines and jenkins, 1968; karlsson and schultz, 1966; sele et al., 2019; vrselja et al., 2019). summary among brain cell-specific membrane structures, volume and geometry changes are a frequent occurrence early in the pmi, and there are several types of common postmortem artifacts in these structures (table 3; figure 5). however, studies using robust visualization methods tend to find that approximate cell membrane location information is still able to be delineated in the early postmortem period. this suggests that the basic connectivity scheme of brain cells can likely be mapped even after postmortem decomposition has begun. feature key points of decomposition patterns brain region differences the cerebellum can have faster decomposition than other regions cell type differences smaller cells may decompose faster than larger cells cell volume cell volumes changes are rapid and occur within minutes. ongoing swelling, shrinkage, and compression processes evolve throughout the pmi dendrite segmental swelling within hours. highly sensitive to visualization method, and appear to be more stable with more robust methods of visualization axon segmental swelling and volume changes, similar to dendrites synapse morphometric changes, including postsynaptic membrane thickening and changes in curvature, occur within minutes. variability based on visualization method, but their overall presence is relatively stable in the early pmi myelin lamellar separation without a break in continuity of the separated lamellae. relatively slow decomposition overall table 3. summary of relative postmortem decomposition rates of different structural features of cell membranes in the brain. figure 5. possible morphometric changes of structural features in the postmortem brain. a-b: ultrastructural diagram of a human synapse at an earlier pmi (1 hour; a) with insignificant postmortem changes. compared to a later pmi (12 hours; b), at which point there are several changes, including vesicle clumping and loss, increased synaptic density with possible difficulty identifying the cleft, swelling of surrounding processes, and mitochondrial damage. these diagrams were adapted from (anders, 1977) (figure 2). c: segmental swelling of axons, adapted from an image of bielschowsky silver-stained ischemic rat brain tissue from (pantoni et al., 1996) (figure 6). d: myelin lamellae splitting, adapted from an electron microscopy image of postmortem human brain tissue from (glausier et al., 2019) (figure 1). variables modifying decomposition rates temperature the temperature at which the brain is stored during the pmi has a very well-replicated role in mediating the rate of postmortem decomposition. mechanistically, cold storage temperature slows down diffusion, decreases enzymatic activity, and inhibits microbial growth, all of which delay decomposition (schavemaker et al., 2018). low temperature storage refers to temperatures down to 0 °c, as below causes ice crystal formation and accompanying tissue damage. storage temperature has been reported to have the strongest effect on the rate of brain protein breakdown of any modifying variable (ferrer et al., 2007). one study in sheep brains shows that decreasing the storage temperature dramatically delays the average onset time of a sudden concentration change in several metabolites, attributed to the initiation of putrefaction, from 30 h pmi at 26 °c to 700 h pmi at 4 °c (ith et al., 2011). many studies find that the storage temperature plays a substantial role in the retention of cell membrane structure during the pmi. in their study of synapses in human brains, kay and colleagues report that they can perform effective ultrastructural analyses on tissue with pmis of up to 100 h (kay et al., 2013). they note that a key factor for ultrastructure tissue preservation is whether the cadaver is stored at cold temperature of 4-6 °c to reduce structural degradation. de wolf and colleagues found that advanced necrosis developed in isolated rat brain tissue at 36 h pmi when stored at 37 °c, compared to 2 months pmi when stored at 0 °c (de wolf et al., 2020). hukkanen and röyttä found that the white matter of isolated human surgical specimens showed degradation patterns at 6 h of pmi when stored at 25 °c, resembling those seen at 24 h of pmi when stored at 4 °c (hukkanen and röyttä, 1987). the environmental temperature at the time of death can also be relevant which has practical implications. for example, more rapid postmortem changes in the brain have been associated with death in summer months, while hypothermia at death is associated with slower postmortem decomposition (albrechtsen, 1977b; kitamura et al., 2005). some brain bankers record the refrigeration delay in addition to the pmi, which is the interval of time between death and when the body was stored at 4 °c (torres-platas et al., 2014; vonsattel et al., 2008). a formula that predicts the amount of brain decomposition based on storage temperature could allow for more accurate estimation of the impact of pmi. for example, the q10 rule suggests that the speed of chemical reactions in biological systems increases two-fold or slightly more with each 10 °c rise in temperature (vass, 2011). in the forensic literature, there is a related metric of “accumulated degree days” (vass, 2011). if the q10 rule holds, then reducing the storage temperature from a typical room temperature of 20-24 °c to 0-4 °c would be expected to decrease the decomposition rate by approximately 4-fold. a challenge is that brain temperature does not immediately equilibrate with the environment. even when the brain is stored at refrigerator temperatures of 8 °c, it can take 20-30 h for the tissue itself to actually reach that temperature, because of the slow rate of heat conduction (perry et al., 1977). as a result, the effects of refrigerator storage are nonlinear. consistent with this, studies investigating postmortem effects at earlier pmi time points, such as 4-12 h of pmi, tend to report smaller effects of refrigeration (hilbig et al., 2004). for the same reason of the low thermal conductivity of brain tissue, refrigerator storage is also expected to have a larger effect on the surface of the brain tissue, in smaller brains, and in isolated brain tissue. acidity and agonal damage across tissues, high acidity is strongly associated with faster decomposition rates in the pmi. for example, in a study on fish, postmortem decomposition was the fastest in the gastrointestinal tract and slowest in muscle tissue, with the brain somewhere in the middle, as expected based on their relative phs (george et al., 2016). across donated brains, those with relatively lower ph values have been found to have more postmortem structural breakdown following a given pmi (albrechtsen, 1977a; glausier et al., 2019). a major reason that lower ph is associated with faster postmortem decomposition is that acidic conditions activate certain autolytic enzymes, such as those in capthesin family (kies and schwimmer, 1942; albrechtsen, 1977a; compaine et al., 1995). postmortem acidic conditions in the brain are usually attributed to an increase in the production of lactic acid as a result of anaerobic glycolysis in the absence of oxygen delivery (powers, 2005). anaerobic glycolysis, in turn, has been reported to occur predominantly in two time periods: (a) the agonal phase and (b) the first few hours of the pmi. the agonal phase is the period during which a person is gravely ill and under profound physiologic stress, usually associated with cerebral hypoxia (lewis, 2002). for some brain donors, the agonal phase can last for only minutes or less prior to death, while for others, it can last for hours or days (li et al., 2004). numerous studies have found that a lower brain tissue ph is strongly correlated with a longer agonal phase (hardy et al., 1985; johnston et al., 1997; li et al., 2004). the agonal phase can contribute to damage measured postmortem in two ways. first, brain tissue can decompose during the agonal phase itself, leading to a higher total amount of decomposition after any given amount of pmi. damage associated with prolonged agonal states can be immense, which is why it is considered extremely important in mediating the degree of tissue decomposition in brain banking (ohm and diekmann, 1994; waldvogel et al., 2006; mccullumsmith et al., 2014; glausier et al., 2019). second, because brain cells are hypoxic during the agonal phase, they will produce excess lactic acid during this period, leading to lower ph levels and a faster rate of autolysis once the pmi commences. in addition to the agonal phase, ph has also been found to decrease over the first several hours of the pmi. for example, one study found that there was a significant inverse relationship of pmis between 1.5 and 4.5 h and brain tissue ph (beach et al., 2008). this is likely because some brain cells still have intact metabolic function in this early time range and therefore are still able to perform anaerobic glycolysis. on the other hand, it has been reported that brain ph does not continue to decrease in the pmi after the initial several hours (albrechtsen, 1977a; lewis, 2002). taken together, because of the complex associations of low ph with postmortem decomposition, tissue acidity is a critical variable to be cognizant of when evaluating the literature on postmortem decomposition. for example, ph has been proposed to be the most decisive determinant of postmortem necrosis in the granule cell layer of the cerebellum (albrechtsen, 1977a). hydration the water content of the brain has implications for postmortem decomposition rates. mechanistically, an increased water content can increase biochemical reaction rates (vass, 2011). across tissues, atmospheric humidity rates of greater than 85% are associated with an increase in decomposition rates (vass, 2011). because the brain is encased in the calvaria, atmospheric humidity likely does not substantially affect decomposition speed in the relatively early pmi ranges most relevant to brain banking. however, when the brain is taken out of the calvaria, either as a part of the preservation procedure or for ex situ storage in a time series study, investigators need to choose how to store the tissue. the brain is typically moist in its in situ location, bathed in cerebrospinal fluid (csf). one option for storage is to submerse tissue in a solution that matches the composition of the csf, although this risks worsening fluid shifts and associated artifacts such as vacuolization (fix and garman, 2000). alternatively, the brain can be stored in the dry air, although this is likely to lead to severe tissue damage due to dehydration (budday et al., 2015). additionally, the brain’s relatively high water content, which has been found to increase in the postmortem period, may play a role in its relatively faster rate of postmortem autolysis compared to some other tissues (ansari et al., 1976b; zhou and byard, 2011). as a result, premortem conditions in brain donors that promote water accumulation, such as cerebral edema, may predispose to a faster decomposition rate during the pmi. oxygen content the atmospheric oxygen content also affects the rate of postmortem decomposition across tissues (cockle and bell, 2015; vass, 2011). mechanistically, high oxygen content leads to oxidative damage to biomolecules as well as increased bacterial growth, thus promoting putrefaction (cockle and bell, 2015). the brain is relatively insensitive to changes in atmospheric oxygen content in the early stages of the pmi, due to its protection in the calvaria and the relative isolation of the brain from bacteria in the gastrointestinal tract. however, the brain is likely much more susceptible to changes in atmospheric oxygen content in the later postmortem period when bacteria have had time to proliferate in the brain tissue. this may help to explain the relatively good long-term preservation of brains stored in environments with low oxygen content, such as the body described in a case study that was immersed underwater for 10 weeks prior to autopsy (mackenzie, 2014). additionally, when the brain is taken out of the calvaria without protection from oxygen, it is likely to be more susceptible to oxidative damage and putrefaction due to the increased exposure to atmospheric oxygen. putrefaction to what extent there are microbes present in the brain under normal conditions remains an open question (link, 2021). regardless, there is evidence that proliferation of putrefactive bacteria is not a typical occurrence in the early pmi. instead, autolysis is thought to be the main contributor to early decomposition within at least the first day of the pmi (ith et al., 2011). various factors could modify the rate at which putrefaction initiates, such as isolation from the gastrointestinal tract, the presence of premortem fever, sepsis, or bacterial encephalitis, and environmental storage conditions (zhou and byard, 2011). at later pmis, the potential for putrefaction is a critical variable to consider. for example, finnie and colleagues noted that there was no confounding bacterial invasion for the full 4 weeks of postmortem decomposition that they studied (finnie et al., 2016). in situ versus ex situ brain storage one of the key distinctions in the methodologies of the studies we identified is whether the brain is stored in situ or excised to be stored ex situ during the pmi. although the decomposition rates between these two conditions were not directly compared among the included studies, this has been found to affect the rate of liver decomposition. specifically, removal of liver fragments from its original location has been found to substantially accelerate the postmortem decomposition kinetics of the liver compared to leaving it in situ (nunley et al., 1972). in studies of neuronal ischemia, morphological damage and disruption of the cytoskeleton have also been found to occur more quickly and intensely in slices than cells in situ (lipton, 1999). in addition to differences in the other modifying variables between the in situ and ex situ conditions, other factors will also affect the rate of decomposition in these two much different environmental contexts. one possible mechanism is that removal of the brain could significantly affect the tissue’s mechanical state, such as its hydraulic permeability, which may affect decomposition rates (jamal et al., 2021). variation across species in metabolic rate several studies noted differences in the decomposition rates between species (lucassen et al., 1995; martin et al., 2003). one notes that postmortem decomposition is likely to be faster in the smaller brain of a rodent and slower in the larger brain of a human (roberts et al., 2014). others suggest that human or bovine brain tissue appear to degrade relatively more slowly postmortem than rodent brain tissue (ansari et al., 1975; schwab et al., 1994). the most likely reason for species differences is that the metabolic rate varies, for example being much faster in rodents, which means that there may be a higher density of catabolic enzymes present in the postmortem period (demetrius, 2005). consistent with this, lesion evolution following cerebral infarction also appears to be markedly faster in rats than humans (mena et al., 2004). the infarct process in general has been reported to occur faster in animals that are smaller with higher metabolic rates (kloner et al., 2018). across tissues, autolysis has been suggested to be faster in malignant tissues with higher metabolic rates (lesnikova et al., 2018). as a result, studies using rodent models of human decomposition may overestimate the rate. this may account for the generally faster rates of decomposition reported in time series studies, which are primarily on rodents, compared with correlational studies, which are predominantly on human brains. premortem metabolic state lindenberg suggest that premortem metabolic state plays an important role in mediating postmortem decomposition rates (lindenberg, 1956). they report that if antemortem hypoxia lasted for more than 60 min, then brain cells retained their microscopic structure for at least 18 h pmi, even if kept at the relatively high temperature of 37 °c. on the other hand, if there was no antemortem hypoxia, then brain cells lost their structure much more quickly. their explanation is that cells exposed to antemortem hypoxia have already depleted and eliminated metabolic products that might otherwise contribute to structural damage. this hypothesis is inconsistent with many other studies finding that more severe agonal damage is associated with worse brain cell morphology (williams et al., 1978; glausier et al., 2019). however, because the underlying biology is complex and poorly understood, it remains true that certain antemortem metabolic states could be protective against postmortem decomposition, requiring further investigation. for example, premortem administration of metformin, a chemical that alters metabolism, has also been shown to decrease the rate of postmortem nuclear swelling (dehghani et al., 2018). age the evidence for the interaction of age with pmi effects is mixed and may depend on the structural feature considered. williams and colleagues found that in general, the effects of the pmi were the same on neuronal morphology in juvenile (14 d old) and adult (60 d old) mice (williams et al., 1978). buell also reported that neuronal morphology in human brains with substantial pmis was not dependent on age (buell, 1982). however, itoyama and colleagues, comparing young (7 d old) and adult rats, found that young rats had more perikaryal swelling of oligodendrocytes, more fragmentation of oligodendrocyte processes, and more myelin vacuolization at the same pmi (itoyama et al., 1980). additionally, mori and colleagues found that blood vessels from older (32-month-old) rats were found to leak proteins at shorter pmis than younger (3-month-old) rats (mori et al., 1991). myelin is known to have delayed development, while blood vessels are known to accumulate damage with age, which may make these features more vulnerable to differential decomposition in the pmi based on age. premortem pathology the presence of premortem brain pathology may affect the rate of postmortem decomposition. this could occur due to factors affecting the cohesiveness or catabolic rate in the brain. in one case study, severe autolysis of granule cells and purkinje cells in the cerebellum occurred after only 6 h of pmi, enormously faster than typical, which was attributed to severe diabetic ketoacidosis (suárez-pinilla and fernández-vega, 2015). death due to uncontrolled diabetes has been associated with higher rates of necrosis of the granule cell layer of the cerebellum in other studies as well (albrechtsen, 1977b). more generally, obesity, hyperglycemia, and ingestion of certain substances have been reported to promote the rate of postmortem decomposition of cadavers and may also therefore influence the rate of brain decomposition (zhou and byard, 2011). on the other hand, some conditions have been found to not increase the rate of postmortem decomposition, such as rabies infection (monroy-gómez et al., 2020). some types of premortem pathology could even predispose for stability in the pmi. certain pathologic proteins in the brain, such as abnormal prion protein and amyloid beta, can be detected via ihc for months after death (scudamore et al., 2011). in the paleoanthropology literature, tissue from a 2600-year-old brain was found to be partially intact, likely due to an aggregate of intermediate filaments, which may have been related to premortem brain pathology (petzold et al., 2020). the possibility of brain pathology affecting postmortem decomposition rates is especially important if investigators adjust for the effects of pmi on cell morphometry with a linear model. in this setting, if there is an interaction effect that is not considered, the investigator might conclude that brain pathology is associated with a particular aspect of cell morphometry, when the difference between groups could instead be due to an artifact of the pmi. summary there are many ways that modifying variables can interact with pmi to affect the visualization of cell morphometry (table 4). well-controlled studies will account for this possibility. these factors also can help to explain heterogeneity between studies. modifying variable reported effect on the rate of postmortem changes storage temperature lower temperatures (above 0°c) are clearly associated with slower decomposition tissue acidity lower ph values are associated with faster decomposition and may be partially a marker for more damage in the agonal state tissue hydration atypically high or low hydration levels may be associated with faster decomposition rates oxygen content higher oxygen content is associated with oxidative damage to biomolecules and more rapid putrefaction putrefaction generally thought to make minimal contributions in the early days of the pmi, but can be more rapid in certain circumstances storage location there is evidence that brains stored outside of the calvaria (ex situ) have more rapid decomposition compared to being stored inside the calvaria (in situ) species there is evidence that smaller animals with faster metabolic rates, such as rodents, have faster decomposition rates premortem metabolic state complex effects of premortem metabolic state on decomposition rate that require further study before conclusions can be drawn age mixed evidence depending on the structural feature studied, with some showing no age dependency, and others, such as myelin and blood vessels, having some indication for an interaction premortem pathology certain pathologies, such as uncontrolled diabetes, are associated with faster postmortem decomposition rates table 4. summary of variables potentially modifying the postmortem decomposition rate. interactions between postmortem changes and preservation methods many studies have noted that the quality of perfusion as compared to immersion fixation could vary based on the pmi. indeed, there is good reason to think that perfusion quality can be limited in the pmi, for example due to a loss of vascular patency, the perivascular accumulation of water, and/or postmortem clot formation (cammermeyer, 1960; de la torre et al., 1992; garcia et al., 1978; hansma et al., 2015; kloner et al., 2018). vascular abnormalities are thought to be the key limitation preventing the brain from tolerating ischemic episodes, thus being a primary cause of death, which makes the difficulty of postmortem perfusion-based preservation unsurprising (jenkins et al., 1979). one study that performed postmortem perfusion, routtenberg and tarrant acknowledged that theoretically perfusion fixation may be lower quality in postmortem cases, and therefore delays in time prior to perfusion fixation may lead to changes in fixation quality rather than pure decomposition effects (routtenberg and tarrant, 1974). in their case, perfusing brains at up to 10 min pmi, they reported that perfusion quality was unlikely to have affected their results, because markers of high-quality perfusion, namely tissue hardness and clearing of blood vessels, were still observed. koenig and koenig used perfusion fixation on guinea pig brains at up to 23.5 h pmi and did not note decreased perfusion quality as an issue that affected their results (koenig and koenig, 1952). consistent with the known benefits of perfusion fixation on tissue quality, several studies note that this method yields substantially fewer postmortem artifacts than immersion fixation (mcfadden et al., 2019). bywater and colleagues, in a study of monkey brains, performed both perfusion and immersion fixation at the different time points studied (bywater et al., 1962). they noted that brains with a pmi of 1 h preserved via perfusion fixation had similar postmortem artifacts as brains immersion fixed immediately after death. liu 1950 noted that immersion fixed brains demonstrate widespread artifacts similar to postmortem artifacts found in brains with extended pmis, which they attribute to inadequate fixation by immersion (liu and windle, 1950). lavenex and colleagues reported that the difference between immunostaining patterns for smi-32 was striking between perfusion fixed brains at 0 h pmi and immersion fixed brains at 2 h of pmi, but that there was no substantial difference between immersion fixed brains preserved at 2 h and 48 h of pmi, with postmortem storage at 4 °c (lavenex et al., 2009). as a result, studies of pmi effects that do not account for the possibility of worse preservation with immersion as compared to perfusion fixation, for example using perfusion fixation as the baseline timepoint of zero pmi and immersion fixation at subsequent pmi timepoints, are susceptible to bias (airaksinen et al., 1991; garcia et al., 1978; geddes et al., 1995; terstege et al., 2022). another important distinction is the differences in fixation as compared to freezing, with several studies noting relative advantages of each. for example, schulz and colleagues noted that dark neurons, a well-known fixation artifact, are seen in small tissue blocks that were fixed but not ones that were frozen, while frozen tissue blocks had freezing artifacts such as cytoplasmic vacuolization (schulz et al., 1980). itoyama and colleagues found a differential effect of a postmortem delay of 20 h when immunostaining for myelin basic protein in frozen as compared to immersion fixed tissue (itoyama et al., 1980). they reported that myelin sheaths were slightly distorted following the pmi in immersion fixed tissue but had substantially worse quality in frozen tissue following the same pmi compared to the baseline state. freezing artifacts could theoretically be worse in cases of longer pmi because more free water and weaker cellular structures may predispose to more mechanical ice damage, which warrants further investigation. finally, the fixative used can also influence observed pmi artifacts. one study found differential effects of a 24 h pmi on immunostaining properties for several antigens in blocks of human brain tissue based on the fixative used for preservation (sillevis smitt et al., 1993). when the tissue was preserved using bouin and b5 fixatives, they found no effect of a 24 h pmi on the quality of immunostaining with smi-32 and bf-10, which are two antibodies that recognize neurofilament. on the other hand, the immunostaining with these antibodies was diminished by a 24 h pmi when the tissue was preserved with a different fixative, sensofix. it may be that fixation needs to be stronger in the presence of a longer pmi to prevent damage during dehydration and embedding. morphological staging of decomposition we present a model of how cell morphometry changes during different stages of the oncotic necrosis pathway in the postmortem brain (figure 6). of note, the timeline of cellular events depicted here likely varies between cells, as these are rough guidelines, not absolute. additionally, the intracellular changes shown, which are not a focus on this review, are primarily based on other sources (choe et al., 1995; majno and joris, 1995; suzuki, 1987). figure 6. model of the oncotic necrosis pathway of cell membrane deterioration in the brain. stage zero is the antemortem cell, which shows intact cell membrane and normal sized structures. stage one is oncosis, wherein ischemia leads to loss of atp, loss of ion pump activity, breakdown of biomolecules, bleb formatting, vacuolization, and other changes. the asterisk (*) indicates that while cell volume increases are possible, no change or shrinkage is also possible. stage two is early necrosis, wherein the physical disruption of the cell membrane causes focal loss in cell membrane shape and leakage of intracellular contents. there is also severe swelling of surrounding processes, potentially compressing cellular structures such as neurites and synapses. from this point on, the cell has necrotic morphology. stage three is late necrosis, wherein gel-like networks break down, eventually leading to a complete cellular dissolution, and only cell fragments or debris may remain. the main figures used for adaptation in building this model were: (majno and joris, 1995) (figure 9), (suzuki, 1987) (figure 2.7), (fricker et al., 2018) (figure 3), and (trump et al., 1984) (figure 8). in this model, oncosis is initiated by a lack of blood flow, leading to atp depletion, a rise in the intracellular concentrations of na+ and ca2+, fluid shifts, and the initiation of biomolecular breakdown and redistribution. while cell swelling is a classical finding in oncosis, it may not occur, or shrinkage can occur instead, depending upon the cell and surrounding milieu. cell volume can also evolve over the course of the pmi. processes surrounding cells also tend to swell, which is an early event in the pmi. finally, cell membranes can form blebs as well. early necrosis is initiated by focal disruptions of the cell membrane, leading to leakage of intracellular contents. the remaining cell membranes tend to become indistinct, although still visible. processes around cells can become extremely swollen, in some cases larger than many cells themselves. note that while these swollen cell processes are usually astrocyte processes, other studies also describe swelling of dendrites, axons, and oligodendrocyte processes (rees, 1977; gibson and tomlinson, 1979). as a result, we describe these agnostically as cell processes. early necrosis is followed by late necrosis, which is characterized by further disruption of gel-like networks leading to the loss of the original cell shape. the ultimate speed of dissolution of cell structures likely depends upon the initial strength of the gel-like networks. for example, the strong compaction of myelin may help to explain why this structure tends to be highly stable in the postmortem period. the structures that last the longest are also the ones that are the most slowly catabolized in the postmortem period. the ultimate outcome of late necrosis is cellular liquefaction and coming to an equilibrium with the environment. an implication of this model is that the transition which leads to loss of cell morphometry information – making the postmortem tissue no longer useful for studying cell morphometric alterations in neurobiological disorders – is most likely the transition from early necrotic to late necrotic, when the gel-like networks maintaining cell shapes are degraded. the transitions between these stages are not discrete but continuous, which may be a useful model for investigators studying postmortem brain tissue. we also present the histologic findings expected regarding cell morphometry at each of the stages (table 5). stage of decomposition cell shape cell size cell membrane findings common artifacts antemortem intact no change not applicable not applicable oncotic largely intact mild to moderate swelling or shrinkage is likely may have blebbing or compression pericellular and perivascular rarefactions; vacuolization early necrotic largely intact some cells, especially astrocytes, are excessively enlarged, while others begin to shrink cell membranes may be indistinct, blurry, or partially damaged above artifacts, but more widespread and severe; intracellular contents may be seen in extracellular space late necrotic altered cells shrink as they dissolve cells are dissolved washed out tissue areas devoid of cells table 5. typical cell morphometric findings at different stages of postmortem decomposition. implications for human brain mapping to map the brain across large areas of brain tissue, surgically extracted tissue will not be sufficient. this draws attention to the inevitable pmi in autopsy brain samples which are of particular interest for designing brain mapping studies. one relevant lesson is that postmortem brain mapping will have higher fidelity to in vivo states for some structural features compared to others. cell volumes and feature morphometry such as synapse size are likely to be altered rapidly in the pmi. cell membrane topography can also be altered relatively early due to the heterogeneous formation of vacuoles, blisters, and compression. on the other hand, cell membrane topological relationships are likely to be maintained across longer pmis. as a result, it may be possible to map connectivity and circuitry, as well as circuit properties such as the rough degree of axon myelination, even after a relatively longer degree of postmortem decomposition. however, there is considerable uncertainty about how broadly this applies across brain regions and cell types. it is unknown what degree of postmortem decomposition can be tolerated before topological cell membrane relationships are also lost, requiring further research. another lesson for postmortem brain mapping is that, compared with animal brains preserved at the time of death, some visualization procedures will be substantially more robust than others. as previously discussed, ihc studies can be sensitive to the pmi, because certain antigens can be lost relatively early in the pmi. if ihc is to be used in postmortem tissue, then labeling antigens that are robust to postmortem decomposition is essential. based on the studies we reviewed, synaptophysin (liu and brun, 1995; sarnat et al., 2010), gfap (blair et al., 2016), and neurofilaments (lavenex et al., 2009; blair et al., 2016) are relatively stable in the postmortem period in visualizing cell morphometry, although this certainly warrants further investigation. some morphological stains have also been shown to be resistant to postmortem decomposition, although there remain questions of how widely they can be used and the degree to which their neuronal mapping is unbiased. for example, golgi-cox staining only visualizes a small subset of neurons, although its resistance to postmortem decomposition makes it a valuable staining method. while some antigens are sensitive to postmortem decomposition, others may be particularly stable in ways that could prove valuable. for example, ischemic fluid shifts such as severe dendrite swelling have been found to be partially reversible if there is a physiological correction within 20-60 minutes, which has been attributed to the resilience of the cytoskeleton (zhang et al., 2005). this suggests that staining and mapping cytoskeletal biomolecules may be a way to infer cellular morphometry even in the setting of volume changes that might affect morphological stains. studies using electron microscopy to map the postmortem brain require particular attention to sample preparation. for example, some studies have found that synapses decrease in number during extended pmis, although this is certainly not a universal finding (huttenlocher, 1979; petit and leboutillier, 1990; roberts et al., 1996). as discussed, several lines of evidence suggest the loss of synapse visualization may be due to compression or loss of synaptic markers, rather than frank synaptic fragmentation or dissolution (gibson and tomlinson, 1979; petit and leboutillier, 1990). as a result, the identification of synapses would likely be less affected by postmortem decomposition if they were visualized with a robust staining procedure and/or volumetric imaging. multiple authors reported optimizing the standard electron microscopy procedures for using on postmortem human brains (sele et al., 2019; tang et al., 2001). in addition to optimizing tissue processing, data analysis methods can also aid in reconstructing the original state of structures affected by postmortem artifacts. in reconstructive brain mapping, common artifacts could be computationally reversed – with some degree of fidelity to the original state – via deterministic or stochastic algorithms. for example, if myelin lamellae have split, it may be possible to infer the shape in which they were originally compacted. as an early example in this field, one study performed a 3d reconstruction of brain tissue based on em images while using a set of models to correct the extracellular space volume fraction for shrinkage attributed to hypoxia, fixation, and dehydration (kinney et al., 2013). it may also be possible to map the decomposed biomolecules that originally comprised a structural feature if the structure itself has fragmented. this could never be done perfectly but could potentially infer the original structure to a sufficient degree of accuracy in many cases. however, because of the ubiquity of non-gaussian diffusion, computational reversal of postmortem biomolecule redistribution is likely a very challenging problem. it would require mapping multiple biomolecules and triangulating their predicted diffusion patterns, while accounting for fluid shifts, biomolecular degradation, and other factors. decomposed biomolecules that have diffused about and partially degraded may be among the last remnants of an in vivo structure before the information about it is totally lost, thereby defining classical physical limits to the potential for reconstructive brain mapping. further studies of postmortem changes a time series study in which the pmi is experimentally manipulated yields the most precise measurements of postmortem changes. however, when time series studies are used as a proxy for what happens in postmortem human brains, they often lack ecological validity. for example, studies on isolated surgical biopsy tissue introduce a dramatically different metabolic and biophysical state than that of the postmortem brain, which may limit the validity of these studies in predicting how decomposition occurs in an intact human brain postmortem. to address this, time series studies could measure and/or manipulate modifying variables, such as the location (in situ or ex situ), temperature, metabolic state, and tissue humidity. it would also be beneficial to perform time series studies that can help to account for variability across the existing studies, as reviewed here. one of the least widely studied areas is variability across brain regions. as we approach the ability to achieve brain-wide cellular mapping in the coming decades, there is a clear need to know whether some brain regions are more susceptible to postmortem decomposition than others, but this knowledge is currently limited. regarding correlational studies, scaling the sample size is likely to be helpful. larger studies will be helpful to account for measured and unmeasured confounders, in addition to including these variables as covariates where available. with larger sample sizes also comes the possibility of unsupervised machine learning studies. most of our existing knowledge of postmortem changes is based on subjective semi-quantitative scoring, but this is biased towards pre-existing knowledge. unsupervised machine learning studies, as have recently begun to be performed in studying the neurobiology of disease, may also help to uncover biomarkers of postmortem decomposition (mckenzie et al., 2022). developing an objective measure of histologic decomposition, analogous to the rna integrity number (rin) for rna preservation quality, would be valuable as a quality metric in brain banking. the status quo involves investigators judgments subject to reporting bias. for example, the same cell could look “distorted” to one person but “largely preserved” to another, partially reflecting their prior expectations. this may be one reason for the heterogeneity in the decomposition outcomes we observed between studies. more quantitative histologic metrics, for example using morphology comparison algorithms, would help to mitigate reporting biases and make comparisons across studies more precise (costa et al., 2016). among the most reliable histologic findings available today to measure the extent of postmortem decomposition are perivascular rarefaction, pericellular rarefaction, and vacuolization on morphologically stained tissue, such as h&e. several studies describe these as common postmortem changes that become more prominent with increasing pmi (de groot et al., 1995; hilbig et al., 2004; shepherd et al., 2009; monroy-gómez et al., 2020). however, the appearance and enlargement of these postmortem artifacts appears to have a non-linear relationship with pmi. moreover, the kinetics of their appearance and the variables modifying them are poorly mapped out. therefore, there is a need for additional research and validation prior to the routine use of these findings as measures of postmortem decomposition. comparison to other reviews to our knowledge, there has not been a previous comprehensive review focused solely on the topic of brain cell morphology changes in the pmi, although several publications have touched on it as a part of a broader discussion. oehmichen reviewed the extant literature on postmortem alterations in the histochemistry of cns tissue (oehmichen, 1980). they concluded that cell structures seen under the light microscope were effectively unchanged up to 6-8 h pmi and that after this, autolytic processes commence. lewis, in a broader review of human brain research, included a discussion of pmi (lewis, 2002). they noted that any pmi threshold for inclusion in a study, such as less than 24 h, would be arbitrary, and that tissue quality is highly dependent on antemortem factors. they also pointed out that postmortem effects can vary based on the feature measured and the brain region. finally, this review noted that it can be difficult to distinguish differences between brains due to disease as opposed to postmortem artifact. ravid and colleagues noted that the distribution of receptors tends to be stable in the postmortem period (ravid et al., 1992). they also discussed the importance of developing profiling techniques for longer pmis. wohlsein and colleagues described common postmortem artifacts in animal brains, such as vacuolization, shrinkage of glial cells, hemolysis, and putrefactive decomposition in advanced stages (wohlsein et al., 2013). hostiuc and colleagues performed a systematic review on ultrastructural changes as a marker of the pmi in various organs; however, they used different search and selection criteria than our review and identified just two studies from the brain (hostiuc et al., 2018). a forensic neuropathology textbook by oehmichen and colleagues discusses histologic changes in the pmi (oehmichen et al., 2006, p. 74). in their assessment, neuronal swelling is the most fundamental postmortem change, causing cells to lose their contours and become spherical. they note that oligodendrocytes and astrocytes can also have postmortem swelling, but that these changes are slight compared to neuronal changes and less specific. ramirez and colleagues reported that there is no correlation between the pmi and tissue quality in brain banking samples (ramirez et al., 2018). they note that many investigators are rigid about the pmi, despite this number not accurately reflecting the degree of metabolic changes that have occurred in the preand post-mortem tissue. there have also been reviews on cell morphology changes in brain death, which results from the same underlying process as in the pmi, namely a global lack of blood flow to the brain (oehmichen, 1994; folkerth et al., 2022). notably, the kinetics of decomposition are faster in brain death because a relatively higher body temperature is maintained. strengths and limitations of this review a strength of this review is that, to the best of our knowledge, this is the largest collection of studies yet assembled on this topic. another strength is that a semi-quantitative grading system was developed and implemented on the time series studies. finally, we have attempted to integrate the literature from studies using both light and electron microscopy to share insights between these two interrelated fields. in addition, this review has several limitations. first, the decomposition grades in the time series studies may give a false sense of precision. instead, the purpose of these grades is to aid in conceptualization and visualization. second, we largely did not consider disease state among the correlational studies which may have contributed to variations in the effects of pmi. finally, there is no doubt that we missed a substantial set of the literature. this includes the non-english literature, especially the relatively large german and russian language literatures. also, we did not systematically evaluate references or citing articles of all included articles, which further limited the literature identified. nevertheless, we expect that the studies included are a representative sample. conclusions a reductionist focus on the numerical pmi, while convenient, does not account for variability based on the visualization method used, feature analyzed, region or cell type, and variables that modify the decomposition speed. indeed, there is no obvious single pmi threshold at which cell morphometry is clearly lost. instead, research is likely to be more fruitful in understanding how the actual amount of decomposition in each brain can be measured, which structural features are likely to be altered sooner than others, and which visualization methods are most robust to postmortem changes. in biorepository contexts, it may make the most sense to cast a wider net on the inclusion of brain tissue, as opposed to having strict pmi inclusion requirements. on the other hand, it is essential to recognize that decomposition starts in the first minutes and is clearly occurring throughout the pmi. therefore, there is a need to minimize postmortem decomposition by expediting processing procedures to the extent possible. more research into knowledge gaps regarding the postmortem decomposition of brain cells will help to further elucidate this critical barrier to studying human neurobiology using donated brains. abbreviations atpase adenosine triphosphatase, csf cerebrospinal fluid, d day, em electron microscopy, er endoplasmic reticulum, gfap glial fibrillary acidic protein, gfralpha glial cell line-derived neurotrophic factor family receptor alpha, h hour, h&e hematoxylin and eosin, icc intraclass correlation, ihc immunohistochemistry, lfb luxol fast blue, map2 microtubule associated protein 2, min minute, nf-h neurofilament heavy chain, nf-l neurofilament light chain, nf-m neurofilament medium chain, neun neuronal nuclear protein, pmi postmortem interval, rameses realist and meta-narrative evidence syntheses evolving standards, rin rna integrity number, rrna ribosomal rna, ssdna single-stranded dna. author contributions m.k., k.f., j.c., and a.m. conceptualized the study. s.w. advised on the search strategy and performed the database searches. a.m. performed abstract and article screening. m.k. and a.m. performed data extraction and review from the individual studies. m.k., j.k., and a.m. performed grading of the studies. a.s. and k.c. performed electron microscopy studies. a.m. wrote the initial draft of the manuscript. all authors reviewed the manuscript and approved the final manuscript. acknowledgements we would like to thank rebecca folkerth, etty cortes, and thomas beach for helpful personal communications regarding this topic. the icahn school of medicine at mount sinai provided access to library resources. electron microscopy tissue preparation and imaging were performed at the microscopy and advanced bioimaging core at the icahn school of medicine at mount sinai. funding the study was supported by nih grants p30ag066514, rf1mh128969, r01ag054008, rf1ns095252, rf1ag060961, r01ns086736, r01ag062348, and the rainwater charitable foundation. the funders had no role in the design of the study or in the collection or interpretation of the data. data availability the generated database of studies studying postmortem changes in brain cell morphometry is freely available at https://github.com/andymckenzie/postmortem_interval. this repository also contains the code required to reproduce the figures created in r. supplementary files supplementary file 1. rameses checklist for the review (pdf) supplementary file 2. supplementary methods including database search methods (pdf) supplementary file 3. export of covidence files containing study screening results (zip file) 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an inherent, natural part of human brain aging: an integrated perspective feel free to add comments by clicking these icons on the sidebar free neuropathology 3:17 (2022) review alzheimer’s disease is an inherent, natural part of human brain aging: an integrated perspective isidro ferrer department of pathology and experimental therapeutics, university of barcelona; emeritus researcher of the bellvitge institute of biomedical research (idibell); biomedical research network of neurodegenerative diseases (ciberned); institute of neurosciences, university of barcelona; hospitalet de llobregat, barcelona, spain corresponding author: i. ferrer · department of pathology and experimental therapeutics · university of barcelona · campus bellvitge · carrer feixa llarga sn · 08907 hospitalet de llobregat · spain 8082ifa@gmail.com submitted: 25 february 2022 accepted: 21 june 2022 copyedited by: félicia jeannelle published: 08 july 2022 https://doi.org/10.17879/freeneuropathology-2022-3806 keywords: alzheimer’s disease, human brain aging, genetics, epigenetics, new therapies abstract alzheimer disease is one of the most challenging demons in our society due to its very high prevalence and its clinical manifestations which cause deterioration of cognition, intelligence, and emotions – the very capacities that distinguish homo sapiens from other animal species. besides the personal, social, and economical costs, late stages of ad are vivid experiences for the family, relatives, friends, and general observers of the progressive ruin of an individual who turns into a being with lower mental and physical capacities than less evolved species. a human brain with healthy cognition, conscience, and emotions can succeed in dealing with most difficulties that life may pose. without these capacities, the same person probably cannot. due, in part, to this emotional impact, the absorbing study of ad has generated, over the years, a fascinating and complex story of theories, hypotheses, controversies, fashion swings, and passionate clashes, together with tremendous efforts and achievements geared to improve understanding of the pathogenesis and treatment of the disorder. familal ad is rare and linked to altered genetic information associated with three genes. sporadic ad (sad) is much more common and multifactorial. a major point of clinical discussion has been, and still is, establishing the differences between brain aging and sad. this is not a trivial question, as the neuropathological and molecular characteristics of normal brain aging and the first appearance of early stages of sad-related pathology are not easily distinguishable in most individuals. another important point is confidence in assigning responsibility for the beginning of sad to a few triggering molecules, without considering the wide number of alterations that converge in the pathogenesis of aging and sad. genetic risk factors covering multiple molecular signals are increasing in number. in the same line, molecular pathways are altered at early stages of sad pathology, currently grouped under the aegis of normal brain aging, only to increase massively at advanced stages of the process. sporadic ad is here considered an inherent, natural part of human brain aging, which is prevalent in all humans, and variably present or not in a few individuals in other species. the progression of the process has devastating effects in a relatively low percentage of human beings eventually evolving to dementia. the continuum of brain aging and sad implies the search for a different approach in the study of human brain aging at the first stages of the biological process, and advances in the use of new technologies aimed at slowing down the molecular defects underlying human brain aging and sad at the outset, and transfering information and tasks to ai and coordinated devices. index summary 1. introduction 2. β-amyloid and tau 2a. β-amyloid (aβ) 2b. tau 3. familial ad (fad; early-onset familial alzheimer’s disease: eofad), and the β-amyloid cascade hypothesis 4. sporadic ad (sad; late-onset alzheimer disease: load) 5. nfts and sps in non-human brain aging 6. synapses 7. neurotransmitters, neuromodulators, and related receptors 7a. acetylcholine (ach) and acetylcholine receptors (achr) 7b. glutamate and glutamate receptors (glurs) 7c. γ-aminobutyric acid (gaba) and gaba receptors 7d. serotonin and 5-hydroxytryptamine (5-ht) receptors 7e. noradrenergic system 7f. adenosine receptors 7g. endocannabinoids and cannabinoid receptors (cbrs) 8. trophic factors and receptors 9. endoplasmic reticulum stress 10. failure to remove debris: the ubiquitin-proteasome system (ups) and autophagy in sad 11. granulovacuolar degeneration (gvd) 12. glial alterations in aging and sad 12a. astrocytes 12b. microglia 12c. oligodendrocytes 13. the neurovascular system in ad 14. purine and pyrimidine metabolism in sad 15. epigenetics in brain aging and sad 15a. histone modifications, dna methylation, and hydroxymethylation 15b. non-coding rnas 16. microorganisms and sad 16a. microorganisms in the brain and oral cavity 16b. gut microbiota 17. seeding and spreading of β-amyloid and tau 17a. seeding β-amyloid 17b. tau seeding 17c. multiple seeding foci of β-amyloid and tau pathology; vulnerable and resistant populations to tau seeding in brain aging and sad 18. neuronal death 19. neuronal connectivity networks in brain aging and sad 20. human brain aging and preclinical ad 21. primary age-related tauopathy (part), rapidly progressive sad, and sad resilience 21a. part 21b. rapidly progressive ad 21c. sad resilience 22. biochemical changes beyond tau and β-amyloid at the the first stages of nft pathology 22a. aberrant cell-cycle re-entry, and altered adult neurogenesis 22b. brain lipids 22c. lipid rafts and cell membranes 22d. mitochondria 22e. oxidative stress damage 22f. inflammation 22g. protein synthesis impairment 22h. dysregulated protein phosphorylation 23. concluding comments abbreviations funding acknowledgements references summary this is a comprehensive historical and up-dated review on the pathogenesis of alzheimer’s disease (ad) in relation to intrinsic process of natural brain aging. the study covers, in addition to β-amyloid and tau pathology, alterations in multiple merging molecular pathways and sub-cellular structures underpinning brain aging and ad. familial ad (fad) is rare and linked to altered genetic information associated with three genes. sporadic ad (sad) is much more common and multifactorial. a major point of clinical discussion has been, and remains, establishing the differences between brain aging and sad. this is not a trivial question, as the neuropathological and molecular characteristics of normal brain aging and the first appearance of early stages of sad-related pathology are not easily distinguishable in most individuals. another important point is confidence in assigning responsibility for the beginning of sad to a few triggering molecules, without considering the wide number of alterations that converge in the pathogenesis of aging and sad. recognized genetic risk factors covering multiple molecular signals are increasing in number. molecular alterations of lipid rafts, protein synthesis from the nucleolus to the ribosome, protein phosphorylation, kinase activation, purine metabolism, epigenetic regulation of dna and rna, mitochondria and energy metabolism, inflammation, oxidative stress, cell-cycle re-entry, and cell death precede, in some regions (i.e., frontal cortex), abnormal tau deposition and amyloid plaques. human brain aging and sad do not follow a linear logic based on the assumption that a cause results in one or several effects; several separate alterations converge and potentiate each other to incorporate anomalies in additional pathways. tau seeding and spreading are active intercellular and intracellular processes that explain, only in part, disease progression. cell and region vulnerability are essential elements. brain aging with neurofibrillary tangles (nfts) restricted to the temporal lobe and selected nuclei of the brain stem, primary age-related tauopathy, preclinical ad, mild cognitive impairment (mci) of alzheimer type, typical ad, rapid progressive ad, and ad subtypes, are forms of sad modulated by individual genetic and molecular factors. as in atherosclerosis, the progression of the process has devastating effects in a relatively low percentage of human beings. future modulation of human brain aging and sad will require the combined application of artificial intelligence, brain dna editing, external electrical or wave-based signals to reduce energy consumption, and optimization of mitochondrial function, together with implantation of microdevices, to facilitate cooperative human-machine operation, pharmacological protection of lipid-protein interactions, high-throughput molecular technology, and resetting during sleep stages. 1. introduction the clinical and neuropathological characteristics and clinical correlates of alzheimer disease (ad) have been described in several recent reviews (1-9). however, the study of ad has generated a fascinating and complex compendium of theories, hypotheses, controversies, fashion swings, and passionate clashes, together with tremendous efforts and achievements geared to improve understanding of the pathogenesis and treatment of the disorder. the present paper is a critical review of brain aging and ad that includes molecular abnormalities and early metabolic alterations beyond β-amyloid and tau pathology. these changes, together with genetic factors, converge in the pathogenesis of ad. learning about early molecular modifications preceding by many years the appearance of clinical symptoms, when present, will serve to improve understanding of brain aging and the ad continuum. figure 1: dystrophic neurites of sps and nfts in the frontal cortex of a 76-year-old woman with dementia. paraffin section, gros-bielschowsky silver method without counterstaining, black and white figure, bar = 25μm. until the beginning of the last century, cognitive impairment and dementia were considered natural features of old age. multiple brain infarcts were common in old people, and vascular dementia due to arteriopathy was thought to be the main cause of senile dementia. however, microscopic study of post-mortem brains stained with the dyes available at that time revealed the presence of certain structural anomalies in aged individuals. paul block and georges marinesco (10) described “amas ronds”, and emil redlich (11) “miliare sklerose” in the neuropil, interpreted at that time as nodules of glial sclerosis, which we now know as senile plaques (sps). the introduction of the max bielschowsky silver method allowed visualisation of argyrophilic structures in neurons. using this method, alois alzheimer described for the first time large numbers of argyrophilic neurofibrillary tangles (nfts) and aggregates of dystrophic neurites in the brain of a 51-year-old woman who had suffered from progressive dementia and hallucinations in the previous four and half years (12). other cases were published shortly afterwards (13). the term alzheimer’s pre-senile dementia was introduced by emil kraepelin (14) to define the combination of pre-senile (before the age of 65) dementia in individuals with the morphological lesions described by alzheimer. oskar fischer (15), using the same method, described the presence of ‘drusen’ or ‘drusige nekrosen’ in 16 cases of senile dementia characterized by loss of memory and sense of location, disorientation, and confabulation. subsequent fischer reports (16, 17) detailed the morphology of abnormal fibrils and abnormal neurites, and their stages of formation, in a large series of older individuals. the term ‘‘senile plaque’’ (sp) for these structures was proposed by simchowitz (18). fischer also described “drusige entartung der gefässe” which corresponds to amyloid angiopathy. interestingly, fischer also reported and illustrated the presence of nfts in the same cases with dementia (19). hundreds of articles appeared in the succeeding years. alzheimer focused on nfts as the main cause of dementia, whereas fischer thought that sps were the main substrate of dementia in older cases. moreover, alzheimer contemplated nfts as aggregates of abnormal neurofibrils, while fischer considered dystrophic neurites of sps composed of abnormal neurofibrils, and nfts a particular abnormality of nerve cells (19). bielschowsky proposed a link between tangles and neuritic changes (20) (figures 1 and 2). nfts and sps are now considered ad-related pathology or ad-neuropathologic change (adnc) (https://www.alz.org/media/documents/alzheimers-facts-and-figures.pdf). the term pick’s disease (pid) was coined in 1926 to distinguish ad from pid primary frontotemporal degenerative atrophy (21). as late as the 1960s, ad and pid were considered early dementias, whereas pure senile dementia, vascular dementias, and mixed (vascular and degenerative) were classified as dementias in old age (22). the frontiers between ad and pure senile dementia were not clear, as the onset of clinical symptoms in many cases classified as ad was after the age of sixty (23). it was not until the 1970s that alzheimer’s pre-senile dementia and senile dementia with changes of alzheimer type were considered to be within the same spectrum (24-27). the inclusive term “alzheimer-fischer dementia” was never contemplated. figure 2: neurofibrillary tangles in the ca1 region of the hippocampus of a man aged 69 years with no apparent cognitive impairment. paraffin section, gallyas staining, lightly counterstained with haemtoxylin, bar = 25μm. the first approach toward a clinical consensus on ad was made in 1984; clinical diagnosis of ad was set up in three categories – possible, probable, and deﬁnite (requiring neuropathological verification) (28). definite ad was fixed as a neurodegenerative disease manifested by progressive dementia with a neuropathological substrate characterized by brain atrophy, neuronal death, and a particular distribution of abundant sps and nfts in the brain. box 1: clinical classification of alzheimer’s disease (https://www.alz.org/media/documents/alzheimers-facts-and-figures.pdf). preclinical alzheimer’s disease in this phase, individuals may have measurable brain changes that indicate the earliest signs of ad (biomarkers), but they have not yet developed symptoms such as memory loss. mild cognitive impairment due to alzheimer’s disease people with mci due to ad have biomarker evidence of alzheimer’s brain changes plus new but subtle symptoms such as problems with memory, language and thinking. these cognitive problems may be noticeable to the individual, family members and friends, but not to others, and they may not interfere with individuals’ ability to carry out everyday activities. mild alzheimer’s dementia in the mild stage of alzheimer’s dementia, most people are able to function independently in many areas but are likely to require assistance with some activities to maximize independence and remain safe. handling money and paying bills may be especially challenging, and they may need more time to complete common daily tasks. they may still be able to drive, work and participate in their favorite activities. moderate alzheimer’s dementia in the moderate stage of alzheimer’s dementia, which is often the longest stage, individuals experience more problems with memory and language, are more likely to become confused, and find it harder to complete multistep tasks such as bathing and dressing. they may become incontinent at times, and they may start having personality and behavioral changes, including suspiciousness and agitation. they may also begin to have problems recognizing loved ones. severe alzheimer’s dementia in the severe stage of alzheimer’s dementia, individuals’ ability to communicate verbally is greatly diminished, and they are likely to require around-the-clock care. because of damage to areas of the brain involved in movement, individuals become bed-bound. being bed-bound makes them vulnerable to physical complications including blood clots, skin infections and sepsis, which triggers body-wide inflammation that can result in organ failure. damage to areas of the brain that control swallowing makes it difficult to eat and drink. because of this, food particles may be deposited in the lungs and cause lung infection. in contrast to ad dementia, well-tolerated progressive slower processing, memory loss particularly related to recent events, more trouble multitasking, slight cognitive decline, sleep disorder, emotional changes, slight or moderate depression, and bilateral brain activation for memory functions developing around the sixties are all consistent with “normal brain aging”. neuropathological alterations in normal old-aged individuals are nfts in the hippocampus, entorhinal cortex, and inferior temporal cortex, and very rarely in the frontal neocortex; the distribution of sps, if present, is more heterogeneous (29-35). clinical and neuropathological criteria to identify borderline cases between ad and cognitive impairment due to normal brain aging yielded only a limited consensus (36, 37). a few years later, cerad proposed a neuritic plaque score based on the number of plaques per mm2 and the age of the individual to categorize ad in comparison to normal brain aging (38, 39). evidence of a clinical progression and post-mortem neuropathological observations showing a concatenation of ad-related changes in old age and sad (29-32, 40-43) prompted a clinical redefinition of ad at the beginning of the second decade of this century. a crucial approach was the combination of clinical criteria, biochemical biomarkers in body fluids, and neuroimaging techniques to define the diagnosis of preclinical ad, mild cognitive impairment (mci) due to ad, and ad (44-50). more precise clinical definitions have been proposed to categorize different stages of ad (51). the classification shown in box 1 is a summarized transcription of the alzheimer’s association report: 2022 alzheimer’s disease facts and figures (https://www.alz.org/media/documents/alzheimers-facts-and-figures.pdf). the american academy of neurology estimates that mci is present in about 8% of people age 65 to 69, in 15% of 75to 79-year-olds, in 25% of those age 80 to 84, and in about 37% of people 85 years of age and older. about 7.5% will develop dementia in the first year after diagnosis of mci; about 15% will develop dementia in the second year; about one third will develop dementia due to ad within five years (52, 53). the prevalence of dementia in 65-69-year-olds is approximately 0.01% of individuals; the prevalence of dementia doubles with increments of five years; thereby, between 25% and 50% of individuals over the age of 85 suffer from dementia (2). it is estimated that between 50% and 80% of cases with dementia have ad (2). age, gender, race, living conditions, and genetic factors mark differences in the duration of preclinical and dementia stages in sad (54, 55). 2. β-amyloid and tau electron microscopic studies revealed that nfts were composed of paired helical filaments (phfs) that disrupted the architecture of the cyto-skeleton. sps were forged from a core of compact fibrils consistent with amyloids surrounded by dystrophic neurites filled with altered mitochondria, vesicles, numerous pleomorphic residual bodies, and phfs (56-61) (figure 3). figure 3: electron microscopy of an sp showing the central core of amyloid fibrils (asterisk) and peripheral dystrophic neurites (black arrows) filled with vesicles, dense bodies, abnormal mitochondria, and paired helical filaments; bar = 5μm. 2a. β-amyloid (aβ) subsequently, molecular studies identified β-amyloid as the main component of cerebral amyloid in β-amyloid angiopathy and sps (62-66). the amyloid precursor protein (app) is a transmembrane protein which modulates brain cell adhesion, synaptic plasticity, and multiple intracellular signaling through the small endodomain of the molecule. app processing is regulated by cytoplasmic phosphorylation (67). cleavage of app occurs through the combined action of α-, β-, and δ-secretases. β-secretase (bace) is a gpi-anchored aspartyl protease (68). γ-secretase is a coprotein complex mainly composed of presenilin 1 (psen1) and presenilin 2 (psen2); components of the γ-secretase complex aph-1 homolog a; γ-secretase subunit (aph1a); aph1b; nicastrin (nct/ncstn); and presenilin enhancer γ-secretase subunit (pen2/psenen), together with the modulators neprilysin (nep/mme) and insulin-degrading enzyme (ide). the γ-secretase complex is considered the “proteasome of the membrane” because of its capacity to act as a protelytic enzyme on more than 90 substrates (69-71). cleavage of app through αand δ-secretase leads to the non-amyloidogenic pathway of app degradation, whereas the combined action of βand δ-secretases generates small truncated c-teminal peptides at positions 42 (aβ1-42 or aβ42) or 40 (aβ1-40 or aβ40), depending on the thickness of the membrane, and many other small forms are amyloidogenic as well (72-75). local cholesterol content affects the various secretase activities (76), including cholesterol derived from astrocytes (77). low physiological concentrations of aβ seem necessary for long-term potentiation induction and for memory formation, probably acting on camp and cgmp (78). however, in aging and ad there is not only abnormal production of β-amyloid. aβ is aggregated and accumulates in the extracellular space due to its hydrofobicity, facility for oligomerization, and transformation from an α-helix to a β-sheet conformation (66, 73, 74). several enzymes can degrade β-amyloid such as neprilysin, plasmin, endothelin-converting enzymes, angiotensin-converting enzymes, insulin-degrading enzyme, several matrix proteinases, and cathepsins a and b (79, 80). soluble aβ is drained across the lymphatic wall, binding to low-density lipoprotein receptor-related protein (lrp-1). the expression levels of some of these enzymes and transporters are reduced in ad (81-85). impaired lymphatic drainage and altered blood vessel walls impair the elimination of soluble β-amyloid via the circulatory system (86, 87) (see section 13). astrocytes followed by neurons are the main source of clusterin in brain; clusterin is then released to the extracellular space (88). clusterin expression is increased in ad (87, 88), and co-localizes with β-amyloid deposits (91), more specifically with aβ1-40 (92). clusterin may act as an extracellular chaperone (93) and it contributes to early stages of β-amyloid plaque pathology (94). in addition to being involved in aβ aggregation and clearance and in the modulation of aβ transport across the blood brain barrier (bbb) (95-97), clusterin is also known to reduce aβ toxicity (98, 99). in addition, clusterin seems to interact with bridging integrator protein 1 (bin1) and tau (100). the main β-amyloid that circulates in brain interstitial fluid and cerebrospinal fluid (csf) is soluble aβ40. β-amyloid deposits in ad are categorized as primitive or immature plaques, mature or neuritic plaques (classical sps), compact or burned-out, cotton-wool plaques, diffuse plaques, subpial β-amyloid deposits, β-amyloid angiopathy, and perivascular plaques (dyshoric angiopathy) (figure 4). β-amyloid can also be found in the cytoplasm of neurons, and in astrocytes at the periphery of sps. β-amyloid is composed of a mixture of peptides of different molecular weight: aβ40 and aβ42 are predominant in sps, while aβ40 is mainly located at the core of sps and aβ42 at their periphery. diffuse plaques contain aβ42 and truncated forms aβ17-42. subpial β-amyloid deposits are mainly composed of amino-terminal truncated species. β-amyloid species have different aggregation properties. n-terminal truncated aβ with pyroglutamate modification at position 3 and aβ phosphorylated at serine 8 show enhanced aggregation into oligomers and fibrils. these forms appear at late stages (biochemical stages 2 and 3) of β-amyloid formation, whereas soluble and insoluble aggregates composed of non-modified aβ are found at early stages (stage 1) (101, 102). figure 4: β-amyloid deposits in the temporal cortex. paraffin section, β-amyloid immunohistochemistry, slight hematoxylin counterstaing, bar = 50μm. soluble β-amyloid oligomers (aβos) and amyloid-β derived diffusible ligands (addls), acting through specific cell surface receptors rather than fibrils, are toxic and cause neurodegeneration (103-115). high-molecular-weight β-amyloid oligomer levels are elevated in the csf in ad (116). yet in the cerebral tissue, the ratio of aβ oligomer levels to plaque density distinguishes demented from non-demented patients (117). several membrane receptors can bind to aβ oligomers. these receptors include the cellular prion protein (prpc); the α7 nicotinic acetylcholine receptor (α7nachr); fcγ receptor ii-b (fcγriib); the p75 neurotrophin receptor (p75ntr); the paired immunoglobulin-like receptor b (pirb); the pirb human orthologue receptor (lilrb2); the β-adrenergic receptors (β-ars); and the eph receptors (118), among others. prpc is one of the binding partners for aβ oligomers (119-125), and prpc mediates impairment of synaptic plasticity by aβ oligomers (124). besides β-amyloid species, several molecules are also components of sps including metal ions, lipids, mucopolysaccharides, immunoglobulins, members of the complement system, molecules linked to lipid metabolism and lipid transport, blood coagulation/haemostasis factors, proteins linked to metabolism and molecular transport, neural, cell adhesion and extracellular matrix proteins, proteoglycans, and other cellular proteins (126, 127). the large amount of proteins in sps is likely the consequence of co-aggregation and alteration of associated biochemical processes by which β-amyloid formation leads to neurodegeneration (127). moreover, prpc co-localizes with aβ in sps (128). β-amyloid plaques are associated with variable alteration of neuronal processes, reactive astrocytes, and microglia. altered synaptic protein deposition with a granular pattern is found in diffuse plaques (129). neurotransmitter-containing and peptidergic dystrophic neurites precede those containing paired helical filaments within sps (130-132). altered neuronal structure, accumulation of abnormal molecules, and abnormal organelles and debris are characteristic of dystrophic neurites of mature sps (133, 135). in addition to synaptic proteins, components of dense-core vesicles accumulate in dystrophic neurites of sps (129, 135-138). immunohistochemical studies have shown that dystrophic neurites of sps contain 3rtau and 4rtau; several phospho-tau species; map2-p; phosphorylated neurofilaments light; medium and heavy chains; and active kinases p38, sapk/jnk, gsk3β, and ck1-δ, in addition to markers of the ubiquitin-proteasome system (ups) and autophagy (139). mitochondria are altered in dystrophic neurites of sps with variable vulnerability of the mitochondrial complexes of the respiratory chain (140, 141). dystrophic neurites are likely derived from axons arising from diverse neuronal populations, as revealed by specific neuronal markers (130-132, 142-146) therefore indicating that neuronal vulnerability is not restricted to a single cellular population. pyramidal cells in the vicinity of sps show distorted dendrites and loss of dendritic spines (147-149). 2b. tau the microtubule-associated protein tau, encoded by mapt, participates in microtubule stability, cellular polarity, and anterograde and retrograde axonal transport of organelles and vesicles. in addition to microtubules and actin, tau interacts with a large number of proteins and lipids in the cytoplasm, cell membranes, and synapses, and with dna and proteins involved in dna protection, among many other substrates (150, 151). the various functions of tau require interaction with multiple partners (151-157). figure 5: neurofibrillary tangles in the ca1 region of the hippocampus. paraffin section, at8 immunohistochemistry, slight haematoxylin counterstaining, bar = 25μm. the main constituent of nfts dystrophic neurites of sps and neuropil threads is abnormal tau (158-173) (figures 5, 6, and 7). a combination of all six hyper-phosphorylated brain tau isoforms (3rtau and 4rtau expressed in brain), generated from alternative tau splicing, is characteristic of ad tau (163, 174, 175). the amount of 3rtau is similar to 4rtau in the human adult brain and in ad. however, possible variations in the ratio of 3rtau/4rtau among cell types in the human brain have not been adequately assessed. abnormal tau in ad includes several species resulting from hyper-phosphorylation at different sites, acetylation, glycosylation, altered confor mation, truncation at glutamic acid 391 and at aspartic acid 421 (mediated by caspase 3), oligomerization, and β-sheet-rich fibril aggregation, among others (171-173, 176-196). the site of tau phosphorylation and other post-translational modifications in tau have commonalities and differences among tauopathies (197, 198). tau inclusions in glial cells are not found in ad, unless accompanied by other tau co-morbidities including aging-related tau astrogliopathy (artag) and argyrophilic grain disease (agd) which are 4rtau-only tauopathies. figure 6: dystrophic neurites of sps in the entorhinal cortex containing hyper-phosphorylated tau. paraffin section, at8 immunohistochemistry, slight haematoxylin counterstaing, bar = 25μm figure 7: immunoelectron microscopy showing phospho-tau deposits (black dots) in paired helical filaments. at8 antibody, bar = 0.2μm. tau hyper-phosphorylation, the first step in nft formation, is geared by the activation of specific kinases, and probably also by accompanying inhibition of phosphatases (171, 199). several kinases are implicated in both the physiological and the pathological phosphorylation of tau, including glycogen synthase kinase 3β (gsk3β); cyclin-dependent kinase 5 (cdk5); protein kinase a (pka); jun n-terminal kinase (jnk); p38; and others (200). co-localization of selected active kinases and tau deposits can be visualized in brain tissue (201-203). g-protein-coupled receptor (gpcr) kinases are also associated with nfts and β-amyloid plaques in ad (204). these alterations are cumulative but not homogeneous. more than one tau species may be present in a particular neuron. furthermore, distinct defects may result depending on the type of accumulated tau, ranging from reversible dysfunction to irreversible disruption of the cytoskeleton, altered axonal transport, undermined cell signaling, synaptic dysfunction, and cell death. tau-linked alterations can be the direct result of toxic species or the interactions of multiple partners (156). soluble and insoluble tau oligomers, both phosphorylated and non-phosphorylated, may be involved in neurodegeneration (191, 205). the association of tau with the plasma membrane is determined by its phosphorylation pattern. tau associated with the plasma membrane can move to the cytosol upon tau hyper-phosphorlation (206, 207). the phosphorylation of tau also depends on phosphatidyl choline and phosphatidyl serine (208). therefore, the composition of lipids at the membrane may modify the phosphorylation of tau and its capacity to shift its binding with actin and cytosolic proteins (209-211). tau interactions with the membrane have several implications (155, 212). in addition to stabilizing membrane-cytosol interactions, tau is secreted associated to vesicles, or vesicle-free, key features in tau transmission (213-215). morphologically, abnormal neuronal tau deposits in ad are manifested as perinuclear tau deposits, granular cytoplasmic deposits, diffuse cytoplasmic deposits (all considered pre-tangle stages), neurofibrillary tangles (classical nfts), ghost tangles (remains of nfts in the neuropil), dystrophic neurites of sps, and neuropil threads. the redistribution of abnormal tau from axons to the somatodendritic compartment of neurons and dendritic spines is a characteristic consequence of tau pathology in ad and other tauopathies. phfs induce tau accumulation into aggresomes that gather misfolded proteins when the protein degradation system is overloaded (216). the structure of tau filaments in the different tauopathies largely depends on tau composition (3rtau and 4rtau) and on post-translational modifications including conformation and truncation, as revealed by transmission electron microscopy, and more recently by optimized cryo-electronmicroscopy and mass spectrometry (193, 217-222). the different structure of tau aggregates in tauopathies indicates the formation of different tau strains which are specific to each tauopathy (223). the accumulation rate of tau aggregates is greater in females and younger β-amyloid-positive subjects (224). increased expression of 19 genes in chromosome x is associated with tau burden and slower cognitive decline in women but not in men, suggesting that specific x chromosome factors could confer risk or resilience in aging and ad (225). in addition to abnormal tau, nfts contain numerous proteins. total tau interacts with a good number of proteins in ad (226, 227). laser-capture micro-dissection of nfts and liquid chromatography-/tandem mass spectrometry (lc-ms/ms) analysis in sad followed by affinity purification mass spectrometry revealed that seventy-five proteins present in nfts interacted with phf1-immunoreactive phosphorylated tau (228). nfts also contain markers of the sequestosome/p62, ubiquitin, and mutant ubiquitin (229, 230). increased prpc expression downregulates tau protein (231-234). conversely, reduction or ablation of prpc levels induces an increase in tau 3rtau/4rtau balance through downregulation of gsk3β activity, thus indicating that prpc plays a role in tau exon 10 inclusion through the inhibitory capacity of gsk3β (235). increased prpc levels at early and middle stages of nft pathology yields lower tau and hosphor-tau. in contrast, prpc levels decrease at advanced stages of nft pathology, which correlates with increased amounts of tau and hosphor-tau. taken together, these observations suggest a protective role for prpc in early stages of ad (236). these observations linking an interaction of prion protein and tau may have implications in certain familial prion diseases grouped under the term gerstmann-sträussler-scheinker disease, in which abundant prpres-amyloid deposits are accompanied by extensive tau pathology (see section 3). 3. familial ad (fad; early-onset familial alzheimer’s disease: eofad), and the β-amyloid cascade hypothesis from the early nineties, mutations in the genes app (β-amyloid precursor protein), psen1 (presenilin1), and psen2 (presenilin2), all of them involved in the production of β-amyloid, have been identified in several families with pre-senile dementia of alzheimer’s type (early-onset familial alzheimer disease: eofad, or fad); increased app dosage was also causative of ad and β-amyloid angiopathy (237-244). recent genetic studies of the first alzheimer’s case identified that the patient carried a mutation in psen1. these groundbreaking discoveries led to the “β-amyloid cascade hypothesis”, which supports the idea that the production of β-amyloid species is the primary factor triggering nft formation and ad progression (245). the amyloid cascade hypothesis was further supported by the production of β-amyloid in transgenic mice bearing human mutations causative of ad. yet mutations in these genes did not result in nft formation in transgenic mice, although a few small hyper-phosphorylated tau deposits did appear in dystrophic neurites around β-amyloid plaques. at most, the joint production of sps and tau deposits similar to nfts in mice requires the cumulative expression of different mutated genes involved in human ad and tauopathies (246-248). however, in vitro and in vivo studies have shown the capacity of β-amyloid to phosphorylate tau and enhance tau aggregation, thus giving a boost to the β-amyloid cascade hypothesis (249). the β-amyloid cascade hypothesis fits with fad linked to mutations of app, psen1, and psen2. another genetic condition linked to increased risk of ad is down syndrome. middle-aged individuals (ma) with down syndrome have neuropathological lesions of ad. amyloid deposits may start as early as 12 years of age and they are universal by the age of 31. nfts appear later in the entorhinal cortex, hippocampus, and neocortex (250-253). the discovery of β-oligomers and cumulative evidence of their toxicity has led to modification of the “β-amyloid cascade” hypothesis, leading to the “amyloid-β oligomer hypothesis” (103-108). according to the new proposal, it is not the presence of fibrillar β-amyloid and deposition into definite aggregates, but rather soluble β-oligomers that are causative of cell damage and that trigger the process of neurodegeneration in ad (103-106, 254-257). however, several points are still obscure. the level of insoluble aβ rises with age and is further increased in ad whereas the total level of aβ40 in both soluble and insoluble fractions and the level of aβ42 in the soluble fraction decline with age before about 50 years. differential production or retention of aβ40 and aβ42 likely contributes to the influence of age on the risk of sporadic ad, but the levels of soluble aβ concentrations, higher in young adults than in older individuals and in subjects with ad, do not match the proposed toxic role of oligomers in ad (258, 259). tau deposits, other than those located in dystrophic neurites of sps, are largely independent of β-amyloid. other factors, including apolipoprotein e (apoe), the endocytic system, cholesterol metabolism, and microglial activation, are regulators of tau pathology (260). neurons derived from induced pluripotent stem cell (ipsc) lines from sad and fad linked to psen1 mutations show increased phosphorylation of tau at different sites, increased levels of active gsk3β, and a significant upregulation of app synthesis and app carboxy-terminal fragment cleavage. however, significantly increased aβ1-42/aβ1-40 ratios are observed in fad but not in sad (261). other amyloids are the main constituents, in combination with nfts, of different genetic neurodegenerative diseases causing dementia. familial british dementia (fbd) and familial danish dementia (fdd) are linked to specific mutations in the bri2 gene; the cleavage of integral membrane protein 2b (bri2) produces abri and adan amyloidogenic peptides, respectively. amyloid plaques and amyloid angiopathy, and nfts with a tau composition identical to ad tau, are found in both diseases (262, 263). gerstmann-sträussler-scheinker disease (gss) is linked to mutations in the prion protein gene (prnp) that cause a prionopathy. depending on the muta-tion, gss is manifested pathologically by a combination of abundant prion-immunoreactive plaques surrounded by dystrophic neurites, together with numerous nfts indistinguishable from ad-nfts (264-266). interestingly, app, bri2, and prion are proteins located at the cell membrane, and they interact with each other in normal conditions. the non-fibrillar, soluble bri2-derived amyloids are also toxic, and probably play a central role in the pathogenesis of bri2-linked dementias (267). the common structure of soluble amyloid oligomers suggests a common mechanism of pathogenesis (109, 113). despite the differing genetic nature of these disorders, plaques and nfts do not appear until middle age. understanding of the mechanisms that control the metabolic pathways, that delay the beginning of the molecular and clinical manifestations of the disease for years, is a major challenge in neurodegenerative diseases linked to mutations in specific genes. in contrast to mutations linked to β-amyloid production, mutations in mapt are causative of familial tauopathy and are never associated with β-amyloid or other amyloid deposits (170, 171). 4. sporadic ad (sad; late-onset alzheimer disease: load) most cases of ad (more than 95%) are sporadic (sad) and occur in older individuals (late-onset alzheimer’s disease: load). sad has an insidious onset and a progressive course leading to death about 10-15 years after the first clinical symptoms of dementia. aging is the main contributory factor. sad is favoured by individual or combined low penetrating genetic factors, mainly allele ε4 of apoe (268-271). genome-wide association studies (gwas) have identified other risk genes of sad: ldl receptor related protein 1 (lrp1); low density lipoprotein protein receptor 1 (ldlr); interleukin 1a; clusterin (clu); phosphatidylinositol binding clathrin assembly protein (picalm); complement component (3b/4b) receptor 1 (cr1); bridging integrator 1 (bin1), involved in synaptic vesicles and endocytosis; triggering receptor expressed on myeloid cells 2 (trem2); sortilin-related receptor 1 (sorl1), involved in endocytosis and sorting; adam metallopeptidase domain 10 (adam10), involved in the cleavage of several proteins; atp binding cassette subfamily a member 7 (abca7); spi-1 proto-oncogene (spi1); paired immunoglobin like type 2 receptor alpha (pilra); membrane-spanning 4-domains subfamily a (msa4), linked to inflammation; cd2-associated protein (cd2ap) that regulates actin cytoskeleton; and ephrin receptor a1 (epha1), among others (272-288). the regional and areal distribution of nfts and sps in the cerebral cortex is not homogeneous. in the hippocampal complex, nfts predominate in the ca1 region and subiculum, the ca2, ca3 and hilus are less affected, and the dentate gyrus is spared in pure sad cases. in the entorhinal cortex, nfts are more abundant in layers ii and v, whereas in the neocortex, nfts predominate in layers iii and v, with marked regional variations (the primary motor and sensory cortices have fewer nfts than the association areas). nfts are found more abundant in the temporal cortex, followed by the frontal and parietal cortex, and the occipital cortex. in subcortical regions, nfts are localized in the basal nucleus of meynert and nuclei of the basal forebrain, amygdala, hypothalamic nuclei, relay neurons within intralaminar and limbic thalamic nuclei, ventral tegmental area, raphe nuclei, locus ceruleus, and olfactory bulb. the cerebellar cortex is spared of nft pathology. cortical neurons with nfts are mainly subpopulations of large pyramidal glutamatergic neurons (289-291). this is consistent with the observation that neurons with high content of neurofilaments are more susceptible to nft formation (290-295). gabaergic neurons are more resistant to nft pathology, although the density of gabaergic neurons decreases and gaba-uptake is impaired in sad (296-300) (see section 6 for details). somatostain, which is expressed in a subpopulation of inhibitory neurons, and somatostatin receptors are also reduced in sad (296, 301, 302). calcium-binding proteins parvalbumin (pv), calbindin d28k (cb) and caretinin (cr) are expressed in subpopulations of gabaergic neurons (303-305). pv-positive neuron numbers in the temporal, visual, and prefrontal cortex are preserved in ad (306-309), but pv-immunoreactive neurons are decreased in the entorhinal cortex and hippocampus in sad (310-316). cb-immunoreactive neurons in the hippocampus, entorhinal cortex, and cortical layers v and vi are vulnerable, whereas cb-positive neurons in the occipital cortex and upper layers of the frontal cortex are resistant (309, 314, 314, 317). cr-positive neurons are not affected in the prefrontal, temporal, and visual cortices (309, 319, 320), but their number is reduced in the hippocampus and entorhinal cortex (314, 321). pv and somatostin, together with neuropeptide y, cholecystokinin and substance p, are found in dystrophic neurites of sps, thus evidencing the involvement of inhibitory and peptidergic neurons in sps (130, 144). neuron loss is negligible in cognitively normal subjects, but the number of neurons decreases in the hippocampus and entorhinal cortex with nft progression (322-324). the rate of this process is extremely variable among individuals. a major achievement in improving our understanding of the progression of sad pathology was the staging of nfts and sps in the post-mortem brain of large cohorts of non-demented and demented individuals covering a natural human population. in the telencephalon, the first nfts appear in the entorhinal and transentorhinal cortex (stages i-ii), followed by the hippocampus, temporal cortex, and other nuclei of limbic system (stages iii-iv), and then continue on to most areas of the neocortex (stages v-vi). the spreading of nfts is accompanied by a dramatic increase in the number of neurons with nft pathology across stage progression (41, 325-327). about 85% of individuals aged 65 have nft pathology, at least restricted to stages i-iii (41, 325, 328, 329). all of them, excluding those having concomitant pathologies, are considered “cognitively normal for age” (330). some individuals at stage iv-v suffer from moderate cognitive impairment; only about 5% have dementia. however, dementia of ad type accounts for about 25%-30% of the population at the age of 85 years, all of them categorized as nft stages v-vi (331). regionaland stage-dependent neuropathological alterations in sad are accompanied by specified patterns of altered gene expression, that extend beyond the genes implicated in tau and β-amyloid pathology (332). the braak staging scheme does not rule out the occurrence of exceptions that do not fulfil the strict neuropathological criteria. these untypical cases are considered ad subtypes: hippocampal sparing, limbic-predominant, and minimal atrophy sad subtypes might account for about 25% of cases (333). in addition, several clinical sad variants including non-amnestic, corticobasal syndromal, primary progressive aphasia, posterior cortical atrophy, behavioral/dysexecutive, and mild dementia variants have been categorized (334). the olfactory bulb and tract, and several nuclei of the brain stem including the raphe nuclei and the locus coeruleus, are affected by nft formation at the first stages of nft pathology. the involvement of the olfactory bulb and tracts may contribute to the altered olfaction arising in sad. damage to selected nuclei of the brainstem, which are the origin of major serotoninergic and noradrenergic innervation of the entire brain, underlies a large series of clinical symptoms including impaired arousal, loss of attention and memory, impaired decision making, apathy, depression, anxiety, and altered reward processing, among others (335-340). considered together, nft generation and neuron loss largely depend on the specific cell and regional vulnerability of specific neuronal populations. moreover, the simultaneous presence of nft lesions in separate brain regions indicates that there is no single origin of nft pathology that spreads through the brain, but rather various and cumulative original sources of tau pathology in the aging brain. additionally, the rates of nft progression, although variable from one individual to another, appear slowly at early nft stages and progress rapidly at advanced stages of the disease (41). the distribution of sps differs from nfts in the cerebral cortex in patients with sad (341). assessing the same series of cases for the study of nft progression evidenced that the localization and distribution of sps largely differ from nft staging. the majority of individuals at nft stages i-ii and almost half of those at stage iii do not have sps or β-amyloid deposits (41, 342). stages 0, a, b, c of braak define the progression of sps through the neocortex. the phases of thal represent, from phase 1 to 6, the progressive and cumulative appearance of sps from the neocortex, allocortex, diencephalic nuclei, striatum, and cholinergic nuclei of the basal forebrain, the brainstem, and the cerebellum (343). the early appearance of tau pathology compared with the later appearance of β-amyloid plaques in a series of 2366 cases from children to centenarians has been recently revisited (344). based on the results of these observations and many previous studies, the paper hypothesizes that tau pathology is an initiating factor in sad (344). indeed, the lack of temporal and regional concordance between nfts and sps in sad is intuitively barely consistent with the β-amyloid cascade hypothesis, unless non-identified soluble or other species of β-amyloid interact with neurons, thus triggering nft pathology (345). these comments do not mean that there is no interaction between the two proteins. tauopathy fuelled by β-amyloid in a synergetic mechanism is well documented in ad (346-348). the arguments between supporters of β-amyloid and of tau as the primal origin of sad have consumed a great deal of effort, time, and financial investment (349). there is no doubt about the new acquisition of knowledge generated regarding sad pathogenesis, but exclusive hypotheses have not produced the anticipated unequivocal results. cognitive impairment and dementia correlate with tau deposition and nft pathology rather than with β-amyloid deposits and sps (331, 350-358). neuron loss occurs largely in parallel with tau pathology rather than with sps in most regions (359). however, neuron degeneration and neuron loss are not restricted to neurons with nfts (see section 10). recently, a classification of ad has been proposed: ad autosomal dominant (fad), apoeε4 sad, and non-apoeε4 sad (360). this categorization is not new, but rather recovers and further emphasizes the well-known relative importance of β-amyloid deposition in the different ad categories depending on genetic factors involved in the production of β-amyloid. 5. nfts and sps in non-human brain aging β-amyloid plaques and β-amyloid angiopathy may be found in old-aged animals in some species including non-human primates, monkeys, dolphins and other cetaceans, dogs, cats, bears, and pinniped species, among others; deposits are usually diffuse whereas core plaques surrounded by tau-containing dystrophic neurites are exceptional (361-372). phosphorylated-tau deposits in neurons are rarely encountered in most mammals, and they usually have the characteristics of pre-tangles rather than nfts, as in a few vulnerable aged mouse lemurs (362). intracytoplasmic tau inclusions in neurons, astrocytes, and oligodendrocytes may occur in aged baboons (373, 374), aged gorillas (369), and chimpanzees (371). tau accumulation in the brain of old sea lions, seals, and walruses forms argyrophilic fibrillar 3rtau and 4rtau aggregates in the neuronal somata and neurites, and olny few tau aggregates are found in oligodendrocytes and microglia (372). importantly, these changes are linked to aging, but they are not the only expression of brain aging (375, 376). hyperphosphorylated tau accumulation in neurons, intraneuronal β-amyloid deposits, and diffuse amyloid plaques may occur in the brain of aged domestic cats (377, 378). the characteristics and distribution of tau lesions in a few cats are reminiscent of sad including the deposition of 4rtau and 3rtau (379). a unique 4rtauopathy without β-amyloid deposits mainly involving neurons of the neocortex but not the hippocampus, accompanied by widespread coiled bodies in the cerebral white matter, has been reported in aged domestic cats (380). these observations show that β-amyloid deposition and tau pathology may occur with high species variability, in aged mammals, and, particularly, in non-human primates and pinnipeds. however, we do not have evidence at present on whether these species show changes in the same way as human beings. in aged cynomolgus monkeys, β-amyloid plaques combine with 4rtau deposits in pre-tangle neurons and coiled bodies in glial cells with a regional pattern reminiscent of progressive supranuclear palsy (370). therefore, the old hypothesis suggesting that sad is a phylogenetic disease (381) has a relative relevance unless applied to the search for mechanisms modulating similarities and differences between non-humans and humans regarding the tremendous prevalence, widespread localization, particular regional distribution, and composition and structure of tau deposits in humans in comparison with other species. 6. synapses synaptic alterations were described in the 1960s in the seminal electron microscopic studies of ad (382). these findings were followed by the observation of decreased numbers of dendritic spines on cortical neurons assessed with the golgi method in post-mortem and biopsy samples at a time when cerebral biopsies were still considered appropriate tools for diagnosis of dementia (383-389). synaptic loss is the major morphological correlate of cognitive impairment (390). for this reason, ad is considered as the consequence of a synaptic failure (391). subsequent studies have refined synaptic alterations using different methods (392; 393), including the use of intraneuronal dyes in post-mortem tissues (394, 395). the golgi method also provided evidence of dendritic degeneration and dendritic sprouting and re-growth in several brain regions in ad (396-401). dendritic sprouting is reinforced by the presence of growth-associated protein 43 (gap-43), a marker of neuritic growth and sprouting around sps (143, 402). aberrant sprouting seems to be triggered by pre-amyloid species and neurotrophic factors (401). aberrant sprouting involves neurites, dendrites, and synapses, and it affects distinct connections in ad (401). cycles of aberrant synaptic sprouting and neurodegeneration are common in ad (403). spine loss occurs mainly in clusters linked to tau pathology (404, 405). immunohistochemistry also reveals altered expression of synaptic markers not only around sps but also in diffuse plaques, suggesting a close relationship between synapses and β-amyloid deposition (129, 406, 407). abnormal preand post-synaptic tau and tau oligomers damage the synapses and produce altered synaptic function (150, 408, 409). double-labeling of neurons also shows a direct relationship between tau deposition and loss of dendritic spines on cortical pyramidal neurons in ad (404). abnormal tau and β-amyloid oligomers act synergistically to disrupt synaptic function (409). however, synaptic loss also appears not to be dependent on fibrillar β-amyloid in a murine model of β-amyloid deposition (149). abnormal neuronal expression of app and cytoskeletal proteins in early stages of the disease might be involved in the mechanisms of synaptic pathology in ad (410). synaptic proteins are important components associated with β-amyloid in sps (127). it has recently been postulated that altered synapses are the origin of amyloid plaques in ad (411; see also section 2a). both β-amyloid and abnormal tau are accumulated at the synapses (412-421). recent neuroimaging studies further support the association of tau pathology, synaptic loss, and altered synaptic function (422). it has been proposed that synaptic tau pathology is an early event, and synaptic tau seeding precedes tau pathology in sad (423). other factors are also important such as cytoskeletal actin dysregulation (424), and oxidative stress lipid and protein damage (425). no less significant is the association between cell-cycle dysfunction and failure of synaptic plasticity in ad (426). synaptic alterations include abnormalities in the synaptic and postsynaptic delivery of neurotransmitters and neuromodulators, and the selective vulnerability and responses of their receptors (see section 7). finally, lipid and protein alterations at the cell membrane, and altered cytoskeletal proteins, may affect synaptic integrity and function (see sections 22c and 22h). in addition, synapses are organelles with high energy consumption, and therefore they are vulnerable to deficits in energy production linked to mitochondrial failure (427) (see section 22d). 7. neurotransmitters, neuromodulators, and related receptors an early relevant biochemical observation was the discovery of the involvement of the meynert nucleus in ad, the correlation of this involvement with the number of plaques and cognitive impairment, and the accompanying impairment of cholinergic innervation in the cerebral cortex (428-430). the “cholinergic hypothesis” stated that ad was a disorder of cholinergic innervations (430, 431). the enthusiasm for the cholinergic theory was supported by the prior discovery of dopamine deficiency in the substantia nigra pars compacta in parkinson’s disease, and the success of l-dopa treatment for this disorder which is still in use 50 years later (432). cholinergic drugs were used although their benefits were clearly lower than initially expected. later, the glutamatergic theory stated that excitotoxicity resulting from excessive synaptic or extrasynaptic activation of n-methyl-d-aspartate (nmda) subtype of ionotropic l-glutamate receptors might enhance vulnerability of neurons in ad (433, 434). the role of glutamate in the pathogenesis of ad was driven, in part, by the discovery of altered glutamate transport and increased excitotoxicity in amyotrophic lateral sclerosis (435), and the interest at that time in excitatory amino acid neurotoxicity in the pathogenesis of neurodegenerative diseases (436). glutamate overload increases mitochondrial ca2+ influx and oxidative stress and leads to mitochondrial dysfunction (437). however, nmda receptor blockers may also have undesirable effects due to their double effects on cell death as well as cell survival and plasticity (438). now, neuroprotective therapies aim to both enhance the effect of synaptic activity and disrupt extrasynaptic nmdar-dependent death signaling (438). cholinergic and glutamatergic neurotransmitter alterations play a significant role in the pathogenesis of brain aging and sad (439-442). other neurotransmitters and receptors are involved as well. the important point is that alterations are not homogeneous; they depend on the type of neurotransmitter, the cells of origin, and the kind of receptor; not all receptors of a given neurotransmitter are equally vulnerable to aging and sad. in addition to the neurotransmitters, neuromodulators, and receptors discussed below, general aspects of gpcr, amylin receptors, netrin receptors, and dopamine receptors in sad are detailed in other reviews (443, 444). endorphins, enkephalins, dynorphins, and endomorphins are endogenous opioid peptides that bind to opioid receptors. β-endorphin has opioid activity through μ-receptors, but α-endorphin and γ-endorphin lack affinity for opiate receptors. endorphins interact with the γaminobutyric acid (gaba), which in turn modulates the release of dopamine. the expression of endogenous opioids and receptors is altered in human brain aging and sad (445-448). many olfactory and taste receptors and molecules involved downstream are expressed in the human brain (449). their ligands and functions remain unknown, although both olfactory and taste receptors might contribute to intercellular and intracellular cell signaling. the expression of some olfactory receptors is altered in sad and other neurodegenerative diseases (450). 7a. acetylcholine (ach) and acetylcholine receptors (achr) ach is synthesized in neurons by choline acetyl transferase (chat). ach acts upon nicotinic acetylcholine receptors (nachrs) and muscarinic acetylcholine receptors (machrs). ach is degraded by acetylcholinesterase (ache). nicotinic receptors are ionotropic ligand-gated receptors, and muscarinic receptors are gpcr (451). the nachrs are arranged into homomeric or heteromeric subunits consisting of a diverse set of complex subtypes including α1-7, α9-10, β14, γ, δ, and ε. allosteric modulation of nachrs increases pre-synaptic ach levels and enhances the cholinergic nicotinic neurotransmission. α7 and α4β2 nachr mediate the presynaptic release of ach. nachrs are also expressed in astrocytes and microglia (452, 453). through its receptors trka and p75ntr, the nerve growth factor (ngf) plays an essential role in the survival and maintenance of cholinergic neurons in the basal forebrain (454-457). loss of basal forebrain cholinergic neurons occurs at early stages of nft pathology (428, 458, 459). pretangle pathology within cholinergic nucleus basalis neurons coincides with local neurotrophic and neurotransmitter receptor gene dysregulation (460). loss of cholinergic neurons in the basal forebrain leads to reduced ach levels and chat upregulation (461). in addition, nachrs, particularly α7nachrs, are altered in sad (462-465). importantly, nicotine and nachrs also participate in the regulation of aβ. on the one hand, nicotine inhibits the formation of aβ1-42 fibrils and disrupts preformed aβ fibrils (466). on the other hand, aβ1-42 binds to α7nachr and inhibits the release of ach (467). finally, α7 nachrs mediate aβ-induced neurotoxicity (468, 469). and aβ-induced tau phosphorylation (470). α7nachrs also participate in microglial activation (471). astrocytic and microglial nachrs modulate aβ phagocytosis and degradation, aβ-related oxidative stress, and neurotoxicity (472). the metabotropic machrs are classified into five m1-m5 subtypes (473). m1, m3, and m5 receptors interact with the gq/11 protein, stimulate phospholipase c (plc), phosphatidylinositol trisphosphate (pi3p), and activate protein kinase c (pkc). m2 and m4 receptors interact with go/i proteins, inhibit adenylyl cyclase (ac) and protein kinase a (pka), and decrease camp levels (474-476). there are no changes in the number of machrs in sad; however, the interaction of gq/11 protein is altered in sad compared with controls (477). m1 muscarinic agonists reduce β-amyloid and tau pathology, whereas m1 muscarinic antagonists or deletion of m1 subtype augment β-amyloid and tau pathology in in vitro and in vivo murine models of ad (478-480). sps have low levels of ache, but the activity of ache increases around β-amyloid plaques (481, 482). ache inhibitors such as donepezil, galantamine, tacrine, and rivastigmine are administered at the initial stages of sad. 7b. glutamate and glutamate receptors (glurs) glutamate is released from synaptic terminals and acts on post-synaptic ionotropic glutamate receptors (iglurs). this mechanism mediates fast excitatory synaptic transmission. glutamate can also act on metabotropic glutamate receptors (mglurs). the mechanism modulates various effects by coupling to g proteins with subsequent recruitment of second messenger systems. there are three families of iglurs: nmda (n-methyl-d-aspartate receptor), ampa (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid), and ka (kainate) receptors: nmdar, ampar, and kar, respectively. nmdars are composed of different subunits encoded by glun1 (nr1), glun2a (nr2a), glun2b (nr2b), glun2c (nr2c), glun2d (nr2d), glun3a (nr3a), and glun3b (nr3b). ampars are composed of combinations of glua1 (glur1), glua2 (glur2), glua3 (glur3) and glua4 (glur4); and kars by gluk1 (glur5), gluk2 (glur6), gluk3 (glur7), gluk4 (ka-1) and gluk5 (ka-2). in nmdars, the binding of glutamate and glycine is necessary to activate glutamate-gated ion channels. the removal of magnesium ions (mg2+) block permits the entry of calcium ions (ca2+) and synaptic signaling (483, 484). both iglurs and mglurs are also localized pre-synaptically acting as auto-receptors and hetero-receptors. this localization facilitates neurotransmission in the short term and depresses neurotransmission in the long term (485). glurs are localized non-synaptically and are also expressed by astrocytes and oligodendrocytes (486). glutamate binding to nmdars, ampars, and kars is reduced in the aging brain and sad mainly in the cerebral cortex and hippocampus. these changes are receptor-, region-, and layer-dependent, thus indicating variable vulnerability. altered expression is likely dependent on several factors and not necessarily correlated with local nfts and sps, although loss of neurons and reduced neuronal connectivity may account, in part, for the decreased receptor expression (440). the localization of the receptors also plays a cardinal role. nmdars containing glun2a subunits are located at synaptic sites and are implicated in the protective pathways. in contrast, glun2b subunits are located mainly at extra-synaptic sites, and they increase neuronal vulnerability. β-amyloid activates glun2b-containing nmdars (487-489). in addition, nmdars are necessary for synaptic targeting of aβ oligomers (490) and neuronal aβ production (491). nmdar alterations are implicated in synaptic dysfunction in sad (492, 493). nmdars also participate in redox-mediated synaptic function impairment in brain aging and sad (494). memantine, an nmdar antagonist, is currently used at the middle clinical stages of ad to reduce the hyperactivity of glutamate, resulting in transient and limited success. ampars are consistently endocytosed. the increase in the rate of ampa endocytosis induces long-term depression and synaptic degeneration (495). soluble aβ oligomers are involved in synaptic damage via the subunit glua3 ampar (496). aβ also induces ampar ubiquitination and degradation (497). glutamate abnormalities in aging and sad are not restricted to alterations in glutamate production and its effects on synaptic receptors in neurons and glial cells. furthermore, glutamate effects in normal and pathological conditions also depend on glutamate transport by specific neuronal and glial transporters (498). excitatory amino acid transporters (eaats) re-uptake glutamate from the synaptic cleft and extra-synaptic sites, and transfer glutamate to glial cells and neurons. vesicular glutamate transporters (vgluts) move glutamate from the cell cytoplasm into synaptic vesicles. eaats can also transport l-aspartate and d-aspartate. eaat1 and eaat2 (slc1a3 and slc1a2, respectively) are localized in astrocytes whereas eeat3 (slc1a1), eaat4 (slc1a6) and vgluts 1/2/3 (slc17a7, slc17a6, slc17a8, respectively) are found in neurons. loss of eaat2 occurs in many neurodegenerative diseases, including sad (see section 12a). abnormal expression of vgluts and eaats may contribute to neuronal excitotoxicity and neuron demise in sad (499-501). mglurs are g-coupled proteins that act upon different effector systems, including plc and ac. mglurs are classified into three groups based on their pharmacological profiles, molecular properties, and transduction mechanisms. group i receptors (mglur1, mglur5) are coupled to plc activation through gq/11 proteins, whereas groups ii and iii are coupled to ac inhibition through gi/o proteins. mglur1/5 are primarily excitatory, and mglur2/3 and mglur4/5/6,7/8 are inhibitory (502-505). mglurs, determined by radioligand binding assays, and expression levels of mglur1, detected by western blotting, are significantly decreased in the frontal cortex in sad. this decrease is already observed at nft stages i-ii and iii-iv not involving the frontal cortex, and further decrease with the appearance of nfts in the frontal cortex with disease progression. the expression levels of phospholipase cβ1 (plcβ1) isoform, which is the effector of group i mglurs, is decreased in parallel. plcβ1 decrease, in turn, is associated with reduced gtpand l-glutamate-stimulated plc activity in sad. these results show that group i mglurs/plc signaling is downregulated and desensitized in the frontal cortex at the first stages of nft pathology, and that these modifications worsen with the progression of sad (506). 7c. γ-aminobutyric acid (gaba) and gaba receptors the inhibitory neurotransmitter gaba is generated by α-decarboxylation from l-glutamate in neurons by the action of glutamic acid decarboxylase (gad). gaba is then incorporated into the synaptic vesicles by vesicular gaba transporter (vgat). after release from synaptic vesicles, gaba binds to ionotropic gabaa and metabotropic gabab receptors. gabaa receptor activation opens chloride ion channels; gabab acts through g proteins, reduces calcium ion channels, and inhibits ac and intracellular production of camp (507-510). gabaa receptors are composed of combinations of five different subunits, α1-α6, β1-β3, γ1-γ3, δ, ε, θ, π, and ρ1-ρ2. the most frequent pentamers are 2α:2β:1γ (507). gabab receptors are heterodimers composed of r1 and r2 subunits (508). gaba at the synapses is picked up by astrocytes that catalyze it to glutamine, which is then transported into neurons and converted to glutamate (511, 512). the involvement of astrocytes in gaba metabolism has suggested a potential role of astrocytes in gaba gliotransmission (513). several studies have shown inconsistent results regarding total gaba levels in sad but there is a trend toward stressing abnormal gabaergic function (514-516). reduced gaba, glutamate, and glutamine levels are observed in individuals with mci and ad as revealed by magnetic resonance spectroscopy (517-520). regarding gaba receptors, electrophysiological studies reveal a reduction in gaba currents in the temporal cortex in sad. this is associated with mrna downregulation of α1 and γ2 subunits and upregulation of α2, β1, and γ1 transcripts (521). α1 and α5 subunit protein-immunoreactive levels are decreased in the ca1 region of the hippocampus (521-524), whereas α3, β1, β2, β3, and γ2 subunits are unaffected. in contrast, α1 subunits are increased in the ca3 region, granule cell layer, and hilus of the dentate gyrus in sad (523-527). β3 subunit expression is decreased in the stratum oriens, radiatum of ca2 and ca3, and stratum moleculare (527), whereas γ1/3 subunits are upregulated in the hippocampus in sad (525, 527). aβ induces the downregulation of gabaa receptors, inhibitory dysfunction, and sprouting of gabaergic axons (528-532). 7d. serotonin and 5-hydroxytryptamine (5-ht) receptors 5-hydroxytryptamine (5-ht) derives from the amino acid tryptophan via the intermediate 5-hydroxytryptophan and decarboxylation to form serotonin. in the brain, 5-ht is mainly produced in the raphe nuclei of the brain stem that constitutes part of the reticular formation. ascending serotoninergic fibres innervate the whole telencephalon. descending projections innervate the cerebellum and the spinal cord (443). serotonin is stored at the synaptic vesicles and released into the synapse, where it binds to post-synaptic and auto-pre-synaptic receptors. serotonin is then re-uptaken via serotonin transporters and reused or degraded by monoamine oxidase. 5-ht receptors are categorized into metabotropic and ionotropic receptors. metabotropic gpcr are 5-ht1, 5-ht2, 5-ht4, 5-ht5, 5-ht6, and 5-ht7. the only ionotropic receptor is 5-ht3, permeable to sodium, potassium, and calcium ions. 5-ht1 and 5-ht5 receptors bind to gαi/o proteins, inhibit ac, and decrease camp levels. 5-ht2 receptors bind with gαq/11, activate plc, generate pi3p, and activate pkc (533-541). 5-ht and receptors interact with the cholinergic, glutamatergic, noradrenergic, gabaergic, endocannabinoid, and glial cell systems (542-545). orexins regulate serotonin neurons in the raphe nucleus (546). the serotoninergic system is altered in aging and sad (443, 547-549). the main alteration of the serotoninergic system in brain aging and sad results from neuronal damage and nft formation in the raphe nuclei in the independent origin of nft pathology at early stages of ad-related pathology. damage to the serotoninergic system contributes to mood changes and depression which are characteristic non-cognitive clinical manifestations of brain aging and sad (443, 550-553). serotonin is linked to decreased β-amyloid production and modulation of soluble β-amyloid precursor protein (sappβ) (554-557). in addition, 5-ht4 receptors inhibit the secretion of β-amyloid peptides (558-560). due to the multiple facets of serotonin, serotonin receptors, and their interaction with other neurotransmitters, agonists, antagonists of the different 5-ht receptors, and principally selective serotonin re-uptake inhibitors are useful pharmacological agents to improve cognition and reduce depression in aging and sad (444, 552, 561-568). 7e. noradrenergic system norepinephrine or noradrenaline is synthesized from dopamine by the enzyme dopamine β-hydroxylase (dbh). norepinephrine is metabolized by mono-amino oxidase (mao) and catechol-o-methyltransferase (comt). norepinephrine is transported from the cytosol to the synaptic vesicles by the vesicular monoamino transporter (vmat) (569, 570). norepinephrine can bind both to metabotropic preand post-synaptic α1, α2, β1, β2, and β3 receptors. α1 are gq-coupled and activate plc; α2 are coupled to gi/g0 proteins and inhibit ac; β1, β2, and β3 are coupled to gs proteins and activate ac (571-574). the locus coeruleus, which contains about 15,000 neurons in primates, is the principal source of brain noradrenaline. noradrenergic terminals innervate the hippocampus, amygdala, cerebral neocortex, and hypothalamus (575-577). post-synaptic α1 receptors are excitatory, whereas perisomatic and pre-synaptic α2 receptors are inhibitory (578). adrenergic receptors are widely distributed in the brain (579, 580). neurons of the locus coeruleus excite the cerebral cortex principally through α1 receptor signaling (581, 582). the locus coeruleus-noradrenergic system has a major role in arousal, attention, and stress responses. in the brain, norepinephrine may also contribute to long-term synaptic plasticity, pain modulation, motor control, and energy homeostasis (583). noradrenergic terminals are also in contact with glial cells and blood vessels (584-586). due to these connections, noradrenergic innervations also modulate inflammation and cerebral blood flow (583, 587-589). in addition, the noradrenergic system interacts with the cholinergic and gabaergic systems (576, 590-592). moreover, the locus coeruleus and the raphe nuclei are interconnected (593, 594). finally, orexin/hypocretin, histamine and noradrenaline converge in the dorsal raphe nucleus (593). the locus coeruleus is damaged at early stages of ad-related pathology (443, 595-598) (see also section 4). at these first stages, nfts accompany neuron loss, but sps only appear in some cases at advanced thal phases of β-amyloid deposition. in contrast to the massive loss of noradrenergic neurons, calbindin-immunoreactive neurons are preserved in the locus coeruleus even at advanced stages of nft pathology (599). early neuronal alterations in the locus coeruleus are accompanied by abnormalities in the ascending noradrenergic system (600-602). increased α2a adrenergic receptor protein occurs in the amygdala and hippocampus in parallel with early nft pathology in the locus coeruleus (338). in contrast, reduced dbh activity is found in the post-mortem hippocampus and neocortex, probably as a compensatory mechanism to noradrenaline lessening (603, 604). dbh levels are also reduced in plasma at early stages of ad (605). moreover, connectivity between norepinephrine and dopamine brainstem centers is disrupted in sad (606). the orexin system is compromised in sad, thus contributing, in combination with the noradrenergic and serotonin decay, to altered sleep in sad (607-611). 7f. adenosine receptors adenosine is transported across the plasma membrane based on: a) its concentration gradients, and b) active na+-dependent transporters that carry adenosine against its concentration gradient. adenosine receptors are purinergic gpcr classified into a1, a2a, a2b, and a3 receptors. a1 and a3 receptors inhibit ac through gi/o proteins, while a2a and a2b receptors stimulate ac through gs proteins (612, 613). adenosine receptors are present in subpopulations of neurons, astrocytes, oligodendrocytes, and microglia (614-616). adenosine receptors modulate the release of glutamate, gaba, acetylcholine, noradrenaline, and serotonin (616-623). early autoradiographic studies showed decreased a1 expression in the hippocampus at advanced stages of sad (624-627). however, a1 receptors accumulate in neurons with nfts in sad (628). more recent studies at first stages of nft pathology have shown upregulation of adenosine receptors and sensitization of their specific signaling pathways preceding nfts and sps in the frontal cortex (629). 7g. endocannabinoids and cannabinoid receptors (cbrs) cbrs are classified as type 1 (cb1r) and type 2 (cb2r) (630). anandamide (n-arachidonoyl ethanolamine, aea) and 2-arachidonoyl glycerol (2-ag) are the main endogenous ligands of cbrs (631-634). both endocannabinoids derive from arachidonic acid (aa). they are synthesized and metabolized by different pathways and induce specific biological functions. in the human brain, cb1rs are mainly expressed in the limbic system. cb1rs localize in the pre-synapses modulating glutamate and gaba neurotransmission (635-641). cb2rs are expressed in microglia, and participate in inflammation and phagocytosis (642, 643). low levels of cb2r expression have also been identified in some neurons (644-646). cannabinoid compounds may also bind to other receptors, such as gpr55, peroxisome proliferator-activated receptors pparα and pparγ, and transient receptor potential vannilloid-1 channels (647, 648). the study of expression of cb1rs and mediators in sad has yielded variable results (649). in contrast, increased cb2r expression in microglia surrounding sps is consistently documented (650, 651). a few studies have shown altered expression levels of endocannabinoids and enzymes linked to their metabolism in sad (652-654). therefore, the endocannabinoid system plays a role in sad although its precise contribution remains largely unknown. treatment with exogenous cannabinoids and modulation of cbrs in murine models of ad has shown beneficial effects including reduction of β-amyloid plaques and β-amyloid burden, reduced tau phosphorylation, reduced inflammation, excitoxicity, mitochondrial dysfunction, and oxidative stress, and relief of cognitive impairment (649). 8. trophic factors and receptors the expression of trophic factors, particularly brain-derived neutrophic factor (bdnf) and nerve growth factor (ngf) and their receptors, is altered in sad (655-659). bdnf mrna and protein are decreased in the frontal cortex and hippocampus in ad (660-662). bdnf immunoreactivity is reduced in tangle-bearing and non-tangle-bearing neurons, whereas immunoreactivity to full-length trkb (the high affinity receptor for bdnf, neurotrophin-3 and neurotrophin-4) is reduced in tangle-bearing neurons. strong bdnf immunoreactivity is observed in dystrophic neurites surrounding sps, and strong trkb in reactive glial cells, including those surrounding sps. truncated trkb immunoreactivity occurs in individual neurons and reactive glial cells in the cerebral cortex and white matter in sad (661). the cause of abnormal trkb immunoreactivity in sad is not known but β-amyloid modulates trkb alternative transcript expression (663). probdnf is increased in sad, and it is modified by reactive oxygen species (ros)-derived advanced glycation end products, which prevent the processing of probdnf to mature bdnf (664). abnormal probdnf/bdnf signaling impairs axonal transport, decreases trophic effects, and increases pathogenicity and cell death (661, 664, 665). in contrast, the expression of ngf is increased in sad (655, 666), but ngf-containing neurons in the basal forebrain are lost in sad (454, 667). prongf is also increased in sad (668, 669). this has relevant implications as prongf induces processing and nuclear translocation of the intracellular domain of p75ntr (the low affinity neurotrophin receptor for ngf, bdnf, neurotrophin 3, and neurotrophin 4) and induces cell death in association with cofactor sortilin, a member of the vps10p sorting receptor family (670-672). 9. endoplasmic reticulum stress the accumulation of abnormally misfolded proteins in the endoplasmic reticulum (er) causes er stress which is manifested by activation of one or more of the three signaling pathways of the misfolded protein response (upr) (673). glucose-related protein 78 (grp78/bip) is the master protein that regulates the upr (674). the cytosolic domains of the transmembrane er proteins pkr-like endoplasmic reticulum kinase (perk), inositol-requiring protein 1 (ire1), and activating transcription factor (atf)-6, trigger specific pathways once activated. perk activates atf-4, and the activation of atf6 involves its displacement from the er to the golgi apparatus to be cleaved into atf6c. ire1 dimers phosphorylate and lead to the production of x-box binding protein 1 (xbp-1) which increases the er capacity of protein folding and the degradation of abnormally folded proteins in the er, thus reducing er stress. ire1 may also bind to the traf2 (tnf receptor-associated factor 2) adaptor molecule and activate the apoptosis signal-regulating kinase 1 (ask1), which in turn causes the phosphorylation of c-jun n-terminal kinase (jnk), thereby triggering cell death. atf-4, truncated atf6, and xbp-1, through downstream target genes, modulate er homeostasis or apoptosis, depending on the saturation of the system (673, 675). the expression of several upr components is altered in aging and sad (676-679). er stress also generates ros which, together with mitochondrial ros, is a major cause of oxidative stress damage. mitochondrial dysfunction and er stress are relevant promoters of apoptosis in sad (680). er stress is also linked to brain inflammation (681, 682). moreover, β-amyloid may activate the upr, either mediated by glutamate receptors and calcium uptake, or linked to mitochondrial dysfunction and ros production; er stress may also be induced by abnormal tau (683-685). moreover, perk, ire1 and atf6 signaling pathways activate autophagy (686-688). 10. failure to remove debris: the ubiquitin-proteasome system (ups) and autophagy in sad autophagy and ups are the two main mechanisms of intracellular protein degradation. there is a certain relationship between these two mechanisms, and there are some molecules in common that initiate compensatory effects to prevent disease progression (689, 690). autophagy includes macroautophagy, microautophagy, and chaperone-mediated autophagy (691). microautophagy and chaperone-mediated autophagy involve lysosomal membrane invagination and chaperon recognition (692). macroautophagy is mediated by autophagosome protein assembly of beclin 1-vps34 lipid kinase, atg9-wipi-1, atg12 conjugation system, microtubule-associated protein light chain 3 (lc3), and unc-51-like autophagy activating kinase 1 protein kinase. lysosomal-associated membrane protein 1 (lamp-1) is a transmembrane glycoprotein enriched in the membrane of lysosomes (693-695). material digested by autophagy is incorporated into lysosomes. autophagy is impaired in brain aging and sad (696-706). altered macroautophagy in sad is manifested in dystrophic neurites of sps, in which mitochondria, dense bodies, and vesicles are common targets, and in synapses and granulovacuolar degeneration (see section 11). autophagy is also involved in β-amyloid metabolism and clearance (707). ups activity is initiated by the conjugation of ubiquitin to the substrate following a three-step cascade to tag the protein into the proteasome. the 20s proteasome is a hetero-oligomer formed by heptameric rings organized into a structure resembling a hollow cylinder which has three main peptidase activities: chymotrypsin-like, trypsin-like, and peptidylglutamyl peptide hydrolyzing activities. the 20s proteasome can associate, in the presence of atp, with two caps or 19s complexes, thereby forming the 26s proteasome complex. the 19s complex serves in the recognition of ubiquitylated proteins, protein unfolding, and translocation of the unfolded polypeptide to the inner chambers of the 20s proteasome for hydrolysis. the 20s proteasome can interact with other complexes. the pa28α/β activator (11s regulator) can also bind to 20s proteasome to form the pa28-proteasome complex. additionally, the three catalytic β subunits of the 20s proteasome in response to γ-interferon are replaced by inducible homologous proteins lmp2, lmp7, and mecl1, forming the immunoproteasome (689). the immunoproteasome has a role in peptide production for antigen presentation by the major histocompatibility complex in most settings, but it is also involved in the clearance of oxidatively damaged proteins (708). the ups is altered in brain aging and ad (709-712). impaired removal of altered proteins is manifested by the deposition of hyper-phospohorylated, abnormally conformed, and truncated ubiquitinated tau species resistant to ups degradation in nfts, dystrophic neurites, and threads (197). in addition to ubiquitin, mutant ubiquitin is expressed in the aging brain and at early stages of sad, whereas misframed ubiquitin contributes to the blockade of the proteasome by nfts and other tau inclusions (229, 230, 713-716). yet, mutant ubiquitin reduces aβ plaque formation (717, 718). the immunoproteasome is activated in ad and app/ps1 double-transgenic mice (719-721). the reasons for immunoproteasome activation are not fully understood, but the presence of advanced glycation end products (ages) appears to induce activation of the immunoproteasome; the inhibition of age receptor (rage), and the downstream signalling jak2 (janus kinase 2)/stat1 (signal transducer and activator of transcription 1) abolishes age-induced activation of the immunoproteasome (722). 11. granulovacuolar degeneration (gvd) gvd is characterized by the presence of vacuolar cytoplasmic lesions with a dense central core, mainly in ca1 hippocampal neurons (18). gvd is common in the hippocampus in the aging human brain and present in the majority, if not all cases, of sad (figure 8). gvd first appears in neurons of the hippocampal subfields ca1 and ca2, and the subiculum; this is followed by the entorhinal cortex, and ca4 neurons in stage 2, temporal neocortex in stage 3, amygdala and/or the hypothalamus in stage 4, and cingulate, frontal, and parietal cortices in stage 5 (723). gvd appears in relation to hippocampal phosphorylated tau accumulation in various neurodegenerative disorders, particularly ad (724). the most widely accepted model of agd generation follows the scheme: abnormal tau in pretangles induces abnormal reticulum stress responses, which in turn trigger endocytic and autophagic pathways in the face of impaired proteolysis and altered function of the ups. this scenario is consistent with the idea that gvd is the final stage of an active process linked to failed degradation of abnormal protein aggregates in the cytoplasm of a subpopulation of neurons (677, 725-727). immunohistochemistry to casein kinase 1δ (ck1-δ) and at8 and laser microdissection have identified the proteomes of neurons containing gvd and nfts, respectively, using label-free lc-ms/ms (728). a significant change in the abundance of 115 proteins in gvd-containing neurons and 197 in nft-containing neurons was observed compared to control neurons (728). differences in protein composition between nfts and neurons with gvd are further supported by the demonstration of different activation of kinases and different profiles of phosphorylated proteins (139). nonetheless, the same study of phosphoprotein expression showed that abnormal phosphorylation of various substrates is common at the first stages of nft and gvd generation (139). these observations suggest that gvd is not restricted to tau pathology but rather involves a varied number of proteins. since gvd is common in the older human population, it is not surprising to find a correlation between gvd pathology and cognitive impairment. however, when analysis is controlled for other associated neuropathologies, the associations between gvd and dementia lose significance (729). figure 8: granulovacuolar degeneration (black arrows) in neurons of the hippocampus. paraffin section, p38-p immunohistochemistry, slight haematoxylin counterstaining, bar = 25 μm. 12. glial alterations in aging and sad glial cells are altered in aging and sad. changes in glial cells have multiple facets, including cell senescence, astrocytic gliosis, microgliosis, activated inflammatory responses, and calcium homeostasis, among others. astrocytes are key players in ad modulating β-amyloid turnover, calcium homeosthasis, tripartite synaptic function, neuroinflammation, oxidative stress responses, and bbb dysfunction (730-734). microglia and astrocytes, together with neurons and blood vessels, participate in the process of activation of inflammatory responses in aging and sad (735-737). moreover, microglia have the capacity to transform a subset of reactive astrocytes through the combination of il-1α, tnf, and c1q (738). both microglia and astrocytes are key participants in neuroinflammation in ad (739). 12a. astrocytes astrocytes are key elements in the maintenance of the central nervous system (cns) due to their role in brain homeostasis at all levels of organization from molecular to the whole organ (740). astrocytes express neurotransmitter receptors, pumps, and transporters at their plasmalemma, along with transporters in the endoplasmic reticulum and mitochondria that regulate the cytosolic levels of ions which underlie most, if not all, astroglial homeostatic functions (741). with aging, astrocytes show accumulation of lipofuscin, hypertrophy of cytoplasmic filaments, increased expression of glial fibrillary acidic protein (gfap), s100β and vimentin, and modifications in morphology and number (742-744). senescent astrocytes also exhibit senescence-associated secretory phenotype manifested by increased production of pro-inflammatory cytokines together with oxidative damage and increased superoxide production (745). perivascular astrocyte senescence leads to altered bbb (746,747). this is accompanied by reduced expression of efflux transporter and increased expression of influx transporter receptors for ages. abnormal transport of proteins through blood vessels affects the transfer of β-amyloid, leading to its accumulation in blood vessels (748, 749). water aquaporin 1 (aqp1) expression in astrocytes is also altered in the frontal cortex at nft stages i-ii, suggesting early impairment of water transport linked to ad-related pathology (750). aqp4 expression is altered with aging and sad; loss of aqp4 is associated with increased levels of β-amyloid and tau, suggesting that loss of aqp4 impairs the bbb and the gliolymphatic barrier (751). astrogliosis and astrocyte atrophy are relatively early events in ad (752-756). reduced branching and reduced connexin 43 expression occur in sad (757) which may compromise the extent of coverage domain and synaptic function in neighboring neurons (758). β-amyloid peptides also induce mitochondrial dysfunction and oxidative stress in astrocytes (759). transcriptomics of laser-captured microdissection using gfap as a marker revealed marked dysregulation of insulin, phosphatidylinositol 3-kinase (pi3k)/akt, and mitogen-activated protein kinase (mapk) signaling pathways at advanced braak stages of the disease; minor and different abnormalities were observed at earlier stages, indicating different responses of astrocytes along disease progression (760). recent transcriptomic studies in sad have shown upregulation of genes related to perisynaptic astrocytic processes and downregulation of genes encoding endolysosomal and mitochondrial proteins; downregulation of astrocytic mitochondrial genes inversely correlates with the disease stages defined by braak and cerad scoring (761). reactive astrocytes are found mainly around aβ deposits in sps and blood vessels (752, 762, 763) and in areas without plaques then distributed in a layered pattern (762, 764). in response to vascular β-amyloid deposition, astrocytes produce cytokines, metabolizing enzymes, and ros which in turn contribute to altered bbb and perivascular astrocytic function (765-769). reactive astrocytes may contain aβ (770-772) and n-terminal truncated β-amyloid (773), and they have the capacity to internalize and degrade β-amyloid fibrils (774). activation of metalloproteinases (765, 775) and lysosomal degradation (776, 777) are the main complementary mechanisms by which astrocytes degrade β-amyloid. in addition, bace may be expressed in astrocytes under appropriate conditions, facilitating the generation of β-amyloid in these cells (752, 778). acquisition of a pro-inflammatory profile along with activation of the β-amyloidogenic pathway further potentiates toxicity of a subpopulation of astrocytes in ad (779). curiously, β-amyloid seems to impair the phagocytosis of dystrophic synapses by astrocytes (780). astrocytes bearing β-amyloid show abnormal calcium homeostasis (758, 759, 781-783). in addition, β-amyloid-induced glutamate release by astrocytes may contribute to neuronal excitatory damage (784). de-regulation of specific metabotropic glutamate receptors in astroglia is also a putative harmful effect of β-amyloid (785). nmdars are expressed in a sub-population of astrocytes in the cortex and spinal cord. these receptors are composed of two glun1, one glun2c or d, and one glun3 subunits. this composition makes astroglial nmdars operational, modulating resting membrane potential (786). eaat2 clears excess extracellular glutamate from the synaptic cleft and extrasynaptic sites via glutamate re-uptake by glial cells and neurons to prevent neuronal hyperexcitability and excitotoxicity. oxidative damage, splice variants, and altered solubility of eaat2 may lead to functional alterations of glutamate transporters in ad (787-789). moreover, eaat2 expression is reduced with disease progression in parallel with increased gfap expression in sad (762, 790, 791). furthermore, astrocytes surrounding sps show altered immunoproteasome markers, and augmented expression of cytokines and mediators of the immune response (721, 735, 792, 793). reactive astrocytes in sad also have higher levels of complement component c3 which is required for both classical and alternative complement activation pathways (794). changes in the morphology of astrocytes are accompanied by alterations in the regulation and expression of gfap and other astrocyte markers in cases with mci and in pre-clinical ad (791, 795-797). these changes are preceded by expression of various cytokines and components of the inflammatory response (793, 798). astrocytic responses are not homogeneous but rather variable, even in the same region (799-801). as for m1 and m2 suggested phenotypes for microglia (see section 12b), a1 and a2 astrocyte phenotypes have been proposed depending on their transcriptional profiles: a1 are neurotoxic and a2 neuroprotective (738, 802). however, this categorization is difficult to apply in the context of sad (801-803). glucose hypometabolism is a characteristic metabolic feature associated with cognitive impairment in ad (804-807). furthermore, neurons utilize lactate as a source of energy, and astrocytes are the main local source of neuronal lactate (808-811). lactate levels are reduced in the brain of ad transgenic murine models (812). in the brain, the local source of ketone bodies results from fatty acid oxidation in astrocytes (813). in addition, astrocytes can produce ketones from amino acids (814). considering that about 20% of cerebral atp is generated from fatty acids (815), it may be inferred that impaired energy metabolism in astrocytes negatively impacts on neuronal energy metabolism, including maintenance of the energy requirements of the synapses (803, 816-818). 12b. microglia microglial cells are multifunctional cells that respond to different stimuli leading to either beneficial or harmful effects depending on the production of specific molecules. several studies have dealt with a proposed polarization of microglia into two types. m1 phenotype is stimulated by interferon-γ (ifn-γ) for the expression of pro-inflammatory cytokines, and m2 phenotype by il-4/il-13 for resolution of inflammation and tissue repair (819). however, the division between m1 and m2 microglial phenotypes with opposing effects in pathological conditions is probably a simplification, as complex microglial responses occur in the same setting, particularly in ad (820, 821). the term neuroinflammation, although widely used, sheds little light on the molecular consequences of microglial (and astrocytic) responses in any particular setting (822-824). microglia are modified in aging and ad (825-828). dystrophic (senescent) microglia precede activated microglia in aging and sad (826). increased numbers of activated microglial cells occur in parallel with the production of β-amyloid and tau pathology (829). microglial cells are found associated with sps in contact with dystrophic neurites and internal to reactive astrocytes (7). diffuse microgliosis involving the cerebral cortex and subcortical white matter occurs as well. microglial activation in ad is associated with upregulation of a large number of cytokines, chemokines, members of the complement system, and other mediators of the immune system (736, 830-837). importantly, the inflammatory response is not homogeneous but is largely region and stage dependent (838). inflammatory responses also occur in transgenic mouse models of ad with specific region and stage profiles. yet, the regulation of different components of brain inflammation and the immune system differ in transgenic mice when compared with sad (838, 839). this is an important point, as inflammatory responses vary in different regions with disease progression; protective and deleterious microglia-associated effects may occur simultaneously in any individual at any stage of the disease. another relevant point is the role of lipids in activated microglia; increased expression of apoe, triggering receptor expressed on myeloid cells 2 (trem2), and lipoprotein lipase (lp2), is found in activated microglia (840). a link between β-amyloid and microglia is well documented (829, 841, 842). a cell surface receptor complex for fibrillary-amyloid mediates microglial activation (843). microglia, in turn, mediates the clearance of soluble aβ through fluid phase macropinocytosis (844). microglia-mediated synapsis loss in ad is enhanced by fibrillar aβ and oligomeric aβ aggregation onto neuronal post-synaptic terminals, complement deposition, c3 receptor (cd11b/cd18) activation, microglial activation, and synapsis phagocytosis (821, 845-849). microglia activation also correlates with tau pathology and nft staging (829, 850-852). positron emission tomography (pet)-based studies have also shown a correlation between tau pathology progression and microglial activation across braak stages (853, 854). abnormal tau at the synapsis also favors synapsis pruning and elimination by micoglial phagocytosis (855). the notion that two types of microglial cells, unrelated to the proposed m1 and m2 subtypes, may play different roles in sad is supported by recent observations. using single nuclei rna sequencing (snrnaseq) of isolated microglial nuclei in sad brains, the abundance of phagocytic/activated named ad1-microglia correlates with tissue β-amyloid load and localizes with β-amyloid plaques; ad2-microglia are more abundant in association with tau pathology (856). microglial and altered expression of inflammatory markers is observed in children with down syndrome, and it is modified in parallel with the appearance of tau and β-amyloid pathology and through disease progression. microglial responses in down syndrome with ad pathology differ from those of sad (857-859). the profile of brain inflammatory responses also differs in human cases with ad-resilient pathology; levels of trophic factors are increased, whereas expression levels of chemokines are decreased (860). the inflammasome is a multi-protein complex containing a member of the nod-like family, such as pyrin-domain containing 3 (nlrp3r) that interacts with the inflammasome-adaptor protein asc. the immune response elicited by β-amyloid functions through complex crosstalk between the toll-like receptor 4 (tlr4), complement, and inflammasome signaling pathways (861). the activation of the inflammasome in microglia triggers a cascade that involves caspase 1, and maturation of several cytokines including il-1β and il-18. the inflammasome in microglia participates in the nucleation of β-amyloid plaques and enhances tau pathology (862, 863). aβ aggregates, and soluble aβ oligomers and protofibrils, activate the nlrp3 inflammasome (864-866). conversely, activation of nlrp3-asc inflammasome aggravates amyloid pathology (867). aggregated tau also activates nlrp3/asc and exacerbates tau pathology (868). loss of nlrp3 inflammasome function reduces tau hyperphosphorylation and aggregation by regulating tau kinases and phosphatases. conversely, tau activates the nlrp3 inflammasome. moreover, the intracerebral injection of fibrillar β-containing brain homogenates has been shown to induce tau pathology in an nlrp3-dependent manner (869). microglial and inflammatory responses are dependent on genetic factors in sad (280, 870-872). apoe status is a modifier of the inflammatory response (873, 874). cd33, trem2, and other genes linked with inflammation are expressed in microglia (871, 875, 876). carriers of ad-associated risk variants in trem2 show a reduction in plaque-associated microglia, and an increase in dystrophic neurites and overall pathological tau compared with ageand disease stage-matched sad patients without trem2 risk variants (877). another study shows that trem+ control cases have no pathological hallmarks of sad, whereas trem2+ sad cases show amoeboid microglia and upregulation of inflammatory markers when compared with trem2+ controls and trem2sad cases. these findings suggest that trem2 influences, but does not trigger, the microglial responses in sad (878). 12c. oligodendrocytes myelin generation and maintenance, and axonal nurture in the cns, are carried by oligodendrocytes. oligodendrocytes may contain abnormal protein deposits in various neurodegenerative diseases with abnormal protein aggregates. yet alterations in oligodendrocytes may occur without accompanying abnormal deposits. oligodendrogliopathy is used to stress the role of altered oligodendrocytes in the pathogenesis of certain neurological diseases with or without abnormal oligodendroglial deposits (879). oligodendrocytes suffer a functional decline in aging and ad as revealed by combined morphological, biochemical, and neuroradiological methods. neuroimaging and neuropathological studies have shown reduced white matter (wm) volume, wm lesions, and altered wm integrity and cortical disconnection in aging human brain (880-884). wm changes are associated with disruption of myelin and axons (885, 886). alterations in the number of oligodendrocytes and oligodendroglial precursor cells (opcs/ng2-positive cells) have been reported in aged primates and rodents (887). neuroimaging studies show reduced wm size, wm hyper-lucencies, and myelin and axon damages in patients with mci and dementia of alzheimer’s type (883, 888-896). wm atrophy, decreased myelin density, and demyelination are also observed in post-mortem neuropathological studies (881, 896, 897). breakdown of wm integrity is considered a contributor to the loss of neuronal tract connectivity in aging and sad (898, 899). recent neuropathological studies have shown preservation of mbp, plp1, cnp, mag, mal, mog, and mobp mrna expression levels in the wm of the frontal cortex at nft stages i-ii/0-a when compared with ma individuals without nft pathology, but a significant decrease at stages iii-iv/0-c. this is accompanied by reduced expression of ng2 and pdgfra (platelet-derived growth factor receptor a) mrna, reduced numbers of ng2-, olig2-, and hdac2 (histone deacetylase 2)-immunoreactive cells, and reduced glucose transporter immunoreactivity at stages iii-iv/0-c. curiously, partial recovery of some of these markers occurs at stages v-vi/b-c. these changes show that myelin loss is accompanied by reduced transcription of myelin-related proteins in the wm of the frontal cortex at middle-stages of ad (342). early myelin loss, decreased numbers of oligodendrocytes, and region-specific alterations, followed by partial reparative responses, also occur in transgenic mouse models of ad (900-903). 13. the neurovascular system in ad the first highlighting of the role of vascular pathology in ad was in 1989 (904). the “vascular hypothesis” of sad has been supported more recently (905-908). interestingly, denervation probably due to the loss of inputs from the locus coeruleus and basal forebrain was initially considered a key factor (909); altered innervation of the cerebral blood vessels linked to cholinergic deficiency is still postulated as contributing to cerebral blood flow (cbf) reduction (910, 911). there are hundreds of papers dealing with β-amyloid angiopathy and its effects on neurovascular function, as well as the role of lipid transporters and vascular receptors in the clearing of β-amyloid in the cerebral blood vessels (912-923). yet, there is also overwhelming evidence of altered neurovascular functioning in sad beyond that expected in association with β-amyloid angiopathy. neurovascular dysfunction in individuals with mci and advanced ad is manifested by reduced cbf (cerebral hypoperfusion), reduced cerebral glucose transport, impaired bbb function, altered lymphatic function, and altered structure of the capillaries (80, 910, 924-935). in addition to atherosclerosis and small blood vessel disease that are common in the elderly, primary involvement of arterioles and capillaries is common in brain aging and sad. primary blood vessel damage in sad is characterized by atrophy and irregularities of capillaries and arterioles, edema and increased numbers of pynocytotic vesicles in endothelial cells and pericytes, atrophy of smooth muscle fibers, thickening and focal disruption of the basal membrane, increase in collagen iv, heparan sulfate, proteoglycans, and laminin in the basal membrane, increased aquaporin expression in perivascular astrocytes, and gliovascular dysfunction, among other defects (910, 936-941). degeneration of endothelial cells is further supported by reduced staining of endothelial cell markers (942) accompanied by aberrant angiogenesis (943). pericytes are decreased in number and show abnormal mitochondria, pinocytotic vesicles, and disorganization and accumulation of osmiophilic material in aging with cumulative damage with sad progression (944-947). aβ oligomers constrict human capillaries in ad by signaling to pericytes (948). pericyte degeneration and impaired bbb function reduce aβ clearance and increase β-amyloid accumulation in the brain. importantly, pericyte alteration occurs at early stages of sad and probably it has a determining role in the altered capillary permeability (949-950). it is difficult to ascertain the weight of each one of the vascular pathologies in the eventual neurovascular failure in a particular individual. atherosclerosis, small blood vessel disease, β-amyloid angiopathy, brain hypoperfusion, altered bbb, and primary ad-linked non-amyloidotic angiopathy have cumulative effects (951, 952). the timing and development of neurovascular failure in sad is not a simple process. the “two-hit vascular hypothesis” suggests that early vascular damage leads to increased accumulation of aβ deposits in the brain, which in turn provokes additional vascular damage. apoe is one of the factors that modulate cerebrovascular integrity (953). impaired glucose uptake is an early event in pre-symptomatic fad (954), and bbb breakdown is an early biomarker of human cognitive dysfunction (955), suggesting that the neurovascular system is dysfunctional at early stages of ad. reduced expression of glucose transporter 1 (glut1) is manifested at early stages of sad, and it impairs blood glucose uptake and vasculo-neuronal function (956, 957). altered glucose transport accounts, in part, for the more complex brain glucose metabolism dysregulation in ad (958). more precise information has been obtained from the study of mouse models of cerebral β-amyloidosis mimicking β-amyloid deposits in ad (959). in the majority of these mice, cerebral blood vessels are altered and there is often impaired bbb at early stages of aβ plaque deposition, usually preceding or in the absence of β-amyloid angiopathy (959). these observations are in line with the role of aβ in the pathogenesis of neurovascular damage in ad (928). unfortunately, little is known about the occurrence of different soluble, insoluble, and oligomeric amyloid species in these models. the possible deleterious effects of abnormal tau on the integrity of the cerebral blood vessels have also been assessed (960). tau expression is accompanied by bbb breakdown in tetracycline-regulable tau-transgenic mice; bbb function is recovered once tau levels are normalized (961). non-structural but functional bbb dysfunction mediated by altered modulation of vasoactive factors including vasoconstrictor endothelin-1 has been suggested (962). altered cbf, impaired bbb, and structural anomalies in the blood vessels also occur in other neurodegenerative diseases such as parkinson’s and huntington’s diseases, amyotrophic lateral sclerosis, and multiple sclerosis (933, 934, 963), in which aβ and ad-tau have no chance to play a causative role. neurons and astrocytes also play a role via glutamate in the regulation of cbf. arteriole constriction depends on the metabolism of arachidonic acid (aa). upon mglur activation, increased aa in the plasma membrane is converted into 20-hydroxyeicosatetraenoic acid and induces vasoconstriction, or it is converted into prostaglandin 2 and produces vasodilatation (964). astrocytes have cardinal functions in the maintenance of bbb (see section 12a). 14. purine and pyrimidine metabolism in sad purines are heterocyclic double-ring aromatic organic molecules. primary purines adenine and guanosine, together with one-ring primary pyrimidine nucleobases cytosine, thymidine, and uracil, are the core of dna, rna, nucleosides, and nucleotides. adenosine and guanosine are purine ribonucleosides resulting from the binding of adenine or guanine to ribose, respectively. when adenine and guanine are attached to a deoxyribose ring, the resulting compounds are deoxyadenosine and deoxyguanosine, respectively. nucleotides result from the incorporation of phosphate groups in nucleosides: adenosine monophosphate (amp), adenosine diphosphate, adenosine triphosphate (atp), guanosine monophosphate (gmp), guanosine diphosphate, guanosine triphosphate, and cyclic forms camp and cgmp are primary purine-derived nucleotides. modified purine nucleobases hypoxantine and xanthine result from the replacement of the amino-group by a carbonyl-group from adenine and guanine, respectively, whereas methyl-guanine results from the incorporation of a methyl group to guanine. corresponding modified purine nucleosides are inosine, xanthosine, and methyl-guanosine, respectively. nucleotides participate in a wide variety of crucial metabolic pathways including energy metabolism and cell signaling. in addition, purine bases are incorporated to other molecules to form cofactors of several enzymatic reactions such as coenzyme a, flavin adenine dinucleotide (fad), nicotinamide adenine dinucleotide (nadþ), nicotinamide adenine dinucleotide phosphate (nadpþ), and the corresponding reduced forms fadh2, nadh, and nadph. s-adenosyl methionine is made from atp and methionine by methionine adenosyltransferase and is involved in the transfer of methyl groups to distinct substrates, including nucleic acids, proteins, lipids, and metabolites (965, 966). in addition, adenosine may act as neuromodulator on specific adenosine receptors (967) (see section 7f). adenosine receptors also modulate the bbb (968). significantly decreased levels of adenosine, guanosine, hypoxanthine, and xanthine are found in the frontal cortex at stages i-ii of nft pathology, but the parietal cortex and temporal cortex show an opposing pattern at advanced stages of sad. the activity of 5’-nucleotidase, which hydrolizes adenosine amp to generate adenosine, is reduced in the frontal cortex mainly at nft stages i-ii, and only at nft stages v-vi in the temporal cortex. adenosine deaminase activity, which synthetizes inosine from adenosine, is decreased in the frontal cortex in sad but is increased at nft stages i-ii in the temporal cortex. finally, purine nucleoside phosphorylase activity, which metabolizes guanosine to guanine, is increased only in the temporal cortex at nft stages i-ii. purine metabolism alterations are regionand stage-dependent and occur independently of nfts and β-amyloid plaques (969). in another study, 23 purine metabolism genes were analyzed with rt-pcr in the entorhinal cortex, frontal cortex area 8, and precuneus in ma individuals without nft pathology and in cases at nft stages i-ii, iii-iv, and v-vi (966). the mrna expression levels of several enzymes were dysregulated at stages iii-iv and v-vi in a region-dependent manner when compared with ma individuals. in addition, liquid chromatography mass spectrometry-based metabolomics in the entorhinal cortex identified decreased levels of xanthosine, guanine, and deoxyguanosine at stages i-ii, followed by dgmp, inosine diphosphate, and glycine at nft stages iii-iv in sad (966). some years ago, studies pointed to s-adenosylmethionine as a complementary candidate for therapeutic intervention in sad (208, 970). more recently, several studies have scrutinized the modulation of purinergic signaling to ameliorate sad (971-976). considering the large number of different enzymes altered, this represents a tremendous endeavor. 15. epigenetics in brain aging and sad epigenetic regulation plays a crucial role in the final transcription of genes (977, 978). three central mechanisms are briefly discussed: modifications of histones, dna methylation and hydroxymethylation, and non-coding rnas. 15a. histone modifications, dna methylation, and hydroxymethylation nucleosomes consist of dna wound around a protein octamer composed of two molecules of the histones h2a, h2b, h3, and h4. the n-terminal of histones (histone tail) extends out from the surface of the nucleosome. acetylations by histone acetyltransferases (hats) and histone deacetylases (hdacs), and methylation mediated by histone methyltransferases (hmts) and histone demethylases (hdms), respectively, occur at the histone tails (979, 980). histone acetylation decreases the interactions of histones with dna, relaxes heterochromatin to euchromatin, and enables gene transcription (981-984). methylation of histones can either increase or decrease gene transcription, depending on which amino acids in the histones are methylated, and on the number of methyl groups involved. methylations, which permit dna uncoiling facilitate the access of transcription factors and rna polymerase. the trimethylation of histone 3 at lysine 4 (h3k4m3) activates transcription, whereas dimethylation of histone h3 at lysine 9 (h3k9me2) is inhibitory. likewise, methylation of lysine 4 of histone 3 (h3k4me1) facilitates gene transcription (985-988). dna methylation and dna hydroxymethylation are biological processes by which methyl or hydroxymethyl groups are added to the dna. methylation and hydroxymethylation can change the activity of a dna segment without changing the sequence. the methylation of a 5′-cytosine in cytosine-guanine-rich regions (cpg islands) of dna promoters results mainly in the inability of transcription factors to bind dna and compact heterochromatin; as a result, gene transcription is silenced. four types of dna methyltransferases, dnmt1, dnmt2, dnmt3a, and dnmt3b, carry out the process of dna methylation. s-adenosyl-l-methionine (sam) is the donor of methyl groups. dna hydroxymethylation can occur as a result of oxidative stress or the action of ten-eleven-translocation-1 (tet1) proteins (989-994). sam which is generated by adding adenosine to methionine, a reaction catalyzed by s-adenosyl-methionine transferase, transfers the methyl group to dna methylation by dna methyltransferase. brain aging and sad is accompanied by epigenetic dna modifications (995-1002). dna modifications involve genes linked to β-amyloid production (1003-1006), apoe-ε4 (996), tau phosphorylation (1007-1009), ribosomes (1010), bdnf (1011), and inflammation (1012, 1013). a large number of unrelated genes has also been assessed, showing either changes or no modifications in the methylation of dna promoters (1014-1023). the expression of adenosine receptor a2a is modulated by dna methylation in the gene promoter region (1024-1026). therefore, epigenetic changes in dna in aging and sad are not widespread but selective for particular genes. in monozygotic twins discordant for ad, significantly reduced levels of dna methylation were observed in the neuronal nuclei of temporal neocortex in the ad twin (991). unfortunately, most studies are carried out on samples at advanced stages of sad which precludes learning whether epigenetic changes in dna are primary or secondary events (1027). in addition to modifications in genomic dna, increased mitochondrial 5-methylcytosine in most cpg and non-cpg sites in the d-loop region of mitochondrial dna (mtdna) has been identified in the entorhinal cortex at nft stages i-ii and iii-iv compared with control samples (1028). these are relevant data as they indicate early mtdna methylation linked to the pathogenesis of ad-related neuropathologic change (1028). 15b. non-coding rnas non-coding rnas do not encode proteins, but most modulate protein translation targeting mrnas. transfer rnas (trnas) and ribosomal rnas (rrnas); small non-coding (snrnas) such as micrornas (mirnas), small interfering rnas (sirnas), piwi-interacting rnas (pirnas), small nuclear rnas (snrnas), small nucleolar rnas (snornas), extracellular rnas (exrnas), small cajal-body specific rnas (scarnas); and long non-coding rnas (lncrnas) are the principal types. mirnas bind to complementary un-translated regions (3’-utrs) of mrnas to regulate target genes, resulting in translational repression or degradation (1029-1031). in animals, mirnas are synthesized from primary mirnas by the action of two rnase iii-type proteins: drosha, in the nucleus, and dicer in the cytoplasm. argonaute (ago) subfamily proteins bind mature mirnas to target mrnas (1032, 1033). thousands of human genes are mirna targets (1034, 1035). ago2:rna interactions using hits-clip have been used to generate a transcriptome-wide map of mirna binding sites in the human brain. about 7,000 stringent ago2 binding sites were highly enriched for conserved sequences corresponding to abundant brain mirnas. this interactome points to functional mirna: target pairs across about 3,000 genes (1036). dysregulation of mirnas is involved in the pathogenesis of sad (1037-1040). several mirnas target genes, many of them abnormally regulated in ad, are associated with β-amyloid (1041-1060), tau phosphorylation, and cytoskeleton (1061-1074). it is difficult to ascertain which mirna alterations are primarily altered or secondary to β-amyloid and tau pathology in sad (1075). several mirnas can bind to specific mrnas, modulating gene transcription differentially depending on the triggering factor (1038). mirna 163, mirna30a 5p, and mirna206 bind to bdnf mrna and modulate altered bdnf expression in sad (1040, 1055, 1076). furthermore, one mirna can bind to different mrnas resulting in various functions. for example, reduced mirna 132 is associated with downregulation of chat immunoreactivity in the nucleus basalis of meynert (1061) and to ache upregulation (1077, 1078). in addition, mirna 132 targets creb and promotes neuritogenesis and synaptic activity (1079). finally, mirna dysregulation is stageand region-dependent. for example, in the locus coeruleus, mirna-27a-3p, mirna-124-3p, and mirna-143-3p show a trend to increase at nft stages i-ii and are significantly upregulated at nft stages iii-iv when compared with ma individuals without nft pathology. in the entorhinal cortex of the same cases, only mirna-143-3p is upregulated at stages iii-iv. the expression levels of mirna-27a-3p, mirna-124-3p, and mirna-143-3p are not modified in ca1 at any stage, whereas mirna-124-3p is significantly downregulated in the dentate gyrus at nft stages i-ii (1080). the interpretation of these results is puzzling: downregulation of mirna124 results in β-amyloid accumulation (1007); mirna143-3p inhibition promotes neuronal survival in sad (1081); and mirna 27a-3p regulates the expression of intercellular junction proteins at the brain endothelium, while its downregulation increases bbb permeability (1082, 1083). 16. microorganisms and sad the possibility that microorganisms participate in the pathogenesis of ad was suggested in a seminal publication in 1907 (1084). the role of bacteria, viruses, and fungi has been proposed to the present (1085-1088). 16a. microorganisms in the brain and oral cavity more than thirty years ago, borrelia burgdorferi was reported in the brains of sad cases (1089), although other studies did not validate this finding (1090). however, spirochetes, including borrelia and treponema, were postulated as contributing factors in the pathogenesis of the disease (1091-1094). rrna sequencing has shown the presence of bacteria in alzheimer’s post-mortem brain (1095). β-amyloid exhibits potent antimicrobial activity against candida albicans and some bacteria, pointing to the possibility that secretion of aβ is triggered by microbial infection (1096). numerous periodontal bacteria have also been implicated in sad in the context of chronic periodontitis (1097-1099). the association between periodontal inflammatory disease and sad has raised much concern mainly because of the possibility of rapid therapeutic intervention (1100-1102). increased levels of anti-helicobacter pylori-specific antibodies were also reported in the csf and serum of sad patients (1099). histological and immunohistochemical techniques have detected fungi and bacteria in the brain of sad (1103-1105). pcr analysis revealed various fungal species in the brain and csf in sad (1106-1108). a combination of bacteria and fungi was observed in several cases, and several fungi were identified in different brain regions in the same individual, suggesting multi-fungal infection (1107). the corpora amylacea are particularly enriched in fungal and bacterial peptides, acting as scavengers of microbial debris (1109). the presence of fungi is not restricted to sad as they have also been identified in the cns and csf in amyotrophic lateral sclerosis (1110, 1111). herpes viruses were proposed as participants in the pathogenesis of sad about four decades ago (1112, 1113). since then, several studies have implicated herpes viruses in sad (11141116). herpes simplex virus 1 (hsv-1) has received much attention due to the presence of high expression levels in the brain and hsv-1 igg antibodies in the csf (1117-1121). interestingly, hsv-1 dna is localized in senile plaques (1122). the link between herpes zoster and sad is still controversial (1123). human cytomegalovirus (cmv) seropositivity was associated with a risk of sad (1124). human herpes virus 6 (hhv-6) has focused on several studies (1125-1128). viral associations have also been reported, including hsv-1 and cmv, hsv-1 and hhv-6, hhv-6 and ebv (epstein-barr virus), and hhv-6 and hhv-7 (1115, 1126, 1129, 1130). a relationship is established between β-amyloid and herpes virus, suggesting a mechanism against brain infection (1131). since differences in sad and controls are not always significant, it is still difficult to conclude that a direct causal relationship exists between herpes virus and sad. the hypothesis of multi-pathogen infections in the pathogenesis of sad has recently been discussed (1085). moreover, exposure to systemic infections in 5xfad transgenic mice, carrying mutated genes associated with fad, causes neurodegeneration in brain regions displaying β-amyloid pathology and high local microglia density (1132). together, the available information shows that the human brain, particularly the aged brain, accommodates viruses, bacteria, and fungi, together with the debris of these microorganisms over time. whether brain and oral microorganisms might contribute to “neuroinflammation” and facilitate the course of sad remains obscure. nonetheless, a recent review postulates a link between the β-amyloid cascade hypothesis and chronic infection in sad: β-amyloid deposition is produced in response to brain microbial inflammation (1133). 16b. gut microbiota in recent years, several studies have implicated the “gut-brain-microbiota axis” in the pathogenesis of sad; altered gut microbiota may promote neuroinflammation, aβ aggregation, and oxidative stress (1134-1140). the putative mechanisms producing effects include changes in the systemic metabolism that may influence brain functions, transfer of microbiota metabolites to the brain through the bbb, and direct impact of microbiota reaching the brain parenchyma. serotonin may play a role in the gut-microbiota axis (1141). the opioid system has been postulated to be contributory as well (1142). conversely, the infusion of tracers into the lateral ventricle of rats has permitted their visualization in the nasal cavity, nasal pharynx, soft palate, and esophagus, thus suggesting that at least rats can swallow waste from the brain (1143). 17. seeding and spreading of β-amyloid and tau 17a. seeding β-amyloid the intracerebral injection of diluted extracts from ad brains or from old ad-like transgenic mice accelerates β-amyloid deposition in transgenic mice bearing app and/or psen1 mutations (1144-1148). soluble forms of aβ are particularly effective at inducing plaque formation (1149). plaques not only develop locally but extend as well to the cerebral cortex distant from the injection site. these findings support the possibility that once the first β-amyloid deposits appear they may accelerate the accumulation of additional seeding in other parts of the brain, thereby contributing to the exponential deposition of β-amyloid (1150). neuronal activity regulates the regional vulnerability to β-amyloid deposition (1151). β-amyloid seeding and spreading may also occur following peripheral inoculation of β-amyloid seeds (1152). however, the intracellular mechanisms linked to the formation and fibrillisation of host β-amyloid and recruited β-amyloid spreading need clarification. intraventricular injection of aβ oligomers in cynomolgus macaques leads to diffusion into the brain, causing tau hyperphosphorylation, nft formation, synaptic loss, and astrocyte and microglial activation in regions of the macaque brain where aβ oligomers are abundant (1153). β-amyloid deposits were also observed in the brains of patients with iatrogenic creutzfeldt-jabob disease (icjd) secondary to cadaveric dura mater grafts, treatment with cadaveric human growth hormone obtained from hypophysis of cjd-affected donors, or contaminated neurosurgery early in life (1154). 17b. tau seeding physiological release of neuronal tau is stimulated by neuronal activity and extracellular tau in vivo (1155-1158). intercellular tau transmission may have physiological functions that permit the transfer of tau status information from one neuron to another, enabling them to modify the status of the host tau according to this information. this mechanism may lie behind the activation of post-translational modifications of tau in the host neuron following the transfer of pathological tau in models of tau seeding and spreading. tau is secreted by exosomes, and the extracellular appearance of tau does not depend on neuronal death, but rather physiological tau transfer from one neuron to another (1159). tau phosphorylation facilitates tau transmission and propagation (1160). several studies have suggested that the progression of sad and other neurodegenerative diseases with abnormal protein aggregates occurs in a similar way to prions in prion diseases (1161-1164). in favor of this hypothesis, many experimental designs in mouse and rat models have provided evidence that cells have the capacity to transfer tau from one cell to another. the over-expression of human tau p301l restricted to the entorhinal cortex shows the progression of tau without detectable transgene expression anterogradely from the entorhinal cortex to the dentate gyrus and ca1 region of the hippocampus and subiculum, and retrogradely to scattered neurons in the perirhinal and secondary somatosensory cortex (1165, 1166). using a lentiviral-mediated rat model, tau protein is axonally transferred from hippocampus neurons to neurons of distant brain regions such as the olfactory and limbic systems (1167) several mechanisms of tau release and uptake have been proposed, mainly in the context of synaptic transmission. however other processes may occur as well. tau release may occur via (snap: soluble nsf attachment protein receptor) snare-mediated exocytosis, release by secretory vesicles from lysosomes, microvesicle shedding, and direct plasma membrane crossing. tau uptake may occur via endocytosis, adsorptive endocytosis, macropinocytosis, transmembrane diffusion, and nanotunneling. binding of phosphorylated tau to ampa and nmda receptors is another putative mechanism (1168-1179). tau seeding and spreading is produced following the intracerebral inoculation of synthetic tau fibrils (1180, 1181), and the inoculation of fibrillar-enriched fractions from human and mouse brain homogenates of tauopathies, including sad, containing hyper-phosphorylated tau (1182-1186). tau seeding and spreading in neurons also occur following intracerebral inoculation of similar tau aggregates in wild type mice (wt) and in transgenic wt mice (1183, 1187-1194). propagation in these models occurs through connectivity rather than proximity (1182). in addition to neurons, deposits may also occur in astrocytes, and the morphology of glial inclusions appears to mimic the glial aggregates of the corresponding human tauopathies (1183, 1188, 1189, 1195). differences in the type of inoculum that produce different protein aggregate deposits in the host and variable involvement of astrocytes have led to the proposal that tau strains are behind the different phenotypes and progression of human tauopathies (1161, 1196-1198). more refined situations probably occur in human neurodegenerative diseases. for example, sad brain contains different tau strains with particular properties (1199); different tau strains may contribute to the clinical heterogeneity of ad (1200). moreover, tau seeding and spreading also occur in oligodendrocytes in wt mice following inoculation of sarkosyl-insoluble fractions in the hippocampus and corpus callosum of human brain homogenates from sad and cases with primary age-related tauopathy (part) (1191-1193). yet, tau deposits in oligodendrocytes never occur in humans affected by sad and part without co-morbidities (see section 21 for part details). these discrepancies between human diseases and mouse models may be explained by the differences between human and murine tau (1201, 1202). whether different types of tau occur in neurons and glial cells, or even in different neuron populations granting selective vulnerability, deserves further research. tau seeding and spreading have also been generated in rhesus monkeys after the inoculation of adeno-associated virus expressing a double-4rtau mutation in the left hemisphere (1203). tau spreading was accompanied by robust trem2+ microglial proliferation (1203), an unexpected observation not seen in other models. another curious observation that deserves validation is the triggering of tau deposits in tau transgenic mice following peripheral administration of tau aggregates (1204). neuropathological studies in humans reveal the beginning of tau seeding in the telencephalon in sad in the transentorhinal and entorhinal cortex, and from these brain regions tau spreads to others (1205, 1206). neuroimaging analysis further support tau spreading along functional connectivity networks (1207). however, combined post-mortem regional seed amplification and pet studies in living individuals suggest that from nft braak stage iii onward, local replication rather than spreading between regions is the main mechanism to explain progressive tau burden in sad (1208). together, tau seeding and spreading differ from seeding and spreading of prions. grossly, tau seeding and spreading circumvent certain regions that should be involved according to the hypotheses of either neuronal connectivity or neuronal proximity. most importantly, once up-taken by the receptive neuron, abnormal tau triggers a series of molecular processes that include activation of several tau kinases, post-translational modifications of tau—for instance, hyperphosphorylation, nitration, and altered conformation; recruitment of additional substrates in tau deposits; and modifications in the ratio of 3rtau/4rtau in the host neuron (1209), which implies the capacity of foreign abnormal tau to modulate exon 10 splicing in mapt in the host (1190, 1191, 1202, 1209). 17c. multiple seeding foci of β-amyloid and tau pathology; vulnerable and resistant populations to tau seeding in brain aging and sad another important point is the characteristics of tau spreading in the human brain that seem to skip over obligate regions on the basis of cell connectivity. the dentate gyrus does not show tau pathology in ad and other 3r+4r tauopathies without co-morbidities, but the dentate gyrus is affected in 3r-tauopathies and 4r-tauopathies. alternative pathways from the entorhinal cortex to the hippocampus proper are possible but there is no clear explanation for this particular pattern in ad. it may be suggested that certain populations are able to transfer abnormal tau from one neuron to the next in the connecting pathway without recruiting host tau and forming local aggregates within themselves. regional vulnerability plays a cardinal role in tau spreading in ad-related animal models (1210). in summary, tau seeding and spreading is not the only cause of tau progression in sad and other tauopathies. cellular vulnerability, including neuronal, astroglial, and oligodendroglial, together with regional vulnerability, already highlighted several years ago, are also key points to understanding tau progression in sad and other tauopathies. finally, several studies implicate microglia in the process of tau seeding with apparent enhancing and mitigating effects (868, 1211-1216). another study concludes that microglia have a complex role; they are capable of taking up and breaking down seed-competent tau, but do so inefficiently and could hardly play a role in the spread of tau pathology (1217). the capacity for seeding of β-amyloid and tau does not imply a unique origin of β-amyloid and tau pathology in sad. neuropathological studies at early stages of nft and sp pathology have shown that independent tau deposits are localized in separate regions such as the raphe nuclei and locus ceruleus, transentorhinal and entorhinal cortex, and olfactory bulb, in addition to isolated tau-positive neurons in other parts of the brain. similarly, β-amyloid deposits in the brain parenchyma are not connected; likewise there is not an intimate connection of blood vessels affected by β-amyloid angiopathy. aβ and tau pathology, like other molecular alterations in brain aging and sad, have multiple and separate foci; lesions in aging and sad do not start in a single region only to progress from that site to the whole brain. 18. neuronal death neuronal depletion in ad is the result of cell death and lack of neuronal renewal. apoptosis was postulated as a major mechanism of neuronal death in ad and most neurodegenerative diseases based on the positivity of cells stained with the method of in situ end-labeling of nuclear fragmentation, and immunoreactivity to cleaved caspase 3 (1218-1220). caspase 3-mediated and caspase 3-non-mediated apoptosis is also supported by studies in cell lines (1221-1224). however, dna fragmentation occurs during the agonic state and post-mortem delay, and active caspase-3 participates in tau truncation during the genesis of nfts. therefore, the weight of apoptosis as a cause of neuron demise in ad is probably unevenly represented. necroptosis is another type of programmed cell death, frequently linked to inflammation that depends on the kinase activity of receptor interacting protein 3 (rip3) and rip1, and the subsequent activation of the mixed lineage kinase domain-like protein (mlkl). this pathway is activated in ad and other neurodegenerative diseases (1225-1227). neuronal degeneration and demise in ad is not correlated with tau pathology (1228, 1229), although ghost tangles are not rare in advanced stages of ad. intraneuronal accumulation of β-amyloid is involved in synaptic dysfunction, cognitive impairment, and the formation of amyloid plaques in ad (416, 1230, 1231). in addition, excessive production of aβ peptides and app activates death signaling pathways including apoptosis, necrosis, necroptosis, oxytosis (gsh-mediated oxidative cell death), pyroptosis (inflammasome-mediated cell death), and autophagy in vitro (1232). in summary, although β-amyloid and tau pathology may induce cell death in sad, they are not the only factors triggering active cell death pathways. other alterations, such as aberrant cell-cycle re-entry, energy metabolism failure, oxidative stress damage, and altered cell membrane signaling, are relevant contributory factors (see section 21). 19. neuronal connectivity networks in brain aging and sad the notion that clinical manifestations in brain aging may correspond solely to early stages of ad-related pathology restricted to the hippocampus, inner regions of the temporal cortex and selected nuclei of the brain stem is simplistic, and probably untrue. functional neuroimaging techniques have shown selective modifications of the intrinsic connectivity network in aging that exceed the domains of these regions. the default mode, salience, dorsal attention, fronto-parietal control, and auditory, visual and motor networks decline by middle adulthood, but the motor network shows increased connectivity in middle adulthood, followed by a lessening (1233-1237). disruption of neuronal connectivity networks appears in aging and is intensified in ad (1237-1243). however, functional connectivity alterations do not always correlate locally to tau and β-amyloid deposition; hyperand hypo-connectivity cycles can occur repeatedly at different stages of the disease (1244). moreover, greater segregation of functional connections into distinct large-scale networks is associated with cognitive resilience at early stages of sad (1245, 1246). additional studies suggest a compensation phase followed by a degenerative phase in aging, and in early, preclinical ad (1247). this is important information, as plasticity changes may circumvent, at least temporarily, deficient functioning during brain aging and early stages of sad. plasticity may explain the particular capacities of the adult brain to manage information in creating new networks, manifested as “experience”. considering the areas affected in functional neuroimaging studies, it may be inferred that molecular changes take place in the aging brain in regions other than those affected by tau and β-amyloid pathology. 20. human brain aging and preclinical ad the combination of clinical manifestations and complementary biomarkers has established consensus criteria to define different clinical phases of ad (see box 1). at preclinical stages, individuals may have measurable brain changes that indicate the earliest signs of ad (biomarkers), but they have not yet developed symptoms such as memory loss (https://www.alz.org/media/documents/alzheimers-facts-and-figures.pdf). the preclinical phase is further categorized into three stages. preclinical ad stage 1 includes cognitively normal individuals with abnormal β-amyloid markers and no neurodegeneration; stage 2, individuals with abnormal β-amyloid and neurodegeneration as revealed by magnetic resonance imaging (mri); and stage 3, individuals with abnormal β-amyloid deposition and neurodegeneration, and “subtle” cognitive changes (2, 48, 1248-1252). the refinement of the diagnosis of preclinical stages has improved with the acquisition of new neuroradiological methods, and optimization of csf and blood biomarkers (1253-1256). however, improvement in detection using csf, blood, and plasma biomarkers largely depends on the availability of optimized instruments and markers. current csf and plasma biomarkers used in the diagnosis of ad are levels of β-amyloid, phospho-tau, tau, and phospho-tau ratio, neurofilaments, synaptic proteins, markers of activated astrocytes, and inflammatory markers. at present, the available methods cannot detect differential levels of tau, phospho-tau, β-amyloid, and structural and synaptic proteins unless the degenerative process is at least at undetermined middle stages of ad neuropathological change. hippocampal atrophy, as revealed by computed tomography (ct) and mri, and currently used to correlate cognitive impairment and altered neuropathological substrate, is a late marker of ad that is only positive when there is advanced nft pathology and neuron loss in the hippocampus. a recent meta-analysis to estimate the prevalence of aβ pathology as measured with biomarkers in participants with normal cognition, subjective cognitive impairment, or mild cognitive impairment revealed that about 25-35% of cognitively normal older adults harbored a significant amount of β-amyloid (1257). further studies have shown that β-amyloid csf-based estimates using adjusted data-driven cutoffs are up to 10% higher than pet-based estimates in people without dementia thus suggesting that preclinical ad may be more prevalent than previously suspected (1258). in contrast, tau pet imaging using optimized tracers provides more precise information in vivo at the different stages of nft progression (855, 1259-1267). improved neuroimaging methods corroborate earlier neuropathological observations in post-mortem brains. individuals without cognitive impairment may have tau or β-amyloid positive signal alone, or β-amyloid and tau pathology, or tau and β-amyloid pet negativity (1268-1270). neuroimaging studies have also identified tau pathology in the entorhinal region and mesial temporal cortex in normal aged individuals with or without minimal cognitive impairment (1271-1276). tau pet observations are also useful to show that tau deposits appear before β-amyloid deposits in most individuals. finally, neuroimaging allows the visualization of a continuum of lesions from “normal” brain aging to alzheimer’s type dementia in sad and fad (1277, 1278). therefore, neuroimaging observations further suggest that tau pathology is predominant in normal brain aging that overlaps with tau pathology in individuals with preclinical ad. clinical criteria of ad are biased by the consideration that aβ pathology precedes tau pathology in sad. in other words, the conception of the β-amyloid cascade hypothesis permeates the diagnosis of sad. in addition to clinical and biomarker criteria, new neuropathological standards, based on the study of a wide number of selected brain regions stained with selected immunohistochemical markers, take into consideration ad pathology progression and the presence of comorbid pathologies were proposed by the national institute on aging–alzheimer’s association (nia-aa) in 2012 (49, 50). the main considerations are a) recognition that ad neuropathologic changes may occur in the apparent absence of cognitive impairment, (b) consideration of an “abc” score for ad neuropathologic change that incorporates histopathologic assessments of β-amyloid deposits (named a, based on thal phases), staging of neurofibrillary tangles (named b, based on braak stages), and scoring of neuritic plaques (named c, based on cerad), and (c) assessment of co-morbid conditions such as lewy body disease, vascular brain injury, hippocampal sclerosis, and tdp-43 proteinopathy; also including artag and agd. at present, the post-mortem neuropathological examination is still the most precise approach to identify the morphological changes linked to a brain aging and sad (49, 50, 325) (table 1). “a” thal phase for aβ plaques “b” braak and braak nft stage “c” cerad neuritic plaque score 0 0 0 none 0 none 1 1 or 2 1 i or ii 1 sparse 2 3 2 iii or iv 2 moderate 3 4 or 5 3 v or vi 3 frequent table 1: “abc” score for ad neuropathologic change modified from ref. 49 and 50. national institute on aging–alzheimer’s association (nia-aa) guidelines for the neuropathologic assessment of alzheimer’s disease. following this classification, the score levels of ad neuropathologic change are classified as “not”, “low”, “intermediate”, and “high” (table 2). the presence of aβ is mandatory. it is also stated that “medial temporal lobe nfts in the absence of significant aβ or neuritic plaques occurs in older people and may be seen in individuals without cognitive impairment, with mild impairment, or with cognitive impairment from causes other than ad” (49, 50). to support this assertion, a previous work is cited: “brains that have many nfts in medial temporal lobe structures (braak stage iii or iv) but no cortical sps may be a diagnostic dilemma; they also raise questions about the amyloid cascade hypothesis of ad in which nft development is thought to occur downstream of the development of amyloid plaques” (1279). more recently, the introduction of the term part has been used to name cases with nft pathology without sps (see section 21a). ad neuropathologic change b a c 0 or 1 2 3 0 0 not not not 1 0 or 1 low low low 2 or 3 low intermediate intermediate 2 any c low intermediate intermediate 3 0 or 1 low intermediate intermediate 2 or 3 low intermediate high table 2: “abc” score level of ad neuropathologic change. aβ/amyloid plaques (a), nft stage (b), and neuritic plaque score (c). the combination of a, b, and c scores is designated as “not”, “low”, “intermediate” or “high” ad neuropathologic change. “intermediate” or “high” ad neuropathologic change is considered sufficient for dementia. national institute on aging–alzheimer’s association (nia-aa) guidelines for the neuropathologic assessment of alzheimer’s disease. a key point of this neuropathologic classification is the notion that β-amyloid deposition is the first “sine-qua-non” condition to consider the possibility of ad. the occurrence of nfts without β-amyloid plaques is not consistent with ad at any stage. the neuropathological proposal of the national institute on aging-alzheimer’s association assumes the “amyloid cascade hypothesis” as the cause of sad. yet the cognitive status correlates with nft burden rather than with β-amyloid plaques (331). 21. primary age-related tauopathy (part), rapidly progressive sad, and sad resilience 21a. part in 2014, the term part, a common pathology associated with human aging, was proposed to categorize a subpopulation of individuals with normal cognition or with mci showing nft pathology at stages i-iv of braak and no sps, and slower progression to the clinical stage of dementia in some individuals (1280-1283). the concept of part derives from the interpretation of the “β-amyloid cascade hypothesis” as the cause of ad. in other words, it is assumed that the presence of nfts without aβ deposition is not ad and therefore, cases with nfts and without sps do not match with the neuropathologic assessment of ad proposed by the nia-aa. part includes the majority of individuals aged 65 or older with first stages of nft pathology and without sps. the individuals represent about 85% of human beings at the age of 65 (328, 329, 1284), and are considered clinically affected by “normal brain aging”. therefore, part appears to be a predominant disorder until the emergence of sps (41, 329, 336, 1284). once a substantial number of sps appears in the brain, ad is overriding, whereas dementia with only tangles (or tangle-predominant dementia) which would be the logical progression of part decays and becomes rare (1285). it has been stated that the frequency of part increases at the age of 85 years (1286). however, most individuals with nft pathology at stages i-iii without sps are younger in largest series of cases (41, 329, 1284). the distribution of tau pathology in ca1 and ca3 regions of the hippocampus is reported to be different in part and sad. however, in one series, the mean age of the individuals was about 88 but the nft stages varied in the samples with part and sad (1287). in another series (mean age 84.3 ± 9.4 years), tau pathology was high in both ca1 and subiculum, followed by ca2/3, entorhinal cortex, ca4, and dentate gyrus in sad. in part, the severity of tau pathology in ca1 and subiculum was high, followed by enthorinal cortex, ca2/3, ca4, and dentate gyrus (1288). differences appear to be minimal and were probably modified by the presence of tau pathology in sps in sad. the proposal of part as a new tauopathy is not widely accepted. part has also been interpreted as being within the spectrum of sad (a part of sad) in cases with a particular genetic background characterized by lower prevalence of apoeε4, ptk2b, bin1, and cr1 genes, and higher prevalence of apoeε2 (1282, 1289, 1290). in contrast, tangle-predominant dementia has been associated with mapt h1 haplotype (1291). 21b. rapidly progressive ad patients with rapidly progressive ad (rpad) are younger and have a median survival time after diagnosis of about 7-10 months (1292); the neuropathological hallmarks and peripheral biomarkers are similar to those seen in current sad excepting for a low frequency of apoeε4 allele and increased serum levels of specific pro-inflammatory cytokines (1293, 1294). yet aβ42 oligomers in rpad have distinct properties which promote the faster spread of aβ42 pathology (1295). the composition of sps also differs in rpad, with significantly higher levels of neuronal proteins, decreased levels of astrocytic proteins, and particular abundance of synaptic-derived proteins (1296). moreover, high-density oligomers of the prion protein and a significant 1.2-fold decrease in di-glycosylated prp isoforms occur in rpad. fifteen proteins appear to interact with prpc while only two proteins, 3/4histone h2b-type1-b and zinc alpha-2 protein3/4, are specifically bound with the prp isoform isolated from rpad cases (1297). abnormal prp isoforms in rpad are accompanied by altered localization of distinct interactors including the growth arrest-specific 2-like protein and associated end-binding protein 1, α-tubulin, and β-actin (1298). molecular profiles of β-amyloid proteoforms differ in rpad when compared with typical sad (1299). moreover, the presence of highly hydrophobic aβ seeds in rpad brains that seeded reactions at a slower pace in comparison to typical sad has been validated (1299). another anomaly is the downregulation and dislocalization from the nucleus to the cytoplasm of the splicing factor proline and glutamine rich (sfpq), its colocalization with tia-1 in stress granules, and its association with tau oligomers in the brain of rpad (1300). 21c. sad resilience centenarians have resistance to sad or very low progression to advanced braak stages of tau and β-amyloid pathology (1301-1303). curiously, ad-related changes in the oldest-old population also show a particular neuropathological distribution including high densities of nfts, mainly in the hippocampus, without apparent major clinical deficiencies (1302-1307). multiple factors are associated with increased or reduced structural and behavioral patterns linked to cognition in the elderly and sad. the cognitive reserve, linked to educational and occupational acquisition, social networks, and leisure activities in later life, have a protective effect (1308-1312). glycolitic dysfunction reduces resilience (1313) whereas coffee and cacao favour protection from sad (1314). comorbidities, including tdp-43 proteinopathy and mesial sclerosis, reduce resilience (1315-1317). genetic factors also participate in cognitive resilience (1318, 1319). a greater number of genes apply in women (225, 1320). a top variant on chromosome 18 upstream of atp8b1 is significantly associated with methylation in prefrontal cortex tissue at multiple cpg sites, including one just upstream of atpb81, and present in individuals with unimpaired cognition (1321). a rare variant in the 3'-utr of rab10 is protective for sad (1322). hdac4, rest, and gi intracellular signaling are sad-specific pathways involved in regulating the onset of memory deficits (1323). molecular modulators of neuronal vulnerability, such as rar related orphan receptor b (rorb), and glial dysfunctions are also involved in neuronal vulnerability (1324). the myocyte-specific enhancer factor 2c (mef2c) is upregulated in a subpopulation of glutamatergic neurons in resilient individuals. over-expression of mef2a/c in the ps19 transgenic mouse model of tauopathy improves cognitive flexibility and reduces hyperexcitability (1325). the profiles of brain inflammatory responses also differ in resilient sad cases, with expression levels of chemokines decreasing and trophic factors increasing (860, 1326). yet a consensus is needed on how to define the concepts of cognitive reserve, resilience, and resistance linked to cognition in aged individuals (1327). the genetic and molecular factors linked to the ad spectrum are listed in table 3. fad mutations in the genes app, psen1 (presenilin1), and psen2; increased app dosage sad ε4 of apoe, lrp1, ldlr, interleukin 1a, clu, picalm, cr1, bin1, trem2, sorl1, adam10, abca7, spi1, pilra, msa4, cd2ap, and epha1 part lower prevalence of apoeε4, ptk2b, bin1, and cr1 genes, and higher prevalence of apoeε2 rpad low frequency of apoeε4 allele; increased inflammation; different aβ oligomers; different amyloid‑β proteoforms; different seeding capacities of β-amyloid; high-density prp oligomers; decreased prp di-glycosylated isoforms; specific prp isoform; altered localization of the growth arrest-specific 2-like 2 protein (g2l2), α-tubulin and β-actin; downregulation and dislocalization from the nucleus to the cytoplasm of sfpq, their colocalization with tia-1 in stress granules, and their association with tau oligomers resilient ad variant of chromosome 18 upstream of atp8b1; rare variant in the 3'-utr of rab10; mef2c upregulation in a subpopulation of glutamatergic neurons; decreased expression levels of chemokines and increased levels of trophic factors tangle-predominant dementia association with mapt h1 haplotype table 3: genetic and molecular factors linked to the ad spectrum (see sections 3, 4 and 21 for references). considering the previous data, and the preponderance of tau pathology over β-amyloid deposits at the first and middle stages of ad-related pathology (344), a modification to the nia-aa “abc” score level of ad neuropathologic is hereby proposed. the added value is the reflection of tau pathology at the same diagnostic value as β-amyloid (table 4). ad neuropathologic change a c b 0 0 0 or 1 2 3 0 0 low low intermediate 1 0 or 1 low low intermediate 2 or 3 low intermediate intermediate 2 any c low intermediate intermediate 3 0 or 1 low intermediate intermediate 2 or 3 low intermediate high table 4: modified “abc” score level of ad neuropathologic change. aβ/amyloid plaques (a), nft stage (b), and neuritic plaque score (c). the combination of a, b, and c scores is designated as “not”, “low”, “intermediate” or “high” ad neuropathologic change. “intermediate” or “high” ad neuropathologic change is considered sufficient for dementia. here, nfts are considered with equal relevance to sps in the progression from normal brain aging with nft changes, part, preclinical ad, and sad (including ad variants, rpad and adnc in centenarians). 22. biochemical changes beyond tau and β-amyloid at the the first stages of nft pathology in addition to the classical neuropathological hallmarks restricted to the inner region of the temporal lobe and selected nuclei of the brain stem which characterize the first stages of nft pathology, there is cumulative evidence of molecular and biochemical alterations in other regions in the same brains. several brain regions such as the frontal cortex, which does not show nft pathology at stages i-iii (and very rarely, if present, β-amyloid deposition at these stages), show altered lipid and protein membrane composition, abnormal mitochondrial function, oxidative stress damage, altered protein synthesis, activation of kinases, dysregulated protein phosphorylation, and abnormal inflammatory responses, among other disruptions. all of these alterations advance in parallel with the progression of nft pathology; most of them have implications in tau and β-amyloid pathology, and they all persist or increase at middle and advanced nft stages and sad. the terms nft and sad in these sections are those used in the original publications. 22a. aberrant cell-cycle re-entry, and altered adult neurogenesis the nervous system has plastic capacities and this is manifested in many ways in sad. one of these is the activation of pathways geared to activate cell cycle re-entry. however, neurons are post-mitotic cells that activate cell death programs at g1/a and g2/m points in response to cell cycle reactivation (1328). aberrant neuronal cell-cycle re-entry, as revealed by the expression of various proteins involved in the activation and progression of the cell cycle, is produced in a subpopulation of neurons in sad (1329-1340). the expression of cell-cycle-related proteins occurs before the appearance of nfts and sps and may trigger programmed cell death or the activation of kinases leading to oxidative stress damage, tau hyperphosphorylation, and activation of β-amyloid pathways (1341-1346). the expression of p75ntr in doublecortin (dcx)-immunoreactive dentate gyrus progenitors is reduced in ad and related transgenic models. the inoculation of pro-ngf neutralizing antibodies into the dentate gyrus restores memory performance of app/ps1 animals and significantly increases the percentage of dcx+ progenitors in the dentate gyrus of these animals, thus suggesting that impaired prongf-p75ntr signaling blocks adult neurogenesis in ad (1347). moreover, the prongf/p75ntr signaling pathway blocks adult neurogenesis and neuron cell death in ad (670-672). altered adult neurogenesis is not restricted to sad, as it occurs as well in the dentate gyrus in other neurodegenerative diseases such as huntington’s disease, amyotrophic lateral sclerosis, lewy body disease, and frontotemporal dementia (1348). 22b. brain lipids the brain is the second richest organ in lipids after the adipose tissue. cholesterol accounts for 20-25% of the total lipids in the plasma membrane of neurons; most local cholesterol is synthesized by astrocytes in the adult brain (1349). glycerophospholipids are the main phospholipid components ubiquitously found in cell membranes, and they are found in abundance in membranes from neural cells. lipids carry out, in addition to structural functions, the roles of mediators or second messengers. these are lipophilic molecules involved in signal transduction processes. lipid mediators are derived from the enzymatic degradation of glycerosphingolipids, sphingolipids, and cholesterol by phospholipases, sphingomyelinases, and cytochrome p50 hydroxylases, respectively. eicosanoids such as prostaglandins, leukotrienes, and lipoxins are derived from oxidation of the aa. docosanoids, including neuroprotectins, resolvins, and maresin, are mediators of docosahexanoic acid (dha). these mediators are important modulators of oxidative damage, inflammation, and apoptosis. other glycerophospholipid-derived lipid mediators are diacylglycerols (dags) phosphatidylinositol 1,4,5-triphosphates, platelet-activating factor, lysophosphatidic acid, and endocannabinoids (1350). degradation of sphingolipids also results in the generation of mediators, such as ceramide, ceramide 1-phosphate, sphingosine, and sphingosine 1-phosphate. these mediators are involved in differentiation, growth, cell migration, and apoptosis. cholesterol-derived lipid mediators, including 24and 25-hydroxycholesterol, produce apoptosis (1351-1354). the physical-chemical properties of the membrane bilayer and the chemical reactivity of fatty acids determine their susceptibility to oxidative damage (1355-1357). ros and reactive nitrogen species (rns) are more soluble in the fluid lipid bilayer than in the aqueous solution (1358, 1359). more importantly, polyunsaturated fatty acid (pufa) residues of phospholipids are very sensitive to oxidation (1360). as a result, neural cell membrane lipids become primary targets of oxidative damage and lipid peroxidation. in the aging brain, there is a progressive decrease in the levels of cholesterol, phosphatidylethanolamine, phosphatidyl inositol, phospholipid, ethanolamine plasmalogen, and sphingomyelin (1361-1366). increasing age is associated with progressive modifications in the composition of pufa, including dha and aa levels (1367). the age-related reduction in pufas is inversely correlated with stearoyl-coa desaturase expression and activity, resulting in higher levels of monounsaturated fatty acids (mufas). lipofuscin located in secondary lysosomes increases with age in neurons and glial cells (1368, 1369). the “aging pigment” is composed of two-thirds protein and one third lipids (1370, 1371). proteins in lipofuscin belong to the cytoskeleton, mitochondrial bioenergetics, synapse, and membrane receptors (1372). this proteome is practically identical to the proteome derived from lipoxidation reactions identified in the frontal cortex of aged humans (1373). levels of glycerophospholipids, sphingolipids, dags, and plasmalogens are altered in the brain in sad (1374-1379). the disturbance of human brain lipid content in sad pathology may be categorized into four main groups (i) decreased expression of phospholipids, specifically plasmalogen pe and plasmalogen pc, due in part to abnormal peroxisome activity; (ii) reduced sulfatide content; iii) increased levels of ceramides; and (iv) increased lipoxidative stress (1354). plasmalogen pe plays a particular role as anti-oxidant (1380). moreover, in gray matter, the major ppe molecular species are enriched in dha and aa. modifications in dha, aa, and pufas in sad produce an imbalance between their protective role (the adaptive responses derived from their lipid mediators) and a deleterious role (derived from their susceptibility to oxidation) (1381, 1382). lipid peroxidation is an early event and a major cause of oxidative stress damage in sad progression (1373, 1381). β-amyloid plays an important part since sps are always surrounded by oxidized lipids, as revealed using fourier transform infrared microscopy (1383). one mechanism to modulate oxidative damage is mediation by the upregulation of dha synthesis, which occurs in selected regions at early stages of sad; this is followed by an adaptation, and then decreased dha contents. this change is not uniform, but rather regionand stage-dependent. dha content in sad progression is like a shock wave manifested in three steps: upregulation, adaptation, and depletion. crucial information connecting lipid alterations and sad comes from genetic data. in addition to allele ε4 of apoe (268-271, 1384, 1385), many genes involved in sad are linked to cholesterol and lipid metabolism (284, 1386-1391). most of them increase the risk of β-amyloid deposition but apoeε2 allele appears to decrease it (1392, 1393). 22c. lipid rafts and cell membranes lipid rafts are microdomains of cell membranes enriched in glycosphingolipids, cholesterol, and protein receptors which favor multiple cell signaling interactions at the cell membrane, necessary for signal transduction (1394, 1395). the exoplasmic leaflet is enriched with glycosphingolipids and sphingomyelin, the cytoplasmic leaflet is enriched with glycerolipids; cholesterol is present in both. the lipid composition makes this system highly dynamic in that several proteins act as structural proteins, transmembrane signaling proteins, and protein anchors linked to protein-protein interactions (1396). cholesterol in lipid rafts facilitates the clustering of αand β-secretases. although cholesterol does not have a direct effect on γ-secretases, the increment of local cholesterol facilitates the production of β-amyloid (1397, 1398). the lipid composition of membranes is altered in sad. β-amyloid oligomers and peptides are recruited in lipid rafts (1399-1402). altered lipid raft composition also occurs in app/ps1 double-transgenic mice, a model of familial cerebral β-amyloidopathy. altered composition of lipid rafts is observed at the age of three months in parallel with the appearance of the first plaques in these mice (1403). prion protein expression in sps further supports alteration of lipid raft-enriched cell membrane in association with β-amyloid deposition at advanced stages of sad (128, 1404, 1405). lipid raft alteration in ad does not occur abruptly. the composition of lipid rafts changes with age in a gender-dependent manner. the main changes affect levels of plasmalogens, polyunsaturated fatty acids (especially dha and aa), total polar lipids (mainly phosphatidylinositol, sphingomyelin, sulfatides, and cerebrosides), and total neutral lipids (particularly cholesterol and sterol esters) (1406). importantly, the lipid composition of lipid rafts in the entorhinal cortex and frontal cortex, but not the cerebellum, is already altered in human brain at nft stages i-ii without β-amyloid deposits; lipid rafts at these stages display higher anisotropy, indicating that lipid changes in brain at nft stages i-ii increase membrane order and viscosity in these domains (1407). among other alterations, the structure of lipid rafts at stages i-ii is associated with increased bace1/aβpp interaction (1408). there is also a close functional relationship between cytoskeletal proteins and cell membranes through protein-protein interactions, electrostatic interactions with lipid membranes, and lipid tails. these complementary interactions are ruined once one of the components is altered. altered composition of lipid rafts and membrane proteins increases aβ pathways and tau fibrilisation. at the same time, tau monomers and β-sheet-rich tau structures disrupt cell membranes (156, 212, 1389, 1409-1413). tau connections with lipid tails depend on electrostatic interactions and phospholipid composition with high affinity for anionic lipids and anionic vesicles (1414, 1416). tau monomers may concentrate at the membrane and form oligomers and fibrils under pathological conditions. these β-sheet-rich tau structures are capable of disrupting membrane organization and function (212). deposition of abnormal tau at the preand post-synaptic membranes may appear prior to the appearance of nfts, and it contributes to early synaptic dysfunction (409). conversely, there is also the possibility that alterations at the cell membrane involving the lipid composition and post-translational modifications of membrane proteins trigger abnormal phosphorylation of tau and induce formation of tau fibrils (1417) (see section 21h). plasma membrane specializations containing caveolin are invaginated and form caveolae which are closely related to lipid rafts (1418). caveolae in sad participate in a wide number of processes including internalization of tau oligomers and β-amyloid metabolism (1419-1421). other factors influence membrane damage, in particular, microglial pro-inflammatory mediators generating membrane damages (1422). 22d. mitochondria mitochondrial alterations were identified in the pioneering ultrastructural research of ad and were later sustained by functional studies (1423-1427). mitochondrial alterations in sad led to the formulation of the “mitochondrial cascade hypothesis”, proposing that mitochondrial alterations and failed energy metabolism trigger sad (1428, 1429). recent reviews have stressed the importance of mitochondrial failure in the pathogenesis of ad (1430-1432). functional defects in atp-synthase are considered a main contributory factor to explain failure of energy production in mitochondria (1433). other studies have stressed the impairment of complex i in mild cases of ad (1434). besides energy metabolism deterioration, abnormal mitochondrial function also causes increased production of ros (1435-1437). mitochondrial atp-synthase is a target of oxidative damage in the entorhinal cortex at stages i-ii of nft pathology; total levels of atp-synthase are preserved but atpase function is impaired (1438). altered mitochondrial dna methylation is manifested in the entorhinal cortex at stages i-ii of nft pathology and advances with disease progression (1028). increased mitochondrial 5-methylcytosine levels are found in the d-loop region of mtdna in the entorhinal cortex. interestingly, this region shows a dynamic pattern in the content of mitochondrial 5-methylcytosine in app/ps1 transgenic mice in parallel with the progression of β-amyloid pathology in these mice (1028). aβ deposits have been considered a main cause of mitochondrial dysfunction in ad (1439). additionally, mitochondrial abnormalities are observed in tau transgenic mice (1440). aβ and tau pathology have synergistic effects on mitochondria in triple transgenic mice (1441). it has been proposed that there are mitochondrial links between brain aging and ad (1442). the mitochondria-associated lipid raft-domain of the er in close contact with the mitochondria, called mam (mitochondria-associated er membrane), facilitates functional and biochemical interaction between these structures, mainly linked to metabolism of the cholesterol, phospholipids, glucose, fatty acids, and calcium signaling (1443, 1444). mam functions are altered at early stages of sad (1445, 1446), thus prompting the “mam-hypothesis” as a determinant in the pathogenesis of sad (1447). there is growing evidence of impaired physical and proteome crosstalk between er and mitochondria in sad (1444). 22e. oxidative stress damage the mitochondria respiratory chain generates ros which participate in cell signalling under physiological conditions. peroxisomes, er, microsomes, nucleus and plasma membrane are potential sources of ros. excess production of ros and deficient anti-oxidant responses lead to oxidative stress damage to dna, rna, carbohydrates, lipids, and proteins. ion-catalyzed oxidation of some amino acid residues may result in the production of carbonyl derivatives such as glutamic semialdehyde and aminoadipic semialdehyde. in addition to direct effects, oxidative modifications may induce the production of reactive carbonyl species such as glyoxal, glycoaldehyde, methylglyoxal, malondialdehyde (mda), and 4-hydroxynonenal (hne), derived from the oxidation of carbohydrates and lipids. carbonyl species react with lysine, arginine, and cysteine residues, leading to the formation of advanced glycation and lipoxidation end-products (age/ales) in proteins. typical ages/ales adducts are carboxymethyl-lysine (cml), carboxyethyl-lysine (cel), and mda-lysine (mdal), among others (1357, 1373, 1381, 1448, 1449). regarding rns, nitric oxide damage to thiols, amines, and hydroxyls leads to nitrosative damage. reactions with rns lead to the formation of 3-nitrotyrosine (nitration) and to oxidation of distinct substrates. reactive peroxinitrite is able to nitrate tyrosine residues and to oxidize methionine residues of proteins (1450). oxidative stress damage is a major component of brain aging. protein oxidative and glycoxidative damage significantly increases with age; 60 years of age is the breakpoint of human frontal cortex aging (1451). oxidative stress damage in the aging brain is regionand age-dependent (1452). regional vulnerability to neurodegeneration based on energy demands, oxidative stress, and other metabolic factors can predict neurodegeneration (1453). for example, lower energy demand, lower mitochondrial stress, and one-carbon metabolism (particularly restricted to the methionine cycle), together with lower target of rapamycin (tor) signaling and better antioxidant capacity, occur in the frontal cortex compared with the entorhinal cortex and the hippocampus. these differences suggest that the frontal cortex is relatively resistant to stress compared to the entorhinal cortex and hippocampus (1454). however, this hypothesis does not apply universally when assessing different brain regions of individual brains in parallel (1455). the results are more complex and their interpretation is more difficult. vulnerable cortical and diencephalic regions are in fact more resistant to degeneration in aging (1455). however, differences in vulnerability to protein oxidation are dependent on the subcellular localization, secondary structure, and external exposition of certain amino acids. lipoxidized proteins are mainly those involved in energy metabolism, cytoskeleton, proteostasis, neurotransmission, and o2/co2/heme metabolism (1452). ages are produced in aging and sad, but their levels and the levels of their receptor (rage) do not correlate with aβ levels, tau levels, or dementia (1456, 1457). most studies of oxidative stress damage in sad are centered on stages v-vi (cases with dementia) and iii-iv (cases with mci). proteins involved in glycolysis and energy metabolism, in electron transport chain, oxidative phosphorylation, and other mitochondrial components; structural proteins; chaperones; stress proteins, and stress responses; and proteins of the ups are all targets of oxidative damage (1373, 1458-1467). protein oxidative damage is usually accompanied by decreased functional activity (1468). altered enzymatic activity has been demonstrated for the oxidized protein creatine kinase bb, enolase 1, glutamine synthetase, pin-1, carbonic anhydrase 2, uchl-1, α-enolase, gapdh, gdh, h+ transporting atpase, ldh, atp synthase, and pyruvate kinase in sad. a few studies have included the identification of oxidatively damaged protein, the quantification of total protein levels, and the reduction of enzymatic activity (1381). in an attempt to correlate oxidative stress damage and regional vulnerability in sad, a meta-analysis of mda, hne, protein carbonylation, 8-hydroxyguanine levels and superoxide dismutase, glutathione peroxidase, glutathione reductase, and catalase activities showed that changes linked to oxidative stress were variable from one region to another and dependent on the type of adduct. no correlation was seen between oxidative damage and regional vulnerability (1469). oxidative damage was advocated to be the earliest event in sad (1470). further research revealed that oxidative damage was more marked in younger cases, in cases with rapid disease progression, and in neurons without nfts when compared with neurons with tangles in the same individual (1471). moreover, oxidative stress precedes β-amyloid deposition in pre-symptomatic fad and down syndrome (1472, 1473). early observations suggested that oxidative post-translational modifications might play a role in the formation of sps and nfts (1474). following this argument, aggregation of aβ and tau was considered a compensatory response to underlying oxidative stress (1475). however, β-amyloid is also a cause of oxidative stress, thereby potentiating the loop (1476). oxidative stress generates mitochondrial dysfunction by damaging structural proteins and components of the mitochondrial respiratory chain in sad (1438, 1477-1479). oxidative damage also causes synaptic dysfunction (425). as indicated in previous paragraphs, oxidative damage of atp-synthase resulting in the loss of its function occurs at stages i-ii of nft pathology (1438). mitochondrial dysfunction, abnormalities in lipid rafts, and oxidative stress damage potentiate each other and are major players in neuronal energy failure at the first stages of nft pathology (1480). another relevant consequence of oxidative stress is the effect of advanced glycation end products on cell-cycle re-entry and arrest, also occurring at the first stages of sad (1481). 22f. inflammation aging is accompanied by low levels of activated innate inflammatory responses (1482, 1483). the role of microglia and inflammation in the aging brain and sad has been discussed in previous paragraphs. here, the focus is on the relevance of inflammatory changes in brain aging and at early stages of ad pathology. several reports in the 1990s described a protective effect of non-steroidal anti-inflammatory drugs used for the treatment of autoimmnune diseases on the manifestation and progression of sad (1484-1490). further studies delineated their positive effects when the treatment was initiated before the appearance of cognitive impairment, but the effects of the same treatments were minimal when administered in advanced ad. these observations show that inflammatory responses modulate the history of ad at the early stages of the process. more importantly, there is also a chance for anti-inflammatory drugs in the treatment of sad when administered at the appropriate time (1491-1493). molecular studies disclosed that multiple cytokines are involved at the early/middle stages of ad (1494). more detailed studies in ma and in cases at first (i-ii), middle (iii-iv), and advanced (v-vi) nft stages examined the expression of cytokines and mediators of the immune system in different regions progressively affected in sad: the entorhinal cortex, orbitofrontal cortex, and frontal cortex area 8 (838). changes in mrna expression correlated with the corresponding protein levels as revealed by immunohistochemistry and western blotting (838). moreover, gene regulation at first stages of ad pathology (nft i-ii, sps: 0) was not related to nfts, β-amyloid plaques, concentration of aβ40 and aβ42, or membrane-bound fibrillar β-amyloid in the frontal cortex (838). these observations do not contradict previous studies showing a relationship between membrane-associated β-amyloid and inflammatory changes in cases with more advanced preclinical ad and sps in the cerebral cortex (101), but they highlight the observations that: (a) inflammatory markers appear at the first stages of nft pathology (stages i-iii) in regions with no nfts and sps; (b) inflammatory changes are modified with disease progression; and (c) different inflammatory responses occur simultaneously in different regions in the same individual. 22g. protein synthesis impairment protein synthesis is altered in sad, and this is due to multiple alterations at different subcellular levels from the nucleolus to the ribosome. the relation nuclear organizer region (nor) surface/total nucleus surface is reduced; the rdna promoter is hyper-methylated, and dimethylated histone h3k9 and acetylated histone h3k12 are decreased in the ca1 region of the hippocampus; nuclear tau declines; specific transcription factors are abnormally regulated; rrna levels are decreased and rna is oxidatively damaged; the expression of nucleolar proteins as well as the expression of rnas involved in the generation of ribosomal proteins decreases; the expression levels of translation initiation and elongation factor of the protein synthesis in ribosomes is dysregulated; and the capacity of isolated ribosomes to incorporate s35 methionine into protein is impaired (1010, 1473, 1495-1506). tau protein disrupts nucleocytoplasmic transport in ad (1507), but the role of tau, if any, in the other steps of the protein synthesis pathway is not known. alterations of protein synthesis pathways are already identified in the hippocampus at the first stages of nft pathology. nucleophosmin 1 (npm1) mrna is significantly increased, and upstream binding transcription factor rna polymerase i gene (ubtf) mrna and 28s rrna significantly decreased in ca1, but not in the dentate gyrus at nft stages i-ii. dimethylated histone h3k9 (h3k9m2) immunoreactivity is reduced in neurons of the dentate gyrus and ca1 at nft stages i-ii. mrna expression of ribosomal proteins rpl23a, rpl26, rpl31rps5, rps6, rps10, and rps13 is significantly reduced in the dentate gyrus at stages i-ii when compared with ma individuals without nft pathology. in contrast, rpl5 and rpl26 mrna expression is increased, and rps5 and rps6 mrna decreased in ca1 at the same stages i-ii (1508). these results show early alterations not only in the ca1 region, which will be involved in nft pathology, but also in the dentate gyrus, which does not contain abnormal tau deposits at any time in the process. seven upregulated mirnas (mir-125b, mir-146a, mir-200c, mir-26b, mir-30e, mir-34a, and mir-34c) and three downregulated mirnas (mir-107, mir-210, and mir-485), all of which associated with oxidative stress, are found in vulnerable brain regions of sad at the clinical prodromal stage (1501). together, these observations show that alterations in protein synthesis pathways appear at early stages of ad-related pathology and they are not linked to tau and β-amyloid deposits. disruption at many steps of the protein synthesis pathway increases in all regions with sad progression (1508), making it difficult to establish a causative relationship between tau and β-amyloid pathology and the progressive decay of protein synthesis at advanced stages of sad. 22h. dysregulated protein phosphorylation early phosphoproteomics studies identified a few abnormally phosphorylated proteins in the hippocampus and cerebral cortex in small numbers of cases with sad compared with controls (1509-1511). subsequent work with more accurate methods in advanced sad identified a large number of abnormally phosphorylated proteins, some of them with increased phosphorylation, and others with decreased phosphorylation. abnormally phosphorylated proteins corresponded to cytoskeletal proteins, integral membrane proteins, synaptic proteins, adhesion molecules, serine/threonine kinases, transport/cargo proteins, heat-shock proteins, and others mostly involved in cell growth and/or maintenance, cell communication, and metabolism (1417, 1513, 1514). multi-omics integration highlighted relevant altered networks including amyloid cascade, inflammation, complement, wnt protein signaling, transforming growth factor-β and bone morphogenic protein signaling, lipid metabolism, iron homeostasis, and membrane transport (1515). recent studies performed in the entorhinal cortex and frontal cortex in human brain aging and sad at different nft stages identified sixty-five dysregulated phosphoproteins in the entorhinal cortex, and eighty-one phosphoproteins in the frontal cortex at nft stages i-ii when compared with ma individuals without nft pathology. dysregulated protein phosphorylation of selected proteins occurs in parallel with the appearance of nfts in the entorhinal cortex but precedes the appearance of nfts and sps in the frontal cortex. the number of dysregulated phosphoproteins increases in both regions with nft stage, most of them added to those already dysregulated at stages i-ii. considering the total number of identified dysregulated phosphoproteins, the most active period corresponds to nft stages iii-iv, at a time when a subpopulation of people might be clinically categorized as suffering from mci (1417). the main group of dysregulated phosphoproteins at nft stages i-ii are membrane proteins; proteins of the cytoskeleton; proteins of the synapses and dense core vesicles; proteins linked to membrane transport and ion channels; kinases; proteins linked to dna and protein deacetylation; proteins linked to gene transcription and protein synthesis, and proteins involved in energy metabolism (1417). altered phosphorylation of selected proteins, accomplished by activation of several kinases, may alter membrane and cytoskeletal function, among these synaptic transmission and membrane/cytoskeleton signaling, in addition to energy metabolism, protein synthesis, and dna homeostasis (1417). dags are constituents of cell membranes that participate in intermediate lipid metabolism, and they are key components in lipid-mediated signaling. in neurons, dags modulate several signal transduction proteins linked to the activation of protein kinases, traffic and fusion of synaptic vesicles, ion channels, axonal guidance, and cytoskeletal homeostasis, among others (1516-1518). tau and β-amyloid phosphorylation may also be mediated by dags and pkc (1519). dag levels are increased in the sad frontal cortex (1520, 1521). further studies are needed to learn about possible links between dags and abnormal phosphorylation of cell membrane proteins. 23. concluding comments linear logic based on the assumption that a cause results in one or several effects does not explain complex biological phenomena such as brain aging and sad; there is no single cause of aging and sad. rather, the biological processes involved in generation, development, living, decline, and death are complex concatenations of complementary, disruptive, and adaptive responses. not surprisingly, the mutually exclusive hypotheses formulated to explain sad are not satisfactory. genetic studies in sad have also provided a wealth of information identifying genetic risk factors which principally cover cholesterol and lipid metabolism, inflammation, and cell membranes and synapses (1390). a scheme of the several factors involved in brain aging and sad is presented in figure 9. in addition to genetic factors, there is an interaction between molecular alterations in cellular structures that may favor the production of β-amyloid and abnormal tau. conversely, the presence of β-amyloid and abnormal tau has a negative effect on the majority of cellular structures, contributing to a detrimental loop in sad pathogenesis. figure 9: factors involved in brain aging and sad. in addition to genetic factors, molecular alterations in cellular structures may induce the production of β-amyloid and abnormal tau. conversely, the presence of β-amyloid and abnormal tau has a negative effect on the majority of cellular structures, contributing to a harmful loop in sad pathogenesis. molecular alterations are similar and cumulative in brain aging and sad. therefore, a continuum of brain aging and sad primarily based on an archetypal distribution of nfts usually followed by the deposition of β-amyloid plaques is a factual possibility. the pathogenesis and evolution of arteriosclerosis is another example of cumulative age-related degenerative changes in blood vessels. almost all people are affected to some degree by the age of 65. however, the first lesions in atherosclerosis, characterized by macrophage infiltration and intracellular lipid accumulation in the blood vessel wall, may occur in the first or second decade; inflammation, endothelial and perycite damage, altered metabolism of muscle fibers, extracellular lipid cores and atheromatous plaques develop later. clinical manifestations may appear at advanced stages of fibroatheroma, as well as complicated lesions in ma individuals. thus, the atheromatous plaque, the characteristic lesion of atherosclerosis, is not the first requirement to identify atherosclerosis as a biological process which is expressed by cumulative stages of blood vessel damage leading or not to clinical manifestations of variable severity. searching possible therapies for sad has been constant over the years. cholinesterase inhibitors, nmda receptor antagonists, membrane protectors, anti-oxidants, and anti-inflammatory agents, all assayed at middle and advanced clinical stages of sad, have proven poorly effective. other treatments are directed to reducing β-amyloid or tau accumulation. several aβ-protein-targeted drugs, including β-secretase inhibitors, γ-secretase inhibitors and modulators, α-secretase activators, direct inhibitors of aβ aggregation, and immunotherapy have been assayed or are under different phases of clinical trial (1522). β-amyloid immunization in humans has been successful in the sense that sps are largely reduced in the brain of treated patients; yet the abundance of tau deposits and the progression of the disease are not substantially modified by β-amyloid immunization (719, 1523, 1524). the clinical improvement seen with β-amyloid immunotherapy has been null or negligible, or at best arguable (1525-1530). treatment with a bace1 inhibitor has yielded very limited neuropathological improvement (1531). trials with new generation aβ immunotherapy are in progress. following the same line of thinking, tau immunization, although putatively preventing tau accumulation, is unlikely to be a unique alternative treatment (1532-1534). various tau-based therapies have been developed or are under development. these therapies include the use of inhibitors of tau phosphorylation, glycosylation, and acetylation; microtubule stabilizers; inhibitors of tau aggregation; and anti-tau immunotherapy (1535-1538). various tau therapies based on active and passive immunization are effective in murine and primate models. however, some of these attempts have failed in sad (1539). although encouraging, it is not clear how such treatments will reduce the different forms of abnormal tau and decrease aβ burden. in short, there is no evidence that merely lessening the abnormal levels and deposits of tau or β-amyloid at middle and advanced stages of the illness will cure sad. therefore, it is crucial to re-consider the optimal age to start combined anti-tauand anti-β-amyloid-based treatments to combat two main representative components of ad. the identification of new putative targets for therapeutic intervention before the appearance of tau and amyloid deposits is a promising endeavor. gene therapy has also been assessed in transgenic mouse models with variable success using both vector-based therapies and genetically modified cell replacement (1540-1542). studies in humans are limited, at this time, to upregulated ngf in sad patients, with little evidence of benefit (1543-1545). exosomes are a class of membrane vesicles derived from endolysosomal compartment implicated in cell-cell communication by shuttling different lipids, protein, and rnas between cells. the use of shuttles including exosomes to deliver selected molecules into the cells is still at the early stages. the meager success of available therapies to curb the effects of human brain aging and sad is a matter of concern. besides disease modification and symptomatic therapies for sad (1546-1549), other relevant and long-term ventures may also be considered. one of these is a revision of animal models used to test sad therapies, because they have failed when applied to humans (1550). to make this clear: for 25 years, many ad-like mouse models have been “cured” in the lab, only for the treatments to fail in clinical trials in humans (1551, 1552). it seems that mice have the ability to remodel complex metabolic defects once one of the factors involved in the process is normalized. this does not occur in humans. these considerations are made yet more compulsory because a number of molecular function derangements take place before or concurrent with the appearance of β-amyloid and tau pathology morphologically manifested as nfts and sps. altered lipid and protein composition of cellular membranes accompanied by impaired subcellular cell signaling from the cell membrane to the er, mitochondrial membranes, altered synapses, altered mitochondria and energy metabolism, and reactivation and abortion of developmental programs leading to neuronal death, together with dysfunctional bbb and singular brain inflammatory expression, all point to sad as a human brain age-related disorder of convergent mechanisms that shatters brain self-organization (329, 1343, 1553, 1554). a great data analysis covering the tremendous body of information on ad must be updated in real time. we need to think of the coming decades as an opportunity to take advantage of the rapid growth of artificial intelligence (ai) and cell reprogramming. we will be able to redesign some aspects of the human brain in the near future using advanced technologies. to this end, we also need to identify the main targets and appropriate intervention times. timing is crucial since it will be difficult to reprogram molecular pathways in old-adult individuals that have already suffered brain deterioration. brain reprogramming would likely be undertaken before the beginning of the slow-pace functional decline in ma adults. brain reprogramming may cover different areas including brain dna editing, utilization of external electrical or wave-based signals to reduce energy consumption of basic neuronal networks, optimization of mitochondrial function, implanting of microdevices to facilitate cooperative human-machine function, pharmacological combined protection of lipid-protein interactions, and program resetting during stages of sleep. improvement of brain function in aging and sad has a chance in the application of high-throughput molecular technology, ai, and robotics (1394, 1555-1561). this new era is certain to present formidable ethical challenges. abbbreviations 2-ag arachidonoyl glycerol, 5-ht 5-hydroxytriptamine, 3’-utr complementary un-translated regions, aa arachidonic acid, aβ beta amyloid, abca7 atp binding cassette subfamily a member 7, aβos soluble β-amyloid oligomers, ac adenylyl cyclise, ach acetylcholine, ache acetylcholinesterase, ad alzheimer disease, adam10 adam metallopeptidase domain 10, addls amyloid-β derived diffusible ligands, adnc ad-neuropathologic change, aea n-arachidonoyl ethanolamine, agd argyrophylic grain disease, ages advanced glycation end-products, ago argonaute subfamily proteins, ai artificial intelligence, ales advanced lipoxidation end-products, amp adenosine monophosphate, ampa α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, ampar α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor, aphh1a α-secretase subunit a, apoe apolipoprotein e, app amyloid precursor protein, aqp aquaporin, artag aging-related tau astrogliopathy, asc inflammasome-adaptor protein, atf-6 activating transcription factor 6, atp8b1 atpase phospholipid transporting 8b1, atp adenosine triphosphate, bace β-secretase, β-ar β-adrenergic receptor, bbb blood brain barrier, bdnf brain-derived neurotrophic factor, bin1 bridging integrator 1, c1qtnf7 complement c1q tumor necrosis factor-related protein 7, c3ar1 complement c3a receptor 1, ca2+ calcium ion, camp cyclic adenosine monophosphate, cb calbindin d28k, cbf cerebral blood flow, cbr cannabinoid receptor, cd2ap cd2-associated protein, cdk5 cyclin-dependent kinase 5, cerad consortium to establish a registry for alzheimer’s disease, cel carboxyethyl-lysine, cgmp cyclic guanosine monophosphate, chat choline acetyl transferase, ck1-δ casein kinase i isoform delta, clu clusterin, cml carboxymethyl-lysine, cmv human cytomegalovirus, cnp 2',3'-cyclic-nucleotide 3'-phosphodiesterase, cns central nervous system, comt catechol-o-methyltransferase, cpg cytosine-guanine-rich regions , cr calretinin, cr1 complement component (3b/4b) receptor 1, creb camp response element-binding protein, csf cerebrospinal fluid, csf1r colony stimulating factor 1 receptor, csf3r colony stimulating factor 3 receptor, ct computed tomography, dag diacylglycerol, dbh dopamine β-hydroxylase, dcx doublecortin, dha docosahexanoic acid, dnmt dna methyl transferases, gsh glutathione, eaat excitatory amino acid transporter, ebv epstein-barr virus, eofad early onset alzheimer disease, eph ephrin, epha1 ephrin receptor a1, er endoplasmic reticulum, exrnas extracellular rnas, fad familial alzheimer disease, fad flavin adenine dinucleotide, fbd familial british dementia, fcγriib fcγ, receptor ii-b, fdd familial danish dementia, gaba γaminobutyric acid, gabaa receptors ionotropic gabaa receptors, gabab receptors metabotropic gabab receptors, gad glutamic acid decarboxylase, gap-43 growth-associated protein 43, gdh glutamate dehydrogenase, gfap glial fibrillary acidic protein, glurs glutamate receptors, glut 1 glucose transporter 1, gmp guanosine monophosphate, gpcr g-protein-coupled receptor, grp78/bip glucose-related protein 78, gsk3β glycogen synthase kinase 3β, gss gerstmann-sträussler-scheinker, gvd granulovacuolar degeneration, gwas genome-wide association studies, h3k4me1 methylation of lysine 4 of histone 3, h3k4m3 trimethylation of histone 3 at lysine 4, h3k9me2 dimethylation of histone h3 at lysine 9, hat histone acetyltransferase, hdac histone deacetylase, hdm histone demethylase, hhv-6 human herpes virus 6, hmt histone methyltransferase, hne 4-hydroxynonenal, hsv-1 herpes simplex virus 1, icjd iatrogenic creutzfeldt-jabob disease, ide insulin degrading enzyme, ifn-γ interferon γ, iglur ioinotropic glutamate receptor, il interleukin, ipsc indiuced pluripotent stem cells, ire 1 inositol-requiring protein 1, jak2 janus kinase 2, ka kainate, kar kainate receptor, lamp-1 lysosomal-associated membrane protein 1, lc3 protein light chain 3, lc-ms/ms liquid chromatography (lc) tandem mass spectrometry (ms), lcnrnas long non-coding rnas, ldh lactate dehydrogenase, ldlr low density lipoprotein protein receptor 1, lilrb2 pirb human orthologue receptor , load late onset alzheimer disease, lp2 lipoprotein lipase 2, lrp1 hldl receptor related protein 1, ma middle-aged, machr muscarinic acetylcholine receptor, mag myelin associated glycoprotein, mal myelkin and lymphocyte protein, mams mitochondria-associated er membranes, map2 microtubule associated protein 2, mapk mitogen-activated protein kinase, mapt microtubule associated protein tau, mapt h1 microtubule-associated protein tau haplotype 1, mao mono-amino oxidase, mbp myelin basic protein, mci mild cognitive impairment, mda malondialdehyde, mdal mda-lysine, mef2c myocyte-specific enhancer factor 2c, mg2+ magnesium ion, mglur metabotropic glutamate receptor, mirna microrna, mlkl mixed lineage kinase domain-like protein, mobp myelin-associated oligodendrocyte basic protein, mog myelin-oligodendrocyte glycoprotein, mri magnetic resonance imaging, msa4 membrane-spanning 4-domains subfamily a, mtrnas mitochondrial rnas, mufa monounsaturated fatty acids, nachr nicotinic acetylcholine receptor, nadþ nicotinamide adenine dinucleotide, nadpþ nicotinamide adenine dinucleotide phosphate, nct/ncstn nicastrin, nep/nme neprelysin, nft neurofibrillary tangle, ng2 neural/glial antigen 2, ngf nerve growth factor, nia-aa national institute on aging–alzheimer’s association, nlrp3r pyrin-domain containing 3, nmda n-methyl d-aspartate, nmdar n-methyl-d-aspartate receptor, nor nuclear iorganizer region, npm1 nucleophosmin 1, nrnas small non-coding rnas, opc oligodendroglial precurosor cells, part primary age-related tauopathy, p75ntr p75 neurotrophin receptor, low affinity nerve growth factor receptor, pdgfra platelet-derived growth factor receptor a, pen2/psenen presenilin enhancer γ-secretase subunit, perk pkr-like endoplasmic reticulum kinase, pet positron emission tomography, phf paired helical filament, pi3k phosphatidylinositol 3-kinase, pi3p phosphatidylinositol trisphosphate, picalm phosphatidylinositol binding clathrin assembly protein, pid pick disease, pilra paired immunoglobin like type 2 receptor alpha, pirb paired immunoglobulin-like receptor b, pirnas piwi-interacting rnas, pka protein kinase a, pkc protein kinase c, plc phospholipase c, plp1 proteolipid protein 1, ppar peroxisome proliferator-activated receptors, prnp prion protein gene, prpc cellular prion protein, psen1 presenilin 1, psen2 presenilin 2, ptk2b protein tyrosine kinase 2 beta, pufa polyunsaturated fatty acids, pv parvalbumin, rab10 ras-related protein rab-10, rage age receptor, rest re1-silencing transcription factor, rip3 receptor interacting protein 3, rns reactive nitrogen species, rorb rar related orphan receptor b, ros reactive oxygen species, rp ribosomal protein, rpad rapidly progressive ad, rrna ribosomal rna, sad sporadic alzheimer disease, sam s-adenosyl-l-methionine, sapk/jnk stress-activated protein kinase/jun n-terminal kinase, scarnas small cajal-body specific rnas, sfpq splicing factor proline and glutamine rich, sirnas small interfering rnas, snap soluble nsf attachment protein receptor, snare snap receptor protein, snrnaseq single nuclei rna sequencing, snrna small nuclear rnas, snornas small nucleolar rnas, sorl1 sortilin-related receptor 1, spi1 spi-1 proto-oncogene, sp senile plaque, stat1 signal transducer and activator of transcription 1, tet1 ten-eleven-translocation-1 protein, tgf transforming growth factor, tlr toll like receptor, tnf tumor necrosis factor, tor target of rapamycin, traf2 tnf receptor-associated factor 2, trem2 triggering receptor expressed on myeloid cells 2, trka specific ngf receptor, trna transfer rnas, ubtf upstream binding transcription factor rna polymerase i gene, uchl-1 ubiquitin c-terminal hydrolase l1, upr misfolded protein response, ups ubiquitin proteasome system, vgat vesicular gaba transporter, vglut vesicular glutamate transporter, vmat vesicular monoamino transporter, wm white matter, wt wild type, xbp-1 x-box binding protein 1. funding the project leading to these results received funding from the “la caixa” foundation (id 100010434) under the agreement lcf/pr/hr19/52160007, hr18-00452. i thank the cerca programme of the generalitat de catalunya for institutional support. acknowledgements this work is, in part, the result of the personal experience of 40 years in the study of ad and other tauopathies. to name all the people i would like to thank for their help, support, and satisfactory collaboration is practically impossible. i appreciate the aid of ciberned, and i am extremely grateful to tom yohannan for his continual editorial assistance. references duyckaerts c, dickson dw. neuropathology of alzheimer’s disease and its variants. in: neurodegeneration: the molecular pathology of dementia and and movement disorders; second edition. dickson dw, wetter ro (eds) international society of neuropathology, blackwell publish ltd, 2011, pp: 62-91. https://doi.org/10.1002/9781444341256.ch10 knopman d. clinical aspects of alzheimer’s disease. in: neurodegeneration, the molecular pathology of dementia and movement disorders. dickson dw, weller ro (eds) wileyblackwell, 2011, pp: 39-50. 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